CN112813082A

CN112813082A - Gene for expressing telomerase, recombinant plasmid, recombinant cell and application

Info

Publication number: CN112813082A
Application number: CN202011599625.6A
Authority: CN
Inventors: 奚绪光
Original assignee: French National Research Centre; Northwest A&F University
Current assignee: French National Research Centre; Northwest A&F University; Centre National de la Recherche Scientifique CNRS
Priority date: 2020-12-29
Filing date: 2020-12-29
Publication date: 2021-05-18

Abstract

The invention provides a gene for expressing telomerase, a recombinant plasmid, a recombinant cell and application, belonging to the technical field of genetic engineering; the genes include a first gene or a second gene, the first gene expressing a first telomerase; the second gene expresses a second telomerase; the amino acid sequence of the first telomerase is shown in SEQ ID NO. 1; the amino acid sequence of the second telomerase is shown in SEQ ID NO. 2. The first telomerase is derived from Candida tropicalis (Candida tropicalis); the second telomerase is derived from candida albicans (candida albicans). The gene for expressing the telomerase can be expressed in a large amount in escherichia coli, saccharomycetes and Sf9 cells, and the expressed telomerase has biological activity.

Description

Gene for expressing telomerase, recombinant plasmid, recombinant cell and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a gene for expressing telomerase, a recombinant plasmid, a recombinant cell and application.

Background

Telomeres are small DNA-protein complexes present at the end of the linear chromosomes of eukaryotic cells and serve to maintain chromosomal integrity and control the cell division cycle. The length of telomeres reflects the history and replication potential of the cell. Telomeres have important functions of maintaining chromosome stability and cell activity in cells of different species, and telomerase can prolong shortened telomeres (the shortened telomeres are limited in cell replication capacity), so that the proliferation capacity of cells in vitro is enhanced. Telomerase has important effects in keeping telomere stable, complete genome, long-term activity of cells, potential continued proliferation capacity and the like. The prior art discloses a technical scheme for expressing yeast telomerase in yeast, but the process of obtaining the yeast telomerase by the method has the technical problem of low yield.

Disclosure of Invention

The invention aims to provide a gene for expressing telomerase, a recombinant plasmid, a recombinant cell and application.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a gene for expressing telomerase, wherein the gene comprises a first gene or a second gene, and the first gene expresses a first telomerase; the second gene expresses a second telomerase; the amino acid sequence of the first telomerase is shown in SEQ ID NO. 1; the amino acid sequence of the second telomerase is shown in SEQ ID NO. 2.

Preferably, the nucleotide sequence of the first gene is shown as SEQ ID NO. 3; the nucleotide sequence of the second gene is shown as SEQ ID NO. 4.

The invention provides a recombinant plasmid containing the gene of the scheme.

Preferably, the backbone plasmid for construction of the recombinant plasmid includes pET15b-s μmo, pPICZA, pPICZaA or pTrix.

The invention provides a recombinant cell comprising the recombinant plasmid of the scheme.

Preferably, when the skeleton plasmid for constructing the recombinant plasmid is pET15b-s mu mo, the original cells of the recombinant cells comprise Escherichia coli; when the skeleton plasmid for constructing the recombinant plasmid is pPICZA or pPICZaA, the primary cell of the recombinant cell comprises yeast; when the backbone plasmid for construction of the recombinant plasmid is pTrix, the original cells of the recombinant cells include Sf9 cells.

The invention provides application of the gene or the recombinant plasmid or the recombinant cell in the scheme in preparing telomerase.

Preferably, when the original cell of the recombinant cell is Escherichia coli, the application comprises the following steps:

1) sequentially carrying out activation culture and amplification culture on the recombinant cells to obtain a bacterial liquid; OD of the bacterial liquid₆₀₀The value is 0.5 to 0.7; the culture mediums of the activation culture and the amplification culture respectively comprise LB liquid culture mediums containing ampicillin; the concentration of the ampicillin in the LB liquid culture medium is 80-120 mug/mL;

2) adding isopropyl thiogalactoside into the bacterial liquid until the molar concentration of the isopropyl thiogalactoside is 0.2-0.4 mM, and inducing telomerase to express for 12-18 h under the conditions of 100-200 rpm and 15-20 ℃ to obtain an inducing liquid;

3) centrifuging the inducing solution, and collecting the precipitate; dissolving the precipitate by using an imidazole aqueous solution to obtain a dissolved solution; the ratio of the volume of the imidazole aqueous solution to the mass of the precipitate is 8-12 mL: 1g of a compound;

4) crushing the dissolved solution under high pressure under the condition of 800-1200 bar, and then performing ultrasonic crushing under the condition of 300-500W to obtain a crushed solution;

5) centrifuging the crushed solution for 30-50 min at 10000-15000 rpm, collecting supernatant, filtering the supernatant by using a 0.45-micrometer filter head, and collecting first filtrate; and (3) loading the first filtrate to an NI chromatographic column, eluting effluent liquid by eluent, and collecting components corresponding to a third peak to obtain a crude extract containing telomerase.

Preferably, the high-pressure crushing time in the step 4) is 3-5 times, and the interval time between two adjacent high-pressure crushing times is 2-5 min; the ultrasonic crushing times are 2-4 times, the ultrasonic treatment time is 6-8 seconds, the interval is 2-4 seconds, and the ultrasonic treatment time is 2-4 min each time.

Preferably, the loading speed in the step 5) is 1 ml/min; the elution rate was 2 ml/min.

Preferably, after the crude extract is obtained in step 5), the purification of the crude extract is further included, and the purification method comprises the following steps:

6) mixing the crude extract with s mu mo enzyme, carrying out s mu mo enzyme digestion to obtain a first protein, adjusting the salt concentration of the first protein to 280mM, filtering by using a 0.45 mu m filter head, and collecting a second filtrate;

7) loading the second filtrate into an SP chromatographic column, eluting effluent liquid of eluent, collecting components corresponding to an elution peak, and performing SDS-PAGE electrophoresis, wherein the protein with the size of 100KD is a second protein;

8) reversely hanging the second protein by passing through an anti-EDTANI column at the speed of 1mL/min, and carrying out first concentration by adopting a 30KD concentration tube at the temperature of 1-5 ℃ and at the speed of 3000-4000 rpm until the volume after concentration is 0.5-1 mL to obtain a first concentrated solution;

9) loading the first concentrated solution to an S200 chromatographic column, eluting effluent liquid of eluent, collecting components corresponding to an elution peak, and performing SDS-PAGE electrophoresis, wherein the protein with the size of 100KD is a third protein;

10) and secondly concentrating the third protein by adopting a 30KD concentration tube at 1-5 ℃ and 3000-4000 rpm until the volume of the concentrated third protein is 0.5-1 mL, thus obtaining the purified telomerase.

The invention has the beneficial effects that: the invention provides a gene for expressing telomerase, wherein the gene comprises a first gene or a second gene, and the first gene expresses a first telomerase; the second gene expresses a second telomerase; the amino acid sequence of the first telomerase is shown in SEQ ID NO. 1; the amino acid sequence of the second telomerase is shown in SEQ ID NO. 2. The first telomerase is derived from Candida tropicalis (Candida tropicalis); the second telomerase is derived from candida albicans (candida albicans). The gene for expressing the telomerase can be expressed in a large amount in escherichia coli, saccharomycetes and Sf9 cells, and the expressed telomerase has biological activity.

Drawings

FIG. 1 shows the results of the crude purification by Ni column in example 2;

FIG. 2 shows the results of purification of SP column in example 2;

FIG. 3 shows the purification result of the S200 column in example 2;

FIG. 4 shows the concentration result of the purified protein on the S200 column in example 2;

FIG. 5 is the telomerase activity assay of example 3;

FIG. 6 is a diagram showing the results of electrophoresis of pPICZA vector in example 6;

FIG. 7 is a graph showing the results of electrophoresis of the pPICZaA vector of example 6;

FIG. 8 shows the results of NI column purification in example 6;

FIG. 9 shows the results of purification of SP column in example 6;

FIG. 10 shows the purification result of S200 in example 6;

FIG. 11 shows the concentration results in example 6;

FIG. 12 shows the results of the activity assay in example 6;

FIG. 13 shows the results of NI column purification in example 8;

FIG. 14 shows the results of purification of SP column in example 8;

FIG. 15 shows the purification result of S200 in example 8;

FIG. 16 shows the concentration results in example 8;

FIG. 17 shows the results of activity measurement in example 8.

Detailed Description

In the invention, the nucleotide sequence of the first gene is shown as SEQ ID NO. 3; the nucleotide sequence of the second gene is shown as SEQ ID NO. 4.

The first telomerase is derived from Candida tropicalis (Candida tropicalis); the second telomerase is derived from candida albicans (candida albicans). The gene for expressing the telomerase can be expressed in a large amount in escherichia coli, and the expressed telomerase has biological activity.

The invention provides a recombinant plasmid containing the gene of the scheme.

In the present invention, the backbone plasmid for construction of the recombinant plasmid preferably includes pET15 b-s. mu. mo, pPICZA, pPICZaA or pTrix.

In the present invention, when the backbone plasmid for construction of the recombinant plasmid is pET15b-s μmo, the gene is preferably inserted between EcoR I and XhoI cleavage sites of pET15b-s μmo; when the gene is the first gene, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 5; when the gene is the second gene, the nucleotide sequence of the recombinant plasmid is shown as SEQ ID NO. 6.

In the present invention, when the backbone plasmid for construction of the recombinant plasmid is pPICZA or pPICZaA, the gene is preferably inserted between NdeI and SalI cleavage sites of pPICZA or pPICZaA.

