摘要
采用融合PCR方法将猪圆环病毒2型(PCV2)Cap基因和人单纯疱疹病毒Ⅰ型病毒(HSV-1)VP22蛋白转导域串联得到Cap-VP22基因,将其插入到杆状病毒转移载体pFastBac Dual构建pFast-Cap-VP22重组转移载体,通过转化含有Bacmid的DH10Bac感受态细胞,经3种抗性和蓝白斑筛选,获得含Cap-VP22基因的重组穿梭质粒rBac-2Cap-VP22。重组穿梭质粒转染昆虫细胞(Sf9),获得重组杆状病毒。重组杆状病毒感染Sf9细胞,通过PCR、间接免疫荧光(IFA)、电镜观察对表达产物进行检测。结果表明,成功构建含双拷贝Cap-VP22基因的杆状病毒载体,IFA证实重组蛋白在Sf9细胞中获得正确表达,与PCV2阳性血清具有良好的反应原性;电镜观察结果显示细胞上清中的目的蛋白可装配形成直径约17nm的病毒样颗粒(VLPs);表明C端融合VP22蛋白转导域序列并不影响Cap蛋白的组装。本研究为进一步研制安全、高效的PCV2疫苗奠定基础。
The Cap gene of procine circovirus type 2 （PCV2） and the VP22 transduction domain derived from HSV-1 were fused into Cap-VP22 gene by SOE-PCR and cloned into the multiple cloning site of the pFastBac Dual transfer vector to get recombinant plasmid pFast-Cap-VP22, then was transformed into DH10Bac which contains a baculovirus shuttle vector（Bacmid） and a helper plasmid,after three antibiotics and bule-white plaque selection, the recombinant expression bacmid rBac-2Cap VP22 was obtained and then was transfected into insect cell Sf9 to get recombinant baculovirus. The expression product of recombinant baculovirus in Sf9 cells was detected by PCR,indirect immunofluorescence assay （IFA） and electron microscope. The results indicated that the recombinant baculovirus vectors containing two copies of Cap-VP22 gene was successfully established. IFA showed that the Cap VP22 protein was expressed in Sf9 cells and reacted with PCV2 positive serum. Furthermore, the result of electron microscopy showed the recombinant protein was released into culture medium and self-assembled into virus-like particles（VLPs） to a diameter of about 17 nm,indicated that the C terminal fusion VP22 protein transduction domain sequence had no influence on the assembly of Cap protein. These studies laid a foundation for development of safe and highly effective vaccines.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2017年第2期218-223,共6页
Chinese Journal of Veterinary Science
基金
广西自然科学基金资助项目(2014GXNSFAA118120)
广西科学研究与技术开发资助项目(桂科合14125008-2-6)
广西重点实验室建设资助项目(14-045-31-A-5)
关键词
猪圆环病毒2型
重组杆状病毒
VP22蛋白转导域
病毒样颗粒
porcine circovirus 2 （PCV2）
recombinant baculovirus
VP22 protein transduction do-main
virus-like particles （VLPs）
