CN1300315C - Prothoracic gland hormone gene of calf of milk cow and its cloning method and use - Google Patents

Abstract

The calf prothymosin gene of dairy cows and its cloning method and application belong to the fields of molecular biology and biotechnology. The invention separates the gene encoding cow prothymosin from cow thymus tissue. Specific primer pairs for reverse transcription and PCR amplification were designed, and the gene encoding prothymosin was isolated from cows by reverse transcription-polymerase chain reaction. The gene sequence length was 330 bases, and the expression vector was further constructed , to transform the host cell. The prothymosin protein is obtained in the host cell culture solution, and the obtained prothymosin protein has relatively high activity. The recombinant gene of the present invention can be used in the industrialized production of medical product-thymosin.

Description

Calf prothymosin gene of dairy cow and its cloning method and application

(1) Technical field

The invention relates to isolating prothymosin α gene from cow thymus tissue, its cloning, recombination and expression method and the application of the expression product, and belongs to the field of molecular biology and biotechnology.

(2) Background technology

The thymus is the central organ of the body's immune system. The thymosin secreted by it is a group of active peptide substances, which are divided into α and β group thymosins. In the treatment of diseases, α-thymosin is mainly used. Thymosin mainly acts on T cells, promotes the differentiation and maturation of T cells, regulates the production level of B cell antibodies, maintains the balance of the body's immune system, improves the activity of killer cells and macrophages, and promotes the production of interleukin-2 and interferon-α. Synthesis etc. Prothymosin α has multiple and approximately the same effects as thymosin α1, such as the treatment of systemic lupus erythematosus, tumors, hepatitis B and C, other viral diseases, autoimmune diseases and congenital immunodeficiency diseases, etc. . Thymosin (thymosin) combined with hepatitis B vaccine has immune regulation and antiviral effects. The author believes that the application of thymosin hepatitis B vaccine can enhance the anti-apoptosis ability of patients' liver cells and reduce the degree of necrosis of liver cells, thereby improving the immune function of patients, enhancing the inhibitory effect on HBV, and reducing the immune damage of liver cells. Promote liver cell repair and improve protein metabolism. At present, a kind of ideal antiviral drug has not been developed in the world, so the only way to prevent viral diseases is to improve the body's resistance and vaccine-specific vaccination. Years of clinical practice have proved that thymosin has a certain effect on viral diseases. It is also one of the antiviral drugs currently used in combination with interferon, and they have played a huge role in the treatment of hepatitis B, hepatitis C and tumors.

At present, thymosin α1 is mainly produced by chemical synthesis in foreign countries. The synthetic thymosin α1 developed by Italian company Sciclone has been put on the market, but domestic thymosin α1 has not yet been industrialized. Thymosin α1 sold in the market is all artificially synthesized, and the cost is high. Tissue extraction is limited by the source of materials, and the output is very low, the price is relatively expensive, and the application is limited.

(3) Contents of the invention

Aiming at the deficiencies of the prior art, the invention provides a calf prothymosin gene of a dairy cow, a cloning method and application thereof. In the invention, the full-length cDNA encoding cow prothymosin is isolated from cows for the first time, connected to an expression vector, and then transformed into a yeast strain, and the expression product has higher activity.

The calf prothymosin gene of a dairy cow has the following sequence:

1.SEQ ID NO.1: DNA sequence

(1) Sequence features

* Length: 330 base pairs

*Type: nucleic acid

* Chain type: double chain

*Topology: Linear

(2) Molecular type: cDNA

(3) Original source: Cow thymus tissue

(4) Sequence description:

