PRRSV核衣壳蛋白基因在杆状病毒中的表达

根据已发表的猪生殖 -呼吸道综合征病毒 ( PRRSV) CH-1 a分离株核衣壳 ( N)蛋白基因核苷酸序列和杆状病毒转移载体 p Blue-Bac-His B多角体蛋白阅读框架 ,设计合成了 1对特异性引物 P7S1 /P7R1。应用PCR对重组质粒 p UC1 8-ORF7扩增 ,获得了 CH-1 a株 N基因的片段。经 Hind 和 Bgl 双酶切 ,将其定向克隆到同样双酶切的 p Blue-Bac-His B的 PH启动子下游 ,获得转移载体 p Blue-Bac-His B-ORF7。将转移载体p Blue-Bac His B-ORF7与苜蓿银蚊夜蛾多核型多角体病毒 ( Ac MNPV,简称杆状病毒 )线性化 DNA ( Bac-N-Blue TMDNA)共转染 Sf9细胞 ,经过蓝斑筛选和蚀斑纯化 ,获得重组病毒 r Bac7。经用引物 P7S1 /P7R1和杆状病毒多角体蛋白基因通用引物 PCR鉴定 ,证明目的基因已插入到杆状病毒中。将重组病毒接种于对数生长期的 Sf9细胞 ,分别于感染后 2 4、4 8、72、96、1 2 0 h收集感染细胞 ,经 SDS-PAGE和 Western blot分析 ,结果表明 ,细胞接种重组病毒 2 4 h即开始表达重组蛋白 ,至 96h达到峰值 ,占整个细胞蛋白的 8.4 % ,此后开始下降。表达产物为融合蛋白 ,大小约 2 0 0 0 0。将重组病毒感染的 Sf9细胞用抗 PRRSV N蛋白的单克隆抗体SDOW-1 7进行间接免疫荧光试验 ,结果在细胞浆和细胞核中观察到了特异性的亮绿

Expression of the Nucleocapsid Protein Gene of Porcine Reproductive and Respiratory Syndrome Virus Strain CH 1A in Baculovirus

The genome of PRRSV contains 8 open reading frames(ORF),and ORF7(N gene) encodes for the nucleocapsid protein,a relatively conservative viral protein In order to amplify N protein gene of strain CH 1a,a set of oligonucleotide primers was designed according to the published sequences of PRRSV strain CH la and multiple cloning sites of pBlueBacHis B transfer vector A fragment of 0 4 kb amplified by PCR was gel purified and cloned into the transfer vector,resulting in a transfer plasmid pBlueBacHisB ORF7 A recombinant baculovirus,designated as rBac7,was obtained by co transfection of Sf9 cells with Bac N Blue TM DNA,Bsu361 linearized AcMNPV,and pBlueBacHisB ORF7 The expression of N gene in baculovirus was confirmed by indirect immunofluorescence,SDS PAGE and Western blotting The results showed that the expressed product had a molecular mass of approximate 20 000 It started expression at 24 hours post inoculation(p i ),and reached peak value by 96 hours p i The expression level was up to 8 4% of the total cell proteins Specific bright green fluorescence was observed across the plasmas and around the nuclei of the cells infected with rBac7 when tested with a monoclonal antibody(SDOW 17) against PRRSV N protein