猪生殖和呼吸综合征病毒中国分离株B_(13)株ORF_5基因在杆状病毒系统中的表达  

Expression of ORF_5 Gene of Porcine Reproductive and Respiratory Syndro me Virus B_(13) Strain in a Baculovirus Expression System

作　　者：赵耘[1] 罗长宝 林志雄 陈茹 李树根 陈博文 刘尚高[3] 

机构地区：[1]中国兽药监察所,北京100081 [2]广州动植物检疫局,广州510406 [3]中国农业大学,北京100094

出　　处：《农业生物技术学报》2000年第3期211-215,共5页Journal of Agricultural Biotechnology

摘　　要：本研究将PRRSV B13株ORF5基因克隆到杆状病毒转移载体质粒pVL1393中，构建了重组转移载体质粒pVL1393-ORF5。用该重组转移载体质粒与线性化苜蓿丫纹夜蛾核型多角体病毒(AcMNPV-SVI－G)基因组DNA(BaculoGold Linearized Baculovirus DNA)共转染草地夜蛾(Spodoptera frugiperda,sf9)细胞，得到重组病毒AcMNPV-OCC－- ORF5 。在感染了重组病毒的sf9细胞中成功地检测到分子量为25 kD的ORF5基因表达产物，其能被猪抗PRRSV B13株多克隆血清特异识别。表明sf9细胞表达了所期望的产物，进而也说明基因片段的正确性。猪抗PRRSV VR-2332株多克隆血清对ORF5基因的表达产物无反应,而猪抗LV株多克隆血清则能特异识别,说明PRRSV中国分离株B13株在抗原性上更接近欧洲亚群分离株LV株的抗原性,进一步证实了PRRSV中国分离株B13株属于欧洲亚群。ORF5基因表达产物与天然蛋白分子量十分接近，说明杆状病毒表达系统能较适当的进行合成蛋白的加工和修饰。本研究的结果为PRRS基因工程疫苗的研究奠定了基础。The ORF5 gene of B13 of PRRSV was cloned into the transfer p lasmid vector pVL1393 , the recombinant transfer plasmid vector pVL139 3-ORF5 was obtained.After cotransfecting the cultured sf9 cells by pVL 1393-ORF5 and the linearized genome DNA of AcMNPV-SVI—G and TnNPV-SVI —G respectively,the recombinant virus AcMNPV-OCC—-ORF5 was obtained. In the sf9 cells infected with the recombinant virus ,the expression p roduct of the ORF5 gene of B13 with molecular weight about 25 kD was s uccessfully detected by Western-blot using anti- B13 of PRRSV polyclon al sera.The result showed that sf9 cells expressed the predicted produ ct and also confirmed the reality of the gene fragment. Anti-VR-2332 p olyclonal sera could not react specially with ORF5 gene expression pro duct.Anti-LV polyclonal sera could react specially with expression pro duct.It showed that Chinese isolate B13 of PRRSV is similar to LV of E uropean subgroup in antigenity.The size of expression product is same as the size of nature protein ,it showed that Baculovirus expression s ystem could process and modify properly to composed protein.The result of this research lays the foundation for the research of PRRS recombi nation vaccine.

关 键 词：PRRSV ORF5基因 杆状病毒表达载体 表达 猪病毒 

分 类 号：Q939.4[生物学—微生物学] S858.285.3[农业科学—临床兽医学]