In the present invention, when the backbone plasmid for construction of the recombinant plasmid is pTrix, the gene is preferably inserted between NdeI and SalI cleavage sites of pTrix. The construction method of the recombinant plasmid is not particularly limited in the invention, and the conventional method in the field can be adopted.

In the present invention, when the backbone plasmid for construction of the recombinant plasmid was pET15b-s μmo, the original cells of the recombinant cells included E.coli; when the skeleton plasmid for constructing the recombinant plasmid is pPICZA or pPICZaA, the primary cell of the recombinant cell comprises yeast; when the backbone plasmid for construction of the recombinant plasmid is pTrix, the original cells of the recombinant cells include Sf9 cells. The method for constructing the recombinant cell is not particularly limited, and a conventional method in the art can be adopted.

In the present invention, when the original cell of the recombinant cell is Escherichia coli, the application comprises the steps of:

Preferably, the high-pressure crushing time in the step 4) is 3-5 times, and the interval time between two adjacent high-pressure crushing times is 2-5 min; the ultrasonic crushing is carried out for 2-4 times, the ultrasonic treatment is carried out for 6-8 s at intervals of 2-4 s, and the time of each ultrasonic treatment is 2-4 min.

According to the invention, firstly, a recombinant cell is required to be constructed, and after the recombinant cell is obtained, the recombinant cell is subjected to activation culture and expansion culture in sequence to obtain a bacterial liquid. In the invention, the temperature of the activation culture and the temperature of the amplification culture are respectively preferably 37 ℃, the rotating speed is respectively preferably 150-200 rpm, and more preferably 180 rpm; the activation culture time is preferably 5-8 h, and more preferably 6 h; the time of the amplification culture is determined by the OD of the bacterial liquid₆₀₀The value is 0.5-0.7, preferably 0.6; the culture mediums of the activation culture and the amplification culture respectively comprise LB liquid culture mediums containing ampicillin (Amp); the concentration of the ampicillin in the LB liquid culture medium is preferably 80-120 mu g/mL, and more preferably 100 mu g/mL.

After obtaining a bacterial liquid, adding isopropyl thiogalactoside (IPTG) into the bacterial liquid until the molar concentration of the isopropyl thiogalactoside is 0.2-0.4 mM, preferably 0.3mM, and inducing telomerase to express for 12-18 h under the conditions of 100-200 rpm and 15-20 ℃ to obtain an inducing liquid; the induction speed is preferably 150rpm, the temperature is preferably 18 ℃, and the time is preferably 15 h.

After obtaining the inducing liquid, centrifuging the inducing liquid, and collecting the precipitate; dissolving the precipitate by using an imidazole aqueous solution to obtain a dissolved solution; the ratio of the volume of the imidazole aqueous solution to the mass of the precipitate is 8-12 mL: 1g, preferably 10 mL: 1g of a compound; in the invention, the rotation speed of the centrifugation is preferably 4000-5000 rpm, preferably 4500rpm, and the temperature is preferably 18 ℃. In the present invention, the formulation of the aqueous imidazole solution is preferably: 500mM NaCl, 20mM Tris-HCl, pH 7.5, 10% glycerol, 5mM imidazole.

After a dissolving solution is obtained, crushing the dissolving solution at 800-1200 bar, preferably at 1000bar under high pressure, and then performing ultrasonic crushing at 300-500W, preferably at 400W to obtain a crushing solution; the high-pressure crushing frequency is preferably 3-5 (the escherichia coli can crack all cells after 3-5 times of high-pressure crushing, and the cells become clear after crushing compared with the cells before crushing), and more preferably 4 times; the interval time between two adjacent high-pressure crushing is preferably 2-5 min; the number of times of ultrasonic crushing is preferably 2-4, more preferably 3, ultrasonic treatment is carried out for 6-8 s at intervals of 2-4 s, and the time of each ultrasonic treatment is preferably 2-4 min, more preferably 3 min.

After obtaining the crushing liquid, centrifuging the crushing liquid for 30-50 min, preferably 40min, at 10000-15000 rpm, preferably 12000rpm, collecting the supernatant, filtering the supernatant by using a 0.45-micron filter membrane, and collecting a first filtrate; adding the first filtrate into an NI chromatographic column, eluting effluent liquid by eluent, and collecting components corresponding to a third peak to obtain a crude extract containing telomerase, wherein the step is used for carrying out crude purification on target protein; in the present invention, the loading speed is preferably 1 ml/min; the NI chromatography column preferably has a size of 5 mL.

In the specific implementation process of the invention, after the first filtrate is loaded to the NI chromatographic column, 50ml of 5mM imidazole binding buffer (imidazole binding solution: formulation: 500mM NaCl, 20mM Tris-HCl, pH 7.5, 10% glycerol, 5mM imidazole), 20-fold volume of 30mM imidazole Washbuffer (imidazole Elution solution), and 5% to 80% B (B: 500mM imidazole Elution buffer) are sequentially added, wherein the formulation is 500mM NaCl, 20mM HCl, pH 7.5, 10% glycerol, 500mM imidazole) are delayed by 20-fold volume (100ml), the Elution speed is preferably 2ml/min, and 1.8ml/tube receives each collection component.

After the crude extract is obtained, the method adopts the s mu mo enzyme to be mixed with the crude extract for s mu mo enzyme digestion to obtain a first protein, the salt concentration of the first protein is adjusted to 280mM, a 0.45 mu m filter head is adopted for filtering, and a second filtrate is collected, wherein the step is used for cutting off a label at the front end of a target protein; the reagent used to adjust the salt concentration of the first protein to 280mM is preferably 60ml of a reduced salt buffer.

In the invention, the s μmo enzyme digestion system is as follows: according to the following steps: mu. mo enzyme was added at a mass ratio of 100(suom enzyme: target protein), and the cleavage was performed overnight at 4 ℃.

In the present invention, the salt-reducing buffer preferably comprises the following components: 50mM NaCl, 20mM Tris-HCl, pH 7.5, 10% glycerol, 2mM EDTA and 1mM DTT.

After the second filtrate is obtained, the second filtrate is loaded to an SP chromatographic column, the effluent liquid of eluent elution is collected, the components corresponding to the elution peak are collected and subjected to SDS-PAGE electrophoresis, the protein with the size of 100KD is the second protein, and the step has the effects of improving the protein purity and removing part of impure proteins.

In the present invention, the procedure of the SDS-PAGE electrophoresis is preferably: 80V, 20 min; 130V and 1 h. A15. mu.L sample was taken for electrophoresis.

In the specific implementation process of the invention, the loading speed is preferably 1 ml/min; the flow rate of the elution is preferably 2ml/min, 1.8ml/tube receiving; elution was performed with 10% (volume percent) B biningbuffer for 10 column volumes at a flow rate of 2mL/min, followed by a delay of 100mL for 15% B to 60% B.

After the second protein is obtained, the second protein is reversely hung on the EDTANI column at the speed of 1mL/min, a 30KD concentration tube is adopted, the first concentration is carried out under the conditions of 1-5 ℃ and 3000-4000 rpm until the volume after concentration is 0.5-1 mL, and a first concentrated solution is obtained, wherein the step has the effect of removing the protein which is not cut through the reverse hanging, and the preparation is carried out for the next step of passing through a molecular sieve after concentration; the temperature of the first concentration is preferably 4 ℃ and the rotation speed is preferably 3500 rpm.

After the first concentrated solution is obtained, loading the first concentrated solution to an S200 chromatographic column, eluting effluent liquid by eluent, collecting components corresponding to an elution peak, performing SDS-PAGE electrophoresis, taking the protein with the size of 100KD as a third protein, further improving the protein purity by a molecular sieve, and simultaneously obtaining uniform protein; the loading rate is preferably 0.6ml/min, 1ml/tube receiving.

After the third protein is obtained, carrying out second concentration on the third protein by adopting a 30KD concentration tube at the temperature of 1-5 ℃ and under the condition of 3000-4000 rpm until the volume of the concentrated third protein is 0.5-1 mL, thus obtaining purified telomerase; the temperature of the second concentration is preferably 4 ℃ and the rotation speed is preferably 3500 rpm.

After obtaining the purified telomerase, the invention also comprises the step of performing cryopreservation on the purified telomerase; the temperature of the cryopreservation is preferably-80 ℃.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the invention, the process steps of recombinant cell construction for expressing two kinds of telomerase, telomerase purification and the like are the same.

Example 1

PCR amplification of the sequence of interest:

50 μ l system:

the primers (primer (F/R)) used therein were as follows:

Trop-F：

tagGAATTCCATATGCAGACCATCTCTCTGCTGCAGTACGTT，SEQ ID NO.7；

Trop-R：tcgctcgagttagtcgacGATGGTCAGGGTGTACAGCCATTCGA，SEQ ID NO.8；

CN F：taggaattccatatgaccgtgaaagtgaacgaaaagaaaaccctgctgcagta，SEQ ID NO.9；

CN R：tcgctcgagttagtcgactatattcaatgtttgtacccactcgactatttc，SEQ ID NO.10。

and (3) PCR circulation: at 98 ℃ for 10 s; at 62 deg.C, 10s, 72 deg.C, 2min30s, 72 deg.C, 7min, 30 cycles; 16 ℃ and infinity;

after the amplification, electrophoresis detection is carried out, 1% agarose, 90V, 50min, 50. mu.l of sample and 5. mu.l of 10x loading are carried out, then gel cutting recovery is carried out, and after recovery, enzyme digestion is carried out by using EcoR I and XhoI.

