鸡补体受体2基因克隆、蛋白表达纯化及其多克隆抗体的制备和鉴定

doi:10.16431/j.cnki.1671-7236.2020.01.024

Abstract

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

CLC Number:

S831

KONG Zimeng, JIANG Bo, CAI Yunhong, HE Houjun, LI Yongqing, JIN Huan. Cloning,Protein Expression and Purification of Chicken Complement Receptor 2 Gene and Preparation and Identification of Its Polyclonal Antibody[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(1): 201-208.

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

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