CN111748543A - Immunomodulatory protein mutant and its nucleotide sequence, recombinant plasmid vector, engineering bacteria, construction method and application - Google Patents

Abstract

The invention is applicable to the technical field of genetic engineering, and provides an immunomodulatory protein mutant and a nucleotide sequence, a recombinant plasmid vector, an engineering bacterium, a construction method and application thereof, wherein the amino acid sequence of the immunomodulatory protein mutant is obtained by mutating phenylalanine at the 8 th position into tryptophan on the basis of the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO.1, and the mutated amino acid sequence is SEQ ID NO. 2. On the basis of ensuring that the immunoregulation activity and the antitumor activity are basically unchanged, the thermal stability of the recombinant LZ-8 and the stability of the recombinant LZ-8 medicament are further improved, on one hand, the thermal stability of the immunoregulation protein mutant is greatly improved, and compared with the wild type LZ-8, the phase transition temperature Tm of F8W is increased by 1.86 ℃, and the phase transition enthalpy value delta H is increased by 39.19 kJ/mol; on the other hand, the immunomodulatory protein mutant has immunomodulatory activity and anti-tumor activity consistent with wild type LZ-8, and has application and development values compared with the prior art.

Description

Immunomodulatory protein mutant and its nucleotide sequence, recombinant plasmid vector, engineering Bacteria, construction methods and applications

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to an immunomodulatory protein mutant and its nucleotide sequence, recombinant plasmid vector, genetically engineered bacteria and applications.

Background technique

Ganoderma lucidum immunomodulatory protein LZ-8 is a dimer protein with immunomodulatory activity abundant in Ganoderma lucidum fruiting bodies. Preclinical studies have confirmed that LZ-8 has a variety of immunomodulatory and antitumor activities, such as the ability to promote mouse Spleen lymphocyte proliferation; promotes T cells to secrete various cytokines; can inhibit the growth of lung cancer, gastric cancer and liver cancer in vitro and in vivo in mice; can significantly improve the anti-tumor activity of tumor DNA vaccines; can reduce local and systemic allergic reactions; can slow down diabetes progression; able to alleviate graft-versus-host reactions, etc. Therefore, LZ-8 has great development potential in the field of tumor therapy and autoimmune disease therapy. Wild Ganoderma lucidum has low yield and high price, and is not suitable for the production of Ganoderma lucidum immunomodulatory protein LZ-8. Artificial cultivation of Ganoderma lucidum has a long production cycle and complex process, which cannot meet the needs of industrial production. With the rapid development of genetic engineering and biopharmaceutical engineering technology, the use of genetically engineered strains to strengthen the transcription and translation of target genes can achieve high-efficiency expression and active secretion of target proteins, and can effectively increase the production scale of Ganoderma lucidum immunomodulatory protein LZ-8.

However, in the production process of Ganoderma lucidum immunomodulatory protein LZ-8, due to the existence of mechanical crushing, stirring, cross-flow membrane filtration and other operations, the local heating of the protein solution is inevitably caused, causing protein unfolding and even denaturation, which seriously affects the final product. Therefore, improving the thermal stability of LZ-8 molecule is of great practical significance for the production of LZ-8 molecule with reliable quality and suitable for new drug application. One of the solutions is to seek LZ-8 mutants with improved thermal stability and unchanged or improved pharmacological activity through molecular engineering technology. In the previous work, the researchers performed heterologous expression of the Ganoderma lucidum immunomodulatory protein LZ-8 in yeast, and obtained a recombinant LZ-8 molecule with immunomodulatory activity and antitumor activity. However, the existing technology cannot further improve the thermal stability of the recombinant LZ-8 on the basis of ensuring that the biological activity of the recombinant LZ-8 remains unchanged or improved, resulting in poor stability of the recombinant LZ-8 medicine.

SUMMARY OF THE INVENTION

The purpose of the embodiments of the present invention is to provide an immunomodulatory protein mutant, which aims to solve the problem that the thermal stability of the recombinant LZ-8 cannot be further improved on the basis of ensuring the biological activity of the recombinant LZ-8 remains unchanged or improved in the prior art. , leading to the problem of poor stability of recombinant LZ-8 medicines.

