Recombinant rubella virus protein and applications thereof

A technology of rubella virus and protein, applied in the field of rubella virus protein and its application, can solve the problems of false positive, poor specificity, and lower detection specificity, and achieve strong immunogenicity, high specificity, and strong specificity

Active Publication Date: 2014-02-05
英诺特(唐山)生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the RV protein antigens used in detection kits are derived from virus culture or recombinantly expressed single protein fragments, or due to low purity and poor specificity of protein antigens, false positives are caused and the detection specificity is reduced.
Although the preparation of detection reagents by combining multiple single fragments can avoid the above problems, it will inevitably require multiple extraction and purification processes, and the purification technology requirements for small molecular proteins or peptides are higher

Method used

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  • Recombinant rubella virus protein and applications thereof
  • Recombinant rubella virus protein and applications thereof
  • Recombinant rubella virus protein and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of recombinant rubella virus protein

[0043] 1.1 Rubella virus protein epitope screening and target gene cloning

[0044] The strong antigenic epitope of rubella virus protein was screened by computer analysis of the entire amino acid sequence of rubella virus, including 152 amino acids from N-terminal 175 to 326 of Rubella virus E1, and 104 amino acids from N-terminal 5 to 108 of C amino acids. The DNA sequence of the recombinant protein is shown in SEQ ID No.1. Among them, the DNA sequence of the E1 target peptide is from the 523rd to the 978th ​​nucleotide of E1, and the DNA sequence of the C target peptide is from the 13th to the 324th nucleotide of C.

[0045] 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 523rd to 978th ​​nucleotides of E1...

Embodiment 2

[0064] Example 2 Preparation and Performance Test of Rubella Virus IgG Antibody Detection Kit

[0065] 2.1 Preparation of human rubella virus IgG antibody detection kit

[0066] 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.

[0067] The kit was developed and used as follows:

[0068] (1) Principle of the kit

[0069] This product adopts the principle of immunological indirect method, uses anti-human IgG antibody (Beijing Baian Tiandi Pharmaceutical Technology Co., Ltd.) to coat nitrocellulose membrane, and colloidal gold-labeled genetically engineered recombinant rubella virus (RV) antigen 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 ...

Embodiment 3

[0124] Example 3 Performance Verification of Rubella Virus IgG Antibody Detection Kit (ELISA)

[0125] 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.

[0126] 3.1 The principle and components of the kit are as follows:

[0127] 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 indirect method Detection of anti-rubella virus IgG antibodies in human serum or plasma.

[0128] 2) The main components of the kit:

[0129] Pre-coated plate, enzyme conjugate, negative control, positive control, concentrated washing solution (20×), substrate solution A, substrate solution B, stop solution.

[0130] 3.2 Kit Performance Verification

[0131...

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Abstract

The invention provides a rubella virus protein and also provides applications of the rubella virus protein in preparing a detection kit. Compared with similar kits in the market, a diagnostic kit prepared by adopting the rubella virus protein provided by the invention has the advantages of strong specificity, high sensitivity and the like, thereby, the requirement of the clinical diagnosis of rubella virus infection is well met.

Description

technical field [0001] The invention relates to the fields of genetic engineering, vaccine development, diagnostic reagents and the like, in particular to a 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) 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, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/40C12N15/70C07K14/19C12N1/21G01N33/68G01N33/569C12R1/19
Inventor 张秀杰张晓刚陈廷友
Owner 英诺特(唐山)生物技术有限公司
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