Tandem expression of universal epitopes of feline calicivirus gi and gii strains and establishment of an indirect ELISA method

A feline calicivirus and antigenic epitope technology, applied in the application of reagents, the field of indirect ELISA for detecting FCV antibodies, can solve the problems of scattered virus risk, complicated preparation of monoclonal antibodies, time-consuming and laborious, etc., and achieves strong clinical practicability. , Good specificity, strong broad-spectrum effect

Active Publication Date: 2022-08-09
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
The preparation of monoclonal antibody is cumbersome and time-consuming, and cannot be completed in a short time

Method used

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  • Tandem expression of universal epitopes of feline calicivirus gi and gii strains and establishment of an indirect ELISA method
  • Tandem expression of universal epitopes of feline calicivirus gi and gii strains and establishment of an indirect ELISA method
  • Tandem expression of universal epitopes of feline calicivirus gi and gii strains and establishment of an indirect ELISA method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] According to the selected FCV different strains of VP1 common B cell epitope region information as shown in Table 1, use a flexible linker sequence to connect the different regions in series, and introduce at both ends of the sequence Bam HI restriction sites and Eco RI restriction enzyme cut sites to obtain recombinant DNA. The schematic diagram of the corresponding amino acid site is as follows figure 1 As shown, 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] Table 1 Selected epitope region information

[0034]

[0035] 2) Construction of positive recombinant plasmid pCold I △FCV VP1

[0036] Link the desired B cell epitope region with Linker, and synthesize it in the company after codon optimization. Bam HI and Eco RI was used to synthesize restriction enzyme sites into the prokaryotic expression vector pCold I and sent to Shanghai Bioengineering Co., Ltd. for ...

Embodiment 2

[0043] Example 2 Establishment of ELISA method

[0044] The purified recombinant protein was used as the coating antigen to prepare an ELISA plate to detect the level of FCV antibody in cat serum, and to optimize the selection of various conditions affecting the experiment. in particular:

[0045] a) Antigen coating concentration and optimal dilution of serum

[0046] The matrix titration method was used, and the antibody dilution (1:100, 1:200, 1:400, 1:800, 1:1000, 1:1200, 1:1400, 1:1600) was selected in the horizontal row, and the antigen concentration was selected in the vertical row. (1ug / mL, 2ug / mL, 4ug / mL, 8ug / mL), each dilution was repeated 3 times, and the average value was taken. As a result, when the antigen concentration is 2 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 2 ug / mL, and the optimal antibody dilution is 1:1000. As shown in table 2.

[0047] Table 2 Determina...

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Abstract

The present invention provides a tandem expression of common antigenic epitopes of feline calicivirus GI and GII strain groups and the establishment of an indirect ELISA method. Using a prokaryotic expression vector, multiple B cell antigens of feline calicivirus VP1 protein were expressed in tandem. The expressed recombinant protein was purified and used as a coating antigen for the detection of feline calicivirus antibodies. A parallel comparison with the method of encapsulating whole virus showed that the value of detecting negative and positive sera was highly consistent. The antigen processing of the invention is convenient, the test time is shortened, and the operation steps are simpler. The invention establishes an indirect ELISA method for detecting the antibody level of feline calicivirus in cat serum, has the characteristics of good repeatability and high specificity, and can be used for feline calicivirus serological investigation. Therefore, the feline calicivirus indirect ELISA detection kit based on the tandem expression of the VP1 protein B cell antigen provided by the present invention is very suitable for the detection of large clinical samples, and is suitable for large-scale promotion.

Description

technical field [0001] The invention belongs to the field of detection and diagnosis of biological vaccines and microorganisms, and more particularly relates to a tandem expression of a recombinant feline calicivirus variant strain and a common antigenic epitope of a classic strain VP1, and an indirect ELISA for detecting FCV antibodies using the same method. The present invention also relates to the application of the recombinant feline calicivirus VP1 protein in preparing feline calicivirus VP1 subunit vaccine and preparing reagents for diagnosing or detecting feline calicivirus infection. Background technique [0002] Feline Calicivirus (FCV) is an important pathogen of cats and other felines. It is a member of the Caliciviridae family, and has only one serotype. FCV mostly infects kittens under one year old. The incubation period is 2~3 days. It usually causes feline oral and upper respiratory tract diseases. Clinical symptoms include rhinitis, conjunctivitis and oral u...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K1/22C12N15/62C12N15/70G01N33/569G01N33/543A61K39/12A61P31/14
CPCC07K14/005C12N15/70G01N33/56983G01N33/54306A61K39/12A61P31/14C12N2770/16022C07K2319/00C12N2800/22C12N2800/101G01N2333/08G01N2469/20A61K2039/53Y02A50/30
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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