10. mu.l of digestion system:

after enzyme digestion, putting the mixture into a 65 ℃ oven for inactivation for 20min, then connecting the template with a pET-15b-s mu mo carrier,

ligation system 10. mu.l:

ligation was carried out in a 15 ℃ water bath for 3h, after which 10. mu.l of the ligation product was transferred in its entirety to 2984. After colony PCR detection, the quality-improved grains are shaken for enzyme digestion identification. The final plasmids were all correct and transformed into BL 21.

Example 2

1. Activating the recombinant cell strain: selecting a single clone for activation, namely 10mL of LB liquid culture medium +10 mu l of Amp + the recombinant cell (pET-15b-s mu mo-Trop-BL21) prepared in the example 1, wherein the Amp concentration is 100 mu g/mL, and activating for 6 hours at 37 ℃ and 180rpm in a constant temperature shaking table;

2. and (3) amplification culture: antibiotic Amp was added to LB medium to a final concentration of 100. mu.g/mL, at a ratio of 1: adding bacterial liquid in a volume ratio of 500 for amplification culture (1.5L LB/bottle), and performing amplification culture in a constant temperature shaking table at 37 ℃ and 180 rpm;

3. IPTG induction: monitoring OD600 value about 0.6, adding 450 μ l 1M IPTG (isopropyl thiogalactoside) to make its final concentration be 0.3mM, 150rpm, inducing at 18 deg.C for 15h, and inducing expression of target protein;

4. and (3) centrifugal bacterium collection: after centrifugation (18 ℃, 4500rpm), an amount of 11.97g of microbial biomass was recovered, and a 5mM imidazole solution was used as a 1: 10 (mass-to-volume ratio (g/mL), namely the mass of the thalli and the volume ratio of a solution to be added), dissolving the thalli, and carrying out high-pressure crushing for 4 rounds and ultrasonic crushing for 3 rounds (4 rounds under 1000bar, 400W, ultrasonic treatment for 7s, 3s interval, 3min round ultrasonic treatment, and 3 rounds of total ultrasonic treatment); centrifuging at 12000rpm for 40min, filtering with 0.45 μm membrane, and loading;

5. and (3) crude purification by using a Ni column: the filtered supernatant was applied to a 5ml NI pre-packed column at a rate of 1ml/min, and then 50ml of 5mM imidazole binding buffer (imidazole binding solution: formulation: 500mM NaCl, 20mM Tris-HCl, pH 7.5, 10% glycerol, 5mM imidazole), 20-fold volume of 30mM imidazole Washbuffer (imidazole eluting solution), and 5% to 80% B (B: 500mM imidazole Elution buffer) formulation: 500mM NaCl, 20mM Tris-HCl, pH 7.5, 10% glycerol, 500mM imidazole) were delayed by 20-fold volume (100ml), Elution rate was 2ml/min, and 1.8ml/tube were used to collect each collected fraction, and this step was intended to perform crude purification of the target protein. The crude purification result of Ni column is shown in FIG. 1, most of the eluted first two peaks are hetero-proteins, and the third peak (corresponding to 20-45 tubes) is the target protein.

6. S mu mo enzyme is used for removing the tag: the 20 th to 45 th tubes in FIG. 1 were digested with s μmo enzyme. The enzyme digestion system of the s mu mo is as follows: according to the following steps: mu. mo enzyme was added at a mass ratio of 100(suom enzyme: target protein), and the cleavage was performed overnight at 4 ℃.

7. SP column purification: after digestion of 47.5mL of the sample s μmo, 60mL of a salt reducing buffer was added to reduce the salt concentration to 280mM, the solution was filtered through a 0.45 μm filter and applied to an SP column at a rate of 1mL/min, 10 column volumes were eluted at a flow rate of 2mL/min using 10% (volume percent) of B biningbuffer, and then 100mL of elution was delayed by 15% B to 60% B. See figure 2 for the results of SP column purification. The higher peak in the middle of FIG. 2 is the protein of interest (corresponding to tubal proteins 26-46), and the other peaks are the mixture of the protein of interest and the hetero-protein.

8. anti-EDTA NI column purification: collecting the No. 26-46 tube protein shown in figure 2, passing through an anti-EDTANI column at a speed of 1mL/min for reverse hanging, and then concentrating, specifically concentrating with a concentration tube with a pore diameter of 30KD at 4 ℃ and 3500rpm until the volume is 0.5 mL. The purpose of this step is to remove the non-cleaved protein by reverse-hanging, and to prepare it for the next step of molecular sieving after concentration.

9. S200 column purification: the protein sample from step 8 was collected and applied to the S200 column at a rate of 0.6ml/min, and received at 1ml/tube, and the purification results are shown in FIG. 3. The middle peak in FIG. 3 is the target protein (corresponding to tube No. 22-31), and the other peaks include the target protein and the hetero-protein. This step further improves protein purity by molecular sieves while obtaining uniform protein.

10. Concentration: collecting No. 22-31 collected liquid in figure 3, and concentrating, specifically concentrating with a concentration tube with a pore diameter of 30KD at 4 deg.C and 3500rpm until the volume is 0.5 mL. The protein can be frozen after concentration. The final concentration results are shown in FIG. 4.

Example 3

1. Telomerase activity identification reaction solution

40m M Tris-HCl，pH7.8；100m M NaCl；20mM KCl；2mM MgCl₂；2mM Mg(AC)₂(ii) a 1mM DTT; 0.2mM dNTP; 1 μ M TERT telomerase (purified protein of interest); 1.2. mu.M RNA; 1. mu.M of 5' -FAM-labeled primer (primer sequence: TTAGTGTAAGGA, SEQ ID NO. 11); 2X reaction stop buffer; 1M urea (urea); 0.05% xylene blue;

2. the specific method comprises the following steps:

telomerase primer extension experiments were performed in a 25. mu.L system using 40mM Tris-HCl, pH 7.8, 2mM MgCl₂100mM NaCl, 20mM KCl, 1mM DTT, 0.2mM dNTPmix (0.2 mM each of dTTP, dATP, dCTP, dGTP), 2mM Mg (AC)₂Sequentially adding 1.0 mu M telomerase, 1.2 mu M RNA and 1 mu M fluorescent primer into a PCR tube, uniformly mixing, carrying out a light-shielding reaction at 37 ℃ for 1h, then adding 10 mu L of 8M urea as loading, adding a reaction termination buffer to terminate the reaction,electrophoresis was performed using 15% urea gel (7M, 19: 1) at 500V for 4h, and after electrophoresis was completed, exposure analysis was performed by a Berlol gel imaging system.

3. The identification result is shown in FIG. 5, wherein M is marker and Trop is the target protein. As can be seen from FIG. 5, the resulting target protein had an elongation activity.

Example 4

The procedure for PCR amplification of the desired sequence was as in example 1.

After the amplification, electrophoresis detection is carried out, 1% agarose, 90V, 50min50 mu l of sample and 5 mu l of 10x loading are carried out, then gel cutting and recovery are carried out, and Nde I and SalI enzyme digestion are carried out after recovery.

10. mu.l of digestion system:

inactivating in a 65 ℃ oven for 20min after enzyme digestion, then connecting the template with pPICZA and pPICZaA vectors,

ligation system 10. mu.l:

ligation was carried out in a 15 ℃ water bath for 3h, after which 10. mu.l of the ligation product was transferred in its entirety to 2984. After colony PCR detection, the quality-improved grains are shaken for enzyme digestion identification. The final plasmids were all correct.

Example 5

1. And (3) plasmid linearization: the plasmid constructed in example 4 was linearized by SacI single digestion, and run electrophoresis detection at 1% agarose, 100V, 40min 5. mu.l sample + 0.5. mu.l 10 × loading.

2. Alcohol precipitation of linearized plasmid: the plasmid after complete linearization was added with 0.3M sodium acetate (pH5.2) at final concentration and two times the volume of absolute ethanol, and left to stand at-20 ℃ for more than 1 h. Centrifuging at high speed 12000rpm/10min, washing precipitate with 70% ethanol twice, oven drying the precipitate at 65 deg.C, and adding appropriate amount of ddH₂O redissolve the DNA pellet (total greater than 5. mu.g).

3. Yeast competence preparation: single colonies of yeast recipient bacteria were picked and inoculated into 5mLYPD medium and shaken overnight at 30 ℃. Inoculating 100mLYPD liquid culture medium with 1% inoculation amount, and shaking overnight at 30 ℃ until OD is 1.3-1.5. Centrifuge at 5000rpm for 5min at 4 deg.C, and discard the supernatant. The cells were resuspended in 40ml of a pretreatment solution (0.1mol/L LiAC, 0.01mol/L DTT, 0.6mol/L sorbitol, 0.01mol/L Tris-HCl7.5), and the cells were left at room temperature for 30min, centrifuged at 4 ℃ at 5000rpm for 10min, and the supernatant was discarded. Then washed 3 times with 5mL of 1mol/L sorbitol, centrifuged at 5000rpm at 4 ℃ for 10min, and the supernatant was discarded. Dissolve in 300. mu.L of 1mol/L ice-cold sorbitol for conversion.

4. Yeast electrotransformation: 80 μ l of competence was mixed with 5-10 μ g of linearized plasmid and transferred to an ice-cold electric rotor. Incubate for 5min on ice. The shock was selected with the appropriate parameters (1500V, 25uF, 200 Ω, 0.2cm cup). 1ml of 1M ice-cold sorbitol was immediately added and transferred to a 15ml tube. Standing and incubating for 1-2 h at 30 ℃, taking 200 mu L of plated YPDs (added with 100ug/ml Zeocin), and slightly centrifuging the rest and then plating YPDS plates. Culturing at 30 ℃. (36h to see the colony)

5. High copy screening: selecting 60-70 monoclonals, and respectively dissolving in 1ml of sterile ddH₂And O, mixing uniformly. 0.5 mu L of the culture medium is respectively spotted on 500ug/ml and 1000ug/ml Zeocin YPD plates for culture at 30 ℃, the growth state of the bacteria is observed at intervals, and clones which grow rapidly and can grow on a high-concentration antibiotic plate are selected.

