猪繁殖与呼吸综合征病毒N蛋白的表达与纯化  

Expression and Purification of Nucleocapsid Protein of Porcine Reproductive and Respiratory Syndrome Virus(PRRSV)

作　　者：姚瑶[1] 宋杰[1] 赵宝华[1] 赵洪明[2] 

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024 [2]石家庄市畜牧水产局,河北石家庄050056

出　　处：《河北师范大学学报（自然科学版）》2012年第3期301-306,共6页Journal of Hebei Normal University：Natural Science

基　　金：石家庄市科技攻关项目(20100210)

摘　　要：根据GenBank报道的PRRSV VR2332基因序列设计引物,经RT-PCR扩增,得到大小约为390 bp的阳性产物;利用BamHⅠ,EcoRⅠ位点将N蛋白基因片段克隆到pET-28a载体,构建原核重组表达质粒pET-N,转化BL21(DE3)并进行SDS-PAGE,Western blot分析.结果表明:克隆的N蛋白基因与GenBank报道的VR2332基因同源性为93.83%;重组菌株经IPTG诱导后,N蛋白基因得到了高效表达;经筛选得到最佳诱导条件,即1 mmol/L IPTG诱导6 h,蛋白表达量可达菌体蛋白总量的52.635%,SDS-PAGE后切胶回收可得到纯化的N蛋白,为进一步制备免疫胶体金试纸条或ELISA试剂盒提供基础.On the basis of the PRRSV VR2332 sequences reported by GenBank,ORF7 gene of porcine reproductive and respiratory syndrome virus（PRRSV） were obtained by RT-PCR after designing one pair of primers and the cDNA fragment was 390 bp approximately.cDNA fragment was cloned into the multiple cloning site（BamH ⅠEcoR Ⅰ） of pET28a（＋） vectort and then transformed into E.coli BL21（DE3）.The results were tested by SDS-PAGE and Western blot.The experimental results demonstrated that the cDNA sequence had 93.83 % homology compared to VR2332＇s.The optimum condition of recombinant strain was induced by 1 mmol/L IPTG in 6 h and accumulation of the N protein was about 52.635 % of total cellular protein.The N protein which has been purified after SDS-PAGE will be used for the further preparation of immunocolloidal gold test strip or the provision of basic ELISA Kit.

关 键 词：N蛋白 诱导条件 WESTERN BLOT 

分 类 号：S852.659.6[农业科学—基础兽医学]