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.
出处
《生物工程学报》
CAS
CSCD
北大核心
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 )~~
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
infectious bursal disease virus, polyprotein, attenuated Salmonella typhimurium, DNA vaccine delivery vector