6. Extracting genome DNA: selecting 15-20 clones with faster growth, scraping rice-grain-sized thalli on a flat plate, and suspending the rice-grain-sized thalli in 1ml of ddH₂And O, washing and centrifuging to remove the supernatant. 500 μ L sorbitol buffer was resuspended and 50 μ L helicase was added and digested at 37 ℃ for 1-4 h. Mix 1 time every 30 min. 4000rpm, 10min, discard the supernatant and collect the precipitate. 500 μ L Tris Na₂EDTA washes 1 time. 500 μ L Tris Na₂EDTA heavy suspension, adding 50 u L10% SDS, 65 degrees, 40min, every 10min mixing 1 times. Adding 200 μ L of 5M KAC, mixing, standing on ice for 1 hr, high speed 10min, collecting supernatant, adding equal volume of isopropanol, mixing, and standing at-20 for more than 1 hr. The speed is increased for 10min, the supernatant is discarded, and the precipitate is washed with 80% ice-cold ethanol. Air drying in ddH₂And (4) re-dissolving the O.

6. And (3) PCR identification: 5 'AOX, 3' AOX universal primer PCR identification. And selecting a positive recombinant strain.

Example 6

1. Activating the recombinant strain: selecting 6-8 recombinant bacteria prepared in example 5 for monoclonal activation, performing activation on 10mL of BMDY liquid medium + recombinant bacteria (pPICZA-Trop-GS115) in a constant temperature shaking table at 30 ℃ and 180rpm for 24 hours; to OD₆₀₀The value is 2 to 6.

2. And (3) collecting thalli: centrifuging at 3000rpm for 5-10 min, and collecting thallus;

3. methanol induction: resuspending the cells to OD with BMMY medium containing 0.5% -1% methanol (volume fraction, 99mL of medium added with 1mL of methanol, namely 1%)₆₀₀1 is ═ 1; the cultivation was continued at 180rpm in a constant temperature shaker at 30 ℃. 1ml of bacterial liquid is collected every 12h, and 0.5-1% of methanol is supplemented every 24 h.

4. And (3) centrifugal bacterium collection: taking 1ml of culture solution every 12h, centrifuging (18 ℃, 4500rpm), collecting the thallus by the pPICZA carrier recombinant bacteria, and collecting the supernatant by the pPICZaA carrier recombinant bacteria.

pPICZA vector recombinant bacteria: the cells were resuspended in 250. mu.Lbindingbuffer. Liquid nitrogen-55 ° -liquid nitrogen (cold-hot-cold cycle 3 times). 60-100 w, performing two ultrasonic cycles (over 3s, stopping for 7s, and total working time of 4min), centrifuging at 12000rpm/min, adding a loading buffer to the supernatant and the precipitate respectively, boiling for 10min, and running glue. (Total protein)

The electrophoresis results are shown in FIG. 6. From the electrophoresis results, it can be seen that the recombinant plasmid successfully expresses the target protein in yeast.

pPICZaA vector recombinant bacteria: the cell fractions were frozen at-20 ℃ for a while, and the supernatant was precipitated by adding 0.43g (1ml) of ammonium sulfate and allowed to stand on ice for 2 hours. At high speed, the supernatant was discarded and the pellet resuspended in 10. mu.L buffer. Dialyzing with 0.25 μm microporous filter membrane, and detecting by running gel. The results of the measurements are shown in FIG. 7.

The pellet was reconstituted with buffer (500mM NaCl, 20mM Tris-HCl, pH 7.5, 10% glycerol) and examined by SDS-PAGE, and the reconstituted supernatant was found to contain the protein of interest, indicating successful expression of the protein of interest in yeast on the recombinant plasmid.

The purification process of the recombinant bacterium telomerase of the pPICZA vector is the same as that of escherichia coli, the specific process refers to the purification steps of the escherichia coli in the example 2, and the following specific purification results are shown: the results of NI column purification are shown in fig. 8. See figure 9 for the results of SP column purification. The purification results of S200 are shown in FIG. 10. The concentration results are shown in FIG. 11. The results of the activity assay are shown in FIG. 12.

Example 7

After the amplification, electrophoresis detection is carried out, 1% agarose, 90V, 50min 50. mu.l of sample + 5. mu.l of 10x loading are carried out, then gel cutting and recovery are carried out, and Nde I and Sal I enzyme digestion are carried out after recovery.

10. mu.l of digestion system:

after enzyme digestion, putting the mixture into a 65 ℃ oven for inactivation for 20min, then connecting the template with a pTrix vector,

ligation system 10. mu.l:

Example 8

And 5 mu L, Bacmid 2.5.5 mu L of the recombinant plasmid prepared in the embodiment 7 and 100 mu L of sterile water are taken and mixed uniformly, 5 mu L of transfection reagent is added and mixed uniformly, the mixture is placed still for 10 to 30min and then is added into the cultured Sf9 cells uniformly, and the recombinant baculovirus is formed in the host cells through homologous recombination. Cell culture supernatants were collected 3-5 days after transfection as stock solutions for recombinant baculoviruses. Sf9 cells were infected with the virus stock solution, cultured for 3 days, and then the lesion cell culture solution was centrifuged at 4000rpm for 10min to collect cells. The cells were resuspended in 1mL bindingbuffer. And (4) repeatedly freezing and thawing liquid nitrogen. After cell lysis, centrifugation is carried out at 12000rpm for 30min, the supernatant is taken to pass through an NI column, the rest steps are the same as the purification of escherichia coli, and finally protein expression is detected by SDS-PAGE.

The subsequent purification process was the same as that of E.coli, and the specific purification procedure was as in E.coli purification process in example 2. The NI column purification results are shown in fig. 13. See figure 14 for SP column purification results. The purification results of S200 are shown in FIG. 15. The concentration results are shown in FIG. 16. See figure 17 for activity assay results.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> northwest agriculture and forestry science and technology university

<120> telomerase expression gene, recombinant plasmid, recombinant cell and application

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 881

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gln Thr Ile Ser Leu Leu Gln Tyr Val Lys Asp Asn Ser Ser Asp

