A kind of recombinant N protein antigen of vesicular stomatitis virus and its preparation method

A technology of vesicular stomatitis and protein antigen, which is applied in the field of genetic engineering, can solve the problems of poor specificity and achieve the effects of simple operation, low detection cost and high sensitivity

Inactive Publication Date: 2017-04-19
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The invention with application number 201010182429.9 discloses a colloidal gold detection test strip for vesicular stomatitis virus antibody and its preparation method. Although the invention is for detecting vesicular stomatitis virus antibody, the antigen used is the whole virus antigen of VSV, and the specificity poor

Method used

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  • A kind of recombinant N protein antigen of vesicular stomatitis virus and its preparation method
  • A kind of recombinant N protein antigen of vesicular stomatitis virus and its preparation method
  • A kind of recombinant N protein antigen of vesicular stomatitis virus and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1: the expression of the VSV N albumen of truncating

[0022] 1. Epitope analysis and codon optimization of VSV N protein: by searching the VSV N gene sequence and corresponding amino acid sequence (GenBank: J02428.1) in the NCBI database, and using online software to analyze its main epitope, determine the selected antigen The epitope is located at the 88th to 374th amino acid in the N protein, its amino acid sequence is SEQ ID No.1 in the sequence table, and its corresponding nucleotide sequence (GenBank: J02428.1) is the 325th to 1185nt nucleotide, See the sequence listing SEQ ID No.5.

[0023] 2. VSV N gene codon optimization and artificial synthesis: codon optimization, that is, according to the preference of the expression system for amino acid codons, the gene sequence is redesigned, and the codons with low utilization or rare codons in the gene sequence are modified to express The system uses frequent codons to ensure that the amino acid sequence of ...

Embodiment 2

[0037] Example 2: Establishment and evaluation of VSV antibody indirect ELISA method

[0038] 1. Evaluate the immunological activity of VSV N protein by indirect ELISA. The ELISA plate was coated with gene-expressed VSV recombinant N protein, and an indirect ELISA antibody detection method was established. The specific operation method is as follows:

[0039] ①. Dilute the purified recombinant protein to 5 μ / mL with a coating solution (carbonic acid buffer solution at pH 9.6), coat a 96-well ELISA plate at 50 μL / well, and place it at 4°C for overnight.

[0040] ②. Discard the coating, and wash 3 times with PBST (20mmol / L PBS+0.5% Tween 20). Add BSA solution with a concentration of 10 g / L, 250 μL / well, and block at 37° C. for 2 h. Discard the blocking solution and wash 3 times with PBST.

[0041] ③. Add positive serum, negative serum and serum to be tested to the ELISA plate, 50μL / well, 37°C, react for 30min.

[0042] ④ Wash 3 times with PBST, add 1:5000 dilution of HRP-la...

Embodiment 3

[0048] Embodiment 3: Preparation and evaluation of colloidal gold immunochromatography test strip

[0049] 1. The vesicular stomatitis virus recombinant N protein antigen of embodiment 1 is used as the detection line reagent, and the rabbit anti-SPGIgG is used as the contrast reagent simultaneously to make colloidal gold immunochromatography test strips, detection line and control line reagent coating amount They are 0.175μg / bar and 0.35μg / bar respectively.

[0050] 2, the specificity of test strip: detect respectively VSV standard positive serum sample, other relevant virus comprise O type, A type and Asian I type foot-and-mouth disease virus (FMDV), Akabane disease virus (AKV) with the test strip among the embodiment 2 ), bluetongue virus (BTV), bovine viral diarrhea virus (BVDV) and sheeppox virus (CPV) positive serum samples and negative control samples. The results showed that the test strip was positive when detecting VSV-positive serum samples, and was negative when de...

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Abstract

The invention belongs to the technical field of gene engineering, and more specifically relates to a vesicular stomatitis virus recombinant N protein antigen, and a preparation method thereof. The amino acid sequence of the vesicular stomatitis virus recombinant N protein antigen is represented by SEQ ID No.1; and the nucleotide sequence of a gene used for coding the vesicular stomatitis virus recombinant N protein antigen is represented by SEQ ID No.2. The vesicular stomatitis virus recombinant N protein antigen is used for preparing test strips used for rapid detection of vesicular stomatitis virus antibodies; specificity and sensitivity are high; detection is rapid; operation is simple; detection sample amount is small; detection cost is relatively low; and an excellent detection method is provided for vesicular stomatitis virus antibody detection and vesicular stomatitis epidemiological investigation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant N protein antigen of vesicular stomatitis virus and a preparation method thereof. Background technique [0002] Vesicular stomatitis (VS) is a major zoonotic disease caused by vesicular stomatitis virus (VSV) belonging to the genus Vesicularvirus in the Rhabdoviridae family. The World Organization for Animal Health (OIE) listed VS as a notifiable infectious disease. Currently, VSV is divided into two serotypes, namely, Indiana (IN type) and New Jersey (New Jersey, NJ type). The infection spectrum of VSV is very wide, and cattle, pigs, horses, sheep and other wild animals can be infected, and humans can also be infected with VSV, and have symptoms similar to acute febrile flu. After the onset of VSV infection in animals, the clinical symptoms are very similar to the symptoms of foot-and-mouth disease (FMD), and it is difficult to make a diff...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/544
CPCG01N33/544G01N33/56983
Inventor 孙洁杨俊兴廖立珊刘建利花群义吕建强张彩虹曾少灵阮周曦陶虹
Owner SHENZHEN AUDAQUE DATA TECH
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