绵羊子宫内膜上皮细胞的培养鉴定及enJSRV囊膜蛋白及其受体Hyal2的瞬时表达

Culturing and identification of uterus endometrial epithelial cells of sheep and transient expression of enJSRV envelope protein and its receptor

为了分离培养绵羊子宫内膜上皮细胞并瞬时表达内源性绵羊肺腺瘤病毒(enJSRV)囊膜蛋白及其受体(Hyal2),采用酶消化法分离培养绵羊子宫内膜上皮细胞,差速消化法纯化细胞。应用角蛋白抗体对细胞进行免疫细胞化学鉴定。扩增enJSRVenv基因及其受体Hyal2基因,并构建真核表达质粒,同时在Hela细胞中瞬时表达。结果显示,细胞在相差显微镜下呈明显的上皮样细胞形态,同时细胞角蛋白染色阳性细胞占90%以上。成功构建了真核表达质粒pcDNA4-enJSRVenv及pcDNA4-Hyal2。Western-blot结果显示,真核表达质粒在HeLa细胞中被成功表达。本研究建立了一套高效、简便的子宫内膜上皮细胞培养方法,同时为进一步探究enJSRV囊膜蛋白的结构和功能奠定了试验基础。

In order to isolate and culture sheep uterus endometrial epithelial cells and transiently express endogenous jaagsiekte sheep retrovirus（enJSRV）envelope protein with its receptor（Hyal2）,this study adopted enzyme digestion to isolate and culture sheep uterus endometrial epithelial cells,differential digestion purified cells.Meanwhile,immunocytochemistry identification was carried out on the endometrial epithelial cells by applying the epithelial cytokeratin antibody.The enJSRVenv gene and its receptor Hyal2 gene were amplified,and their eukaryotic expression plasmids were constructed and transiently expressed in HeLa cells.The epithelioid cell morphology was obviously observed under the phase contrast microscope.Cytokeratin positive cells were more than 90%.The eukaryotic expression plasmids pcDNA4-enJSRVenvand pcDNA4-Hyal2 were constructed successfully.Western-blot showed that the eukaryotic expression plasmid successfully expressed in HeLa cells.This study result established a set of efficient uterus endometrial epithelial cell culture method and provided an experimental basis for further exploring the structure and function of enJSRV envelope protein.