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

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

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。

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.