The embodiments of the present invention are implemented in this way, an immunomodulatory protein mutant, the amino acid sequence of the immunomodulatory protein mutant is based on the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO. The phenylalanine at position 8 is mutated into tryptophan, and the amino acid sequence after the mutation is SEQ ID NO.2.

Another object of the embodiments of the present invention is a nucleotide sequence of an immunomodulatory protein mutant, the nucleotide sequence is shown in SEQ ID NO.4.

Another object of the embodiments of the present invention is a recombinant plasmid vector containing the nucleotide sequence.

Another object of the embodiments of the present invention is a genetically engineered bacterium expressing the immunomodulatory protein mutant.

Another object of the embodiments of the present invention is a construction method of the immunomodulatory protein mutant, the construction method comprising:

Based on the amino acid sequence and nucleotide sequence of the immunomodulatory protein LZ-8, the primer F8W-F/R for site-directed mutagenesis was designed; the sequence of the primer F8W-F is shown in SEQ ID NO. 5, and the primer F8W-R The sequence is shown in SEQ ID NO.6;

According to the primers for site-directed mutagenesis, the plasmid carrying the keratinase LZ-8 gene is subjected to site-directed mutagenesis PCR to obtain the target mutant plasmid;

The target mutant plasmid is subjected to PCR amplification to obtain a recombinant plasmid;

The recombinant plasmid is transformed into fungi, and the mutant protein is expressed.

Another object of the embodiments of the present invention is the application of the immunomodulatory protein mutant in the fields of immunomodulator preparation, immune adjuvant preparation, tumor treatment or autoimmune disease treatment.

The amino acid sequence of the immunomodulatory protein mutant provided in the embodiment of the present invention is based on the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO. The latter amino acid sequence is SEQ ID NO.2; on the one hand, the thermal stability of the immunomodulatory protein mutant is greatly improved. Compared with the wild-type LZ-8, the F8W phase transition temperature Tm increases by 1.86 °C, and the phase transition enthalpy value ΔH was increased by 39.19kJ/mol; on the other hand, the immunomodulatory protein mutant had the same immunomodulatory activity as wild-type LZ-8, and had a mitogenic effect on Balb/c mouse splenocytes, and the maximum mitogenic dose was 0.3125μg /ml, in addition, it also has the same anti-tumor activity as wild-type LZ-8, and has a growth inhibitory effect on Hela cells cultured in vitro, and its IC50 dose is 2.292 μg/ml; that is, the immunomodulatory protein mutant provided by the present invention. On the basis of ensuring that the immunomodulatory activity and antitumor activity are basically unchanged, the thermal stability of the recombinant LZ-8 and the stability of the recombinant LZ-8 drug are further improved, which has more application and development value than the prior art.

Description of drawings

Fig. 1 is the simulated steady-state conformation diagram of wild-type Ganoderma lucidum immunomodulatory protein LZ-8 provided in the embodiment of the present invention at different temperatures;

Fig. 2 is the temperature sensitive area map of B-factor prediction of Ganoderma lucidum immunomodulatory protein LZ-8 provided in the embodiment of the present invention;

Fig. 3 is the SDA-PAGE gel electrophoresis image of the purified protein of Ganoderma lucidum immunomodulatory protein mutant provided in the embodiment of the present invention;

4 is a graph showing the immunomodulatory activity assay curve of Ganoderma lucidum immunomodulatory protein LZ-8 and its various mutants provided in the embodiment of the present invention;

Fig. 5 is a graph showing the anti-tumor activity of the Ganoderma lucidum immunomodulatory protein LZ-8 and its mutants provided in the embodiment of the present invention.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

