China Animal Husbandry and Veterinary Medicine ›› 2020, Vol. 47 ›› Issue (1): 201-208.doi: 10.16431/j.cnki.1671-7236.2020.01.024

• Preventive Veterinary Medicine • Previous Articles     Next Articles

Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody

KONG Zimeng1,2, JIANG Bo2, CAI Yunhong2, HE Houjun1, LI Yongqing2, JIN Huan2   

  1. 1. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang 330045, China;
    2. Research Center for Infectious Diseases in Livestock and Poultry, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
  • Received:2019-06-24 Online:2020-01-20 Published:2020-01-17

Abstract: In order to identify chicken complement receptor 2 (ChCR2) gene,we designed specific primers and amplified ChCR2 gene by RT-PCR.The prokaryotic expression vectors pGEX-6P-1-ChCR2-△TM and pET-32a-ChCR2-△TM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM were constructed by homologous arm ligation.The two prokaryotic plasmids were transferred into E.coli expression system to express GST-ChCR2-△TM and His-ChCR2-△TM proteins.The polyclonal antibody was prepared by immunizing BALB/c mice with GST-ChCR2-△TM protein.The antibody titer was detected by ELISA.Indirect immunofluorescence test and Western blotting were used to identify the specific binding of antigen and antibody.The results showed that a ChCR2 gene with a size of 1 113 bp was amplified.The prokaryotic expression vectors pGEX-6P-1-ChCR2-ΔTM and pET-32a-ChCR2-ΔTM and the eukaryotic expression vector pCMV-HA-ChCR2-△TM with the transmembrane region removed were successfully constructed.The GST-ChCR2-△TM protein was soluble at low temperature (16 ℃),while His-ChCR2-△TM protein was in the form of inclusion body.The serum titer of BALB/c mice immunized with GST-ChCR2-△TM protein was 1:512 000 detected by ELISA.The results of indirect immunofluorescence assay and Western blotting showed that the polyclonal antibody against GST-ChCR2-△TM protein could specifically bind to ChCR2 protein.The results of this study provided a reference for further identification of ChCR2 gene and related immunological research of chicken and even poultry.

Key words: ChCR2 gene; expression and purification of protein; polyclonal antibody; ELISA; indirect immunofluorescence assay; Western blotting

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