摘要
为了研究gB蛋白在细胞内的作用位点,试验以猪伪狂犬病病毒(Pseudorabies virus,PRV)SA株为模板,设计特异性引物,扩增gB基因保守片段;以pEGFP-N1质粒为模板扩增EGFP基因;以gB基因与EGFP基因胶回收产物为模板,用gB基因上游引物及EGFP基因下游引物通过重叠延伸PCR(SOE PCR)技术获得gB/EGFP融合基因;构建pcDNA3.1/gB/EGFP重组表达质粒,转染乳仓鼠肾细胞(BHK细胞),采用Western-blot技术及荧光显微镜检测荧光蛋白在细胞内的表达情况。结果表明:试验成功扩增到300bp的gB基因、760bp的EGFP基因及1060bp的gB/EGFP融合基因;成功构建出pcDNA3.1/gB/EGFP重组表达质粒并转染BHK细胞;Western-blot技术及荧光显微镜检测显示,融合蛋白在BHK细胞中成功表达。说明通过荧光蛋白研究目的蛋白的作用位点是可行的。
In order to study the site of gB protein in the cell, the Pseudorabies virus(PRV) SA strain was used as a template, and specific primers were designed to amplify the gB conservative fragments in this experiment. The EGFP gene was amplified with the pEGFP-N1 plasmid as a template at the same time. Using gB gene and EGFP gene gel recovery product as template, gB/EGFP fusion gene was obtained by using gB gene upstream primer and EGFP gene downstream primer by overlapping extended PCR(SOE PCR) technique.pcDNA3.1/gB/EGFP eukaryotic expression vector was constructed and transfected into BHK cells. Western-blot and fluorescence microscopy were used to detect the expression of fluorescent protein in cells. The results show that 300 bp gB gene, 760 bp EGFP gene and 1 060 bp gB/EGFP fusion gene are successfully amplified. The eukaryotic expression vector of pcDNA3.1/gB/EGFP is successfully constructed and transfected into BHK cells. Western-blot and fluorescence microscopy show that the fusion protein is successfully expressed in BHK cells. It shows that it is feasible to study the site of action of target protein by fluorescent protein.
作者
韩强
郭广君
吕素芳
沈志强
HAN Qiang;GUO Guangjun;LV Sufang;SHEN Zhiqiang(Shandong Lvdu Bio-Science & Technology Co.,Ltd.,Binzhou 256600,China;Key Laboratory of Shandong Provincial Veterinary Biotechnology,Shandong Binzhou Animal Husbandry & Veterinary Medical Academy,Binzhou 256600,China;Research and Development Center of Shandong Binzhou Animal Propolis Vaccine,Binzhou 256600,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2019年第11期77-80,181,共5页
Heilongjiang Animal Science And veterinary Medicine
基金
山东省自然科学基金项目(ZR2012CQ012)
山东省畜牧兽医科技服务业沈志强创新团队项目(鲁发服务[2014]01041号)
山东省现代农业产业技术体系生猪创新团队项目(SDAIT-08-17)