In order to solve the problem that the existing technology cannot further improve the thermal stability of recombinant LZ-8 on the basis of ensuring the biological activity of recombinant LZ-8 remains unchanged or improved, resulting in poor stability of recombinant LZ-8 medicine, Provided is an immunomodulatory protein mutant whose amino acid sequence is based on the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO. The amino acid sequence of F8W is SEQ ID NO.2; on the one hand, the thermal stability of the immunomodulatory protein mutant is greatly improved. Compared with the wild-type LZ-8, the phase transition temperature Tm of F8W increases by 1.86℃, and the phase transition enthalpy value ΔH On the other hand, the immunomodulatory protein mutant has the same immunomodulatory activity as wild-type LZ-8, and has a mitogenic effect on Balb/c mouse splenocytes, and the maximum mitogenic dose is 0.3125μg/ ml, in addition, has the same anti-tumor activity as wild-type LZ-8, and has a growth inhibitory effect on HeLa cells cultured in vitro, and its IC50 dose is 2.292 μg/ml.

It is worth noting that the current existing technology can only increase the phase transition temperature Tm by 0.9 °C and the phase transition enthalpy ΔH by 23.14 kJ/mol, which cannot further improve the thermal stability of the recombinant LZ-8. -8 has the problem of poor stability of finished medicines, while the immunomodulatory protein mutant provided by the present invention can further improve the thermal stability of the recombinant LZ-8 and improve the The stability of the recombinant LZ-8 drug has more application and development value than the existing technology.

The embodiment of the present invention provides an immunomodulatory protein mutant, the amino acid sequence of the immunomodulatory protein mutant is based on the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO. Alanine is mutated into tryptophan, and the mutated amino acid sequence is SEQ ID NO.2.

SEQ ID NO. 1:

MSDTALIFRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAYTYRVAVSGRNLGVKPSYAVESDGSQKVNFLEYNSGYGIADTNTIQVFVVDPDTNNDFIIAQWN

SEQ ID NO. 2:

MSDTALIWRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAYTYRVAVSGRNLGVKPSYAVESDGSQKVNFLEYNSGYGIADTNTIQVFVVDPDTNNDFIIAQWN

In the examples of the present invention, a site-directed mutant was constructed based on the amino acid sequence (P14945) of the wild-type Ganoderma lucidum immunomodulatory protein LZ-8 published on the NCBI website.

的水溶剂箱內，加入Na+平衡电荷；(2)能量最小化：用sander模块进行3000步能量最小化，执行最陡下降法500步后转位共轭梯度法，以消除不合理的能量势垒；(3)加热：对系统加热使其从0K升温至303K、313K和323K，并在保持体积不变的状态下运行50ps带有位置限制的动力学，将非键相互作用阈值设为

使用弱耦合的算法来控制温度；(4)平衡：在常压条件下系统分别处在303K、313K和323K条件下，进行500ps的分子动力学模拟，以平衡系统；(5)动力学模拟：恒温恒压系统下进行分子动力学模拟，所用步长为2fs，以每1000步采集一次能量与坐标信息。分析动力学模拟过程中结构的均方根偏差值(Root Mean Square Deviation，RMSD)，再选择RMSD值平衡阶段的数据，计算每个氨基酸的均方根涨落值(Root Mean Square Fluctuation，RMSF)，形成轨迹文件，最后通过VMD软件分析该模拟轨迹，计算不同环境温度下LZ-8分子的稳态构象。如图1所示，随着环境温度由0K逐渐上升至303K、313K和323K，LZ-8分子的N端α螺旋逐渐解构，表明LZ-8分子N端α螺旋对温度敏感。Specifically, taking the crystal structure of Ganoderma lucidum immunomodulatory protein LZ-8 (PDB ID: 3F3H) as the initial structure at 0K, the NAMD software was used to calculate the system temperature from 0K to 303K (30°C), 313K (40°C) and 323K. (50°C), the all-atom motion trajectory of LZ-8 monomer, and then the steady-state conformation of LZ-8 monomer at three temperatures of 30°C, 40°C and 50°C was simulated by VMD software (Fig. 1). The specific molecular dynamics calculation conditions are as follows: (1) Simulation system construction: The CHARMM force field is used, the TIP3P octahedral water molecular box model is used, and the protein molecules are placed in the