1 5 10 15

Gln Ser Val Gln Leu Pro Ser Phe Ile Asn Tyr Leu Lys Ser Val Asn

20 25 30

Leu Ile Glu Ile Thr Lys Pro Asn Lys Leu Pro Arg Ile Thr Ser Asp

35 40 45

Thr Ser Tyr Gln Asp Phe Ile Asn Asn Leu Ile Thr Glu Leu Val Leu

50 55 60

Arg Lys Ser Gln Asn Val Leu Val Ser Gly Tyr Lys Ser Thr Ile Glu

65 70 75 80

Asp Ser Lys Val Ala Leu Pro Cys Val Asn Leu Asp Gly Asn Phe Leu

85 90 95

Pro Thr Asn Leu Asn Asp Ser Asn Trp His Arg Val Phe Thr Leu Leu

100 105 110

Gly Ser Glu Lys Phe Leu Asn Leu Leu Ile Asn His Lys Gly Phe Leu

115 120 125

Lys His Lys Asn Gly Gln Gln Ile Gln Ile Phe Gly Asp Ile Thr Ser

130 135 140

Tyr Ala Lys Pro Ala Ala Lys Ala Val Leu Pro Ile Pro Lys Trp Ile

145 150 155 160

Tyr Lys Phe Asp Val Leu Tyr Gln Ser Arg Asn Pro Arg Tyr Arg Asn

165 170 175

Tyr Glu Ile Ile Arg Thr Asn Val Gln Glu Val Leu Ala Glu Ile Val

180 185 190

Ser Pro Ser Thr Lys Asn Pro Asn Ser Glu Asn Pro Ala Val Lys Lys

195 200 205

Arg Phe Arg Gly Ile Lys Asn Val Val Ser Arg Ile Ile Ser Asn Asp

210 215 220

Lys Lys Cys Arg Tyr Asp Leu Ile Tyr Asn Lys Tyr Leu Ser Ser Ser

225 230 235 240

Asp Thr Arg Lys Leu Lys Thr Met Ile Asp Tyr Ser Thr Lys Phe Asn

245 250 255

Arg Val Val Glu Val Val Leu Ile Ile Met Gly Lys Leu Leu Pro Leu

260 265 270

Asp Ala Trp Gly Gly Thr Glu Asn Lys Lys Val Ile Gln Asp Arg Ile

275 280 285

Val Asp Phe Leu Arg Leu Gly Ala Asn Glu Arg Leu His Leu Asp Asp

290 295 300

Val Leu Ser Gly Ile Lys Leu Ser Lys Phe Lys Trp Leu Gly Val Gly

305 310 315 320

Asn Asn Ile Ser Ser Gln Gln Asp Phe Gln Ile Arg Lys Arg Leu Leu

325 330 335

Glu Gly Tyr Ile Asn Trp Val Phe Ile Ser Leu Val Lys Asn Ile Val

340 345 350

Arg Ala Phe Trp Tyr Val Thr Glu Ser Ser Asn Met Asp Arg Ser Lys

355 360 365

Leu Phe Tyr Phe Thr His Ser Ile Trp Asn Glu Leu Ser Ser Asn Trp

370 375 380

Ile Thr Lys Tyr Ala Lys Gly Asn Leu Val Gln Val Val Ser Pro Glu

385 390 395 400

Ser Lys Gly Gln Phe Thr Asn Gly Lys Ile Lys Leu Ile Pro Lys Arg

405 410 415

Gly Gly Phe Arg Val Ile Cys Val Pro Leu Lys Gln Ser Leu Tyr Ser

420 425 430

Phe Asn Asn Lys Arg Asn Phe Ala Leu Lys Gln Lys Glu Lys Trp Asp

435 440 445

Tyr Ile Phe Tyr Gln Lys Tyr Thr Leu Ser Pro Val Arg Gln Val Leu

450 455 460

Gln Leu Lys Leu Asn Ala Leu Arg Lys Ser Asp Met Gly His Arg Ser

465 470 475 480

Ser Val Asn Ser Thr Asn Glu Val Ala Asp Arg Ile Leu Thr Phe Arg

485 490 495

Asn Asp Leu Leu Lys Lys Asn Lys Thr Leu Pro Val Leu Tyr Met Ile

500 505 510

Lys Phe Asp Met Lys Glu Cys Tyr Asp Arg Leu Asn Gln Asn Ala Leu

515 520 525

Lys Glu Ser Ile Ala Gly Ile Phe Lys Glu Asp Asn Glu Asn Thr Thr

530 535 540

Tyr His Val Arg Glu Tyr Gly Thr Leu Asp Glu Phe Leu Lys Leu Lys

545 550 555 560

Arg Val Arg Thr Leu Ile Glu Thr Glu Val Gln Asn Phe Asn Ile Ile

565 570 575

Met Asn Ser Lys Asp Glu Ala Glu Ala Gly Ser Arg Ser Tyr Gly Thr

580 585 590

Lys Val Asp Lys Val Lys Thr Leu Ser Ile Ser Lys Asn Lys Ile Ile

595 600 605

Glu Val Cys His Ser Gln Ile Glu Asp Ala Thr Cys Leu Val Lys Asn

610 615 620

Lys Glu Gly Gln Tyr Asp Leu Phe Lys Arg Lys Gln Gly Val Phe Gln

625 630 635 640

Gly Phe Ser Leu Ser Gly Ile Phe Cys Asp Ile Leu Tyr Ser Thr Met

645 650 655

Val Ser Lys Glu Phe Lys Phe Leu Trp Glu Ala Thr Glu Asp Asn Leu

660 665 670

Leu Leu Arg Leu Val Asp Asp Phe Ile Phe Ile Thr Ser Asn Lys Asp

675 680 685

Thr Leu Lys Lys Val Lys Asp Lys Ile Ser Ser Asn Glu Leu Gln Lys

690 695 700

Tyr Gly Ala Phe Val Asn His Glu Lys Thr Val Glu Ile Asn Gly Glu

705 710 715 720

Ala Gly Ser Ser Asn Lys Met Thr Phe Val Gly Leu Asp Ile Asn Cys

725 730 735

Leu Thr Leu Asp Val Lys Lys Asp Ser Ser Gln Phe Ser Arg Pro Thr

740 745 750

Cys Lys Phe Arg Ser Phe Lys Ala Leu Phe Ser Asn Leu Lys Gln Phe

755 760 765

Tyr Cys Ser Asn Leu Ser Glu Phe Leu Leu Asp Phe Ser Ser Asn Ser

770 775 780

Leu Glu Thr Ile Arg Glu Asn Val Asp Ala Ile Leu Lys Leu Thr Phe

785 790 795 800

Glu Ala Ile Gln Thr Ser Phe Ala Thr Ile Ser Lys Gln Asp Ser Phe

805 810 815

Glu Arg Tyr Arg Phe Met Lys Phe Leu His Val Ile Ile Glu Thr Thr

820 825 830

Ile Glu Lys Phe Ala Arg Val Asn Gly Ser Met Glu Gly Val Glu Tyr

835 840 845

Leu Leu Thr Cys Ile Lys Ile Thr Ile Thr Lys Ser Leu Ala Phe Met

850 855 860

Ala Thr Lys Gln Glu Ile Ile Glu Trp Leu Tyr Thr Leu Thr Ile Val

865 870 875 880

Asp

<210> 2

<211> 867

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Thr Val Lys Val Asn Glu Lys Lys Thr Leu Leu Gln Tyr Val Leu

