CN111748543A

CN111748543A - Immune regulatory protein mutant and nucleotide sequence, recombinant plasmid vector, engineering bacterium, construction method and application thereof

Info

Publication number: CN111748543A
Application number: CN202010627080.9A
Authority: CN
Inventors: 黄伟; 蒋振彦; 戴璐; 孙熙麟; 李泓睿; 李柏志; 刘志屹
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2020-07-01
Filing date: 2020-07-01
Publication date: 2020-10-09

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

Immune regulatory protein mutant and nucleotide sequence, recombinant plasmid vector, engineering bacterium, construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an immunomodulatory protein mutant, a nucleotide sequence, a recombinant plasmid vector, a genetic engineering bacterium and an application thereof.

Background

Ganoderma lucidum immunomodulatory protein LZ-8 is a dimeric protein with immunomodulatory activity rich in Ganoderma lucidum fruiting body, and preclinical studies have confirmed that LZ-8 has various immunomodulatory and anti-tumor activities, such as: can promote the proliferation of mouse spleen lymphocytes; promote T cells to secrete various cytokines; can inhibit the growth of lung cancer, gastric cancer and liver cancer in vivo and in vitro of mice; the anti-tumor activity of the tumor DNA vaccine can be obviously improved; can relieve local and systemic anaphylaxis; can slow down the progress of diabetes; can reduce graft versus host response, and the like. Therefore, LZ-8 has great development potential in the fields of tumor treatment and autoimmune disease treatment. The wild ganoderma lucidum has low yield and high price, and is not suitable to be used as a production source of ganoderma lucidum immunomodulatory protein LZ-8. The production period of artificially cultivated ganoderma lucidum is long, the process is complex, and the requirement of industrial production cannot be met. With the rapid development of gene engineering and biological pharmaceutical engineering technology, the gene engineering strain is adopted to strengthen the transcription and translation of target genes, so that the high-efficiency expression and activity secretion of target proteins can be realized, and the production scale of the ganoderma lucidum immunomodulatory protein LZ-8 can be effectively improved.

However, in the production process of the ganoderma lucidum immunomodulatory protein LZ-8, due to the existence of operations such as mechanical crushing, stirring, cross-flow membrane filtration and the like, local temperature rise of a protein solution is inevitably caused, protein unfolding and even denaturation are caused, and the titer and homogeneity of a final product are seriously influenced, so that the thermal stability of the LZ-8 molecule is improved, and the method has important practical significance for producing the LZ-8 molecule which is reliable in quality and suitable for new medicine application. The search for LZ-8 mutant with improved thermal stability and unchanged or improved pharmacological activity through molecular modification technology is one of the solutions. Researchers have carried out heterologous expression of yeast on ganoderma lucidum immunomodulatory protein LZ-8 in earlier work, and recombinant LZ-8 molecules with immunomodulatory activity and anti-tumor activity are obtained. However, the prior art can not further improve the thermal stability of the recombinant LZ-8 on the basis of ensuring the unchanged or improved biological activity of the recombinant LZ-8, so that the stability of the recombinant LZ-8 patent medicine is poor.

Disclosure of Invention

The embodiment of the invention aims to provide an immunomodulatory protein mutant, and aims to solve the problem that the prior art cannot further improve the thermal stability of recombinant LZ-8 on the basis of ensuring the unchanged or improved biological activity of the recombinant LZ-8, so that the stability of the recombinant LZ-8 patent drug is poor.

The embodiment of the invention is realized by an immunomodulatory protein mutant, wherein the amino acid sequence of the immunomodulatory protein mutant is obtained by mutating phenylalanine at position 8 to tryptophan on the basis of the amino acid sequence of immunomodulatory protein LZ-8 of SEQ ID NO.1, and the mutated amino acid sequence is SEQ ID NO. 2.

Another object of an embodiment of the present invention is a nucleotide sequence of an immunomodulatory protein mutant, the nucleotide sequence being set forth in SEQ ID No. 4.

Another object of an embodiment of the present invention is a recombinant plasmid vector containing said nucleotide sequence.