(2) Energy minimization: use the sander module for 3000 steps of energy minimization, and perform the steepest descent method for 500 steps and then the transposition conjugate gradient method to eliminate the unreasonable energy potential (3) Heating: heating the system from 0K to 303K, 313K and 323K, and running 50ps kinetics with position constraints while keeping the volume constant, setting the non-bonding interaction threshold as

A weakly coupled algorithm is used to control the temperature; (4) Equilibrium: Under normal pressure conditions, the system is at 303K, 313K and 323K, respectively, and a molecular dynamics simulation of 500 ps is performed to balance the system; (5) Dynamics simulation: Molecular dynamics simulations were carried out in a constant temperature and pressure system with a step size of 2 fs, and energy and coordinate information were collected every 1000 steps. Analyze the root mean square deviation (Root Mean Square Deviation, RMSD) of the structure during the kinetic simulation process, and then select the data at the equilibrium stage of the RMSD value to calculate the root mean square fluctuation value (Root Mean Square Fluctuation, RMSF) of each amino acid. , a trajectory file was formed, and finally the simulated trajectory was analyzed by VMD software to calculate the steady-state conformation of LZ-8 molecule at different ambient temperatures. As shown in Figure 1, as the ambient temperature gradually increased from 0K to 303K, 313K and 323K, the N-terminal α-helix of LZ-8 molecule was gradually deconstructed, indicating that the N-terminal α-helix of LZ-8 molecule was sensitive to temperature.

Among them, the temperature factor B-factor is often used to reflect the conformational state of protein molecules in crystals. The higher the value of B-factor, the greater the ambiguity, and the more unstable or flexible the conformation of the corresponding part. According to the prediction of the temperature factor B-factor, there are three groups of temperature-sensitive sites in LZ-8 (Fig. 2), which are: (1) the 8th and 9th amino acids on the N-terminal α-helix; (2) the β4 sheet Amino acids 63-71 on the LOOP loop between layer and β5 sheet; (3) amino acids 103 and 104 on the β7 sheet. Among them, the N-terminal α-helix is the common temperature-sensitive region predicted by B-factor and NAMD molecular simulation, so we located the amino acid site to be mutated to this region. As shown in Figure 2, on the N-terminal α-helix of LZ-8 molecule, the B-factor values of amino acid residues at positions 8 and 9 are higher than those of amino acid residues at other positions. It is speculated that they are more sensitive to temperature than other amino acid residues. It is more sensitive. In the prior art, researchers often select the 8th and 9th amino acid residues to carry out double-site mutation on this basis. The present invention takes into account the similarity of point mutation amino acid structure and strong hydrophobicity. Weakly and innovatively selected the 8th amino acid residue for single-site mutation.

In the embodiment of the present invention, the nucleotide sequence encoding the immunomodulatory protein LZ-8 is shown in SEQ ID NO.3.

SEQ ID NO. 3:

ATGTCTGATACCGCTTTAATCTTTAGATTGGCCTGGGATGTTAAAAAATTATCTTTTGATTACACACCTAATTGGGGTCGTGGTAACCCTAACAATTTCATTGACACTGTTACTTTTCCAAAAGTGTTGACTGATAAGGCCTATACCTATCGAGTTGCAGTTTCTGGCCGTAACCTTGGCGTGAAACCCTCATATGCCGTTGAATCTGATGGTTCTCAGAAAGTGAACTTCTTGGAGTACAACAGTGGCTACGGTATTGCAGACACCAATACCATCCAAGTCTTTGTCGTCGACCCAGATACGAACAATGATTTTATTATCGCCCAGTGGAAC

The embodiment of the present invention also provides a nucleotide sequence of an immunomodulatory protein mutant, and the nucleotide sequence is shown in SEQ ID NO. 4.

