Prokaryotic expression,purification and antigenicity analysis of the Cap protein of porcine circovirus type 2

YANG Yingying;LIU Yonggang;HAO Lisha;WANG Gang;TU Yabin;TONG Jie;TIAN Lihong;CAI Xuehui;State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences;College of Wildlife Resource,Northeast Forestry University;

PCV-2 ORF2;;Cap protein;;Escherichia coli;;optimized expression;;ImageJ software;;antigenicity

To obtain massive recombinant porcine circovirus type 2( PCV- 2) Cap proteins with good antigenicity and develop a test kit for detection of PCV- 2 antibody,a full- length PCV- 2 ORF2 gene was amplified by PCR,and then was cloned into p ET- 30 a to construct recombinant plasmid p ET- 30a- Cap. The recombinant plasmid was transformed into E. coli BL21( DE3). By using the SDS- PAGE analysis,as well as Image J and Graphpad prism 5 softwares,the prokaryotic expression conditions were optimized for the recombinant bacteria E. coli BL21( p ET- 30a- Cap),and then its antigenicity was identified by SDS- PAGE and Western- blot after the induced product was purified. The results showed that the optimized expression conditions of the recombinant Cap protein were as follows: when the OD600 of the recombinant bacteria reached 1. 0 to 1. 5,and IPTG was added in the final concentration of 0. 2 mmol / L,and the shaking culture was carried out in a thermostatic shaker at 37 ℃ and 175 r / min for 5 h. And then the recombinant Cap protein with high purity was obtained using purification techniques,and the protein had a good reactogenicity. The results indicate the recombinant Cap protein has good antigenicity,and can provide the diagnostic antigen for the development of indirect ELISA kit for detection of PCV- 2 antibody.