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

Another object of an embodiment of the present invention is to provide a method for constructing the immunomodulatory protein mutant, the method comprising:

designing a primer F8W-F/R of site-directed mutagenesis according to the amino acid sequence and the nucleotide sequence of the immunoregulation protein LZ-8; the sequence of the primer F8W-F is shown as SEQ ID NO.5, and the sequence of the primer F8W-R is shown as SEQ ID NO. 6;

carrying out site-directed mutagenesis PCR on the plasmid carrying the keratinase LZ-8 gene according to the site-directed mutagenesis primer to obtain a target mutant plasmid;

carrying out PCR amplification on the target mutant plasmid to obtain a recombinant plasmid;

and transferring the recombinant plasmid into fungi to express mutant protein to obtain the mutant protein.

The embodiment of the invention also aims at application of the immunomodulatory protein mutant in the fields of preparation of immunomodulators, preparation of immunoadjuvants, tumor treatment or treatment of autoimmune diseases.

The amino acid sequence of the immunomodulatory protein mutant provided by the embodiment of the invention is that on the basis of the amino acid sequence of immunomodulatory protein LZ-8 of SEQ ID NO.1, phenylalanine at position 8 is mutated into tryptophan, and the mutated amino acid sequence is SEQ ID NO. 2; on one hand, the thermal stability of the immunomodulatory protein mutant is greatly improved, and compared with 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 consistent with wild type LZ-8, has mitogenic effect on splenocytes of Balb/c mice, the maximum mitogenic dose is 0.3125 μ g/ml, and in addition, has anti-tumor activity consistent with wild type LZ-8, has growth inhibitory effect on Hela cells cultured in vitro, and the IC50 dose is 2.292 μ g/ml; namely, the immunomodulatory protein mutant provided by the invention further improves the thermal stability of the recombinant LZ-8 and the stability of the recombinant LZ-8 patent drug on the basis of ensuring that the immunomodulatory activity and the anti-tumor activity are basically unchanged, and has application and development values compared with the prior art.

Drawings

FIG. 1 is a simulated steady state constellation diagram of wild-type Ganoderma lucidum immunomodulatory protein LZ-8 provided in accordance with an embodiment of the invention at different temperatures;

FIG. 2 is a diagram of the temperature sensitive region of B-factor predicted ganoderma lucidum immunomodulatory protein LZ-8 provided by an embodiment of the invention;

FIG. 3 is a SDA-PAGE gel electrophoresis diagram of purified protein of ganoderma lucidum immunomodulatory protein mutant provided by the embodiment of the invention;

FIG. 4 is a graph of the determination of the immunomodulatory protein LZ-8 of Ganoderma lucidum and the immunomodulatory activity of each mutant thereof according to the embodiment of the invention;

FIG. 5 is a graph of the determination of anti-tumor activity of Ganoderma lucidum immunomodulatory protein LZ-8 and its mutants.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In order to solve the problem that the thermal stability of recombinant LZ-8 cannot be further improved on the basis of ensuring the unchanged or improved biological activity of the recombinant LZ-8 in the prior art, so that the stability of the recombinant LZ-8 patent drug is poor, the embodiment of the invention provides an immunomodulatory protein mutant, 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 one hand, the thermal stability of the immunomodulatory protein mutant is greatly improved, and compared with 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 consistent with wild type LZ-8, mitogenic effect on splenocytes of Balb/c mice, maximum mitogenic dose of 0.3125 μ g/ml, and anti-tumor activity consistent with wild type LZ-8, growth inhibitory effect on Hela cells cultured in vitro, and IC50 dose of 2.292 μ g/ml.

It is worth noting that the prior art can only raise the phase transition temperature Tm by 0.9 ℃, and increase the phase transition enthalpy value delta H by 23.14kJ/mol, and the problem that the thermal stability of the recombinant LZ-8 cannot be further improved, which causes poor stability of the recombinant LZ-8 patent drug exists, but the immunomodulatory protein mutant provided by the invention realizes further improvement of the thermal stability of the recombinant LZ-8 and the stability of the recombinant LZ-8 patent drug on the basis of ensuring that the immunomodulatory activity and the anti-tumor activity are basically unchanged, and has application and development values compared with the prior art.