SEQ ID NO. 4:

ATGTCTGATACCGCTTTAATCTGGAGATTGGCCTGGGATGTTAAAAAATTATCTTTTGATTACACACCTAATTGGGGTCGTGGTAACCCTAACAATTTCATTGACACTGTTACTTTTCCAAAAGTGTTGACTGATAAGGCCTATACCTATCGAGTTGCAGTTTCTGGCCGTAACCTTGGCGTGAAACCCTCATATGCCGTTGAATCTGATGGTTCTCAGAAAGTGAACTTCTTGGAGTACAACAGTGGCTACGGTATTGCAGACACCAATACCATCCAAGTCTTTGTCGTCGACCCAGATACGAACAATGATTTTATTATCGCCCAGTGGAAC

The embodiment of the present invention also provides a recombinant plasmid vector containing the nucleotide sequence.

In the embodiment of the present invention, the recombinant plasmid vector is constructed on the basis of any plasmid in the pPICZα series, pPICZ series, pGAPZα series or pPIC3.5K.

The embodiment of the present invention also provides a genetically engineered bacterium expressing the immunomodulatory protein mutant.

In the embodiment of the present invention, the genetically engineered bacteria are yeast or other fungi.

The embodiment of the present invention also provides a construction method of the immunomodulatory protein mutant, the construction method comprising:

Based on the amino acid sequence and nucleotide sequence of the immunomodulatory protein LZ-8, the primer F8W-F/R for site-directed mutagenesis was designed; the sequence of the primer F8W-F is shown in SEQ ID NO. 5, and the primer F8W-R The sequence is shown in SEQ ID NO.6;

SEQ ID NO. 5: ACCGCTTTAATCTGGAGATTGGCCTGGGAT

SEQ ID NO. 6: ATCCCAGGCCAATCTCCAGATTAAAGCGGT

According to the primers for site-directed mutagenesis, the plasmid carrying the keratinase LZ-8 gene is subjected to site-directed mutagenesis PCR to obtain the target mutant plasmid;

The target mutant plasmid is subjected to PCR amplification to obtain a recombinant plasmid;

The recombinant plasmid is transformed into fungi, and the mutant protein is expressed.

The embodiment of the present invention also provides an application of the immunomodulatory protein mutant in the fields of immunomodulator preparation, immune adjuvant preparation, tumor treatment or autoimmune disease treatment.

The immunomodulatory protein mutants and their nucleotide sequences, recombinant plasmid vectors, engineering bacteria, construction methods and applications of the present invention will be further described below in conjunction with specific examples, but the specific implementation methods mentioned in these examples are only for the purposes of the present invention. The enumeration and explanation of the technical solutions do not limit the scope of implementation of the present invention, and all improvements and substitutions on the basis of the present invention based on the above principles should fall within the protection scope of the present invention.

Example 1: Construction of Ganoderma lucidum immunomodulatory protein site-directed mutants

As shown in Figure 2, the letters indicate the name of the amino acid residues in the heat-sensitive region, and the numbers indicate the order of the amino acid residues in the amino acid sequence. On the N-terminal α helix of the LZ-8 molecule, the 8th and 9th positions The B-factor value of the amino acid residue at the position is higher than that of the amino acid residues at other positions. The present invention selects the amino acid residue at the 8th position for single point mutation, and combines it with the selection of the single point mutation of the amino acid residue at the 9th position and the selection of the 8th amino acid residue at the same time. The three mutants were named as F8W, R9K and DM in turn.

According to the sequence of LZ-8 (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.3), primers for site-directed mutagenesis (Table 1) were designed respectively. The gene plasmid LZ-8/pGAPZα was subjected to site-directed mutagenesis PCR to obtain the target mutant plasmid, which was transformed into DH5α E. coli competent cells. After the sequencing was correct, the target plasmid was amplified, and the linearized plasmid was electroporated into yeast X-33 strain. expression of a mutant protein. details as follows:

Table 1 Primer sequences of different Ganoderma lucidum immunomodulatory protein site-directed mutants

According to the operating instructions of the Muta-direct ^TM site-directed mutagenesis kit, use the site-directed mutagenesis primers in Table 1 and Muta-direct ^TM enzyme to carry out full-length PCR on the template plasmid. Mutazyme ^TM enzyme re-digests the unmutated template plasmid, and the remaining PCR-amplified The full-length mutant plasmid was transformed into DH5α competent cells, several clones were picked, and the plasmid was extracted in small quantities for sequencing. After amplifying the correctly sequenced mutant plasmid, according to the pGAPZα plasmid instructions, linearize the plasmid, and electro-transform the yeast X33 strain to obtain a strain capable of expressing the mutant. Three recombinant yeast strains of F8W, R9K and DM.

