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O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof

A foot-and-mouth disease virus and particle technology, applied in the fields of genetic engineering and immunology, can solve the problems of poor safety and unsatisfactory use effect, and achieve the effect of good stability, favorable for popularization and use, and good safety.

Inactive Publication Date: 2015-09-02
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problems of poor safety and unsatisfactory use effect of existing inactivated foot-and-mouth disease vaccines, the present invention provides a safe and effective O-type foot-and-mouth disease virus-like particle vaccine capable of effectively preventing foot-and-mouth disease virus and its preparation method and application

Method used

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  • O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof
  • O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof
  • O type foot and mouth disease virus-like particle vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the acquisition of O-type foot-and-mouth disease virus antigen VP1 gene

[0039] The O-type foot-and-mouth disease virus antigen VP1 gene was constructed by PCR amplification. First, the upstream and downstream primers were synthesized, and the primer sequences were as follows:

[0040] Upstream primer: 5'-GGGTCGAC ATGACCACTTCGACGGGCGAGTCGGCTG- 3';

[0041] Downstream primer: 5'-CCCTCGAG CAAGGACTGCTTTACAGGCGCCACT- 3'.

[0042] Among the above-mentioned upstream and downstream primer sequences, the underlined part is the sequence complementary to the VP1 gene, and the bold italic part is the introduced XhoI and SalI restriction sites. The specific fragment amplified by PCR is reclaimed after 1% agarose gel electrophoresis to obtain the O-type foot-and-mouth disease virus antigen VP1 gene fragment with XhoI and SalI restriction sites. The nucleotide sequence of the VP1 gene fragment is as follows: Sequence 2 in the sequence listing (the nucleotide sequ...

Embodiment 2

[0043] Example 2: Construction of recombinant expression vector plasmid pET30-FMD-VP1

[0044] The O-type foot-and-mouth disease virus antigen VP1 gene fragment (that is, the complete reading frame containing the O-type foot-and-mouth disease virus VP1 gene) obtained in Example 1 with XhoI and SalI restriction sites is inserted into the prokaryotic treated by XhoI and SalI double enzyme digestion On the multi-cloning enzyme cutting site of the expression vector pET30a, the ligation product was transformed into Escherichia coli DH5α, and the white clone was picked from the resistance plate for SalI / XholI double enzyme digestion sequencing identification, the results are as follows figure 2 As shown, it was confirmed by sequencing that the connection was successful, that the VP1 virus coding frame was correct, and that the restriction site used for the connection was mutated, and that the sequenced correct clone was named the recombinant expression vector plasmid pET30-FMD-VP1. ...

Embodiment 3

[0045] Embodiment 3: VP1 protein expression, identification and purification

[0046] Transform the correct recombinant expression vector plasmid pET30-FMD-VP1 obtained in Example 2 into Escherichia coli BL21 (DE3) to obtain the recombinant genetically engineered bacteria, and inoculate the obtained recombinant genetically engineered bacteria into the LB liquid with kana resistance In the culture medium, shake culture to about OD600=0.6, add IPTG to a final concentration of 1mmol / L, induce the expression of recombinant genetically engineered bacteria, the induction conditions are: temperature 37°C, rotation speed 180r / min, induction 6h; The bacterial cells were collected, and the expression of the target protein was detected by SDS-PAGE electrophoresis; the expression of the bacterial protein induced by IPTG and the negative control (uninduced empty cells) were subjected to SDS-PAGE electrophoresis detection. The result is as image 3 As shown, compared with the uninduced rec...

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Abstract

The invention discloses an O type foot and mouth disease virus-like particle vaccine as well as a preparation method and an application thereof, and belongs to the fields of genetic engineering and immunology, aiming at solving the problems of an existing inactivated foot and mouth disease vaccine which is poor in safety and unsatisfying in use effect. The amino acid sequence of the vaccine is shown as SEQ ID NO: 1 in a sequence list, and a nucleotide sequence for encoding the amino acid sequence is shown as SEQ ID NO: 2 in the sequence list. After being immunized, the vaccine can induce an animal to simultaneously generate an O type foot and mouth disease resistant antibody; the vaccine exists in the form of virus-like particles, and foot and mouth disease virus VP1 epitope locates on the surface of the virus-like particles. According to the invention, the amino acid sequence constituting the virus-like particle vaccine and the nucleotide sequence for encoding the amino acid sequence are provided, and the nucleotide sequence contains all genes of coat protein which can be self-assembled as the virus-like particles in expression. The preparation of the O type foot and mouth disease virus-like particle vaccine disclosed by the invention is good in safety and free from the probability of reversion or toxin dispersion; and the vaccine is long in immunizing validity period and convenient in transportation and storage.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and immunology, in particular to an O-type foot-and-mouth disease virus-like particle vaccine and its preparation method and application. Background technique [0002] Foot-and-mouth disease (FMD) is an acute heat and highly contagious infectious disease caused by foot-and-mouth disease virus (FMDV). famous. FMD has a wide range of hosts, including domestic ruminants, deer and pigs, and more than 70 wild artiodactyls. The World Organization for Animal Health (OIE) lists it as the first class A infectious disease. Once the foot-and-mouth disease breaks out, it often causes a pandemic, which is difficult to control and eliminate, and brings serious economic losses to the animal husbandry in the disease-endemic countries and regions. At present, countries in the world adopt three strategies for the prevention and control of foot-and-mouth disease: extermination and eradication, immune ...

Claims

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Application Information

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IPC IPC(8): A61K39/135C12N15/70A61K9/16A61P31/14A61P37/04
Inventor 郭利杨勇杨艳玲孙娜程世鹏任静强席娜李家伟刘莹林鹏宁浩然
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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