应用不同表达载体表达欧洲型PRRSV ORF5蛋白的研究  

Effects of different expression vector on the ORF5 gene of European-type PRRSV

作　　者：孙文超[1,2] 任静强[3] 温树波[1,2] 赵权[1] 阎富龙[1,2] 刘昊[3] 陆飞[1,2] 靖杰[3] 鲁会军[2] 金宁一[2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国病原生物学杂志》2013年第11期961-965,共5页Journal of Pathogen Biology

基　　金：科技部"863"项目(No.2011AA10A208);吉林省科技发展计划重点资助项目(No.20090235)

摘　　要：目的构建多种欧洲型PRRSV GP5蛋白原核表达载体,并比较其在大肠埃希菌中的表达效率。方法参考GenBank发表的欧洲型PRRSV LV株(GenBank登录号:M96262)ORF5序列,通过序列分析,设计合成ORF5全基因序列扩增引物及信号肽缺失引物。构建原核表达质粒pET-28a-GP5、pET-28a-gGP5、pET-22b-GP5、pET-22b-gGP5、pET-32a-GP5、pET-32a-gGP5、pGEX-4T-GP5、pGEX-4T-gGP5,经IPTG诱导后采用SDS-PAGE电泳分析GP5蛋白在各个原核表达载体的表达情况。结果 pET-28a-gGP5、pET-22b-gGP5、pET-32a-gGP5均没有高效的表达出缺失信号肽的GP5蛋白,缺少信号肽的GP5蛋白在pGEX-4T-1载体中高效表达,目的蛋白分子质量单位为42ku,与理论值相符。没有缺失信号肽的GP5蛋白在pET-28a-GP5、pET-22b-GP5、pET-32a-GP5、pGEX-4T-GP5,均未得到高效表达。结论本实验构建的重组原核表达载体pGEX-4T-gGP5能在大肠埃希菌中高效表达欧洲型PRRSV GP5蛋白,为进一步分析该蛋白的抗原性奠定了基础。Objective To improve the prokaryotic expressions of the GP5 gene of European-type PRRSV with different vectors. Methods In accordance with the genome sequences of European-type PRRSV LV strain in GenBank, two pair of primers were designed to amplify the ORF5 sequence of PRRSV. The amplified GP5 and gGP5 genes were cloned and 8 recombinant plasmids were respectively constructed： pET-28a-GPS, pET 28a-gGP5, pET-22b-GPS, pET 22b gGPS, pET-32a-GP5, pET-32a-gGP5, pGEX-4T-GPS, and pGEX-4T-gGP5. Results After induction with 1. 0 mmol/L IPTG, pET-28a-gGP5, pET-22b-gGP5, and gGP5 protein were not efficiently expressed by pET-32a-gGP5, but the gGP5 gene was successfully expressed by pGEX-4T-gGP5. The GP5 gene without signal peptides was not expressed at high levels in pET-28a-GP5, pET-22b GPS, pET-32a-GPS, or pGEX-4T-GPS. Conclusion Results indicated that the ORF5 gene of European-type PRRSV was successfully expressed with a prokaryotie expression vector, and this provides a good basis for further analysis of its immunogenicity.

关 键 词：欧洲型PRRSV GP5蛋白 原核表达 信号肽 

分 类 号：S852.65[农业科学—基础兽医学]