The embodiment of the invention provides an immunomodulatory protein mutant, wherein the amino acid sequence of the immunomodulatory protein mutant is obtained by mutating phenylalanine at position 8 to tryptophan on the basis of the amino acid sequence of immunomodulatory protein LZ-8 of SEQ ID NO.1, and the mutated amino acid sequence is SEQ ID NO. 2.

SEQ ID NO.1：

MSDTALIFRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAYTYRVAVSGRNLGVKPSYAVESDGSQKVNFLEYNSGYGIADTNTIQVFVVDPDTNNDFIIAQWN

SEQ ID NO.2：

MSDTALIWRLAWDVKKLSFDYTPNWGRGNPNNFIDTVTFPKVLTDKAYTYRVAVSGRNLGVKPSYAVESDGSQKVNFLEYNSGYGIADTNTIQVFVVDPDTNNDFIIAQWN

In the embodiment of the invention, the site-directed mutant is constructed on the basis of a wild type ganoderma lucidum immunomodulatory protein LZ-8 amino acid sequence (P14945) published by NCBI website.

Specifically, the crystal structure (PDB ID:3F3H) of Ganoderma lucidum immunomodulatory protein LZ-8 is used as the initial structure of 0K, the NAMD software is used to calculate the all-atom movement locus of LZ-8 monomer when the system temperature is increased from 0K to 303K (30 ℃), 313K (40 ℃) and 323K (50 ℃), and then VMD software is used to simulate the steady state conformation of LZ-8 monomer at 30 ℃, 40 ℃ and 50 ℃ (FIG. 1). The specific molecular dynamics calculation conditions were as follows: (1) building a simulation system: by usingCHARMM force field, selecting TIP3P octahedral water molecular box model, placing protein molecule in the water molecular box model

Na + is added into the hydrosolvent box to balance the charge; (2) energy minimization: carrying out 3000-step energy minimization by using a sander module, and executing 500-step backward transposition conjugate gradient method of a steepest descent method to eliminate unreasonable energy barriers; (3) heating: heating the system from 0K to 303K, 313K and 323K and running 50ps dynamics with position limitation while keeping the volume constant, setting the threshold of nonbond interaction to

The method comprises the steps of (1) controlling temperature by using a weak coupling algorithm, (4) balancing, namely performing molecular dynamics simulation of 500ps under 303K, 313K and 323K conditions respectively under a normal pressure condition to balance a system, (5) performing dynamics simulation under a constant temperature and pressure system, wherein the step length is 2fs, energy and coordinate information is collected every 1000 steps, analyzing Root Mean Square Deviation (RMSD) of a structure in the dynamics simulation process, selecting data in an RMSD value balancing stage, calculating Root Mean Square Fluctuation (RMSF) of each amino acid to form a track file, and finally analyzing the simulation track by VMD software to calculate steady-state conformations of LZ-8 molecules under different environmental temperatures, wherein α helices of the LZ-8 molecules are gradually decomposed along with the gradual rise of the environmental temperature from 0K to 303K, 313K and 323K, so that the N-end α helices of the LZ-8 molecules are sensitive to the temperature.

Among them, the temperature factor B-factor is often used to reflect the conformational state of a protein molecule in a crystal, and the higher the value of B-factor, the greater the ambiguity, the more unstable or flexible the conformation of the corresponding site. According to the prediction of the temperature factor B-factor, LZ-8 has 3 groups of temperature sensitive sites (FIG. 2), which are: (1)

amino acids

8 and 9 of the N-terminal alpha helix; (2) amino acids 63-71 of the LOOP LOOP between the beta 4 sheet and the beta 5 sheet; (3) amino acids 103 and 104 of the beta 7 sheet. Where the N-terminal alpha helix is the common temperature sensitive region predicted by molecular simulations of B-factor and NAMD, we then localize the amino acid site to be mutated to this region. As shown in figure 2, on the N-terminal alpha helix of the LZ-8 molecule, the B-factor values of the amino acid residues at the 8 th position and the 9 th position are higher than those of the amino acid residues at other positions, and the B-factor values are presumed to be more sensitive to temperature than those of the amino acid residues at other positions.

