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Recombinant rubella virus protein and application

A rubella virus and protein technology, applied in the field of genetic engineering, can solve the problems of low protein antigen purity and reduced detection specificity, and achieve the effects of high sensitivity, strong specificity and strong immunogenicity

Inactive Publication Date: 2012-07-25
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a recombinant rubella virus protein produced by genetic engineering to avoid problems such as false positives caused by protein antigens with low purity or poor specificity and lower detection specificity.

Method used

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  • Recombinant rubella virus protein and application
  • Recombinant rubella virus protein and application
  • Recombinant rubella virus protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of recombinant rubella virus protein

[0025] 1.1 Rubella virus protein epitope screening and target gene cloning

[0026] The dominant antigenic epitope of rubella virus protein was screened by computer analysis of the entire amino acid sequence of rubella virus, including 180 amino acids from N-terminal 147 to 326 of Rubella virus E1 and 165 amino acids from N-terminal 5 to 169 of C amino acids. The DNA sequence of the recombinant protein is shown in SEQ ID No.1, wherein the E1 target peptide DNA sequence is from the 439th to the 978th ​​nucleotide of E1, and the C target peptide DNA sequence is from the 13th to the C 507 nucleotides.

[0027] Design two pairs of PCR primers (P1, P2) and (P3, P4) according to the DNA sequence of the target fragment, that is, the cDNA sequence of the target peptide on E1 and C of RV and the restriction site on the plasmid. P1 and P2 are used to amplify the 439th to 978th ​​nucleotides of E1, and P3 and P4 are u...

Embodiment 2

[0046] Example 2 Preparation and Performance Test of Rubella Virus IgG Antibody Detection Kit

[0047] 2.1 Preparation of human herpes simplex virus IgG antibody detection kit

[0048] The recombinant RV protein prepared in Example 1 was used as the labeled antigen of the kit, and the RV IgG antibody was determined by the colloidal gold method.

[0049] The kit was developed and used as follows:

[0050] 1) The principle of the kit: This product adopts the principle of immune capture method. The nitrocellulose membrane is coated with anti-human IgG antibody, and the colloidal gold-labeled genetically engineered recombinant rubella virus (RV) antigen is used as a tracer. When using, add the serum to be tested. If the sample contains anti-RV specific IgG antibody, it can combine with the anti-human IgG antibody on the membrane surface to form a complex, which will combine with colloidal gold-labeled RV antigen to present a purple band. .

[0051] 2) Kit performance optimizati...

Embodiment 3

[0101] Example 3 Performance Verification of Rubella Virus IgG Antibody Detection Kit (ELISA)

[0102] The recombinant RV protein prepared in Example 1 was used as the substrate for the preparation of enzyme-labeled antigen in the kit, and the RV IgG antibody was determined by ELISA method.

[0103] 3.1 The principle and components of the kit are as follows:

[0104] 1) The principle of the kit: This strain uses a microwell plate coated with anti-human IgG antibody, horseradish peroxidase (HRP) marks the genetically engineered recombinant RV antigen as a tracer, TMB color development system, and applies the principle of capture method Detection of anti-rubella virus IgG antibodies in human serum or plasma.

[0105] 2) The main components of the kit:

[0106] Pre-coated plate, enzyme conjugate, negative control, positive control, concentrated washing solution (20×), substrate solution A, substrate solution B, stop solution.

[0107] 3.2 Kit Performance Verification

[0108]...

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Abstract

The invention discloses a recombinant rubella virus protein, which comprises dominant epitopes E1 fragment and C fragment of the rubella virus protein RV, and is used as a rubella virus antibod IgG immune detection kit for preparing antigen. The invention has the advantages of strong pecificity, high sensitivity and the like compared with the similar kit on market, and better meets the requirements of clinical diagnosis of rubella virus infection. The antigen has high pecificity, strong immunogenicity and complete antigenic determinants as much as possible, and the invention has practical application value in the field of RV vaccine development.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant rubella virus protein and its application. Background technique [0002] Rubella virus is the only member of the genus Rubellavirus in the Togaviridae family. The spherical rubella virus (Rubella virus, RuV or RV) is the pathogen of rubella, and humans are its only host. Rubella is an acute respiratory infectious disease with fever, respiratory tract sputum and rash as the main symptoms. Rubella virus infection in pregnant women, especially in early pregnancy, can lead to fetal congenital rubella syndrome (CRS). Rubella and CRS occur all over the world, and the infection rate is high in Asia. After pregnant women suffer from rubella, especially within the first 3 to 4 months of pregnancy, rubella virus can invade the placenta and infect the fetus, causing congenital rubella as high as 30% to 35%. In addition to stillbirth, miscarriage and premature delivery, pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/19C12N15/40C12N15/70G01N33/569A61K39/20A61P31/14
Inventor 包洪于庭金玉芬常静肖霞高艺航
Owner JILIN UNIV
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