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Duck plague virus (DPV) UL13 intercepted recombinant protein and polyclonal antibody, preparation method and application thereof

A technology of polyclonal antibody and duck plague virus, which is applied in the field of genetic engineering, can solve the problems of complex whole virus antigen purification methods, affecting the accuracy of test results, poor stability, etc., and achieves good product purity and stability, low production cost, highly specific effect

Active Publication Date: 2014-02-05
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the previous ELISA method used the whole virus of DPV as the coating antigen, and the purification method of the whole virus antigen is complicated, and the purity is limited and the stability is poor, which affects the accuracy of the detection results.

Method used

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  • Duck plague virus (DPV) UL13 intercepted recombinant protein and polyclonal antibody, preparation method and application thereof
  • Duck plague virus (DPV) UL13 intercepted recombinant protein and polyclonal antibody, preparation method and application thereof
  • Duck plague virus (DPV) UL13 intercepted recombinant protein and polyclonal antibody, preparation method and application thereof

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Embodiment 1

[0033] Cloning of embodiment 1, DPV UL13 truncated gene

[0034] 1. Primer design and synthesis

[0035] According to the nucleotide sequence corresponding to amino acids 163 to 386 in the UL13 gene coding protein sequence (SEQ ID No.1, named pUL13), the following primers were designed with Oligo6.0 software: Upstream primer: 5'- ggatcc ctggtggctacggagag-3' (SEQ ID NO.3), the underlined part is the BamH I site, this primer is an optimized primer obtained by replacing the original C base with the merged T base; downstream primer: 5'- aagctt ccaagggcgtatatgtc-3' (SEQ ID NO.4), the underlined part is the Hind III site. The primers were synthesized, dissolved in sterilized deionized water to make a solution with a concentration of 10 pmol / L, and stored at -20°C for future use.

[0036] 2. Extraction of DPV genomic DNA

[0037] a. Preparation of duck embryo fibroblasts (DEF): take 10-day-old healthy duck embryos, disinfect the surface of the eggshell with 5% (v / v) iodine and 7...

Embodiment 2

[0043] Embodiment 2, the construction of recombinant prokaryotic expression plasmid

[0044] The pUC19-UL13 plasmid and the pET32c(+) vector were digested with BamH I and Hind III respectively, and the DPV UL13 truncated gene and the pET32c(+) linear fragment were recovered, and ligated overnight at 16°C under the action of DNA ligase. Transform the cloned host strain E.coli DH5α competent cells with the ligation product, inoculate the obtained transformation product on the LB agar plate containing ampicillin (Amp), cultivate overnight at 37°C, pick a single white colony and inoculate it on the LB liquid containing Amp the next day In the culture medium, shake culture in a water bath at 37°C for 18 hours, extract the plasmid, and carry out PCR and enzyme digestion identification. The positive recombinant plasmid obtained is the recombinant prokaryotic expression plasmid, named pET32c-UL13. For enzyme digestion results, see image 3 , it can be seen that the recombinant prokar...

Embodiment 3

[0045] Embodiment 3, the construction of recombinant prokaryotic expression engineering bacteria

[0046] Pick the cloned engineered bacteria DH5α containing the recombinant plasmid pET32c-UL13, streak and inoculate it on the LB agar plate containing Amp, culture it overnight at 37°C, take a single colony and inoculate it in the LB liquid medium the next day, and culture it vigorously for 10-16 hours , extract the plasmid, transform and express the host strain E.coli BL21 (DE3) competent cells, inoculate the obtained transformation product on the LB agar plate containing Amp, cultivate overnight at 37°C, screen positive clones the next day, and obtain the recombinant plasmid pET32c - UL13 expression engineering bacteria BL21 (DE3).

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Abstract

The invention discloses a duck plague virus (DPV) UL13 intercepted recombinant protein of which an amino acid sequence is shown as SEQ ID No.1, an encoding gene, a recombinant expression vector and engineering bacteria of the intercepted recombinant protein, as well as a method for preparing the intercepted recombinant protein by utilizing the engineering bacteria. The method is simple and convenient to operate, low in cost and suitable for industrial large-scale production. The product standardization can be realized, and results in different labs are compared. The obtained intercepted recombinant protein has high immunoreactivity, can be specifically bound to a DPV antibody, can serve as a detection antigen for detecting the DPV antibody, and is high in detection accuracy, high in specificity, free of cross reaction and free of toxic danger. A polyclonal antibody is successfully obtained for an antigen immune animal through the DPV UL13 intercepted recombinant protein, can serve as a detection antibody for detecting the DPV, and has the advantages of high detection specificity, no cross reaction and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a viral truncated recombinant protein and a preparation method thereof, a polyclonal antibody obtained by immunizing animals with the truncated recombinant protein as an antigen, and the truncated recombinant protein and its polyclonal antibody application. Background technique [0002] Duck plague (Duck Plague, DP) is an acute, febrile, contact-infected septicemia infectious disease of waterfowl such as ducks, geese, and swans caused by Duck plague virus (DPV) of the Herpesviridae family. It can lead to a decrease in egg production and death of commercial waterfowl, and it also has different lethality rates for wild waterfowl. In recent years, with the intensive and large-scale development of the duck industry, DP, as one of the most serious contagious diseases threatening the duck industry, has caused huge economic losses. [0003] Clinical and laboratory tests have...

Claims

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Application Information

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IPC IPC(8): C07K14/03C12N15/38C12N15/70C12N1/21C07K16/08G01N33/569G01N33/68C12R1/19
CPCC07K14/005C07K16/085C12N2710/16022G01N33/56994G01N2333/03G01N2469/20
Inventor 汪铭书胡溪霞程安春陈孝跃贾仁勇
Owner SICHUAN AGRI UNIV
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