Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof

A technology of bursal disease and chicken infectivity, applied in the field of cDNA, achieves the effect of low price, preventing loss, and simple cultivation

Inactive Publication Date: 2007-07-04
HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The first technical problem to be solved in the present invention is to overcome the deficiencies of the prior art, carry out molecular transformation and modification of chicken infectious bursal disease virus VP2 cDNA, and provide a kind of chicken infectious bursal disease virus VP2 cDNA suitable for high-efficiency expression in yeast cells Virus artificial VP2 cDNA

Method used

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  • Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof
  • Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof
  • Chicken infectivity bursa of Fabricius virus VP2 cDNA, its expression vector, expressed recombinant protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0050] [Example 1] Synthesis and cloning of recombinant chicken infectious bursal disease virus VP2 cDNA

[0051] 1. Extraction of viral RNA The bursa of SPF chickens infected with chicken infectious bursal disease virus supervirulent Gx (vvIBDV-Gx) strain was ground with a grinder. Take 200 μl of disease material and add TE (PH8.0) to 500 μl, add 5 μl of proteinase K and 50 μl of 10% SDS, and bathe in water at 56°C for 3 hours. Add an equal volume of phenol / chloroform for extraction three times, and an equal volume of chloroform for extraction once. Remove the supernatant to another 1.5ml centrifuge tube, add 1 / 10 volume of NaAc (3M, pH 5.2), and equal volume of isopropanol, and precipitate at -20°C for 2 hours. Centrifuge at 12000 rpm for 15 minutes at 4°C and wash once with 75% ethanol. Dry in vacuo and redissolve RNA in RNase-free deionized water.

[0052] 2. Obtaining the RF243 gene of the genome fragment A. Perform RT-PCR on the extracted viral RNA, and use random pri...

Embodiment 2

[0059] [Example 2] Construction and identification of recombinant yeast vector

[0060] 1. Construction of recombinant yeast vector The optimized VP2 gene and yeast vector were digested with Xhol I and Xba I respectively, and recovered with an agarose gel recovery kit. The vector is dephosphorylated with CIAP. T for foreign fragments and vectors 4 DNA ligase performs the ligation reaction. See Figure 1 for the construction map. The ligation product was transformed into E. coli competent cells.

[0061] 2. Extraction and Identification of Recombinant Plasmids Add corresponding antibiotics to low-salt LB according to the concentration of action (screen pPICZαVP2 plasmid antibiotics and select Zeocin TM ), pick a single colony and inoculate LB. Plasmids were extracted by alkaline lysis. Identify by PCR method, PCR identification primer and condition are the same as embodiment 1, PCR amplifies 1.3Kb band (Fig. According to the results of gene sequencing, the gene insertion ...

Embodiment 3

[0062] [Example 3] Transformation of recombinant yeast vector, detection and screening of recombinant yeast strains

[0063] 1. Transforming yeast competent cells Linearize the recombinant yeast vector with Sac I, transform yeast competent cells, and follow the guidance method of Pichia pastoris electroporation from Invitrogen Company. Yeast after transformation is coated with YPDS plate containing antibiotic (select the recombinant yeast antibiotic that pPICZαVP2 transforms to select Zeocin TM ).

[0064] 2. Screening and identification of yeast recombinants Pick 10 single yeast colonies, inoculate 5ml of YPD containing antibiotics, culture overnight, centrifuge, discard the culture medium, and wash once with 5ml of sterilized deionized water. Apply proteinase K and SDS to digest the yeast overnight, centrifuge, transfer the supernatant to another 1.5ml centrifuge tube, extract 3 times with phenol and chloroform, and precipitate with absolute ethanol. AOX1 5` and 3` primers...

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Abstract

The invention discloses a novel chicken infectious bursal disease virusVP 2c DNA (SEQ ID NO: 1), construction of its expression carrier, expressed recombined VP2 protein and the application of said recombined protein in preparing subunit genetic engineering vaccine against chicken infection bursal disease virus. The chicken infectious bursal disease virusVP 2c DNA can be highly expressed in yeast cell, and expressed recombined protein possesses biological activity and immunogenicity of the chicken infectious bursal disease virus natural protein . The expressed recombined protein can be produced into vaccine, and it is demonstrated through immunity chicken test that: the protein subunit vaccine can effectively induce body to generate spcial humoral immune response, make immunity chicken get 90% protection from deadly attack of highly pathogenic vv IBDV, and effectively prevent virus proliferation in the body.

Description

technical field [0001] The present invention relates to a cDNA, in particular to an artificially transformed chicken infectious bursal disease virus VP2 cDNA, the construction of its expression vector, the expressed recombinant VP2 protein and the recombinant protein used in the preparation of chicken infectious bursal disease virus VP2 cDNA application in disease vaccines. Background technique [0002] Chicken infectious bursal disease is an important viral disease that can cause immune suppression in chickens and seriously endangers the chicken industry in the world. Traditional vaccines have played a very important role in the history of preventing and controlling the disease. With the new changes in the disease in recent years-the appearance of serotype variants and super-virulent strains, the prevention and control of the disease has become more and more important. The more difficult it is, the development speed of traditional vaccines has been somewhat difficult to ke...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33C12N15/81C07K14/005C12P21/02C12N1/19A61K39/12C12Q1/68G01N33/53C12R1/84
Inventor 王笑梅高宏雷付朝阳高玉龙
Owner HARBIN VETERINARY RES INST CHINESE ACADEMY OF AGRI SCI
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