VP22短肽融合猪圆环病毒2型重组病毒样颗粒的构建及其鉴定

发布时间：2018-04-10 00:32

本文选题：猪圆环病毒型　切入点：重组杆状病毒　出处：《中国兽医学报》2017年02期

【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。
[Abstract]:Cap-VP22 gene was constructed by inserting porcine circovirus type 2 PCV2Cap gene and human herpes simplex virus type I virus (HSV-1) VP22 protein transduction domain into the baculovirus transfer vector pFastBac Dual.The recombinant shuttle plasmid rBac-2 Cap-VP22 containing Cap-VP22 gene was obtained by transformation of DH10Bac competent cells containing Bacmid.The recombinant shuttle plasmid was transfected into insect cell line Sf9 and the recombinant baculovirus was obtained.Sf9 cells were infected with recombinant baculovirus. The expression products were detected by indirect immunofluorescence and electron microscopy.The results showed that the recombinant protein was correctly expressed in Sf9 cells and had good reactivity with PCV2 positive serum.The results of electron microscopy showed that the target protein in the supernatant could assemble the virus like particles about 17nm in diameter, indicating that the C-terminal fusion VP22 protein transduction domain did not affect the assembly of Cap protein.This study lays a foundation for the further development of a safe and efficient PCV2 vaccine.
【作者单位】：广西兽医研究所广西畜禽疫苗新技术重点实验室;
【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)
【分类号】：S852.651

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