河南农业科学Issue(10)：123-126,140,5.
猪圆环病毒Ⅱ型河南地方株 ORF3的克隆及原核表达
Cloning and Prokaryotic Expression of ORF3 of Porcine Circovirus Type Ⅱ from Henan Strain
摘要
Abstract
The research aimed to obtain the recombinant ORF3 protein of porcine circovirus typeⅡ(PCVⅡ)to further research the characteristics and functions of ORF3 protein.According to the genomic sequence of PCV Ⅱ from GenBank,a pair of specific primers were designed.The ORF3 of Henan strain was amplified from pathological samples suspected PCVⅡ infection by PCR.Then the gene was cloned into prokaryotic expression vector pET-32a for expression in E. coli BL21(DE3).The expression product was purified by Ni+ NTA and refolded by dialysis.Japan white rabbits were immunized with the refolded recombinant ORF3 protein to generate polyclonal antiserum.The titer and specificity of the antiserum were detected respectively by ELISA and Western blot.The ORF3 of PCV Ⅱ,which was 315 bp in length,was amplified by PCR. Restriction enzyme digestion and sequencing proved that recombinant plasmid pET32a-ORF3 was constructed correctly.SDS-PAGE analysis showed that the ORF3 gene could express in E.coli BL21(DE3)and the expression prudct of ORF3 was about 30 ku,mainly in form of inclusion bodies.The purified recobinant ORF3 protein had good antigenicity.The specificty of polyclonal antiserum was high,and the titer could be above 1 ∶ 12 800.The ORF3 of PCV Ⅱ from Henan strain was successfully cloned and prokaryotic expression,which layed foundation for further research on the characteristics and functions of ORF3 protein.关键词
猪圆环病毒Ⅱ型/ORF3/原核表达/多抗血清Key words
porcine circovirus typeⅡ/ORF3/prokaryotic expression/polyclonal antiserum分类
农业科技引用本文复制引用
郑鸣,李华玮,边传周,王永芬,王老七..猪圆环病毒Ⅱ型河南地方株 ORF3的克隆及原核表达[J].河南农业科学,2014,(10):123-126,140,5.基金项目
河南省高等学校青年骨干教师资助计划项目 （）
