猪繁殖与呼吸综合征病毒ORF5基因不同编码区的表达和反应原性鉴定

为鉴定猪繁殖与呼吸综合征病毒(PRRSV)囊膜糖蛋白GP5编码基因ORF5不同编码区的原核表达能力,利用PCR方法分别扩增ORF5基因缺失信号肽的D片段、缺失信号肽和跨膜区的L片段、缺失信号肽的5′端N片段和缺失跨膜区的3′端C片段,经克隆测序后分别插入原核表达载体pET-32a中进行原核表达,结果显示未缺失跨膜区的D片段未见表达产物,而L片段、N片段和C片段分别表达大小约为33,25,29 kDa的融合蛋白。Western blotting检测结果显示L片段表达蛋白可与PRRS阳性血清发生阳性反应,C片段反应性稍弱,而N片段未出现阳性反应,证实ORF5基因编码产物的C端在与抗体结合的反应原性方面具有重要作用。

Prokaryotic Expression and Immunoreactivity Identification of Different Encoding Regions of the ORF5 Gene of PRRSV

To identify prokaryotic expression of the ORF5 gene of porcine reproductive and respiratory syndrome virus(PRRSV) in different encoding regions,fragments D,L,N and C obtained by deleting the N-terminal signal peptide sequence and/or the transmembrane domain sequence of ORF5 gene were amplified by PCR.Based on cloning and sequence.These fragments were inserted into the prokaryotic expression vector pET-32a respectively.The recombinant expression plasmids contained fragments L,N or C yielded recombinant fusion proteins of the expected sizes(33,25 and 29 kDa respectively),but fragment D yielded no recombinant fusion protein. The recombinant fusion protein of fragment L showed strong positive reaction with PRRSV-infected swine serum by Western blotting,fragment C showed weak positive,whereas fragment N showed no positive.It indicated that the C-terminus of ORF5 encoding protein played an important role in immunoreactivity.