猪圆环病毒2型ORF2基因的原核表达

Expression of PCV2-ORF2 gene in prokaryotic cells

猪圆环病毒是引起猪多系统衰竭综合征的重要病原,全病毒培养十分困难,避开培养病毒,得到大量的检测抗原,对诊断本病有着重要的意义。本研究对疑似猪圆环病毒2型感染猪脾脏提取病毒DNA,进行套式PCR扩增获得了猪圆环病毒2型(PCV2)ORF2基因序列,并将扩增片段定向插入质粒载体pPRO-HTb,转化BL21大肠杆菌后,构建了表达猪圆环病毒2型ORF2蛋白的重组原核表达系统。经IPTG诱导,对表达产物经SDS-PAGE和Western-blot分析,表明ORF2基因在大肠杆菌中得到了表达,表达产物以包涵体形式存在于菌体中,通过提取包涵体,获得了初步纯化的ORF2蛋白,为进一步利用PCV2ORF2蛋白抗原进行PCV血清学检测提供了基础。

Porcine circovirus to avoid culturing PCV, therefore （PCV） was the important pathogen that caused PMWS, and it was very difficult it had the significance to diagnose PCV. This article studied on extracting the virus DNA from the infected pig spleen samples of the suspected PCV2, obtaining quantity detection antigen without culturing PCV, and then PCV2-ORF2 gene was amplified by polymerase chain reaction （PCR）, and at last the PCR products were directional inserted, into plasmid vector pPRO-HTb. The result of pPRO-HTb-ORF2 was transformed into E. coli BL21 cells to generate recombinant prokaryotic expression system of PCV2-ORF2 protein expressing. It was induced by IPTG, the results of SDS-PAGE and Western-blot showed that the ORF2 gene had been expressed, the expression products were inclusion body form which was extracted, obtaining preliminary purified ORF2 protein. This research provided foundation for the further research of PCV2ORF2 protein antigen in order to provide the method for detecting the serology.