A-type foot-and-mouth disease subunit vaccine as well as preparation method and application thereof

A subunit vaccine, foot-and-mouth disease technology, applied in the field of molecular biology, can solve the problems of limited value and prospects, low expression efficiency, etc., to achieve good protection, high immune efficacy, improved stability and protective efficacy.

Active Publication Date: 2020-12-15
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the patent (CN101914501B) discloses the introduction of SUMO into the foot-and-mouth disease structural proteins VPO, VP1, and VP3, and expresses them through three plasmids respectively to prepare Asia1 type foot-and-mouth disease virus-like particles; and the patent (CN104404074B) introduces a small ubiquitin-like fusion protein (SUMO), the co-expression plasmids of SUMO-VP0, SUMO-VP1 and SUMO-VP3 were constructed, and Asia1 type FMD virus-like particles were obtained, but the expression efficiency was low
The highest yield of the purified Asia1 type foot-and-mouth disease virus structural protein prepared by the Escherichia coli expression system is only about 20 mg / L, and at least 50 μg / head is required to produce a fully protected immune antigen, which greatly limits its commercial application value and prospect

Method used

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  • A-type foot-and-mouth disease subunit vaccine as well as preparation method and application thereof
  • A-type foot-and-mouth disease subunit vaccine as well as preparation method and application thereof
  • A-type foot-and-mouth disease subunit vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The preparation of embodiment 1 foot-and-mouth disease virus structural protein composition

[0050] 1. Construction of recombinant expression vector for structural protein of foot-and-mouth disease virus

[0051] (1) Design the gene sequence THS encoding the fusion tag protein composed of the following elements in series, wherein T is the nucleotide sequence of the translation initiation region, H is the nucleotide sequence encoding the histidine tag, and S is the nucleotide sequence encoding the tag containing The nucleotide sequence of Saccharomyces cerevisiae small ubiquitin-like modification protein (SUMO); the nucleotide sequence of THS is shown in SEQ ID NO:11.

[0052] (2) The above-mentioned THS gene sequence was concatenated in sequence with the structural protein genes VPO, VP3, and VP1 encoding the A / GDMM / 2013 strain, respectively, to form three fusion gene sequences THS-VPO, THS-VP3, and THS-VP1. Wherein said coding A / GDMM / 2013 strain structural protein ge...

Embodiment 2

[0075] The expression efficiency of embodiment 2 foot-and-mouth disease virus structural protein composition

[0076] Four kinds of foot-and-mouth disease virus structural protein compositions A / GDMM / 2013, Re / A / GDMM / 2013 / K3118R, Re / A / GDMM / 2013 / K1209R, Re / A / GDMM / 2013 / K3118R+K1209R was identified for prokaryotic expression, and the expression efficiency of different foot-and-mouth disease virus structural protein compositions was analyzed by SDS-PAGE. The experimental results are as follows Figure 5 As shown, neither the single gene mutation of the FMDV structural protein VP3 or VP1, nor the simultaneous mutation of the structural proteins VP3 and VP1 will significantly affect the expression efficiency of the FMDV structural protein composition.

[0077] According to the method described in Example 1, respectively to 4 kinds of foot-and-mouth disease virus structural protein compositions A / GDMM / 2013, Re / A / GDMM / 2013 / K3118R, Re / A / GDMM / 2013 / K1209R, Re / A / GDMM / 2013 / K3118R+K1209R ...

Embodiment 3

[0080] Example 3 Detection of Neutralizing Antibody Response in Animals Immunized with Foot-and-Mouth Disease Subunit Vaccine

[0081] The foot-and-mouth disease virus structural protein compositions A / GDMM / 2013, Re / A / GDMM / 2013 / K3118R, Re / A / GDMM / 2013 / K1209R, Re / A / GDMM / 2013 / K3118R+ K1209R vaccine was prepared with the same antigen dose and immunized in pigs. Blood was collected on days 0, 7, 14, 21, and 28 after immunization, and the serum was separated for detection of neutralizing antibody titers. Test results such as Figure 6As shown, the A-type foot-and-mouth disease virus structural protein composition A / GDMM / 2013, Re / A / GDMM / 2013 / K3118R, Re / A constructed with the A / GDMM / 2013 strain structural protein gene VPO, VP3, VP1 / GDMM / 2013 / K1209R, Re / A / GDMM / 2013 / K3118R+K1209R can produce higher levels of neutralizing antibodies after animal immunization, and the structural protein genes VP3 and VP1 of the A / GDMM / 2013 strain or The type A foot-and-mouth disease virus structural pr...

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Abstract

The invention relates to the technical field of molecular biology, in particular to an A-type foot-and-mouth disease subunit vaccine as well as a preparation method and application thereof. Accordingto the invention, optimization design is performed based on amino acid sequences of three kinds of structural proteins VP0, VP3 and VP1 of an A-type foot-and-mouth disease virus epidemic strain (A/GDMM/2013) in 2013 in China, a single plasmid is screened out by means of small ubiquitin-related modifier (SUMO) fusion protein to simultaneously express the three kinds of structural proteins efficiently, uniformly and solubly in escherichia coli, and the three kinds of virus structural proteins are successfully self-assembled in vitro; and finally obtained target protein accounts for about 30% orabove of total bacterial protein, and the maximum yield of the purified target protein can reach 150 mg/L. The foot-and-mouth disease vaccine prepared with the method disclosed by the invention has agood protection effect on A-type foot-and-mouth disease epidemic viruses in China, and the minimum full-protection immune dose of the vaccine can be as low as 20 micrograms per vaccine.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a type A foot-and-mouth disease subunit vaccine and its preparation method and application. Background technique [0002] Foot and Mouth Disease Virus (FMDV) belongs to the family Picornaviridae and belongs to the genus Aphthovirus. The FMD virion has no envelope, is icosahedral, and is composed of structural proteins VP0, VP3 and VP1, wherein the VP0 protein is composed of VP4 and VP2. The virus mainly infects cloven-hoofed animals such as pigs, cattle, and sheep, and has the characteristics of rapid transmission and strong infection pathogenicity. FMD virus has seven serotypes (O, A, Asia1, SAT1, SAT2, SAT3 and C), and there is no cross-protection reaction among the various types. Currently, type O and type A of foot-and-mouth disease are mainly prevalent in my country. Among them, the Asia Topotype (Asia Topotype) Southeast Asia-97 strain (Sea-97) was introduced i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/135A61K39/39A61P31/14C07K19/00C07K1/22C12P21/06C12N15/62C12N15/70
CPCA61K39/12A61K39/39A61K2039/5258A61K2039/552A61P31/14C07K14/005C07K2319/21C07K2319/95C12N15/70C12N2770/32122C12N2770/32134C12P21/06
Inventor 茹毅刘华南张贵财杨帆李丹郭建宏何继军张娇燕李亚军马坤伍春平郝荣增卢炳州田宏朱紫祥张克山曹伟军刘永杰靳野马旭升党文
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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