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Recombinant syncytial virus protein and application thereof

A technology of syncytial virus and recombination, which is applied in the fields of genetic engineering, vaccine development and diagnostic reagents, can solve problems such as failure to separate successfully, and achieve high sensitivity, easy operation and strong specificity

Inactive Publication Date: 2015-05-06
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is another open reading frame in the M gene and the M2 gene, which encode proteins of 75 amino acids and 990 amino acids respectively, so it is assumed that there may be the 11th and 12th viral proteins, but they have not been isolated so far

Method used

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  • Recombinant syncytial virus protein and application thereof
  • Recombinant syncytial virus protein and application thereof
  • Recombinant syncytial virus protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of syncytial virus protein

[0050] 1.1 Syncytial virus protein epitope screening and target gene cloning

[0051] The strong epitope of the syncytial virus protein is screened out by computer analysis of the entire amino acid sequence of the syncytial virus, and the recombinant protein contains the 628th to the 999th position of the N-terminal in the RSV genome from the N-terminal to the C-terminal in sequence nucleotides, and 515 amino acids from 1140th to 2312th nucleotides. Wherein the DNA sequence of the above recombinant protein is shown in SEQ ID No.2.

[0052] According to the cDNA sequence of the target peptide in the syncytial virus RSV genome and the restriction site on the plasmid, two pairs of PCR primers (P1, P2) and (P3, P4) were designed. p1 and p2 are used to amplify nucleotides 628 to 999 in RSV, and p3 and p4 are used to amplify nucleotides 1140 to 2312 in RSV; primers p1 and p4 have BamH I and Xho respectively The enzyme ...

Embodiment 2

[0073] Example 2 Preparation and Performance Test of Syncytial Virus Antibody Colloidal Gold Detection Kit

[0074] 2.1 Preparation of syncytial virus antibody colloidal gold detection kit

[0075] The recombinant rRSV-58 protein prepared above was used as the labeled antigen of the kit, and the RSV antibody was determined by the colloidal gold method. The kit was developed and used as follows:

[0076] (1) The principle of the kit: According to the principle of the double-antigen sandwich method, the present invention uses the rRSV-58 recombinant antigen to coat the nitrocellulose membrane, and the colloidal gold-labeled genetically engineered recombinant syncytial virus (rRSV-58) antigen is used as a tracer. When using, add the serum to be tested. If the sample contains anti-RSV specific antibody, it can combine with the rRSV-58 recombinant antigen on the surface of the membrane to form a complex. The complex combines with colloidal gold-labeled rRSV-58 antigen to appea...

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Abstract

The invention discloses a recombinant syncytial virus protein rRSV-58 and genes rRSV-58 for expressing the protein. The preparation method comprises the following steps: by taking a syncytial virus culture as a template, amplifying nucleotide fragments from the 628th site to the 999th site in the syncytial virus genome by virtue of primers P1 and P2, and amplifying nucleotide fragments from the 1140th site to the 2312th site by virtue of primers P3 and P4; by taking two fragments obtained in the first round of PCR amplification as a template, adding the primers P1 and P4, and amplifying the rRSV-58 fragments, thereby obtaining the series target gene recombinant fragments; inserting the recombinant fragments into pET-28a, performing transfection to Escherichia coli BL21(DE3), and inducing the expression, thereby obtaining the recombinant syncytial virus protein. A colloidal gold kit prepared by taking the recombinant syncytial virus protein as an antigen is used for detecting the syncytial virus infection and is high in specificity and high in sensitivity. Compared with the test of abroad exported kits, the kit disclosed by the invention has the advantage that the total detection coincidence rate (49+206) / 260 is equal to 98.1 percent.

Description

technical field [0001] The invention relates to the fields of genetic engineering, vaccine development, diagnostic reagents and the like, in particular to a recombined syncytic virus protein, a gene expressing the protein and an application thereof. Background technique [0002] Respiratory syncytial virus (RSV) is abbreviated as syncytial virus, also known as fusion virus. Virus particles are round and vary in size, with a diameter of 100-300nm. The core is a single-stranded RNA, with a nucleocapsid outside, and the outermost layer is an envelope with spiny protrusions. Because it does not contain hemagglutinin, it has no hemagglutination effect. The virus can grow in human respiratory cells and cell cultures such as human kidney and monkey kidney, and cause lesions. There is only one serotype, which can be divided into 4 subtypes. Weak resistance, not resistant to ether, acid, heat and freeze-thaw, pH below 3 and five minutes at 55 ℃ can be inactivated. [0003] epide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/45C12N15/10C07K14/135C12N15/70C12N1/21G01N33/68G01N33/569
Inventor 于庭包洪吴纯常静吴丽霞张晓燕刘树业
Owner JILIN UNIV
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