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E2 recombinant protein and application thereof

A recombinant protein and sequence listing technology, applied to recombinant protein E2 and its application fields, can solve problems such as high mutation, achieve the effects of high accuracy, high sensitivity, and avoid high mutation rate

Pending Publication Date: 2020-02-04
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

E2 protein has neutralizing activity, but has the characteristics of high mutation

Method used

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  • E2 recombinant protein and application thereof
  • E2 recombinant protein and application thereof
  • E2 recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Amplification of E2 protein sequence and construction of prokaryotic expression plasmid pET30a-E2:

[0028] Use the codon optimization software MaxCodonTM Optimization Program (V13) to optimize the amino acid sequence of the E2 protein, and use the whole gene to synthesize the recombinant E2 protein gene. The amino acid sequence list of the E2 recombinant protein is SEQ.ID.No.1, and the recombinant The nucleotide sequence list of the E2 protein gene is SEQ.ID.No.2. Insert the recombinant E2 protein gene into the expression vector pET30a through the restriction sites Nde I and Hind III, and add 4.5 μL of the recombinant E2 protein gene fragment, 0.5 μL of the pET-30a expression vector, and 5 μL of the DNA ligation kit were mixed thoroughly, and placed in a low-temperature ligation instrument at a temperature of 16°C for the ligation reaction for 30 minutes to obtain the ligation product, which was passed through a restriction The accuracy of the final expression vecto...

Embodiment 2

[0052] This embodiment is the application of the recombinant protein E2 described in Example 1, the recombinant protein E2 is used to detect the indirect ELISA kit of bovine viral diarrhea virus antibody, and the indirect ELISA kit includes a coated microplate, negative Control serum, positive control serum, enzyme-labeled secondary antibody, enzyme-labeled secondary antibody diluent, concentrated washing solution, sample diluent, color development solution and stop solution, the coated microtiter plate uses recombinant protein E2 as the coating antigen.

[0053] 1. Preparation of enzyme-labeled secondary antibody: Rabbit anti-bovine IgG (produced by Jackson ImmunoResearch, USA) was diluted 100 times with HRP conjugate stabilizer / diluent I (lettuce from Huzhou Yingchuang Biotechnology Co., Ltd.).

[0054] 2. Preparation of coating buffer, blocking solution, sample diluent, enzyme-labeled secondary antibody diluent, and washing solution:

[0055] Coating buffer is the 0.05M car...

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Abstract

The invention provides an E2 recombinant protein. The E2 recombinant protein has an amino acid sequence of a sequence table SEQ.ID.No.1, and the E2 recombinant protein has a base sequence of a sequence table SEQ.ID.No.2. The invention also provides application to an indirect ELISA kit for detecting bovine viral diarrhea virus antibodies. The E2 recombinant protein provided by the invention has high accuracy, not only is the neutrality of the protein ensured, but also the trouble of high mutation rate is avoided, and the recombinant protein is more beneficial to the development of a BVDV antibody detection technology. The E2 recombinant protein uses a traditional protein prokaryotic expression technology, the cost is low, the expression quantity is large, and after the successfully expressed E2 recombinant protein is subjected to urea gradient renaturation, the West blot detection reactionogenicity is strong. The E2 recombinant protein is applied to the indirect ELISA kit for detectingbovine viral diarrhea virus antibodies, and an established BVDV antibody ELISA detection method is high in sensitivity and good in specificity, is more beneficial to popularization and application ingrass-roots farmers, and is more beneficial to purification of BVDV in cattle herds in China.

Description

technical field [0001] The invention belongs to the technical field of gene recombination, and in particular relates to a recombinant protein E2 and its application. Background technique [0002] Bovine viral diarrhea / mucosal disease (BVDV / MD) is an important infection of pigs, cattle, sheep, camels and other wild animals caused by bovine viral diarrhea virus (BVDV). disease with high morbidity and low mortality. The clinical types include acute infections such as pneumonia, diarrhea, female abortion, stillbirth and teratosis, as well as mucosal diseases and persistent infections, which cause immunosuppression and cause secondary infections and other vaccine immune failures. Persistent infection (PI) is also a form of BVDV that persists in the natural environment, mainly caused by intrauterine infection in cows during the first trimester of pregnancy. At this time, the fetus' immune system is not yet mature, and cannot recognize the foreign non-cytopathic BVDV, so that the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C07K1/22C12N15/40C12N15/70G01N33/569
CPCC07K14/005C12N15/70G01N33/56983C12N2770/24322G01N2333/08G01N2469/20
Inventor 盛金良肖盛中李岩马旭升杨艳张彦红张哲陈创夫
Owner SHIHEZI UNIVERSITY
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