ATGTCAGACG CGGCCGTGGA CAACAGCTCC GAGATCACCA CCAAGGACTT 50

AAAGGAGAAG AAGGAAGTTG TGGAGGAGGC GGAGAATGGG AGAGAGGCAC 100

CTGCAAATGG GAATGCTAAT GAGGAAAATG GGGAGCAGGA AGCAGACAAC 150

GAGGTAGACG AAGAAGAGGA GGAAGGTGGG GAGGAAGAGG AGGAGGAGGA 200

AGGTGACGGT GAGGAAGAGG ACGGAGATGA AGATGAGGAG GCCGAGGCAG 250

CTACGGGCAA ACGGGCAGCT GAAGATGACG AGGATAACGA TGTGGATACC 300

AAGAAGCAGA AGACTGATAA AGATGACTAG 330

2.SEQ ID NO.2: amino acid sequence

(1) Sequence features

*Length: 109 amino acids

*Type: amino acid

* Chain type: single chain

*Topology: Line type

(2) Molecular type: protein

(3) Sequence description

Met Ser Asp Ala Ala Val Asp Asn Ser Ser Glu Ile Thr Thr Lys

1 5 10 15

Asp Leu Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn Gly

20 25 30

Arg Glu Ala Pro Ala Asn Gly Asn Ala Asn Glu Glu Asn Gly Glu

35 40 45

Gln Glu Ala Asp Asn Glu Val Asp Glu Glu Glu Glu Glu Gly Gly

50 55 60

Glu Glu Glu Glu Glu Glu Glu Gly Asp Gly Glu Glu Glu Asp Gly

65 70 75

Asp Glu Asp Glu Glu Ala Glu Ala Ala Thr Gly Lys Arg Ala Ala

80 85 90

Glu Asp Asp Glu Asp Asn Asp Val Asp Thr Lys Lys Gln Lys Thr

95 100 105

Asp Lys Asp Asp 109.

The cloning method of the calf prothymosin gene of dairy cow of the present invention is as follows, comprises the steps:

(1) Design specific primers:

Forward primer 1: CCA TCT TTG CAT TGT TCC

Reverse primer 2: CTC ATA CAA ACC AAC TCA TTA TAG TAC AGC

(2) extract total RNA or mRNA from the calf thymus tissue of dairy cows, then use reverse transcriptase and reverse primer 2 to produce double-stranded mRNA/cDNA hybrid molecules by the mRNA of the prothymosin gene;

(3) using DNA polymerase, forward primer 1 and reverse primer 2 to produce double-stranded cDNA molecules by PCR reaction after step (2);

(4) inserting the obtained double-stranded cDNA molecule into the vector, and transforming the host cell;

(5) A plasmid containing the prothymosin gene is obtained after host cell proliferation:

a) extracting and determining the prothymosin gene in the plasmid,

b) inserting the above-mentioned genes into an expression vector to construct a recombinant expression vector,

c) transforming the host cell with the above-mentioned recombinant expression vector,

d) After the above-mentioned host cells are cultured and multiplied in the medium, they accumulate prothymosin protein.

The prothymosin protein expressed and accumulated in the above step (5) is separated and purified to obtain calf prothymosin protein, which has the amino acid sequence shown in SEQ ID NO.2.

The reaction components of above-mentioned steps (2) are as follows:

Reverse transcriptase 1ul

Reverse primer 2ul

Thymosin gene mRNA 13ul

5 times RT-Buffer 5ul

10mM dNTP 1ul

The reaction components and reaction conditions of above-mentioned steps (3) are as follows:

10x reaction buffer 5 μl

25mM MgCl2 4μl

10mM dNTP 1μl

Forward primer 1 5p mol

Reverse primer 2 5p mol

Template cDNA 2μl

DNA polymerase 0.5μl

Sterilized deionized water 36.5μl

Total volume 50μl

PCR reaction conditions:

94°C for 4 minutes; cycle: 94°C for 45 seconds, 55°C for 50 seconds, 72°C for 40 seconds, a total of 30 cycles, 72°C for 3 minutes.

The PCR amplified product was purified and ligated to a DNA vector. Transform the competent cells with the recombinant DNA vector. The cells containing the recombinant DNA vector are cultured and proliferated in the medium, and positive cultures are screened. After the positive culture was continued to be cultured and proliferated, the plasmid was extracted, identified by electrophoresis, enzyme digestion and PCR amplification, and sequenced. Sequencing results show that the present invention obtains a 330bp DNA fragment, which is confirmed to be the calf prothymosin gene of dairy cows through comparison with the prothymosin gene sequence in the gene bank.

The present invention separates the prothymosin α gene, clones, identifies, and determines the sequence of the prothymosin α gene; constructs a recombinant gene containing the prothymosin α gene; the recombinant gene can accumulate and express the prothymosin protein in host cells ; The recombinant gene is used to produce prothymosin; the prothymosin α protein expressed by gene technology has the biological activity of natural product. The calf prothymosin α gene of the dairy cow has the characteristics of SEQ ID NO.1, and the dairy cow prothymosin α protein produced by expression has the characteristics shown in SEQ ID NO.2. The recombinant gene of the present invention can be used in the industrialized production of medical product-thymosin.