In the embodiment of the invention, the nucleotide sequence of the immunoregulatory protein LZ-8 is shown as SEQ ID NO. 3.

SEQ ID NO.3：

ATGTCTGATACCGCTTTAATCTTTAGATTGGCCTGGGATGTTAAAAAATTATCTTTTGATTACACACCTAATTGGGGTCGTGGTAACCCTAACAATTTCATTGACACTGTTACTTTTCCAAAAGTGTTGACTGATAAGGCCTATACCTATCGAGTTGCAGTTTCTGGCCGTAACCTTGGCGTGAAACCCTCATATGCCGTTGAATCTGATGGTTCTCAGAAAGTGAACTTCTTGGAGTACAACAGTGGCTACGGTATTGCAGACACCAATACCATCCAAGTCTTTGTCGTCGACCCAGATACGAACAATGATTTTATTATCGCCCAGTGGAAC

The embodiment of the invention also provides a nucleotide sequence of the immunomodulatory protein mutant, wherein the nucleotide sequence is shown as SEQ ID NO. 4.

SEQ ID NO.4：

ATGTCTGATACCGCTTTAATCTGGAGATTGGCCTGGGATGTTAAAAAATTATCTTTTGATTACACACCTAATTGGGGTCGTGGTAACCCTAACAATTTCATTGACACTGTTACTTTTCCAAAAGTGTTGACTGATAAGGCCTATACCTATCGAGTTGCAGTTTCTGGCCGTAACCTTGGCGTGAAACCCTCATATGCCGTTGAATCTGATGGTTCTCAGAAAGTGAACTTCTTGGAGTACAACAGTGGCTACGGTATTGCAGACACCAATACCATCCAAGTCTTTGTCGTCGACCCAGATACGAACAATGATTTTATTATCGCCCAGTGGAAC

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

In the embodiment of the invention, the recombinant plasmid vector is constructed on the basis of any one plasmid of pPICZ alpha series, pPICZ series, pGAPZ alpha series or pPIC3.5K.

The embodiment of the invention also provides a genetic engineering bacterium for expressing the immunomodulatory protein mutant.

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

The embodiment of the invention also provides a construction method of the immunomodulatory protein mutant, which comprises the following steps:

SEQ ID NO.5：ACCGCTTTAATCTGGAGATTGGCCTGGGAT

SEQ ID NO.6：ATCCCAGGCCAATCTCCAGATTAAAGCGGT

The embodiment of the invention also provides application of the immunomodulatory protein mutant in the fields of preparation of immunomodulators, preparation of immunoadjuvants, tumor treatment or treatment of autoimmune diseases.

The immunomodulatory protein mutant, the nucleotide sequence, the recombinant plasmid vector, the engineered bacterium, the construction method and the application thereof are further described below with reference to the following embodiments, but the embodiments mentioned in the embodiments are only illustrative and explanatory of the technical scheme of the present invention, and do not limit the scope of the present invention, and all modifications and substitutions based on the above principles should be within the scope of the present invention.

Example 1: construction of site-directed mutant of ganoderma lucidum immunomodulatory protein

As shown in FIG. 2, wherein letters indicate names of amino acid residues in a heat-sensitive region, numbers indicate the sequence of the amino acid residues on an amino acid sequence, and on an alpha helix at the N-terminal of an LZ-8 molecule, the values of B-factor factors of the 8 th amino acid residue and the 9 th amino acid residue are higher than those of the other amino acid residues, the 8 th amino acid residue is selected for single-site mutation, and compared with the single-site mutation of the 9 th amino acid residue and the double-site mutation of the 8 th amino acid residue and the 9 th amino acid residue, the construction of the three mutants is named sequentially: F8W, R9K and DM.