1 5 10 15

Asp Asn Thr Ser Asn Glu Val Pro Leu Leu Pro Ser Leu Lys Glu Tyr

20 25 30

Met Glu Thr Val Leu Val Tyr Gln Ser Ile Lys Arg Pro Leu Pro Ala

35 40 45

Ile Arg Pro Gln Glu Ser Phe Asp Glu Phe Met Lys Glu Leu Val Thr

50 55 60

Arg Leu Val Met Glu Lys Ser Asn Asn Val Ile Ala Tyr Gly Tyr Lys

65 70 75 80

Thr Ser Ala Met Glu Ser Arg Ser Ile Phe Thr Thr Phe His Ser Ser

85 90 95

Gly Asn Phe Ile Leu Thr His Ile Thr Ser His Asn Trp Ser Thr Ile

100 105 110

Phe Ser Leu Leu Gly Pro Lys Lys Phe Leu Glu Leu Leu Val Asn Asn

115 120 125

Lys Gly Phe Val Ser Lys Val Asn Gly Glu Ser Val Gln Ile Phe Gly

130 135 140

Asp Val Asn Ser His Arg Lys Ala Val Val Val Ser Lys Tyr Ile Thr

145 150 155 160

Lys Phe Asn Val Leu Tyr Asn Ser Tyr Ser Arg Asp Phe Ser Arg Phe

165 170 175

Glu Met Ile Arg Pro Ser Ile Gln Thr Ile Leu Gln Asp Ile Leu Ser

180 185 190

Phe Ser Gly Leu Asn Pro Gly Arg Ser Ser Lys Arg Tyr Arg Gly Phe

195 200 205

Lys Ser Leu Leu Ser Arg Ile Ile Ala Asn Asp Lys Lys Cys Arg Tyr

210 215 220

Asp Ile Leu Tyr Ala Lys Phe Ile Gly Thr Ser Lys Cys Asn Phe Ala

225 230 235 240

Asn Val Val Ser Asn Lys Thr Glu Ile Ser Gln Val Ile Gln Phe Val

245 250 255

Leu Leu Val Leu Gly Lys Leu Leu Pro Leu Asp Ala Trp Gly Gly Val

260 265 270

Ser Asn Lys Lys Ile Ile Lys Asp Arg Val Val Asp Phe Leu Leu Leu

275 280 285

Gly Ala Asn Glu Lys Ile His Met Asp Asp Leu Phe Arg Gly Ile Arg

290 295 300

Leu Lys Asp Phe Lys Trp Leu Gly Arg Ala His Gln Ile Ser Ser Lys

305 310 315 320

Gln Asp Phe Glu Leu Arg Thr Ala Phe Leu Lys Gly Tyr Leu Trp Trp

325 330 335

Leu Phe Glu His Leu Leu Lys Asn Ile Leu Arg Ser Phe Trp Tyr Ile

340 345 350

Thr Glu Thr Ser Ser Ile Val Ser Leu Glu Leu Asn Tyr Phe Pro Gln

355 360 365

Tyr Leu Trp Lys Glu Leu Tyr Glu Ser Trp Val Ser Lys Tyr Ala Lys

370 375 380

Asn Asn Leu Val Lys Met Pro Ser Lys Ile Gln Arg Glu Gln Leu Pro

385 390 395 400

Cys Gly Lys Ile Lys Leu Ile Pro Lys Arg Ser Ser Phe Arg Val Ile

405 410 415

Cys Val Pro Ile Lys Arg Ser Leu Lys Leu Leu Asn Lys Lys Leu Glu

420 425 430

Leu Asp Thr Leu Glu Lys Glu Lys Arg Glu Phe Glu Arg Tyr Arg Lys

435 440 445

Glu Val Leu Ser Pro Val Gly Gln Ile Leu Arg Leu Lys Leu Ser Lys

450 455 460

Leu Arg Asp Thr Tyr Glu Ser Tyr Arg Ala Ser Val His Ser Ser Ser

465 470 475 480

Asp Val Ala Glu Lys Ile Ser Asp Tyr Arg Asp Ser Leu Leu Thr Arg

485 490 495

Phe Gly Glu Ile Pro Lys Leu Phe Ile Leu Lys Phe Asp Met Lys Glu

500 505 510

Cys Tyr Asp Arg Leu Ser Gln Pro Val Leu Met Lys Lys Leu Glu Glu

515 520 525

Leu Phe Glu Asn Gln Asp Asn Lys Thr Ser Tyr Tyr Val Arg Tyr Tyr

530 535 540

Ala Gln Leu Asp Ala Ser His Lys Leu Lys Lys Val Lys Thr Thr Ile

545 550 555 560

Asp Thr Gln Tyr His Asn Leu Asn Ile Leu Ser Ser Ser Arg His Leu

565 570 575

Ser Asn Cys Lys Ser Leu Val Asp Lys Thr Lys Thr Ile Ala Leu Gln

580 585 590

Lys Gly Asn Ile Leu Glu Val Cys Arg Ser Gln Ile Tyr Asp Val Val

595 600 605

Gly Ser Val Lys Asp Ala Arg Gly Asn Leu His Leu Tyr Lys Arg Lys

610 615 620

Arg Gly Val Phe Gln Gly Phe Ser Leu Ser Ser Ile Phe Cys Asp Ile

625 630 635 640

Leu Tyr Ser Ala Met Val His Asp Cys Phe Gln Phe Leu Trp Lys Ser

645 650 655

Lys Gln Asp Phe Leu Phe Val Arg Leu Val Asp Asp Phe Leu Leu Val

660 665 670

Thr Pro Asp Ser Asn Ile Tyr Asp Gln Val His Asn Ile Leu Ser Gly

675 680 685

Lys Ile Leu Glu Ser Tyr Gly Ala Phe Val Asn Lys Asp Lys Thr Val

690 695 700

Val Val Asn Gln Thr Thr Thr Lys Pro Ser Ile Asp Phe Val Gly Leu

705 710 715 720

Glu Val Asn Thr Thr Asp Leu Ser Ile Lys Arg Asn Ser Gly Ser Ile

725 730 735

Ser Leu Val Thr Thr Asn Phe Arg Thr Phe Lys Thr Leu Val Lys Tyr

740 745 750

Leu Lys Thr Phe Tyr Gln Leu Asn Leu Glu Gly Phe Leu Leu Asp Cys

755 760 765

Ser Phe Gly Val Leu Glu Asn Val Leu Glu Asn Met Gly Ser Leu Leu

770 775 780

Arg Leu Val Leu Arg Glu Phe Lys Thr Lys Phe Thr Ser Ile Val Lys

785 790 795 800

Tyr Asp Thr Phe His Cys Tyr Lys Phe Ile Lys Phe Leu Tyr Asp Ile

805 810 815

Ser Asn Tyr Thr Ile Val Lys Tyr Val Glu Thr Asn Ser Asp Trp Asp

820 825 830

Gly Ala Pro Glu Leu Leu Asn Cys Ile Lys Gln Ile Ile Val Lys Glu

835 840 845

Phe Ser Ser Phe Glu Ser Tyr Ser Glu Ile Val Glu Trp Val Gln Thr

850 855 860

Leu Asn Ile

865

<210> 3

<211> 2646

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgcagacca tctctctgct gcagtacgtt aaagacaact cttctgacca gtctgttcag 60

ctgccgtctt tcatcaacta cctgaaatct gttaacctga tcgaaatcac caaaccgaac 120

aaactgccgc gtatcacctc tgacacctct taccaggact tcatcaacaa cctgatcacc 180

gaactggttc tgcgtaaatc tcagaacgtt ctggtttctg gttacaaatc taccatcgaa 240

gactctaaag ttgcgctgcc gtgcgttaac ctggacggta acttcctgcc gaccaacctg 300

aacgactcta actggcaccg tgttttcacc ctgctgggtt ctgagaagtt cctgaacctg 360

ctgatcaacc acaaaggttt cctgaaacac aagaatggtc agcagatcca gatcttcggt 420

gacatcacct cttacgcgaa accggcggcg aaagcggttc tgccgatccc gaaatggatc 480

tacaaattcg acgttctgta ccagtctcgt aacccgcgtt accgtaacta cgaaatcatc 540

cgtaccaacg ttcaggaagt tctggcggaa atcgtttctc cgtctaccaa gaatccgaac 600

tctgaaaacc cggcggttaa gaagcgtttc cgtggtatca agaatgttgt ttctcgtatc 660

atctctaacg acaagaagtg ccgttacgac ctgatctaca acaaatacct gtcttcttct 720

gacacccgta aactgaaaac catgatcgac tactctacca aattcaaccg tgttgttgaa 780

gttgttctga tcatcatggg taaactgctg ccgctggacg cgtggggtgg taccgaaaac 840

aagaaggtta tccaggaccg tatcgttgac ttcctgcgtc tgggtgcgaa cgaacgtctg 900

cacctggacg acgttctgtc tggtatcaaa ctgtctaaat tcaaatggct gggtgttggt 960

aacaacatct cttctcagca ggacttccag atccgtaaac gtctgctgga aggttacatc 1020

aactgggttt tcatctctct ggttaagaat atcgttcgtg cgttctggta cgttaccgaa 1080

tcttctaaca tggaccgttc taaactgttc tacttcaccc actctatctg gaacgaactg 1140

tcttctaact ggatcaccaa atacgcgaaa ggtaacctgg ttcaggttgt ttctccggaa 1200

tctaaaggtc agttcaccaa cggtaaaatc aaactgatcc cgaaacgtgg tggtttccgt 1260

gttatctgcg ttccgctgaa acagtctctg tactctttca acaacaaacg taacttcgcg 1320

ctgaaacaga aagagaagtg ggactacatc ttctaccaga aatacaccct gtctccggtt 1380

cgtcaggttc tgcagctgaa actgaacgcg ctgcgtaaat ctgacatggg tcaccgttct 1440

tctgttaact ctaccaacga agttgcggac cgtatcctga ccttccgtaa cgacctgctg 1500

aagaagaaca aaaccctgcc ggttctgtac atgatcaaat tcgacatgaa agaatgctac 1560

gaccgtctga accagaacgc gctgaaagaa tctatcgcgg gtatcttcaa agaagacaac 1620

gaaaacacca cctaccacgt tcgtgaatac ggtaccctgg acgaatttct gaaactgaaa 1680

cgtgttcgta ccctgatcga aaccgaagtt cagaacttca acatcatcat gaactctaaa 1740

gacgaagcgg aagcgggttc tcgttcttac ggtaccaaag ttgacaaagt taaaaccctg 1800

tctatctcta agaataaaat catcgaagtt tgccactctc agatcgaaga cgcgacctgc 1860

ctggttaaga ataaagaagg tcagtacgac ctgttcaaac gtaaacaggg tgttttccag 1920

ggtttctctc tgtctggtat cttctgcgac atcctgtact ctaccatggt ttctaaagaa 1980

tttaaattcc tgtgggaagc gaccgaagac aacctgctgc tgcgtctggt tgacgacttc 2040

atcttcatca cctctaacaa agacaccctg aagaaggtta aagacaaaat ctcttctaac 2100

gaactgcaga aatacggtgc gttcgttaac cacgagaaga ccgttgaaat caacggtgaa 2160

gcgggttctt ctaacaaaat gaccttcgtt ggtctggaca tcaactgcct gaccctggac 2220

gttaagaagg actcttctca gttctctcgt ccgacctgca aattccgttc tttcaaagcg 2280

ctgttctcta acctgaaaca gttctactgc tctaacctgt ctgaatttct gctggacttc 2340

tcttctaact ctctggaaac catccgtgaa aacgttgacg cgatcctgaa actgaccttc 2400

gaagcgatcc agacctcttt cgcgaccatc tctaaacagg actctttcga acgttaccgt 2460

ttcatgaaat tcctgcacgt tatcatcgaa accaccatcg agaagttcgc gcgtgttaac 2520

ggttctatgg aaggtgttga atacctgctg acctgcatca aaatcaccat caccaaatct 2580

ctggcgttca tggcgaccaa acaggaaatc atcgaatggc tgtacaccct gaccatcgtc 2640

gactaa 2646

<210> 4

<211> 2604

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaccgtga aagtgaacga aaagaaaacc ctgctgcagt atgtgctgga taacaccagc 60