The prothymosin gene sequence of the present invention has a length of 330 bases, which is 3 bases missing than the published prothymosin gene of some humans and animals. The nucleotide difference rates with human prothymosin genes AF348514, BC003510, BC022433, BC051265, J04799 and L21693 were 9.8%, 9.8%, 10.5%, 9.8%, 10.6%, 11.2%; The nucleotide difference rate was 18.5%, and the nucleotide difference rate with the mouse M20035 and NM008972 prothymosin genes was 10.1%.

According to the nucleotide sequence of the bovine prothymosin gene obtained in the present invention, the following primers are designed to construct the expression vector.

Forward primer 1': ATG TCA GAC GCG GCC GTG

Reverse primer 2': CTC GAG CTA GTC ATC TTT ATC AGT CTT C

Using the reverse-transcribed cDNA of bovine thymus total RNA as a template, the sequence containing the full-length coding frame was amplified by PCR, the PCR product was digested with XhoI and EcoRI, and the digested fragment was connected to the expression vector. Prokaryotic host cells were transformed by heat shock method, and positive clones were selected for sequencing identification.

The purified recombinant DNA vector is digested and linearized, transformed into yeast cells by the cesium chloride method, and the resistant transformants are screened in the culture medium.

The expression of prothymosin was induced in the medium medium, and 1 ml of the supernatant was taken every day, and the expression was induced for 7 days. The samples were silver-stained after SDS-PAGE. The molecular weight of the expression product determined according to the standard protein was: 1.3×10 ⁴ D.

The biological activity of prothymosin was measured by ³ H-TdR incorporation method. The mouse spleen was ground on a steel sieve, and the mononuclear cells were separated with lymphocyte separation medium, washed with PBS, and suspended in 1640 10% FCS. In the culture medium, 4×10 ⁵ /well spleen cells and 125ug/well ConA were added to the 96-well culture plate. Cultivate in a carbon dioxide incubator at 37°C for 6 hours, then add different concentrations of fusion protein and commercial Tα1, continue to cultivate for 72 hours, add ³ H-TdR185×10 ^-3 GBq to each well and continue to cultivate for 6 hours, harvest the cells on glass fiber filter paper, After drying, detect the cpm value with a liquid scintillation counter, and calculate the proliferation rate according to the following formula:

Proliferation rate (%)=(cpm value of experimental group-cpm value of control group)/cpm value of control group×100%

The results showed that prothymosin had obvious proliferative effect on mouse spleen lymphocytes.

The calf prothymosin cDNA obtained in the present invention is transformed into yeast and expressed through the existing genetic engineering technology, and can be used to produce thymosin that can improve human immunity and has anticancer effects, and can be used in pharmaceutical products production preparation.

The invention uses the gene expression method to produce prothymosin, which not only solves the problem of raw material source, but also solves the bottleneck of industrialized mass production, and can reduce the current market price of thymosin, and open up a wider application range. Due to the role played by thymosin protein in the medical field, the present invention isolates the gene encoding cow prothymosin from cow thymus tissue, designs specific primer pairs for reverse transcription and PCR amplification, and utilizes reverse transcription-polymerization Enzyme chain reaction, isolation of the gene encoding prothymosin from cows. Further construct expression vectors and transform host cells. The prothymosin protein is obtained in the host cell culture solution, and the obtained prothymosin protein has relatively high activity.

(4) Description of drawings

Figure 1 is the PCR electrophoresis pattern of the coding region of the prothymosin gene, wherein lane 1 is the standard DNA molecular weight Mark of 100 bp, and lanes 2 and 3 are the positive PCR of the target gene.

Figure 2 is the plasmid restriction map of the coding region sequence, wherein lane 1 is the standard DNA molecular weight Mark of 100 bp, swimming lane 2 is the standard DNA molecular weight Mark of 1 kb, and swimming lanes 3-6 are the results of positive plasmid digestion.

Fig. 3 is the enzyme digestion map of the expression vector, wherein lane 1 is the standard DNA molecular weight Mark of 1 kb, lane 2 is the negative control, and lane 3 is the positive. From the 330bp band cut out in 3, it can be seen that the expression vector has been successfully constructed.

