减毒沙门氏菌为载体在Vero细胞中表达传染性法氏囊病病毒多聚蛋白基因  

Expression of the Infectious Bursal Disease Virus Polyprotein in Vero Cells Using Attenuated Salmonella typhimurium as Transgenic Carrier

作　　者：李龙[1,2] 方维焕[1] 樊拥军[3] 许健[3] 方立[1] 李建荣[1] 于涟[1] 

机构地区：[1]浙江大学动物预防医学研究所 [2]浙江大学生物医学工程学系,杭州310027 [3]浙江大学生物医学工程学系

出　　处：《生物工程学报》2004年第3期437-440,共4页Chinese Journal of Biotechnology

基　　金：浙江省自然科学基金重大项目基金资助 (No .ZA0 10 5 )~~

摘　　要：用长距离RT PCR扩增了传染性法氏囊病病毒 (infectiousbursaldiseasevirus,IBDV)ZJ2 0 0 0株多聚蛋白基因 ,定向克隆入真核表达载体pCI,电转化dam- 和phoP- 双突变的减毒鼠伤寒沙门氏菌ZJ111株 ,并直接转染Vero细胞。RT PCR和间接免疫荧光试验可从Vero细胞中检测到阳性信号 ,SDS PAGE和West blotting均可检测到 4 1kD的蛋白条带。结果表明减毒沙门氏菌可将外源基因导入Vero细胞 ,并进行转录和表达 ,具有免疫反应性 ,为进一步研制减毒沙门氏菌为载体的IBDV口服DNA疫苗打下基础。To examine if polyprotein gene(VP2/VP4/VP3) of Infectious Bursal Disease Virus (IBDV) could be delivered into mammalian cells and expressed using attenuated Salmonella typhimurium as vector. The IBDV polyprotein gene was amplified by RT-PCR and inserted in to pCI, an eukaryotic expression plasmid. The resulting recombinant pCI-VP2/VP4/VP3 was transformed by electroporation into attenuated Salmonella typhimurium strain ZJ111 (dam - and phoP -), which was then use to transfect the Vero cells. Gene specific RT-PCR revealed that VP2/VP4/VP3 was transcribed into mRNA in the Vero cells. Indirect immunofluorscence assay, SDS-PAGE and Western-blot analysis showed that VP2/VP4/VP3 was expressed and the product was immuno-reactive with anti-IBDV serum. This work provides essential precondition for developing a new oral DNA vaccine against IBDV.

关 键 词：鸡传染性法氏囊病病毒 多聚蛋白 减毒沙门氏菌 基因疫苗载体 

分 类 号：S852.4[农业科学—基础兽医学]