Recombinant feline herpesvirus type 1 gB protein antigen and application thereof to antibody diagnosis and vaccine preparation

A feline herpes virus and recombinant protein technology, applied in the direction of virus antigen components, virus/bacteriophage, virus, etc., can solve the problems of low efficiency of prokaryotic or eukaryotic expression, unsuccessful establishment of antibody detection methods, and the risk of virus shedding, etc. Achieve the effects of strong clinical practicability, short detection time and high positive rate

Active Publication Date: 2022-01-18
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a large amount of virus is required for full virus encapsulation, which is costly and has the risk of spreading the virus
However, the full-length molecular weight of the gB protein is about 130kd, and neither prokaryotic nor eukaryotic expression efficiency is high, and it is more difficult to purify in large quantities. Therefore, an antibody detection method for this protein has not been successfully established.

Method used

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  • Recombinant feline herpesvirus type 1 gB protein antigen and application thereof to antibody diagnosis and vaccine preparation
  • Recombinant feline herpesvirus type 1 gB protein antigen and application thereof to antibody diagnosis and vaccine preparation
  • Recombinant feline herpesvirus type 1 gB protein antigen and application thereof to antibody diagnosis and vaccine preparation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] According to the selected gB dominant epitope region, use a flexible linker sequence to connect different regions in series, and introduce at both ends of the sequence Bam HI restriction site and Eco RI restriction enzyme site to obtain recombinant DNA. The corresponding amino acid sequence is shown in SEQ ID NO.1, and the sequence encoding the recombinant DNA is shown in SEQ ID No.2.

[0033] 2) Construction of positive recombinant plasmid pET-28a △FHV-1 gB

[0034] Connect the required B cell antigen epitope regions with Linker, and synthesize them in the company after codon optimization. Bam HI and Eco The RI restriction enzyme site was synthesized into the prokaryotic expression vector pET-28a, and the PCR amplification of the synthesized prokaryotic expression plasmid was identified as follows: figure 1 Shown, sent to Shanghai Bioengineering Co., Ltd. for sequence determination. The recombinant plasmid identified as positive by sequencing was the positive r...

Embodiment 2

[0041] Embodiment 2 Establishment of ELISA method

[0042] The purified recombinant protein was used to prepare the ELISA plate for the coated antigen, the level of FHV-1 antibody in the cat serum was detected, and various conditions affecting the experiment were optimized. in particular:

[0043] a) Antigen coating concentration and serum optimal dilution

[0044] Use matrix titration method, select antibody dilution in horizontal row (1:100, 1:200, 1:400, 1:800, 1:1000, 1:1200, 1:1400, 1:1600), and select antigen concentration in vertical row (0.25 ug / mL, 0.5 ug / mL 1ug / mL, 2 ug / mL,), each dilution was repeated 3 times, and the average value was taken. As a result, when the antigen concentration is 1 ug / mL and the serum to be tested is diluted 1:1000, the P / N value is the largest. Therefore, the optimal antigen coating concentration is 0.5 ug / mL, and the optimal antibody dilution is 1:1000, such as Table 1 shows.

[0045] Table 1 Determination of optimal antigen coating c...

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Abstract

The invention provides a recombinant feline herpesvirus type 1 gB protein antigen and an application thereof to antibody diagnosis and vaccine preparation. By using a prokaryotic expression vector, dominant epitopes of the feline herpesvirus type 1 gB protein are subjected to tandem expression. The expressed recombinant protein is purified and used as a coating antigen for detecting the feline herpesvirus type 1 antibody. The antigen treatment is convenient, the test time is shortened, and the operation steps are simpler. An indirect ELISA method established by the invention is used for detecting the antibody level of the feline herpesvirus type 1 in feline serum, has the characteristics of good repeatability and high specificity, and can be used for feline herpesvirus type 1 serology investigation. Therefore, an indirect ELISA detection kit for the feline herpesvirus type 1 based on tandem expression of a dominant antigen of the gB protein, provided by the invention, is very suitable for detection of clinical large samples and is suitable for large-scale popularization.

Description

technical field [0001] The invention belongs to the field of detection and diagnosis of biological vaccines and microorganisms, and more specifically relates to a recombinant feline herpes virus gB protein antigen and its application in antibody diagnosis and vaccine preparation, and its use in detecting FHV-1 body Indirect ELISA method. The invention also relates to the application of the recombinant feline herpes virus gB protein in the preparation of feline herpes virus gB vaccine and reagents for diagnosing or detecting feline herpes virus infection. Background technique [0002] Feline herpesvirus type 1 (FHV-1) is an acute, highly contagious upper respiratory disease of cats caused by the family Herpesviridae and the subfamily Alpha-herpesvirus. Kittens aged 2 to 6 months are most susceptible. The morbidity rate is 100%, and the fatality rate can reach 20% to 50%. It is one of the most important feline respiratory diseases known at present. After HV-1 infection, it ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/03C12N15/70C07K1/22G01N33/569G01N33/58G01N33/543A61K39/245A61P31/22
CPCC07K14/005C12N15/70G01N33/56983G01N33/581G01N33/54306A61K39/12A61P31/22C12N2710/16022C12N2710/16034G01N2469/20G01N2333/03A61K2039/53Y02A50/30
Inventor 孟春春刘光清王真真李传峰朱杰汤傲星刘春草
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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