According to the sequence of LZ-8 (the amino acid sequence is shown as SEQ ID NO.1, the nucleotide sequence is shown as SEQ ID NO. 3), respectively designing a primer of site-directed mutagenesis (Table 1), carrying out site-directed mutagenesis PCR on plasmid LZ-8/pGAPZ alpha carrying keratinase LZ-8 gene to obtain a target mutant plasmid, transforming DH5 alpha escherichia coli competent cells, amplifying the target plasmid after the sequence to be detected is correct, and electrically transferring the linearized plasmid into a yeast X-33 strain to express three mutant proteins. The method comprises the following specific steps:

TABLE 1 primer sequences of different site-directed mutants of ganoderma lucidum immunomodulatory protein

According to Muta-direct^TMInstructions for site-directed mutagenesis kits Using the site-directed mutagenesis primers and Muta-direct from Table 1^TMEnzyme-mediated full-Length PCR of the template plasmid, Mutazyme^TMDigesting the non-mutated template plasmid by enzyme, transforming DH5 α competent cells by the residual PCR amplified full-length mutated plasmid, selecting a plurality of clones, sequencing the plasmids by small amount extraction, amplifying the mutated plasmids with correct sequencing, linearizing the plasmids according to pGAPZ α plasmid instruction, and electrically transforming yeast X33 strain to obtain three recombinant yeast strains capable of expressing mutants F8W, R9K and DM.

Example 2: expression and purification of ganoderma lucidum immunomodulatory protein mutant

Yeast X-33 strain expressing the mutant protein was picked up in YPD liquid medium (containing 100. mu.g/ml Zeocin)^TM) Shaking culture at 28-30 ℃ overnight (250-300 rpm); the next day, seeds were inoculated at 5% of the inoculum sizeInoculating the fermentation liquid into a fermentation tank containing YPD liquid culture medium, continuously culturing at 28-30 deg.C, fermenting for 72 hr, and centrifuging at 4 deg.C to collect supernatant of the fermentation liquid.

And purifying the recombinant protein by using an AKTA protein purifier, wherein the temperature in the whole purification process is controlled to be 4 ℃. The method comprises the following specific steps: (1) SP SaPharose XL cation exchange chromatography removes most of miscellaneous proteins, small molecular nucleic acid fragments, pigments and the like, phase A pH3.550Mm NaAc-HAc, phase B pH3.550Mm NaAc-HAc/1M NaCl linear elution; (2) Q-Sapharose Fast Flow anion exchange chromatography middle purification mutant protein, pH7.225mM Tris-HCL A phase equilibrium chromatographic column, pH7.225mM Tris-HCL/0.5M NaCl B phase elution; (3) superdex^TMThe mutant protein was finely purified by using 75 molecular sieves, and eluted at pH 7.250Mm Tris-HCl/0.15M NaCl after loading. The eluted peaks were collected and subjected to SDS-PAGE electrophoresis of the purified protein, as shown in FIG. 3, wherein M represents a protein molecular weight standard, different lane names represent different proteins, and the arrows indicate the positions of the target protein bands.

Example 3: immunomodulatory activity assay

The specific operation method comprises the steps of separating mouse spleen by a sterile operation method, separating single spleen cells by an ophthalmic shearing method, filtering the single spleen cells by a 200-mesh filter screen, collecting residual cells after the action of erythrocyte lysate, counting the cell density, diluting the cells to a proper density by 1640 culture solution containing 2% fetal calf serum, adding 1 × 10 to a 96-hole culture plate in each hole, and detecting the immune regulation activity of the mutant by using a mouse spleen lymphocyte proliferation experiment⁵Adding the protein to be detected at a given final concentration into each cell, and placing at 37 deg.C with 5% CO₂Culturing in an incubator for 24h, and detecting the cell proliferation by a CCK-8 method (figure 4). The results showed that LZ-8 and its mutant proteins had a consistent splenocyte proliferation-promoting response curve with a maximum splenocyte proliferation-promoting dose of 0.3125. mu.g/ml for each.

