猪圆环病毒2型ORF2基因及片段的原核表达

目的表达猪圆环病毒2型ORF2抗原,为研制猪圆环病毒2型血清学诊断方法以及生物制品生物安全性检验,提供技术支持。方法根据猪圆环病毒2型(PCV2)LC株序列,设计合成5对引物,采用PCR方法从全序列重组质粒PCV2-LC中扩增出了ORF2基因和4个不同ORF2基因片段,分别将其克隆到原核表达载体PET-32a上,再分别将各个重组表达质粒转化到大肠杆菌BL21中,用终浓度1 mmol/LIPTG诱导。结果表达产物以包涵体的形式存在,经Western-blot检测表明,表达的重组蛋白ORF2b、ORF2c、ORF2d能够被PCV2阳性血清所识别,具有良好的抗原性。表达的重组蛋白为ELISA诊断试剂盒的研制奠定了基础。

Prokaryotic Expression of ORF2 Gene and Its Four Different Fragments of Porcine Circovirus Type 2

Based on the porcine circovirus type 2(PCV2)LC strain,5 pair of primers were designed according to the Luchuan sequence,the ORF2,ORF2a,ORF2b,ORF2c and ORF2d gene were amplified from the PCV2-LC plasmids by PCR,and inserted into the prokaryotic expression vector PET-32a.The constructed recombinant expression plasmids were transformed into the receptive cells of E.coli BL21 and induced by IPTG with a finial concentration 1 mmol/L,then the recombinant protein was expressed in the form of inclusion bodies.The protein expression could react with PCV2 positive serum by Western-blot,sharing a good antigenicity.This study will provide a good fundament for development of PCV2 diagnostic ELISA diagnosis assay kit.