摘要： 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。
关键词:
猪繁殖与呼吸综合征病毒,
ORF5基因,
原核表达,
反应原性,
跨膜区
Abstract: 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.
Key words:
PRRSV,
ORF5 gene,
Prokaryotic expression,
Immunoreactivity,
Transmembrane domain
中图分类号:
周宏专, 徐福洲, 王金洛, 杨新建, 史爱华, 杨兵. 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[J]. 华北农学报, 2007, 22(3): 12-15. doi: 10.3321/j.issn:1000-7091.2007.03.004.
ZHOU Hong-zhuan, XU Fu-zhou, WANG Jin-luo, YANG Xin-jian, SHI Ai-hua, YANG Bing. Prokaryotic Expression and Immunoreactivity Identification of Different Encoding Regions of the ORF5 Gene of PRRSV[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2007, 22(3): 12-15. doi: 10.3321/j.issn:1000-7091.2007.03.004.