Example 4: detection of antitumor Activity

The anti-tumor activity of the mutant is detected by adopting a Hela cell growth inhibition experiment, and the specific operation method is as follows: digesting the adherent Hela cells into a single cell suspension by using 0.25% pancreatin, stopping the pancreatin digestion by fetal bovine serum, and counting the cellsCell density, in 96-well plates, 5 × 10 cells per well⁴Adding the protein to be detected with a set final concentration into each cell, culturing the cells in an IMDM culture medium containing 2% fetal calf serum at 37 ℃ in a 5% CO2 culture box for 48h, and detecting the cell proliferation by a CCK-8 method (figure 5). The results show that three mutants F8W, R9K and DM have a growth-inhibiting effect on Hela cells cultured in vitro that is consistent with wild-type LZ-8, and that their IC50 doses are, in order: 2.292. mu.g/ml, 2.363. mu.g/ml, 2.407. mu.g/ml and 2.238. mu.g/ml.

Example 5: determination of thermodynamic parameters of protein to be detected

Differential calorimetric scanning analyzer MicroCal VP-DSC (general electric medical treatment, USA) measures the phase transition temperature Tm and phase transition enthalpy value deltaH of LZ-8 and each mutant, and experimental parameters are as follows: LZ-8 or LZ-8 mutant samples were dissolved in 0.01M PBS buffer (pH7.2-7.4) to a final concentration of 1mg/mL, a sample injection volume of 500. mu.L, heating range of 25-55 ℃ and heating rate of 1 ℃/min. Raw data were collected and samples were analyzed for thermodynamic parameters using Origin software (table 2).

TABLE 2 thermodynamic parameters of LZ-8 and its mutants (n ═ 3, mean. + -. SD)

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

To sum up, the amino acid sequence of the immunomodulatory protein mutant provided by the embodiment of the invention is that the phenylalanine at the 8 th position is mutated 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 one hand, the thermal stability of the immunomodulatory protein mutant is greatly improved, and compared with 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 consistent with wild type LZ-8, has mitogenic effect on splenocytes of Balb/c mice, the maximum mitogenic dose is 0.3125 μ g/ml, and in addition, has anti-tumor activity consistent with wild type LZ-8, has growth inhibitory effect on Hela cells cultured in vitro, and the IC50 dose is 2.292 μ g/ml; it is worth noting that the immunomodulatory protein mutant provided by the invention further improves the thermal stability of the recombinant LZ-8 and the stability of the recombinant LZ-8 patent drug on the basis of ensuring that the immunomodulatory activity and the anti-tumor activity are basically unchanged, and has application and development values compared with the prior art.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

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Claims

1. An immunomodulatory protein mutant is characterized in that the amino acid sequence of the immunomodulatory protein mutant is that the phenylalanine at the 8 th position is mutated 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.

2. The mutant immunomodulatory protein of claim 1, wherein the nucleotide sequence encoding the immunomodulatory protein LZ-8 is set forth in SEQ ID No. 3.

3. The nucleotide sequence of the mutant immunomodulatory protein of claim 1 or 2, wherein the mutant immunomodulatory protein has the nucleotide sequence shown in SEQ ID No. 4.

4. A recombinant plasmid vector comprising the nucleotide sequence of claim 3.

5. The recombinant plasmid vector of the nucleotide sequence of claim 4, wherein the recombinant plasmid vector is constructed on the basis of any one of pPICZ α series, pPICZ series, pGAPZ α series, or pPIC3.5K.

6. A genetically engineered bacterium expressing the immunomodulatory protein mutant of claim 1 or 2.

7. The genetically engineered bacterium of the immunomodulatory protein mutant, according to claim 6, wherein the genetically engineered bacterium of the immunomodulatory protein mutant is a yeast.

8. A method for constructing an immunomodulatory protein mutant according to claim 1 or 2, comprising:

9. Use of an immunomodulatory protein mutant according to claim 1 or 2 in the field of immunomodulatory agent preparation, immunoadjuvant preparation, tumor therapy or treatment of autoimmune diseases.