Example 2: Expression and purification of Ganoderma lucidum immunomodulatory protein mutants

The yeast X-33 strain expressing the mutant protein was picked and cultured in YPD liquid medium (containing 100 μg/ml Zeocin ^TM ) at 28-30° C. overnight with shaking (250-300 rpm); the next day, the seed fermentation broth was plated according to 5% of the inoculum. It was connected to a fermenter containing YPD liquid medium, and after continuous culture and fermentation at 28-30 °C for 72 h, the supernatant of the fermentation broth was collected by centrifugation at 4 °C.

The recombinant protein was purified by AKTA protein purifier, and the temperature of the whole purification process was controlled at 4 °C. The specific steps are as follows: (1) SP Sapharose XL cation-exchange chromatography removes most of the miscellaneous proteins and small nucleic acid fragments, pigments, etc., phase A pH3.5 50Mm NaAc-HAc, phase B pH3.5 50Mm NaAc-HAc/1M NaCl Linear elution; (2) Q-Sapharose Fast Flow anion exchange chromatography for intermediate purification of mutant protein, pH7.2 25mM Tris-HCL A phase equilibrium chromatography column, pH7.2 25mM Tris-HCL/0.5M NaCl B phase elution; (3) The mutant protein was purified by Superdex ^TM 75 molecular sieve. After loading, the protein was eluted with pH7.2 50Mm Tris-HCl/0.15M NaCl. The elution peaks were collected and subjected to SDS-PAGE electrophoresis of purified protein, as shown in Figure 3, where M represents protein molecular weight standard, different lane names represent different proteins, and arrows indicate the position of the target protein band.

Example 3: Immunomodulatory activity assay

The immunomodulatory activity of the mutant was detected by the mouse spleen lymphocyte proliferation assay. The specific operation methods are as follows: the mouse spleen was isolated by aseptic surgery, the single splenocytes were isolated by ophthalmic scissors and shearing method, the single splenocytes were filtered through a 200-mesh filter, and the erythrocytes were lysed. After the action of the solution, collect the remaining cells, count the cell density, dilute the cells to an appropriate density with 1640 medium containing 2% fetal bovine serum, and add 1 × 10 ⁵ cells to each well in a 96-well culture plate, and then add the final The concentration of the protein to be detected was cultured in a 37°C 5% CO ₂ incubator for 24 hours, and the cell proliferation was detected by CCK-8 method (Figure 4). The results showed that LZ-8 and its mutant proteins had the same effect curve of promoting splenocyte proliferation, and their maximum dose of promoting splenocyte proliferation was 0.3125μg/ml.

Example 4: Detection of antitumor activity

The antitumor activity of the mutants was detected by the Hela cell growth inhibition assay. The specific operation method is as follows: the adherent Hela cells were digested into a single cell suspension with 0.25% trypsin, the trypsinization was terminated with fetal bovine serum, and the cell density was counted. 96-well culture plate, add 5×10 ⁴ cells to each well, and then add the protein to be tested at a predetermined final concentration. The reaction system is IMDM medium containing 2% fetal bovine serum, and it is placed in a 37°C 5% CO2 incubator for 48 hours. Cell proliferation was detected by CCK-8 method (Figure 5). The results showed that the three mutants F8W, R9K and DM had the same growth inhibitory effect as wild-type LZ-8 on Hela cells cultured in vitro, and their IC50 doses were: 2.292μg/ml, 2.363μg/ml, 2.407μg /ml and 2.238 μg/ml.

Example 5: Determination of thermodynamic parameters of the protein to be tested

Differential calorimetry scanning analyzer MicroCal VP-DSC (GE Healthcare, USA) was used to determine the phase transition temperature Tm and phase transition enthalpy value ΔH of LZ-8 and each mutant. Experimental parameters: LZ-8 or LZ-8 mutant samples Dissolved in 0.01M PBS buffer (pH 7.2-7.4), final concentration 1 mg/mL, injection volume 500 μL, heating range 25-55 °C, heating speed 1 °C/min. The raw data were collected, and Origin software was used to analyze the thermodynamic parameters of the samples (Table 2).

