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Preparation method of leptospira interrogans MAP vaccine

A leptospira and vaccine technology, applied in the field of cellular immunity, can solve the problems of high cost, single antigen, poor immune effect, etc., and achieve the effect of good antigenicity and cross-immunity reactivity

Pending Publication Date: 2019-11-22
ZHEJIANG MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, it has been found that Leptospira contains some genus-specific protein antigens LipL32, OmpLl and Lipl2l for the preparation of general-purpose genetic engineering vaccines. question

Method used

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  • Preparation method of leptospira interrogans MAP vaccine
  • Preparation method of leptospira interrogans MAP vaccine
  • Preparation method of leptospira interrogans MAP vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Epitope prediction and identification

[0016] According to the protein primary structure sequence obtained from the lipL21, lipL32, groEL and loa22 gene sequences of the reference standard strains of Leptospira serotypes in my country, T cells were predicted by using the Propred HLA-DR binding peptide prediction-ProPred server and the ANTIGENIC program in the EMBOSS software package epitopes and B cell epitopes. The predicted epitope sequence was cloned into the phage vector M13KE to display it on the surface of PⅢ protein, and the display product was purified and analyzed for immune activity. The previous research results showed that the epitope composed of amino acids 97-122 and 176-184 of LipL21 protein, LipL32 The epitope composed of amino acids 133-160 and 221-247 of the protein, the epitope composed of amino acids 215-247 of the GroEL protein, and the epitope composed of amino acids 22-90 of the Loa22 protein have strong immune activity. The epitope seq...

Embodiment 2

[0020] Embodiment 2: Construction of gene synthesis and expression vector

[0021] The nucleotide sequence corresponding to each epitope is determined according to the amino acid sequence of the LLGL multivalent epitope and constituent epitope and the preferred codon characteristics of the expression host Escherichia coli, and the flexible peptide linker GGGGS nucleotide sequence ggtggcggtggcagc is used to connect each epitope, and in LLGL The 5' end of the polyvalent epitope coding sequence is the restriction endonuclease site EcoR I and the start codon ATG, and the 3' end is the restriction endonuclease site BglII, the stop codon TAA and BamH I in turn, entrusted Synthesized by Shanghai Invitrogen Company. The nucleotide sequence of the LLGL polyvalent epitope gene is shown below. The synthetic gene fragment was digested and purified by BamH I and EcoR I, then subcloned into E. coli expression vector pET-28a, transformed into E. coli BL21, and constructed a prokaryotic expr...

Embodiment 3

[0024] Embodiment 3: Expression and purification of recombinant protein

[0025] to pick E. coli BL21 pET-28a-LLGL A single colony was inoculated into 10 ml of Kana-resistant LB medium, and cultured overnight at 37°C with shaking. Transfer the culture medium to 10 ml of fresh LB medium resistant to kanamycin at a ratio of 1:100, culture with shaking at 37°C for 3 h, add IPTG to make the final concentration 1 mmol / L, and culture with shaking at 37°C 4 h. Take 1.5 ml of the culture solution and collect the cells by centrifugation, then resuspend them in 150 µl of PBS (pH7.4) buffer solution and perform low-temperature ultrasonication (300 V, 10S x5) to disrupt the cells. After centrifugation at 4°C and 12 000 r / min for 5 min, the supernatant and precipitate were collected for SDS-PAGE analysis. Re-inoculate the high-expression strains into 500 ml LB medium resistant to kanamycin to induce the expression of rLLGL in large quantities. After sonication, the recombinant protei...

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Abstract

The invention discloses a dominant T-and B-cell T-B combined multivalent antigen epitope fusion gene and a prokaryotic expression system thereof by constructing leptospira interrogans LipL21, LipL32,groEL and loa22 proteins. The MAP vaccine is prepared by the following steps of: expressing a multiple antigen peptide (MAP) consisting of antigen epitopes, and artificially crosslinking the MAP witha high polymer core carrier. The prepared MAP vaccine is has good immune effect, is low-cost, and is capable of effectively inducing cellular immunity. Toxic and side effects of the vaccine are reduced, safety is improved, and immune pertinence is enhanced.

Description

technical field [0001] The invention belongs to the technical field of cellular immunity, and in particular relates to a preparation method of a Leptospira interrogans MAP vaccine. Background technique [0002] Leptospirosis, which can be caused by pathogenic Leptospira (hereinafter referred to as Leptospira) infection, is an important natural foci of epidemics, zoonotic infectious diseases in the world [2] . Leptospirosis is not only one of the 13 infectious diseases included in the national monitoring and immunization program, but also one of the four infectious diseases that my country focuses on monitoring and epidemic prevention during natural disasters. Vaccination is the most effective and economical means of preventing and controlling infectious diseases. However, Leptospira has the characteristics of many serogroups and types, and there are obvious differences in the dominant prevalent serogroups in different regions, and the cross-protection effect between differ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61P31/04C07K19/00C12N15/70C12R1/19
CPCA61K39/0225A61P31/04C07K14/20C12N15/70A61K2039/53C07K2319/00C12N2800/22Y02A50/30
Inventor 孙爱华严杰张金良楼宏强
Owner ZHEJIANG MEDICAL COLLEGE
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