Fig. 4 is a graph showing the activity of prothymosin, ie, the effect of expressed prothymosin on the proliferation of mouse spleen lymphocytes. The abscissa is the action time, and the ordinate is the proliferation rate of mouse spleen lymphocytes. "—▲—" indicates the proliferation rate of the genetically engineered bacteria, and "—■—" indicates the proliferation rate of thymosin before commercial products. In the past, thymosin was first incubated with lymphocytes for about 5 hours, and then stimulated with ConA, which produced the best proliferation-promoting effect.

(5) Specific implementation methods

The present invention will be further described below in conjunction with specific examples.

Example 1:

1. Collection and processing of calf thymus tissue from dairy cows

Thymus tissue was collected from a 12-day-old bull calf and stored frozen at -80°C. This calf bull is a Friesian dairy breed. Take 3 grams of thymus tissue stored in a -80°C refrigerator, cut it into pieces, place it in a sterile mortar, add sterile quartz sand for grinding, add 0.01M sterilized phosphate buffer at a ratio of 1:3 to make a cell suspension , stored in -20°C refrigerator for later use.

2. Design of primers for reverse transcription and PCR amplification of cow prothymosin α gene

The prothymosin gene sequences of 6 humans, 1 chicken and 2 mice were downloaded from GenBank, and the accession numbers are AF348514, BC003510, BC022433, BC051265, J04799, L21693 and BM486191. Using biological software to compare and analyze the sequence differences or homology of the above genes, a pair of primers were designed, which is of great significance in the reverse transcription of prethymosin mRNA and cDNA amplification to obtain the prethymosin α gene:

Forward primer 1: CCA TCT TTG CAT TGT TCC

Reverse primer 2: CTC ATA CAA ACC AAC TCA TTA TAG TAC AGC

Reverse primer 2 was used for reverse transcription of prothymosin mRNA; forward primer 1 and reverse primer 2 were used together for PCR amplification of prothymosin α gene.

3. Cloning method of cow prothymosin α gene

1. Extraction of total RNA

a) The calf thymocyte suspension stored in a -20°C refrigerator was repeatedly frozen and thawed twice.

b) Add 450 μl of cell lysate containing β-mercaptoethanol and 450 μl of absolute ethanol to 450 μl of thymocyte suspension.

c) Adsorb nucleic acid with a silica gel column.

d) Wash the silica gel column with buffer.

e) Nucleic acid was eluted with RNase-free sterilized water, centrifuged and collected in a test tube.

f) adding an RNase inhibitor to the above RNA extract.

2. mRNA reverse transcription forms mRNA/cDNA hybrid strand (first strand):

First design the following primers:

Forward primer 1: CCA TCT TTG CAT TGT TCC

Reverse primer 2: CTC ATA CAA ACC AAC TCA TTA TAG TAC AGC

Take 2ug of total RNA, add 5 μl of 5 times RT buffer, 1 μl of 10 mM dNTP, 21 μl of reverse primer, 10 u of reverse transcriptase, react at 42°C for 60 minutes, and react at 72°C for 10 minutes to terminate the reaction.

3. Synthesize the double-stranded cDNA fragment of calf prothymosin gene with forward primer 1, reverse primer 2 and DNA polymerase by PCR method.

(1) Reaction system

10x reaction buffer 5 μl

25mM MgCl 24μl

10mM dNTP 1μl

Forward primer 1 5p mol

Reverse primer 2 5p mol

Template cDNA 2μl

Taq DNA polymerase 0.5μl

Sterile water 36.5μl

Total volume 50μl

(2) PCR reaction conditions

94°C for 4 minutes; cycle: 94°C for 45 seconds, 55°C for 50 seconds, 72°C for 40 seconds, a total of 30 cycles, 72°C for 3 minutes.

4. Gene cloning: Take 2 μl of PCR product and connect it with pGEM-T easy vector, and the specific operation steps are carried out according to the instructions of pGEM-T easy Vector system of Promega company. Then the ligation product was transformed into Escherichia coli DH5a competent strain, grown overnight on the LB plate containing ampicillin coated with 5-bromo-4-chloro-3-indole-D-galactoside and X-gal, picked White single colonies were cultured overnight in LB liquid medium by shaking.

5. Extraction of plasmid DNA: Take 1-2ml of the overnight culture solution and centrifuge at 10000rpm for 1 minute. The cells were collected, and the plasmids were purified with a micro DNA purification kit from Promega, USA, and the purification steps were performed according to the instructions.