Table 2 Thermodynamic parameters of LZ-8 and its mutants (n=3, mean±SD)

*Indicates p<0.05 by t-test compared to LZ-8 group

To sum up, the immunomodulatory protein mutant provided in the embodiments of the present invention has an amino acid sequence based on the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO. amino acid, the mutated amino acid sequence is SEQ ID NO.2; on the one hand, the thermal stability of the immunomodulatory protein mutant is greatly improved. Compared with the wild-type LZ-8, the F8W phase transition temperature Tm increased by 1.86℃, The phase transition enthalpy value ΔH was increased by 39.19kJ/mol; on the other hand, the immunomodulatory protein mutant had the same immunomodulatory activity as the wild-type LZ-8, and had a mitogenic effect on Balb/c mouse splenocytes, with a maximum mitogenic effect. The dose is 0.3125μg/ml, in addition, it also has the same anti-tumor activity as wild-type LZ-8, and has a growth inhibitory effect on HeLa cells cultured in vitro, and its IC50 dose is 2.292μg/ml; It is worth noting that the present invention The provided immunomodulatory protein mutant further improves the thermal stability of the recombinant LZ-8 and improves the stability of the recombinant LZ-8 drug on the basis of ensuring that the immunomodulatory activity and anti-tumor activity are basically unchanged. Technology has more application and development value.

The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the patent of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the appended claims.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

sequence listing

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atcccaggcc aacttccaga ttaaagcggt 30

Claims (9)

1. An immunomodulatory protein mutant, wherein the amino acid sequence of the immunomodulatory protein mutant is based on the amino acid sequence of the immunomodulatory protein LZ-8 of SEQ ID NO. Phenylalanine is mutated into tryptophan, and the mutated amino acid sequence is SEQ ID NO.2. 2 . The immunomodulatory protein mutant according to claim 1 , wherein the nucleotide sequence encoding the immunomodulatory protein LZ-8 is shown in SEQ ID NO.3. 3 . 3 . The nucleotide sequence of the immunomodulatory protein mutant according to claim 1 or 2 , wherein the nucleotide sequence of the immunomodulatory protein mutant is shown in SEQ ID NO. 4. 4 . 4. A recombinant plasmid vector comprising the nucleotide sequence of claim 3. 5. according to the recombinant plasmid carrier of the described nucleotide sequence of claim 4, it is characterized in that, described recombinant plasmid carrier is to construct on the basis of any one plasmid in pPICZα series, pPICZ series, pGAPZα series or pPIC3.5K owned. 6. A genetically engineered bacterium expressing the immunomodulatory protein mutant of claim 1 or 2. 7 . The genetically engineered bacteria of the immunomodulatory protein mutant according to claim 6 , wherein the genetically engineered bacteria of the immunomodulatory protein mutant is yeast. 8 . 8. a construction method according to the described immunomodulatory protein mutant of claim 1 or 2, is characterized in that, described construction method comprises: Based on the amino acid sequence and nucleotide sequence of the immunomodulatory protein LZ-8, the primer F8W-F/R for site-directed mutagenesis was designed; the sequence of the primer F8W-F is shown in SEQ ID NO. 5, and the primer F8W-R The sequence is shown in SEQ ID NO.6; According to the primers for site-directed mutagenesis, the plasmid carrying the keratinase LZ-8 gene is subjected to site-directed mutagenesis PCR to obtain the target mutant plasmid; The target mutant plasmid is subjected to PCR amplification to obtain a recombinant plasmid; The recombinant plasmid is transformed into fungi, and the mutant protein is expressed. 9 . The application of the immunomodulatory protein mutant according to claim 1 or 2 in the fields of immunomodulator preparation, immune adjuvant preparation, tumor treatment or autoimmune disease treatment. 10 .

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