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Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation

A feline parvovirus and recombinant protein technology, applied in the direction of viral antigen components, virus/bacteriophage, complete cells/virus/DNA/RNA components, etc., can solve the problem of low recombinant protein expression, large difference in viral sequence, no tandem expression, etc. problems, to achieve the effect of good specificity, short detection time, and easy operation

Active Publication Date: 2022-01-21
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

Although some researchers have made some progress in the detection of parvoviruses in other animals, although they are targeting the VP2 protein, first of all, the sequences of the viruses are very different, and some of their homology are less than 70%. Only one of the epitopes was taken, and all feasible epitopes were not predicted and verified for tandem expression
However, due to the low expression of the full-length recombinant protein of FP2 VP2 and the difficulty of protein purification, its application was once limited.

Method used

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  • Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation
  • Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation
  • Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation

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Experimental program
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Effect test

Embodiment 1

[0032] First, based on multiple B cell prediction software, we performed prediction statistics on the VP2 gene B cell epitopes isolated from 20 strains of FPV viruses from different ages and countries, and obtained a total of 35 theoretically conserved potential B cell epitopes. Firstly, 35 potential B cell epitopes were synthesized into polypeptides, and coupled with the skeleton protein to form a complete antigen, and after immunizing mice, it was tested whether the B cell epitopes could stimulate the production of specific antibodies. After verification, a total of 11 epitopes with good immunogenicity were obtained, as shown in Table 1.

[0033] According to the selected FPV different strains of VP2 common B cell epitope region information is shown in Table 1, then use a flexible linker sequence to connect different regions in series, and introduce at both ends of the sequence BamHI restriction enzyme sites and EcoRI Restriction enzyme cut sites to obtain recombinant DN...

Embodiment 2

[0044] Embodiment 2 Establishment of ELISA method

[0045] The purified recombinant protein was used to prepare an ELISA plate for the coated antigen, and the level of FPV antibody in the cat serum was detected, and various conditions affecting the experiment were optimized. in particular:

[0046] a) Antigen coating concentration and serum optimal dilution

[0047] Matrix titration method was used to select antibody dilution (1:100, 1:200, 1:400, 1:800) in horizontal row and antigen concentration (0.5 ug / mL, 1 ug / mL, 2 ug / mL, 4 ug / mL in tandem). ug / mL, 8 ug / mL, 16 ug / mL), each dilution was repeated 3 times, and the average value was taken. As a result, when the antigen concentration is 8 ug / mL and the serum to be tested is diluted 1:400, the P / N value is the largest. Therefore, the optimal antigen coating concentration is 8 ug / mL, and the optimal antibody dilution is 1:400, such as Table 2 shows.

[0048] Table 2 Determination of optimal antigen coating concentration and ...

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Abstract

The invention provides a recombinant feline parvovirus VP2 protein antigen and application thereof in vaccine preparation and virus diagnosis. A plurality of B cell antigens of the feline parvovirus VP2 protein are subjected to tandem expression by using a prokaryotic expression vector. The expressed recombinant protein is purified and used as a coating antigen for detecting the feline parvovirus antibody. And compared with a whole virus coating method in parallel, the values of detected positive and negative serum are highly consistent. The antigen treatment method provided by the is convenient, the test time is shortened, and the operation steps are simpler. According to the invention, an indirect ELISA method is established for detecting the antibody level of the feline parvovirus in feline serum, which has the characteristics of good repeatability and high specificity, and can be used for feline parvovirus serology investigation. Therefore, the indirect ELISA detection kit for the feline parvovirus based on the tandem expression of the VP2 protein B cell antigens, provided by the invention, is very suitable for the 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 tandem antigen of recombinant feline parvovirus VP2 protein and an indirect ELISA method using it to detect FPV antibodies. The present invention also relates to the application of the recombinant feline parvovirus VP2 protein in preparing feline parvovirus VP2 subunit vaccine and reagents for diagnosing or detecting feline parvovirus infection. Background technique [0002] Feline parvovirus (Feline parvovirus, FPV) belongs to the family Parvoviridae, the genus Parvovirus, also known as feline panleukopenia virus, feline distemper virus and feline infectious enteritis virus. The virus is a highly fatal contagious disease that is mainly susceptible to young animals. It can infect a variety of animals such as cats and raccoons, among which young cats under the age of 6 months are the most susceptible. Its typical c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00
CPCC07K14/005C12N15/70G01N33/56983G01N33/543G01N33/581A61K39/12A61P31/20C12N2750/14322C12N2750/14334C07K2319/00A61K2039/53G01N2333/015Y02A50/30
Inventor 孟春春刘光清王真真朱杰李传峰汤傲星刘春草
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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