6. Sequence determination: take the purified plasmid and carry out automatic sequencing with vector primers. The detected gene has the following sequence information:

(1) Sequence features

* Length: 330 base pairs

*Type: nucleic acid

* Chain type: double chain

*Topology: Linear

(2) Molecular type: cDNA

(3) Original source: Cow thymus tissue

(4) Sequence description:

ATGTCAGACG CGGCCGTGGA CAACAGCTCC GAGATCACCA CCAAGGACTT 50

AAAGGAGAAG AAGGAAGTTG TGGAGGAGGC GGAGAATGGG AGAGAGGCAC 100

CTGCAAATGG GAATGCTAAT GAGGAAAATG GGGAGCAGGA AGCAGACAAC 150

GAGGTAGACG AAGAAGAGGA GGAAGGTGGG GAGGAAGAGG AGGAGGAGGA 200

AGGTGACGGT GAGGAAGAGG ACGGAGATGA AGATGAGGAG GCCGAGGCAG 250

CTACGGGCAA ACGGGCAGCT GAAGATGACG AGGATAACGA TGTGGATACC 300

AAGAAGCAGA AGACTGATAA AGATGAGTAG 330

7. Homology search: use BLAST software to compare the isolated sequence with the sequence in GENEBANK.

Comparison results: the length of the prothymosin gene sequence of the present invention is 330 bases, which is 3 bases less than the published prothymosin genes of other humans and animals. The nucleotide sequence difference rates with human prothymosin genes AF348514, BC003510, BC022433, BC051265, J04799 and L21693 were 9.8%, 9.8%, 10.5%, 9.8%, 10.6%, 11.2%, respectively; with chicken BM486191 prothymosin gene The nucleotide difference rate of the gene is 18.5%, and the nucleotide difference rate of the mouse M20035 and NM008972 prothymosin genes is 10.1%.

Embodiment 2: Construction of expression vector

1. according to the nucleotide sequence of the cow prothymosin gene that embodiment 1 obtains, design primer:

Forward primer 1': ATG TCA GAC GCG GCC GTG

Reverse primer 2': CTC GAG CTA GTC ATC TTT ATC AGT CTT C

2. Obtaining the Complete Prothymosin Gene

The polymerase chain reaction was carried out using the recombinant plasmid containing the cow prothymosin gene as a template.

(1) Reaction system:

10x reaction buffer 5 μl

25mM MgCl2 4μl

10mM dNTP 1μl

Forward primer 1' 5p mol

Reverse primer 2' 5p mol

Plasmid DNA 2 μl

Taq DNA polymerase 0.5μl

Sterile water 36.5μl

Total volume 50μl

(2) PCR reaction conditions: 94° C. for 3 minutes. Cycle: 94°C for 45 seconds, 51°C for 50 seconds, 72°C for 40 seconds, a total of 30 cycles. Extension at 72°C for 5 minutes.

3. Ligate 600 μg of the digested PCR product to 100 μg of vector, transform the ligated product into Escherichia coli, and screen resistant transformants on LB plates containing G418. Restriction digestion and sequence determination were carried out after the plasmid was extracted. The correctly sequenced strains were stored in glycerol.

4. A large number of recombinant vector plasmids were extracted, purified, linearized, and transformed into yeast strains by LiCl transformation method, and the resistant transformants were screened on the YPD plate containing G418. After the plasmid was extracted, the resistant transformants were further identified by methods such as PCR and enzyme digestion. Yeast strains containing recombinant vectors were stored in glycerol.

Example 3: Induced protein expression and identification of expression products in eukaryotic yeast cells

1. Induce the expression of prothymosin in BMGY medium containing 20ml/L methanol, take 1ml of cell culture medium every day, induce expression for 7 days, and save the samples for future use.

2. Isolation and Purification of Prothymosin Protein

The collected cell culture solution was centrifuged at 9000rpm for 20 minutes, the supernatant was collected, and the protein was purified by Ni ²⁺ -chelatingSepharose affinity column chromatography, and the elution peak was collected for activity determination.

3. Determination of molecular weight: SDS-PAGE urea method. After the sample was subjected to SDS polyacrylamide electrophoresis and silver staining, the molecular weight of the expressed product was determined to be 1.3×10 ⁴ D according to the standard protein.

4. Determination of protein content was carried out by the Lowry method, using bovine serum albumin as the standard protein.

