Duck hepatitis virus polyprotein antigen sense region DHAV-Mag genetic recombination protein and preparation method and application thereof

A duck hepatitis virus and polyprotein technology, applied in the field of biomedicine, can solve the problem of whether the capsid protein is completely cleaved and the quantity cannot be confirmed, and achieves the effects of good reactogenicity, low preparation cost and strong specificity

Inactive Publication Date: 2016-02-17
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Although the full gene sequence of duck hepatitis virus has been published, bioinformatics analysis speculates that the structure and function of its genome may be similar to other picornaviruses, but whether its capsid protein is completely cleaved, whether there is L protein, the number of 2A, etc. It cannot be confirmed yet; although there have been reports on the expression of some proteins, the information of each gene and protein is based on bioinformatics analysis rather than experimental confirmation. Therefore, how to obtain accurate target genes and proteins in a targeted manner becomes a It is an urgent problem to be solved in the study of hepatitis virus; and the in vitro expression of genes with good immunogenicity, and the research on the role of in vitro expressed proteins in detection and immune protection have become a research hotspot for those skilled in the art

Method used

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  • Duck hepatitis virus polyprotein antigen sense region DHAV-Mag genetic recombination protein and preparation method and application thereof
  • Duck hepatitis virus polyprotein antigen sense region DHAV-Mag genetic recombination protein and preparation method and application thereof
  • Duck hepatitis virus polyprotein antigen sense region DHAV-Mag genetic recombination protein and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0054] A duck hepatitis virus polyprotein antigen-dense region DHAV-MAg gene recombinant protein, its amino acid sequence is shown in SEQ ID NO: 1, and its DNA sequence is shown in SEQ ID NO: 2.

[0055] A method for preparing recombinant protein of duck hepatitis virus polyprotein antigen dense region DHAV-MAg gene, comprising the steps of:

[0056] Step 1: DHAV-MAg gene DNA fragment cloning in the polyprotein antigen-dense region of duck hepatitis virus: analysis and determination of the DHAV-MAg gene and restriction sites in the polyprotein antigen-dense region of duck hepatitis virus, according to Genbank accession number JQ316452.1 The whole gene sequence of DHAV-1X strain, the design of specific primers to amplify the DHAV-MAg gene DNA fragment in the polyprotein antigen-dense region of duck hepatitis virus, and the addition of BamHI and XhoI restriction enzymes to the 5' ends of the upstream and downstream primers site, extract duck hepatitis virus RNA, use specific pri...

Embodiment 2

[0103] The expression condition optimization of embodiment 2DHAV-MAg gene recombinant protein

[0104] experimental method:

[0105] 1. Optimization of IPTG concentration

[0106] Inoculate the expressing bacteria into four LB / Amp medium test tubes at a ratio of 1:100, and culture at 37°C until OD 600nm At 0.6, add IPTG to the final concentration: 0mmol / L, 0.4mmol / L, 0.8mmol / L and 1.2mmol / L to induce culture for about 4h, sample treatment, SDS-PAGE electrophoresis.

[0107] 2. Optimization of induction time

[0108] Inoculate the expressing bacteria into 6 LB / Amp medium test tubes according to the ratio of 1:100, and wait until OD 600nm After reaching 0.6, add the optimal IPTG concentration, induce 4h, 6h, 8h, 10h, 12h and no induction respectively, the treatment is the same as above.

[0109] 3. Optimization of induction temperature

[0110] Inoculate the expressing bacteria into 3 LB / Amp medium test tubes at a ratio of 1:100, and wait until OD 600nm After reaching 0.6,...

Embodiment 3

[0113] Example 3 Activity Analysis of DHAV-MAg Gene Recombinant Protein and Preparation, Characteristics and Application of Hyperimmune Serum

[0114] experimental method:

[0115] 1. Preparation of rabbit anti-DHAV-MAg gene recombinant protein hyperimmune serum

[0116] Immunogen preparation: prokaryotic expression of DHAV-MAg gene recombinant protein and purification of DHAV-MAg gene recombinant protein with high purity, stored at -20°C for future use.

[0117] Animal immunization protocol:

[0118] Select young healthy rabbits with appropriate body weight and carry out immunization according to Table 1.

[0119] Table 1 The immune program for the preparation of rabbit anti-DHAV-MAg gene recombinant protein hyperimmune serum

[0120]

[0121] Serum collection and determination of rabbit antiserum titer: 7 days after the last immunization, blood was collected, kept at 37°C for 2 hours, and refrigerated overnight at 4°C after coagulation. Serum was collected, tested for...

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Abstract

The invention discloses a duck hepatitis virus polyprotein antigen sense region DHAV-Mag genetic recombination protein and a preparation method and application thereof, and belongs to the field of biological medicine. The preparation method of the DHAV-Mag genetic recombination protein comprises the following steps of a, DHAV-Mag DNA fragment cloning and expression vector construction; b, DHAV-Mag genetic recombination protein expression; c, DHAV-Mag genetic recombination protein purification. The DHAV-Mag genetic recombination protein is used for successfully obtaining a polyclonal antibody for an antigen immune animal, preliminary study is performed on detection and immune protection effect of the DHAV-Mag genetic recombination protein and the polyclone antibody, and a new approach is provided for detection and prevention of the duck hepatitis virus.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a preparation method and application of a DHAV-MAg gene recombinant protein in a polyprotein antigen-dense region of duck hepatitis virus and a polyclonal antibody. Background technique [0002] Duck viral hepatitis (DVH) caused by duck hepatitis virus (DHV) is one of the most serious infectious diseases that endanger the duck industry. The disease mainly affects ducklings under 4 weeks old. The clinical symptoms mainly manifested as convulsions, opisthotonus and other neurological symptoms, commonly known as "back neck disease". Autopsy showed hepatomegaly and surface bleeding spots of different sizes. Once the disease breaks out, the mortality rate is extremely high, which brings serious economic losses to the global duck industry. There are at least three kinds of viruses that cause DVH, but the current outbreak is duck hepatitis A virus type 1 (DHAV-1), which correspon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/10C12N15/41C12N15/70G01N33/569A61K39/125A61P31/14
CPCA61K39/00C07K14/005C12N2770/32422C12N2770/32434G01N33/56983G01N2469/20
Inventor 汪铭书程安春齐晓燕杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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