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Enterovirus 71 capsid protein 3 recombinant antigen and application thereof

A technology of recombinant antigen and capsid protein, applied in applications, antiviral agents, recombinant DNA technology, etc., can solve the problems of restricting gene expression, affecting application, difficult to high expression, etc., to reduce risk, improve sensitivity, and clarify immunity. The effect of diagnostic significance

Inactive Publication Date: 2012-08-08
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, the wild EV71-VP3 gene is difficult to achieve high expression in Escherichia coli, the main reason is that the wild type VP3 gene contains 46 rare codons of the Escherichia coli expression system, accounting for 18.9% of the entire VP3 amino acid, especially G, R The content of rare codons exceeds 50%, which limits the expression of its genes and directly affects its application in EV71-IgM ELISA detection reagents

Method used

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  • Enterovirus 71 capsid protein 3 recombinant antigen and application thereof
  • Enterovirus 71 capsid protein 3 recombinant antigen and application thereof
  • Enterovirus 71 capsid protein 3 recombinant antigen and application thereof

Examples

Experimental program
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Effect test

Embodiment 1EV7

[0022] Transformation and synthesis of embodiment 1EV71-VP3 gene

[0023] Through bioinformatics software, the amino acid sequence of the EV71-VP3 antigen was reverse-translated into a nucleotide sequence using the dominant codon of Escherichia coli, and the modified EV71-VP3 gene sequence is shown in SEQ ID NO: 1 in the sequence table; consider Up to the present domestic gene primer synthesis efficiency, the above-mentioned gene is divided into 4 sections A, B, C and D to be synthesized respectively, and the end of the four gene fragments has a matching sequence of 16 nucleotides. After further splicing into a complete EV71-VP3 gene sequence, each synthetic primer sequence is shown in Table 1, which is divided into 3 steps:

[0024] Step 1: Synthesis of A, B, C, D gene fragments

[0025] The synthesis of the above genes requires 3 rounds of annealing and extension PCR reactions. The first round of PCR reaction system consists of 25 μl of 2×PCR reaction solution from Biotech,...

Embodiment 2

[0033] Cloning, expression and labeling of embodiment 2EV71-VP3 modified gene

[0034] 1. Construction of EV71-VP3 antigen expression plasmid

[0035] 1.1 PCR product and expression vector pBET-28a double digestion

[0036] Take 30 μl of the gene product synthesized in Example 1 and the pBET-28a expression vector and put them in Eeppendorf centrifuge tubes respectively, add 4 μl of 10×buffer (D), 1 μl of BamHI (10u / μl) and EcoRI (12u / μl) respectively, Add sterilized distilled water to 40 μl, and place in a 37°C water bath for enzyme digestion overnight.

[0037]Agarose gel electrophoresis purification and recovery of enzyme-cleaved products: PCR product and carrier pET-32a are purified with 1.2% agarose gel after double digestion, and the specific method is according to "Molecular Cloning" (Science Press, second version) method. The purified gene is then recovered with a small amount of gel recovery kit produced by Shanghai Huashun Bioengineering Co., Ltd.: cut out the agar...

Embodiment 3

[0044] Example 3 Establishment and Clinical Evaluation of EV71-ELISA-IgM Antibody Detection Technology Based on Recombinant VP3 Antigen

[0045] 1. EV71-VP3 Antibody Detection Technology

[0046] Recombinant VP3 antigen-coated enzyme-linked plate (diluted to 2 μg / ml in PBS), 100 μl per well, discarded after overnight at 4 °C, washed 3 times with distilled water, patted dry, added 100 μl of 1% BSA to each well, room temperature for 2 hours, discarded. After adding 100 μl sample dilute solution to each well, add 10 μl serum of the sample to be tested, 37°C for 30 minutes, discard the solution, wash the plate 5 times with washing solution, discard the solution, pat dry, add horseradish peroxidase-labeled anti-human IgM (1:1000) 100μl, incubate at 37°C for 20min, discard the solution and wash the plate 5 times and pat dry. Add TMB color development solution: 50 μl each of A and B solutions, develop color at 37°C in the dark for 10 minutes, add 50 μl of stop solution to each well,...

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Abstract

The invention discloses an enterovirus 71 capsid protein 3 recombinant antigen belonging to the technical field of virus detection and an application thereof. The gene nucleotide sequence of the recombinant antigen is showed in a sequence table SEQ ID NO: 1. The gene splicing technology is adopted, and through the annealing extending polymerase chain reaction (PCR) technology, a VP3 gene for codon optimization escherichia coli is obtained, the optimization VP3 gene is efficiently expressed in escherichia coli, and a corresponding recombined EV71-VP3 gene is obtained through an affinity chromatography method in purification mode. The obtained EV71-VP3 antigen can have a specific immune response with serum of patients of hand-foot-and-mouth disease, and the recombinant antigen can improve detection sensitivity of hand-foot-and-mouth disease by combining with EV71-VP1 for use and can be used for clinical diagnosis of EV71 acute infection.

Description

technical field [0001] The invention belongs to the technical field of virus detection, and in particular relates to a recombinant antigen of enterovirus 71 capsid protein 3 and an application thereof. Background technique [0002] Enterovirus 71 (Enterovirus 71, EV71) infection can lead to hand, foot and mouth disease, herpetic angina, aseptic meningitis, encephalitis and polio-like paralytic diseases, etc., and severe cases can lead to death of patients. Hand, foot and mouth disease caused by EV71 virus is on the rise in my country in recent years. In 2008, about 50,000 people were infected and 120 people died [Ooi E E, Phoon M C, Ishak B, et al. Seroepidemiology of human enterovirus 71.Emerg Infect Dis. 2002;8:995-997.]. [0003] Early diagnosis of EV71 is of great significance to the prevention and treatment of later diseases. However, the "gold standard method" based on virus culture is difficult to be used clinically. Although RNA detection has been successfully deve...

Claims

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Application Information

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IPC IPC(8): C12N15/41C12N15/63C12N15/70C12N1/21G01N33/68A61K39/125A61P31/14
CPCY02A50/30
Inventor 修冰水张贺秋陈堃戴振华冯晓燕杨锡琴
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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