5. Purity identification Two gradients of 10% and 16% were used for PAGE. In the vertical electrophoresis tank, sequentially add 16% and 10% separation gel to polymerize, then add stacking gel, and insert the sample comb. Sample loading, electrophoresis. Current 20mA. After electrophoresis, the gel plate was fixed with 50%-0.054% formaldehyde solution for 1 hour, then stained with 0.25% Coomassie Brilliant Blue G250 dye solution for 2 hours, and decolorized with 15% methanol-7.5% acetic acid solution.

Example 4: Bioactivity assay

1. Lymphocyte proliferation assay ( ³ H-TdR incorporation method)

Spleen of mice was aseptically collected, ground and pulverized in a mortar with sterile quartz sand, lymphocyte separation medium was used to separate lymphocytes, washed twice with 0.01M PBS pH7.2, and centrifuged. The pellet was resuspended in 1640 medium containing 10% calf serum. Add splenocytes 4×10 ² /well and different concentrations of prothymosin to the 96-well culture plate at different times, culture in a CO ₂ incubator at 37°C for 6 hours, then add ConA at a final concentration of 5 μg/ml, and Set up a control group. After culturing the culture plate at 37°C and 5% CO ₂ for 72 hours, add ³ H-TdR18.5×10 ^-3 GBq to each well and continue culturing for 6 hours. Harvest the cells on glass fiber filter paper, dry them, and count them with liquid scintillation The instrument detects the cpm value. The proliferation rate was calculated according to the formula:

Proliferation rate (%)=(cpm value of experimental group-cpm value of control group)/cpm value of control group×100%

test results:

When splenocytes and prothymosin were pre-acted for 6 hours, the effect of 60-120 μg/ml dose of prothymosin on the proliferation of lymphocytes was the most obvious. High doses of prothymosin (>680μg/ml) will have a certain inhibitory effect.

2. NK cell activity assay

Experimental group: Mouse spleen lymphocytes were treated with different concentrations of prothymosin at 37°C for 3 hours.

Control group: mouse spleen lymphocytes were reacted with culture medium at 37°C for 3 hours.

Then it was washed with culture medium, and the pellet was resuspended in culture medium as effector cells. NK activity was measured by ⁵¹ Cr release method. 100 μl of effector cells at different concentrations and 100 μl of ⁵¹ Cr-labeled YAC-1 target cells were added to a 96-well culture plate, and culture medium was used instead of effector cells as a natural release control. Centrifuge at 1000rpm for 5min, act at 37°C and 5% _CO2 for 6h, and calculate the cytotoxic activity according to the following formula:

Cytotoxic activity (%) = cpm (experimental group - natural release group) cpm (maximum release group - natural release group) × 100%.

The results showed that prothymosin at a dose of 10-1000 μg/ml interacted with lymphocytes could significantly enhance the killing effect of NK cells.

3. IL-1 activity detection

3 days before the experiment, pMφ was induced in aged mice with TG medium, and pMφ was collected according to conventional methods. Prepare a cell suspension with a concentration of 2×106/ml in RPM1640 culture solution, add it to each well of the culture plate, then add LPS and different concentrations of the drug to be tested, act for 24 hours, and harvest the supernatant, which is the IL-1-containing culture solution . Thymocytes of experimental mice were used as target cells, and triple wells were set up, and IL1 activity was measured by conventional 3H-TdR incorporation method. A blank control and a negative control were set up.

The results showed that prothymosin could significantly increase the activity of IL1 secreted by pMφ in aged mice under the stimulation of LPS.

Embodiment 7: the recombinant application of calf prothymosin cDNA

The calf prothymosin cDNA obtained in Example 1 is transformed into yeast and expressed through existing genetic engineering technology methods, and can be used to produce thymosin that can improve human immunity and has an anticancer effect. Production preparation of pharmaceutical products.

Embodiment 8: clinical application of thymosin

1. Malignant tumor

Chemotherapy and radiotherapy for malignant tumors have a negative impact on the immune function of the body. Li Xiuqing et al. (1991) used thymosin as adjuvant therapy for tumor patients with radiotherapy and chemotherapy. Compared with the control group without thymosin, it was concluded that thymosin can regulate and enhance the immune function of tumor patients, and reduce the side effects of chemotherapy and radiotherapy. function, improve curative effect and prolong survival period. Animal experiments have shown that thymosin-treated starved lymphocytes can significantly inhibit the metastasis of pulmonary melanoma. Zheng Jindi et al. (1994) reported that the effective rate of thymosin in the treatment of lung cancer was 64%, which can enhance the resistance of patients and reduce the response to chemotherapy. The drug has almost no irritation and side effects.

