摘要
本研究通过间接免疫荧光技术,确定了EHV-1囊膜蛋白gD单独表达于BHK-21细胞内的亚细胞定位情况,为其亚细胞定位机制及病毒二级囊膜的研究奠定了基础。以EHV-1基因组为模板,利用PCR方法扩增gD部分基因,并构建其原核表达载体pET28-gD,转入BL21(DE3)感受态细胞内进行诱导表达,利用Ni-NTA亲和层析法纯化gD重组蛋白;以纯化的重组蛋白为抗原免疫实验小鼠并制备抗囊膜蛋白gD的多克隆抗体;分别以间接ELISA方法及Western blot方法检测抗体效价及反应性;人工转染pVAX-Kozak-gD重组质粒至BHK-21细胞,利用IF技术对囊膜蛋白gD进行亚细胞定位。结果表明:成功表达了大小约29kD的gD重组蛋白;制备的多克隆抗体效价为1∶102 400,能较好地与囊膜蛋白gD真核表达产物特异性结合;通过IF方法确定囊膜蛋白gD大部分呈斑点状分布于宿主细胞核周围的胞质中,极少量定位于细胞核内。
We aimed to investigate subcellular localization of envelope glycoprotein D （gD） expressed in BHK-21 cells separately through an immunofluorescence method and to lay the foundation for further research of the localization mechanism and secondary envelopment of equine herpesvirus type-1 （EHV-1）. Using the EHV-1 genome as a template, the partial gD gene was amplified by polymerase chain reaction and cloned to the prokaryotic expression vector pET28a to obtain the pET28-gD recombinant plasmid. The latter was transformed into BL21（DE3） competent cells and induced to expression. The recombinant protein was purified by Ni-NTA affinity column chromatography. Laboratory mice were immunized with purified recombinant protein （as antigen） to obtain polyclonal antibody against envelope gD. Antibody titer and specificity were determined with an enzyme-linked immunosorbent assay and Western blotting, respectively. Subcellular localization of gD was observed by an indirect immunofluorescence method in BHK-21 cells transfectedtransiently by the recombinant plasmid pVAX-kozak-gD. The recombinant gD protein, which had a molecular weight of ≈29 kD, was expressed in BL21（DE3） cells. The prepared polyclonal antibody could react well with the eukaryotic products of gD and its titer was 1 ： 102,400. The envelope gD was detected mainly in the cytoplasm around the nuclei of host cells, and a small amount of gD was in the nucleus.
出处
《病毒学报》
CAS
CSCD
北大核心
2017年第3期419-424,共6页
Chinese Journal of Virology
基金
新疆维吾尔自治区高等学校科研计划(项目号:XJEDU2014S024)
题目:马疱疹病毒Ⅰ型囊膜蛋白gD亚细胞定位研究
新疆维吾尔自治区大学生创新项目(项目号:201610758167)
题目:马疱疹病毒Ⅰ型囊膜蛋白gD的原核表达~~
