Foot-and-mouth disease recombinant virus with reduced immunosuppression function as well as preparation method and application thereof

A foot-and-mouth disease virus and recombinant virus technology, applied in the field of bioengineering, can solve problems such as transformation and improvement of targeting 3B protein

Active Publication Date: 2020-10-09
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

In recent years, reverse genetic manipulation technology has been widely used in the transformation of biological characteristics such as virus virulence or virus yield, but currently no reverse genetic manipulation technology has been used to target the virulence of 3B protein to FMDV or the virus A plan to improve biological characteristics such as yield

Method used

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  • Foot-and-mouth disease recombinant virus with reduced immunosuppression function as well as preparation method and application thereof
  • Foot-and-mouth disease recombinant virus with reduced immunosuppression function as well as preparation method and application thereof
  • Foot-and-mouth disease recombinant virus with reduced immunosuppression function as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Immunosuppressive effect of 3B protein

[0058] First, the amino acid sequences of 3B proteins of different serotypes of FMD viruses were compared, and it was found that the three copies of 3B proteins 3B1, 3B2 and 3B3 of different serotypes of FMD viruses were highly homologous, and the results were as follows figure 1 shown. Therefore, in this implementation, only the epidemic strain O / BY / CHA / 2010 strain is taken as an example to study the immunosuppressive function of the 3B protein of the epidemic strain. The specific experiments are as follows:

[0059] 1.1 Effect of 3B protein on innate immune response

[0060] IFN-β, ISRE and NF-kB reporter systems were used to detect the immunosuppressive effect of 3B protein: HEK293T cells were transfected with FLAG-3B plasmid and each reporter system, and after 24 hours of transfection, the model virus SeV was induced with interferon ( Sendai virus) was stimulated, and the expression changes of the reporter system ...

Embodiment 23B

[0071] Example 2 Construction method of 3B protein mutant eukaryotic expression plasmid

[0072] On the basis of Example 1, the inventor mutated the 3B protein of the FMDV O / BY / CHA / 2010 strain, and constructed a eukaryotic expression plasmid for the 3B protein mutant. Using the method described in this example, the same can be constructed Eukaryotic expression plasmids of other serotype foot-and-mouth disease virus 3B protein mutants, the specific process is as follows:

[0073] Mutation strategies such as Figure 5 As shown, on the basis of wild-type FMDV O / BY / CHA / 2010 strain 3B protein (nucleotide sequence as shown in SEQ ID NO.3, amino acid sequence as shown in SEQ ID NO.4), using gene synthesis technology The 17th amino acid of 3B1, the 17th amino acid of 3B2, and the 17th amino acid of 3B3 were all mutated into glutamic acid in the 3B protein gene coding sequence (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO. .2); an...

Embodiment 33B

[0074] Example 3 Effects of 3B protein mutants on the expression of type I interferon

[0075] In order to further test the effect of 3B protein immunosuppressive site elimination mutant 3B-A / E on the expression of type I interferon. Spread HEK293T cells into 6-well plates, and after the cells grow to 70% density, transfect the same amount of FLAG-CMV7.1 vector, FLAG-3B plasmid and FLAG-3B-A / E plasmid respectively, and after 24 hours of transfection, Aspirate the upper culture solution, rinse twice with PBS, inoculate the interferon-induced model virus SeV, and discard the medium after 16 hours of infection, rinse twice with PBS, and collect cell samples. The total RNA of cell samples was extracted with an RNA extraction kit, and cDNA was synthesized by reverse transcription with random primers. Real-time quantitative PCR was used to detect the expression changes of IFN-βmRNA, samples were prepared in parallel, total cell protein was extracted, SDA-PAGE was performed, and the...

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Abstract

The invention belongs to the technical field of veterinary drug biological products, and particularly relates to a foot-and-mouth disease recombinant virus with a reduced immunosuppression function, arecombinant vaccine strain and a preparation method and application thereof. According to the invention, the FMDV 3B protein is firstly found to have an immunosuppression function, and a key site forexerting the immunosuppression function is found; secondly, key site amino acid mutations are respectively introduced into three repeated copies of the FMDV 3B protein, a foot-and-mouth disease recombinant vaccine strain with immunosuppression function loss of the FMDV 3B protein is constructed, the foot-and-mouth disease recombinant vaccine strain is stably subcultured in a foot-and-mouth disease production cell line BHK-21 with interferon pathway defects, virus titer is not affected, poisoning force of the foot-and-mouth disease recombinant vaccine strain in swine-derived host cells (PK-15)is reduced, and higher natural immune response inducibility is achieved; finally, the obtained recombinant vaccine strain is prepared into an inactivated vaccine, early immune response can be promoted, high-level antibody production is induced, biosafety and immune protection efficacy are improved, and good application prospects are achieved.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a foot-and-mouth disease recombinant virus with reduced immunosuppressive function, a recombinant vaccine strain and a preparation method and application thereof. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infectious disease that mainly affects domestic animals and wild artiodactyls. Generally, adult domestic animals will heal themselves after infection, and there is a high incidence rate in natural hosts, showing typical lesions among different species. FMD can cause high mortality rates in juvenile infection or in the context of a pandemic in wild animals. FMD is listed as a class A infectious disease by the World Organization for Animal Health (OIE), and it is also listed as a class I animal disease in my country. [0003] FMD is an acute febrile disease caused by Foot-and-mouth disease virus (FMDV), ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61P37/06A61K39/135A61P31/14C12N7/01C12R1/93
CPCA61K39/12A61P31/14A61K38/162A61P37/06C12N7/00C07K14/005C12N2770/32121C12N2770/32122C12N2770/32134A61K2039/552C12N2770/32151A61K2039/5252A61K39/135A61K2039/525C12N2770/32152C12N2770/32171
Inventor 郑海学杨帆朱紫祥张向乐曹伟军王聪聪李康丽刘会胜田宏张克山刘湘涛
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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