aacgaagtgc cgctgctgcc gagcctgaaa gaatatatgg aaaccgtgct ggtgtataaa 120

agcattaaac gcccgctgcc ggcgattcgc ccgcaggaaa gctttgatga atttatgaaa 180

gaactggtga cccgcctggt gatggaaaag agcaacaacg tgattgcgta tggctataaa 240

accagcgcga tggaaagccg cagcattttt accacctttc atagcagcgg caactttatt 300

ctgacccata ttaccagcca taactggagc accattttta gcctgctggg cccgaaaaaa 360

tttctggaac tgctggtgaa caacaaaggc tttgtgagca aagtgaacgg cgaaagcgtg 420

cagatttttg gcgatgtgaa cagccatcgc aaagcggtgg tggtgagcaa atatattacc 480

aaatttaacg tgctgtataa cagctatagc cgcgatttta gccgctttga aatgattcgc 540

ccgagcattc agaccattct gcaggatatt ctgagcttta gcggcctgaa cccgggccgc 600

agcagcaaac gctatcgcgg ctttaaaagc ctgctgagcc gcattattgc gaacgataaa 660

aaatgccgct atgatattct gtatgcgaaa tttattggca ccagcaaatg caactttgcg 720

aacgtggtga gcaacaaaac cgaaattagc caggtgattc agtttgtgct gctggtgctg 780

ggcaaactgc tgccgctgga tgcgtggggc ggcgtgagca acaagaaaat tattaaagat 840

cgcgtggtgg attttctgct gctgggcgcg aacgaaaaga ttcacatgga tgatctgttt 900

cgcggcattc gcctgaaaga ttttaaatgg ctgggccgcg cgcatcagat tagcagcaaa 960

caggattttg aactgcgcac cgcgtttctg aaaggctatc tgtggtggct gtttgaacat 1020

ctgctgaaaa acattctgcg cagcttttgg tatattaccg aaaccagcag cattgtgagc 1080

ctggaactga actattttcc gcagtatctg tggaaagaac tgtatgaaag ctgggtgagc 1140

aaatatgcga aaaacaacct ggtgaaaatg ccgagcaaaa ttcagcgcga acagctgccg 1200

tgcggcaaaa ttaaactgat tccgaaacgc agcagctttc gcgtgatttg cgtgccgatt 1260

aaacgcagcc tgaaactgct gaacaagaaa ctggaactgg ataccctgga aaaagaaaaa 1320

cgcgaatttg aacgctatcg caaagaagtg ctgagcccgg tgggccagat tctgcgcctg 1380

aaactgagca aactgcgcga tacctatgaa agctatcgcg cgagcgtgca tagcagcagc 1440

gatgtggcgg aaaaaattag cgattatcgc gatagcctgc tgacccgctt tggcgaaatt 1500

ccgaaactgt ttattctgaa atttgatatg aaagaatgct atgatcgcct gagccagccg 1560

gtgctgatga aaaaactgga agaactgttt gaaaaccagg ataacaaaac cagctattat 1620

gtgcgctatt atgcgcagct ggatgcgagc cataaactga aaaaagtgaa aaccaccatt 1680

gatacccagt atcataacct gaacattctg agcagcagcc gccatctgag caactgcaaa 1740

agcctggtgg ataaaaccaa aaccattgcg ctgcagaaag gcaacattct ggaagtgtgc 1800

cgcagccaga tttatgatgt ggtgggcagc gtgaaagatg cgcgcggcaa cctgcatctg 1860

tataaacgca aacgcggcgt gtttcagggc tttagcctga gcagcatttt ttgcgatatt 1920

ctgtatagcg cgatggtgca tgattgcttt cagtttctgt ggaaaagcaa acaggatttt 1980

ctgtttgtgc gcctggtgga tgattttctg ctggtgaccc cggatagcaa catttatgat 2040

caggtgcata acattctgag cggcaaaatt ctggaaagct atggcgcgtt tgtgaacaaa 2100

gataaaaccg tggtggtgaa ccagaccacc accaaaccga gcattgattt tgtgggcctg 2160

gaagtgaaca ccaccgatct gagcattaaa cgcaacagcg gcagcattag cctggtgacc 2220

accaactttc gcacctttaa aaccctggtg aaatatctga aaacctttta tcagctgaac 2280

ctggaaggct ttctgctgga ttgcagcttt ggcgtgctgg aaaacgtgct ggaaaacatg 2340

ggcagcctgc tgcgcctggt gctgcgcgaa tttaaaacca aatttaccag cattgtgaaa 2400

tatgatacct ttcattgcta taaatttatt aaatttctgt atgatattag caactatacc 2460

attgtgaaat atgtggaaac caacagcgat tgggatggcg cgccggaact gctgaactgc 2520

attaaacaga ttattgtgaa agaatttagc agctttgaaa gctatagcga aattgtggaa 2580

tgggtgcaga ccctgaacat ttaa 2604

<210> 5

<211> 8654

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 60

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 120

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 180

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 240

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 300

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 360

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 420

agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 540

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 600

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 660

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 720

accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 840

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 900

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 960

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1020

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1080

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1140

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1200

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1260

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1320

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1380

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1440

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1500

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1560

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1620

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1680

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1740

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1800

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1860

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1920

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1980

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2040

tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2100

gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2160

gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2220

acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2280

cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2340

tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2400

ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2460

tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2520

cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2580

tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2640

ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2700

acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2760

agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2820

gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2880

tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2940

tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3000

tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3060

cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3120

acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3180

ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3240

taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3300

cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3360

aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3420

ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3480

gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3540

cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3600

tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3660

cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 3720

tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3780

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3840

gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3900

ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3960

ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4020

cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4080

gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4140

cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4200

ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4260

gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4320

ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4380

gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4440

caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4500

gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4560

ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4620

cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4680

gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4740

gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4800

caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4860

gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4920

ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4980

ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5040

agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5100

aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5160

cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5220

ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5280

aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat 5340

catcacagca gcggcctggt gccgcgcggc agccatatga gcgatagcga agtgaaccag 5400

gaagcaaaac cggaagtgaa accggaagtg aaaccggaaa cccatattaa cctgaaagtg 5460

agcgatggta gcagcgaaat tttttttaaa attaaaaaaa ccaccccgct gcgccgcctg 5520

atggaagcat ttgcaaaacg ccagggtaaa gaaatggata gcctgcgctt tctgtatgat 5580

ggtattcgca ttcaggcaga tcagaccccg gaagatctgg atatggaaga taacgatatt 5640

attgaagcgc atcgcgaaca gattggcggc gaattccata tgcagaccat ctctctgctg 5700

cagtacgtta aagacaactc ttctgaccag tctgttcagc tgccgtcttt catcaactac 5760

ctgaaatctg ttaacctgat cgaaatcacc aaaccgaaca aactgccgcg tatcacctct 5820

gacacctctt accaggactt catcaacaac ctgatcaccg aactggttct gcgtaaatct 5880

cagaacgttc tggtttctgg ttacaaatct accatcgaag actctaaagt tgcgctgccg 5940

tgcgttaacc tggacggtaa cttcctgccg accaacctga acgactctaa ctggcaccgt 6000

gttttcaccc tgctgggttc tgagaagttc ctgaacctgc tgatcaacca caaaggtttc 6060

ctgaaacaca agaatggtca gcagatccag atcttcggtg acatcacctc ttacgcgaaa 6120

ccggcggcga aagcggttct gccgatcccg aaatggatct acaaattcga cgttctgtac 6180

cagtctcgta acccgcgtta ccgtaactac gaaatcatcc gtaccaacgt tcaggaagtt 6240

ctggcggaaa tcgtttctcc gtctaccaag aatccgaact ctgaaaaccc ggcggttaag 6300

aagcgtttcc gtggtatcaa gaatgttgtt tctcgtatca tctctaacga caagaagtgc 6360

cgttacgacc tgatctacaa caaatacctg tcttcttctg acacccgtaa actgaaaacc 6420

atgatcgact actctaccaa attcaaccgt gttgttgaag ttgttctgat catcatgggt 6480

aaactgctgc cgctggacgc gtggggtggt accgaaaaca agaaggttat ccaggaccgt 6540

atcgttgact tcctgcgtct gggtgcgaac gaacgtctgc acctggacga cgttctgtct 6600

ggtatcaaac tgtctaaatt caaatggctg ggtgttggta acaacatctc ttctcagcag 6660

gacttccaga tccgtaaacg tctgctggaa ggttacatca actgggtttt catctctctg 6720

gttaagaata tcgttcgtgc gttctggtac gttaccgaat cttctaacat ggaccgttct 6780

aaactgttct acttcaccca ctctatctgg aacgaactgt cttctaactg gatcaccaaa 6840

tacgcgaaag gtaacctggt tcaggttgtt tctccggaat ctaaaggtca gttcaccaac 6900

ggtaaaatca aactgatccc gaaacgtggt ggtttccgtg ttatctgcgt tccgctgaaa 6960

cagtctctgt actctttcaa caacaaacgt aacttcgcgc tgaaacagaa agagaagtgg 7020

gactacatct tctaccagaa atacaccctg tctccggttc gtcaggttct gcagctgaaa 7080

ctgaacgcgc tgcgtaaatc tgacatgggt caccgttctt ctgttaactc taccaacgaa 7140

gttgcggacc gtatcctgac cttccgtaac gacctgctga agaagaacaa aaccctgccg 7200

gttctgtaca tgatcaaatt cgacatgaaa gaatgctacg accgtctgaa ccagaacgcg 7260

ctgaaagaat ctatcgcggg tatcttcaaa gaagacaacg aaaacaccac ctaccacgtt 7320

cgtgaatacg gtaccctgga cgaatttctg aaactgaaac gtgttcgtac cctgatcgaa 7380

accgaagttc agaacttcaa catcatcatg aactctaaag acgaagcgga agcgggttct 7440

cgttcttacg gtaccaaagt tgacaaagtt aaaaccctgt ctatctctaa gaataaaatc 7500

atcgaagttt gccactctca gatcgaagac gcgacctgcc tggttaagaa taaagaaggt 7560

cagtacgacc tgttcaaacg taaacagggt gttttccagg gtttctctct gtctggtatc 7620

ttctgcgaca tcctgtactc taccatggtt tctaaagaat ttaaattcct gtgggaagcg 7680

accgaagaca acctgctgct gcgtctggtt gacgacttca tcttcatcac ctctaacaaa 7740

gacaccctga agaaggttaa agacaaaatc tcttctaacg aactgcagaa atacggtgcg 7800

ttcgttaacc acgagaagac cgttgaaatc aacggtgaag cgggttcttc taacaaaatg 7860

accttcgttg gtctggacat caactgcctg accctggacg ttaagaagga ctcttctcag 7920

ttctctcgtc cgacctgcaa attccgttct ttcaaagcgc tgttctctaa cctgaaacag 7980

ttctactgct ctaacctgtc tgaatttctg ctggacttct cttctaactc tctggaaacc 8040

atccgtgaaa acgttgacgc gatcctgaaa ctgaccttcg aagcgatcca gacctctttc 8100

gcgaccatct ctaaacagga ctctttcgaa cgttaccgtt tcatgaaatt cctgcacgtt 8160

atcatcgaaa ccaccatcga gaagttcgcg cgtgttaacg gttctatgga aggtgttgaa 8220

tacctgctga cctgcatcaa aatcaccatc accaaatctc tggcgttcat ggcgaccaaa 8280

caggaaatca tcgaatggct gtacaccctg accatcgtcg actaactcga ggatccggct 8340

gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca ataactagca 8400

taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata 8460

tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat 8520

ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct 8580

gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct 8640

gtcaaacatg agaa 8654

<210> 6

<211> 8612

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 60

aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 120

tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 180

gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 240

tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 300

aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 360

cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 420

agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480

ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 540

tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 600

tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 660

caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 720

accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780

attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 840

ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 900

taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 960

taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1020

aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1080

agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1140

ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1200

ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1260

cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1320

tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1380

tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1440

tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1500

tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1560

ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1620

acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1680

ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1740

gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1800

ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1860

ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1920

taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1980

cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2040

tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2100

gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2160

gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2220

acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2280

cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2340

tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2400

ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2460

tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2520

cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2580

tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2640

ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2700

acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2760

agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2820

gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2880

tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2940

tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3000

tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3060

cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3120

acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3180

ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3240

taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3300

cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3360

aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3420

ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3480

gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3540

cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3600

tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3660

cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 3720

tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3780

gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3840

gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3900

ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3960

ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4020

cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4080

gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4140

cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4200

ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4260

gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4320

ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4380

gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4440

caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4500

gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4560

ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4620

cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4680

gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4740

gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4800

caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4860

gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4920

ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4980

ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5040

agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5100

aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5160

cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5220

ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5280

aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat 5340

catcacagca gcggcctggt gccgcgcggc agccatatga gcgatagcga agtgaaccag 5400

gaagcaaaac cggaagtgaa accggaagtg aaaccggaaa cccatattaa cctgaaagtg 5460

agcgatggta gcagcgaaat tttttttaaa attaaaaaaa ccaccccgct gcgccgcctg 5520

atggaagcat ttgcaaaacg ccagggtaaa gaaatggata gcctgcgctt tctgtatgat 5580

ggtattcgca ttcaggcaga tcagaccccg gaagatctgg atatggaaga taacgatatt 5640

attgaagcgc atcgcgaaca gattggcggc gaattccata tgaccgtgaa agtgaacgaa 5700

aagaaaaccc tgctgcagta tgtgctggat aacaccagca acgaagtgcc gctgctgccg 5760

agcctgaaag aatatatgga aaccgtgctg gtgtataaaa gcattaaacg cccgctgccg 5820

gcgattcgcc cgcaggaaag ctttgatgaa tttatgaaag aactggtgac ccgcctggtg 5880

atggaaaaga gcaacaacgt gattgcgtat ggctataaaa ccagcgcgat ggaaagccgc 5940

agcattttta ccacctttca tagcagcggc aactttattc tgacccatat taccagccat 6000

aactggagca ccatttttag cctgctgggc ccgaaaaaat ttctggaact gctggtgaac 6060

aacaaaggct ttgtgagcaa agtgaacggc gaaagcgtgc agatttttgg cgatgtgaac 6120

agccatcgca aagcggtggt ggtgagcaaa tatattacca aatttaacgt gctgtataac 6180

agctatagcc gcgattttag ccgctttgaa atgattcgcc cgagcattca gaccattctg 6240

caggatattc tgagctttag cggcctgaac ccgggccgca gcagcaaacg ctatcgcggc 6300

tttaaaagcc tgctgagccg cattattgcg aacgataaaa aatgccgcta tgatattctg 6360

tatgcgaaat ttattggcac cagcaaatgc aactttgcga acgtggtgag caacaaaacc 6420

gaaattagcc aggtgattca gtttgtgctg ctggtgctgg gcaaactgct gccgctggat 6480

gcgtggggcg gcgtgagcaa caagaaaatt attaaagatc gcgtggtgga ttttctgctg 6540

ctgggcgcga acgaaaagat tcacatggat gatctgtttc gcggcattcg cctgaaagat 6600

tttaaatggc tgggccgcgc gcatcagatt agcagcaaac aggattttga actgcgcacc 6660

gcgtttctga aaggctatct gtggtggctg tttgaacatc tgctgaaaaa cattctgcgc 6720

agcttttggt atattaccga aaccagcagc attgtgagcc tggaactgaa ctattttccg 6780

cagtatctgt ggaaagaact gtatgaaagc tgggtgagca aatatgcgaa aaacaacctg 6840

gtgaaaatgc cgagcaaaat tcagcgcgaa cagctgccgt gcggcaaaat taaactgatt 6900

ccgaaacgca gcagctttcg cgtgatttgc gtgccgatta aacgcagcct gaaactgctg 6960

aacaagaaac tggaactgga taccctggaa aaagaaaaac gcgaatttga acgctatcgc 7020

aaagaagtgc tgagcccggt gggccagatt ctgcgcctga aactgagcaa actgcgcgat 7080

acctatgaaa gctatcgcgc gagcgtgcat agcagcagcg atgtggcgga aaaaattagc 7140

gattatcgcg atagcctgct gacccgcttt ggcgaaattc cgaaactgtt tattctgaaa 7200

tttgatatga aagaatgcta tgatcgcctg agccagccgg tgctgatgaa aaaactggaa 7260

gaactgtttg aaaaccagga taacaaaacc agctattatg tgcgctatta tgcgcagctg 7320

gatgcgagcc ataaactgaa aaaagtgaaa accaccattg atacccagta tcataacctg 7380

aacattctga gcagcagccg ccatctgagc aactgcaaaa gcctggtgga taaaaccaaa 7440

accattgcgc tgcagaaagg caacattctg gaagtgtgcc gcagccagat ttatgatgtg 7500

gtgggcagcg tgaaagatgc gcgcggcaac ctgcatctgt ataaacgcaa acgcggcgtg 7560

tttcagggct ttagcctgag cagcattttt tgcgatattc tgtatagcgc gatggtgcat 7620

gattgctttc agtttctgtg gaaaagcaaa caggattttc tgtttgtgcg cctggtggat 7680

gattttctgc tggtgacccc ggatagcaac atttatgatc aggtgcataa cattctgagc 7740

ggcaaaattc tggaaagcta tggcgcgttt gtgaacaaag ataaaaccgt ggtggtgaac 7800

cagaccacca ccaaaccgag cattgatttt gtgggcctgg aagtgaacac caccgatctg 7860

agcattaaac gcaacagcgg cagcattagc ctggtgacca ccaactttcg cacctttaaa 7920

accctggtga aatatctgaa aaccttttat cagctgaacc tggaaggctt tctgctggat 7980

tgcagctttg gcgtgctgga aaacgtgctg gaaaacatgg gcagcctgct gcgcctggtg 8040

ctgcgcgaat ttaaaaccaa atttaccagc attgtgaaat atgatacctt tcattgctat 8100

aaatttatta aatttctgta tgatattagc aactatacca ttgtgaaata tgtggaaacc 8160

aacagcgatt gggatggcgc gccggaactg ctgaactgca ttaaacagat tattgtgaaa 8220

gaatttagca gctttgaaag ctatagcgaa attgtggaat gggtgcagac cctgaacatt 8280

taactcgagg atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc 8340

gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg 8400

ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca 8460

agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag 8520

atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa 8580

gcttatcgat gataagctgt caaacatgag aa 8612

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

taggaattcc atatgcagac catctctctg ctgcagtacg tt 42

<210> 8

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcgctcgagt tagtcgacga tggtcagggt gtacagccat tcga 44

<210> 9

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

taggaattcc atatgaccgt gaaagtgaac gaaaagaaaa ccctgctgca gta 53

<210> 10

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tcgctcgagt tagtcgacta tattcaatgt ttgtacccac tcgactattt c 51

<210> 11

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttagtgtaag ga 12

Claims

1. Genes expressing telomerase, the genes comprising a first gene or a second gene, the first gene expressing a first telomerase; the second gene expresses a second telomerase; the amino acid sequence of the first telomerase is shown in SEQ ID NO. 1; the amino acid sequence of the second telomerase is shown in SEQ ID NO. 2.

2. The gene of claim 1, wherein the nucleotide sequence of the first gene is shown as SEQ ID No. 3; the nucleotide sequence of the second gene is shown as SEQ ID NO. 4.

3. A recombinant plasmid comprising the gene of claim 1 or 2, said recombinant plasmid being constructed with a backbone plasmid comprising pET15b-s μmo.

4. The recombinant plasmid according to claim 3, wherein the backbone plasmid for construction of the recombinant plasmid comprises pET15b-s μmo, pPICZA, pPICZaA or pTrix.

5. A recombinant cell comprising the recombinant plasmid of claim 3 or 4.

6. The recombinant cell according to claim 5, wherein when the backbone plasmid for construction of the recombinant plasmid is pET15b-s μmo, the original cell of the recombinant cell comprises Escherichia coli; when the skeleton plasmid for constructing the recombinant plasmid is pPICZA or pPICZaA, the primary cell of the recombinant cell comprises yeast; when the backbone plasmid for construction of the recombinant plasmid is pTrix, the original cells of the recombinant cells include Sf9 cells.

7. Use of the gene of claim 1 or 2 or the recombinant plasmid of claim 3 or 4 or the recombinant cell of claim 5 or 6 for the preparation of telomerase.

8. The use according to claim 7, wherein, when the original cell of the recombinant cell is E.coli, the use comprises the following steps:

5) centrifuging the crushed solution for 30-50 min at 10000-15000 rpm, collecting supernatant, filtering the supernatant by adopting a 0.45-micrometer filter membrane, and collecting first filtrate; and (3) loading the first filtrate to an NI chromatographic column, eluting effluent liquid by eluent, and collecting components corresponding to a third peak to obtain a crude extract containing telomerase.

9. The application of claim 8, wherein the high-pressure crushing time in the step 4) is 3-5 times, and the interval time between two adjacent high-pressure crushing times is 2-5 min; the ultrasonic crushing times are 2-4 times, the ultrasonic treatment time is 6-8 seconds, the interval is 2-4 seconds, and the ultrasonic treatment time is 2-4 min each time.

10. The use according to claim 8, wherein the loading rate in step 5) is 1 ml/min; the elution rate was 2 ml/min.

11. The use according to claim 8, wherein after the crude extract is obtained in step 5), further comprising purifying the crude extract, comprising the steps of: