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Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein

A rotavirus and analysis method technology, applied in the field of immunogenicity analysis and expression, to achieve the effects of controllable gene expression, simple equipment, and stable genetic traits

Pending Publication Date: 2022-05-13
TONGHUA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Since there is no specific drug for the treatment of rotavirus diarrhea, the development of RV (Rotavirus, RV) vaccine is the main task to prevent RV diarrhea

Method used

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  • Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein
  • Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein
  • Method for analyzing expression and immunogenicity of recombinant human rotavirus VP7 protein

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Experimental program
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Effect test

Embodiment Construction

[0017] The expression of recombinant human rotavirus VP7 protein, the analysis method of immunogenicity, its steps are as follows:

[0018] 1 Material method

[0019] 1.1 Sources of strains and viruses

[0020] Escherichia coli pMD18-T and Schizosaccharomyces pombe expression vector pESP-2 were purchased from Takala Biological Company, and Schizosaccharomyces pombe strain SPQ-01 was preserved by the Bioreactor Laboratory of Jilin Agricultural University. Rotavirus disease materials were stored at -70°C by the Department of Infectious Diseases, Tonghua Central Hospital.

[0021] main reagent

[0022] The reverse transcription kit, TaqDNApolymerase, and DNAmarker were purchased from Takala Biological Company; the PCR product recovery and purification kit was purchased from Kangrun Biotechnology; the mouse anti-rotavirus VP7 monoclonal antibody was purchased from Zhuhai Bomei Biotechnology Co., Ltd.; horseradish peroxidation Biozyme-labeled goat anti-human IgG was purchased fr...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to an analysis method for expression and immunogenicity of recombinant human rotavirus VP7 protein. The method comprises the following steps: firstly, designing a primer according to a VP7 complete sequence (NC007571.1) published in GeneBank, carrying out nucleic acid extraction on a disease material, carrying out reverse transcription on cDNA (complementary deoxyribonucleic acid) as a template, cloning a VP7 gene, constructing an expression vector pESP-2-VP7, and transforming schizosaccharomyces pombe; vP7 expression is induced by 1% IPTG (isopropyl-beta-d-thiogalactoside), separation and purification are performed to obtain the VP7 protein, and mice are immunized by the VP7 protein in different immunization modes to evaluate the immune protection effect of the VP7 protein. The results show that the primer design is consistent with the prediction result, the length of the cloned VP7 gene is 1050 bp, and the SDS-PAGE and Western Blot identification results show that the protein size is 63 kDa; after a mouse is immunized by the VP7 protein, IgG, SIgA and neutralizing antibody results show that the immune effect of 50 micrograms of each nasal drop is better than that of 20 micrograms of each nasal drop and an injection group (50, 20 micrograms of each nasal drop); and the nasal drop immunity has a relatively high challenge protection rate of 100%.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, namely the expression of recombinant human rotavirus VP7 protein and the analysis method of immunogenicity. To investigate the expression of rotavirus VP7 protein in Schizosaccharomyces pombe, and identify and evaluate its immune protection effect. Background technique [0002] In the prior art, rotavirus (Rotavirus, RV) rotavirus belongs to the genus Rotavirus of the family Reoviridae, and is an enterovirus. It is the main cause of acute gastroenteritis in infants and young children. About 870,000 infants and young children die from diarrhea caused by rotavirus every year. It is one of the main pathogens causing diarrhea and death. Children under 5 years old are susceptible. According to statistics, about 20 million children under the age of 5 see a doctor and 2 million children are hospitalized every year around the world. In areas where RV vaccination is not widely vaccinated, 40% of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/14C12N15/46C12N15/81C12N1/19C07K16/10G01N33/543G01N33/569G01N33/577A61K39/42A61P31/14A61P1/12C12R1/645
CPCC07K14/005C12N15/815C07K16/10G01N33/56983G01N33/543G01N33/577A61P31/14A61P1/12C12N2720/12322C12N2720/12334G01N2333/14A61K2039/505A61K2039/543
Inventor 王红涛
Owner TONGHUA NORMAL UNIV
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