2. Viral hepatitis and liver cirrhosis

Thymosin can promote the production of IL-2, regulate immune function, and have direct or indirect protective effects on liver cells. Example Dixin et al (1989) treated chronic severe hepatitis with thymosin showed that the survival rate of patients in the treatment group was twice as high as that in the untreated group. Gao Tao et al. (1997) reported that thymosin can regulate the immune function of human cells, can inhibit and eliminate hepatitis B virus, and treat hepatitis B. It can make the negative conversion rate of HbeAg in patients with hepatitis B reach 72.27%, and the clearance rate of hepatitis B virus DNA reaches 54.55%. And no toxic side effects were seen.

3. Kidney disease

Thymosin can increase the absolute number of T cells, activate phagocytes, and offset the side effects of immunosuppressant therapy. Xiang Yi et al. (1991) treated 20 patients with nephropathy with thymosin and found that thymosin could improve clinical symptoms.

4. Flat warts

Rui Jianxin et al. (1991) treated 45 patients with flat warts with thymosin. Compared with levamisole, the cure rate was 21/3 times higher and the effective rate was 14/6 times higher.

5. Recurrent respiratory infections in children

Zheng Jinjuan et al. (1990) used thymosin to treat 100 cases of children with recurrent respiratory tract infection, intramuscular injection 1-3 times a week, 2-3 weeks as a course of treatment. The results showed that the total effective rate was 88%, and the cure rate was 59%, which was obviously superior. in the control group. After treatment, the immune function of the treatment group increased, the body weight increased, and the side effects were minimal.

Sequence Listing

<110> Institute of Biology, Shandong Academy of Sciences

<120> Calf prothymosin gene of dairy cow and its cloning method and application

<140>

<141>

<160>2

<170>Patent In3.1

<210>1

<211>330

<212>DNA

<213> Cow thymus tissue

<400>1

atgtcagacg cggccgtgga caacagctcc gagatcacca ccaaggactt aaaggagaag 60

aaggaagttg tggaggaggc ggagaatggg agagaggcac ctgcaaatgg gaatgctaat 120

gaggaaaatg gggagcagga agcagacaac gaggtagacg aagaagagga ggaaggtggg 180

gaggaagagg aggagggagga aggtgacggt gaggaagagg acggagatga agatgaggag 240

gccgaggcag ctacgggcaa acgggcagct gaagatgacg aggataacga tgtggatacc 300

aagaagcaga agactgataa agatgactag 330

<210>2

<211>109

<212>PRT

<213> Cow thymus tissue

<400>2

Met Ser Asp Ala Ala Val Asp Asn Ser Ser Glu Ile Thr Thr Lys

1 5 10 15

Asp Leu Lys Glu Lys Lys Glu Val Va1 Glu Glu Ala Glu Asn Gly

20 25 30

Arg Glu Ala Pro Ala Asn Gly Asn Ala Asn Glu Glu Asn Gly Glu

35 40 45

Gln Glu Ala Asp Asn Glu Val Asp Glu Glu Glu Glu Glu Gly Gly

50 55 60

Glu Glu Glu Glu Glu Glu Glu Gly Asp Gly Glu Glu Glu Asp Gly

65 70 75

Asp Glu Asp Glu Glu Ala Glu Ala Ala Thr Gly Lys Arg Ala Ala

80 85 90

Glu Asp Asp Glu Asp Asn Asp Val Asp Thr Lys Lys Gln Lys Thr

95 100 105

Asp Lys Asp Asp 109

Claims (5)

1, the calf prothymosin gene of milk cow is characterized in that sequence is as follows:

