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

An immunomodulatory protein and nucleotide sequence technology, applied in the field of genetic engineering, can solve the problems of poor stability and inability to further improve the thermal stability of recombinant LZ-8

Inactive Publication Date: 2020-10-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the embodiments of the present invention is to provide an immunomodulatory protein mutant, which aims to solve the problem that the existing technology cannot further improve the thermal stability of recombinant LZ-8 on the basis of ensuring that the biological activity of recombinant LZ-8 remains unchanged or improved , leading to the problem of poor stability of the recombinant LZ-8 finished drug

Method used

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  • Immunomodulatory protein mutant and nucleotide sequence thereof, recombinant plasmid vector, engineering bacterium, construction method and application
  • Immunomodulatory protein mutant and nucleotide sequence thereof, recombinant plasmid vector, engineering bacterium, construction method and application
  • Immunomodulatory protein mutant and nucleotide sequence thereof, recombinant plasmid vector, engineering bacterium, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of Ganoderma lucidum immunomodulatory protein site-directed mutants

[0052] Such as figure 2 , where the letter indicates the name of the amino acid residue in the thermosensitive region, and the number indicates the order of the amino acid residue in the amino acid sequence. On the N-terminal α-helix of the LZ-8 molecule, the 8th and 9th amino acid residues If the B-factor factor value of the base is higher than that of amino acid residues at other positions, the present invention selects the 8th amino acid residue for a single point mutation, and compares it with the selection of the 9th amino acid residue for single point mutation and simultaneously selects the 8th and 8th amino acid residues. The double-site mutations of the 9 amino acid residues were compared, and the construction of the three mutants were named in turn: F8W, R9K and DM.

[0053] According to the sequence of LZ-8 (the amino acid sequence is shown in SEQ ID NO.1, and the n...

Embodiment 2

[0057] Example 2: Expression and purification of Ganoderma lucidum immunomodulatory protein mutants

[0058] Pick the yeast X-33 strain expressing the mutant protein in YPD liquid medium (containing 100 μ g / ml Zeocin TM ) at 28-30°C for overnight shaking culture (250-300rpm); the next day, according to the inoculum size of 5%, the seed fermentation liquid was connected to a fermenter containing YPD liquid medium, and after continuous culture and fermentation at 28-30°C for 72 hours, 4°C The supernatant of the fermentation broth was collected by centrifugation.

[0059] AKTA protein purifier was used to purify the recombinant protein, 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 to remove most of the miscellaneous proteins and small molecular nucleic acid fragments, pigments, etc., A phase pH3.5 50Mm NaAc-HAc, B phase pH3.5 50Mm NaAc-HAc / 1M NaCl Linear elution...

Embodiment 3

[0060] Example 3: Determination of Immunomodulatory Activity

[0061] The mouse spleen lymphocyte proliferation assay was used to detect the immunomodulatory activity of the mutant, and the specific operation method was as follows: the mouse spleen was isolated by aseptic surgery, a single splenocyte was isolated by ophthalmic shearing, a single splenocyte was filtered through a 200-mesh filter, and red blood cells 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 culture medium containing 2% fetal bovine serum, add 1×10 5cells, then add the protein to be detected at a predetermined final concentration, and place at 37°C in 5% CO 2 After culturing in an incubator for 24 hours, the cell proliferation was detected by the CCK-8 method ( Figure 4 ). The results showed that LZ-8 and its mutant proteins had consistent splenocyte proliferation-promoting effect curves, and their ma...

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Abstract

The invention is applicable to the technical field of gene engineering. The invention provides an immunomodulatory protein mutant and a nucleotide sequence thereof, a recombinant plasmid vector, an engineering bacterium, a construction method and application. The amino acid sequence of the immunomodulatory protein mutant is obtained by mutating phenylalanine at the 8th site into tryptophan on thebasis of the amino acid sequence of immunomodulatory protein LZ-8 shown as SEQ ID NO.1, and the mutated amino acid sequence is shown as SEQ ID NO.2. According to the invention, the thermal stability of the recombinant LZ-8 and the stability of the recombinant LZ-8 patent medicine are further improved on the basis that the immunomodulatory activity and the anti-tumor activity are basically unchanged; on one hand, the heat stability of the immunomodulatory protein mutant is greatly improved, and compared with wild type LZ-8, the F8W phase transition temperature Tm is increased by 1.86 DEG C, andthe phase transition enthalpy value delta H is increased by 39.19 kJ/mol; and on the other hand, the immunomodulatory protein mutant has immunomodulatory activity and anti-tumor activity consistent with those of wild type LZ-8, and has higher application and development value compared with the prior art.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an immunoregulatory protein mutant and its nucleotide sequence, a recombinant plasmid vector, a genetic engineering bacterium and its application. Background technique [0002] Ganoderma lucidum immunomodulatory protein LZ-8 is a dimeric protein rich in immunomodulatory activity in the fruiting bodies of Ganoderma lucidum. Preclinical studies have confirmed that LZ-8 has a variety of immune regulatory and anti-tumor activities, such as: can promote Spleen lymphocyte proliferation; promote T cells to secrete a variety of cytokines; can inhibit the growth of lung cancer, gastric cancer and liver cancer in mice; can significantly improve the anti-tumor activity of tumor DNA vaccine; can reduce local and systemic allergic reactions; can slow down diabetes progress; the ability to mitigate graft-versus-host reactions, and more. Therefore, LZ-8 has great develo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/50C12N15/57C12N15/81C12N1/19A61K38/16A61K39/39A61P37/02A61P35/00
CPCC12N9/63C12N15/81A61K39/39A61P37/02A61P35/00A61K38/00A61K2039/55516
Inventor 黄伟蒋振彦戴璐孙熙麟李泓睿李柏志刘志屹
Owner JILIN UNIV
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