ATGTCAGACG?CGGCCGTGGA?CAACAGCTCC?GAGATCACCA?CCAAGGACTT 50

AAAGGAGAAG?AAGGAAGTTG?TGGAGGAGGC?GGAGAATGGG?AGAGAGGCAC 100

CTGCAAATGG?GAATGCTAAT?GAGGAAAATG?GGGAGCAGGA?AGCAGACAAC 150

GAGGTAGACG?AAGAAGAGGA?GGAAGGTGGG?GAGGAAGAGG?AGGAGGAGGA 200

AGGTGACGGT?GAGGAAGAGG?ACGGAGATGA?AGATGAGGAG?GCCGAGGCAG 250

CTACGGGCAA?ACGGGCAGCT?GAAGATGACG?AGGATAACGA?TGTGGATACC 300

AAGAAGCAGA?AGACTGATAA?AGATGACTAG?330。

2, the cloning process of the calf prothymosin gene of the described milk cow of claim 1 comprises the steps:

(1) design Auele Specific Primer:

Forward primer 1:CCA TCT TTG CAT TGT TCC

Reverse primer 2:CTC ATA CAA ACC AAC TCA TTA TAG TAC AGC

(2) from the calf thymic tissue of milk cow, extract total RNA or mRNA, produce the mRNA/cDNA hybrid molecule of two strands then with ThermoScript II and reverse primer 2 by the mRNA of above-mentioned prothymosin gene;

(3) produce double-stranded cDNA molecule in step (2) back by the PCR reaction with archaeal dna polymerase, forward primer 1 and reverse primer 2;

(4) the cDNA molecule with the two strands of gained inserts in the carrier transformed host cell;

(5) obtain containing the plasmid of prothymosin gene after host cell is bred:

A) prothymosin gene in extraction and the definite plasmid,

B) said gene is inserted in the expression vector, makes up recombinant expression vector, the primer that the target gene PCR that obtains to insert during construction of expression vector is reacted used is as follows:

Forward primer 1 ': ATG TCA GAC GCG GCC GTG

Reverse primer 2 ': CTC GAG CTA GTC ATC TTT ATC AGT CTT C

C) above-mentioned recombinant expression vector transformed host cell,

D) after above-mentioned host cell is cultivated breeding in medium, accumulation prothymosin albumen; Separated and the purifying of cumulative prothymosin albumen, what obtain is calf prothymosin albumen, this albumen has following aminoacid sequence:

Met?Ser?Asp?Ala?Ala?Val?Asp?Asn?Ser?Ser?Glu?Ile?Thr?Thr?Lys

1 5 10 15

Asp?Leu?Lys?Glu?Lys?Lys?Glu?Val?Val?Glu?Glu?Ala?Glu?Asn?Gly

20 25 30

Arg?Glu?Ala?Pro?Ala?Asn?Gly?Asn?Ala?Asn?Glu?Glu?Asn?Gly?Glu

35 40 45

Gln?Glu?Ala?Asp?Asn?Glu?Val?Asp?Glu?Glu?Glu?Glu?Glu?Gly?Gly

50 55 60

Glu?Glu?Glu?Glu?Glu?Glu?Glu?Gly?Asp?Gly?Glu?Glu?Glu?Asp?Gly

65 70 75

Asp?Glu?Asp?Glu?Glu?Ala?Glu?Ala?Ala?Thr?Gly?Lys?Arg?Ala?Ala

80 85 90

Glu?Asp?Asp?Glu?Asp?Asn?Asp?Val?Asp?Thr?Lys?Lys?Gln?Lys?Thr

95 100 105

Asp?Lys?Asp?Asp

109。

3, the cloning process of the calf prothymosin gene of milk cow as claimed in claim 2 is characterized in that, the reactive component of described step (2) is as follows:

ThermoScript II 1ul

Reverse primer 2 1ul

The mRNA 13ul of thymosin gene

5 times RT-Buffer 5ul

10mM?dNTP 1ul。

4, the cloning process of the calf prothymosin gene of milk cow as claimed in claim 2 is characterized in that, described step (3) reactive component and condition are as follows:

10 times of PCR Buffer 5 μ l

25mM?MgCl2 4μl

10mM?dNTP 1μl

Forward primer 15p mol

Reverse primer 2 5p mol

Template cDNA 2 μ l

Archaeal dna polymerase 0.5 μ l

Sterilization deionized water 36.5 μ l

Cumulative volume 50 μ l

The PCR reaction conditions:

94 ℃ 4 minutes; Circulation: 94 ℃ 45 seconds, 55 ℃ 50 seconds, 72 ℃ 40 seconds, totally 30 circulations, 72 ℃ were extended 3 minutes.

5, the calf prothymosin gene of the described milk cow of claim 1 is used to prepare the thymosin pharmaceutical prod.

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