【作者】 叶建强； 秦爱建； 邵红霞； 刘红梅； 金文杰； 刘岳龙；

【Author】 YE Jian-qiang,QIN Ai-jian,SHAO Hong-xia,LIU Hong-mei,JIN Wen-jie,LIU Yue-long (Key Laboratory of Jiangsu Preventive Veterinary Medicine,Yangzhou University,Yangzhou225009,Jiangsu,China)

【机构】 扬州大学江苏省动物预防医学重点实验室；

【摘要】 将J亚群禽白血病病毒囊膜基因（ALV-J env）插入pcDNA3.1真核表达载体,构建成转移载体pcDNA-env,并转染鸡胚成纤维细胞系DF-1,通过Zeocin药物筛选获得转染阳性细胞。细胞经传30代后,冻存。13个月后复苏,置不含药物的培养基中传代培养。连续传40代后,分别用PCR、Southern blot、间接免疫荧光（IFA）及Western blot对该细胞中的env基因进行了检测,并进行了抗病毒感染分析。研究结果表明,通过Zeocin药物筛选获得了稳定表达ALV-J env基因的鸡胚成纤维细胞系,命名为pcDNA-env-DF-1细胞。该细胞在体外抗ALV-J感染试验中,能抵抗1×104个TCID₅₀病毒感染量,提示所构建的细胞系具有良好的抗病毒作用。这一细胞系的构建为研究ALV-J Env蛋白与宿主细胞表面受体相互作用分子机理提供了很好的工具。更多还原

【Abstract】 The recombinant plasmid pcDNA - env was constructed by inserting the env gene of ALV - J into the vector pcDNA3.1. Chicken embryo fibroblast cell line DF-1 was transfected with the recombinant plasmid pcD-NA-env by Lipofectin.Then the transfected DF-1 cells were analyzed for transient ALV-J env expression by indirect immunofluorescence assay（IFA） and were selected for resistant cells to Zeocin.The Zeocin - resistant cells were passaged for 30 generations and then frozen.After 13 months, these cells were refreshed and cultured in the medium free of Zeocin.After another 40 passages,env gene and Env protein in the transfected DF-1 cell were detected by PCR,Southern blot,IFA and Western blot.The results showed that DF - 1 cell transfected with the recombinant plasmid,designated as pcDNA-env-DF-1,could stably express the env gene of ALV-J.The anti-virus assay showed that pcDNA-env-DF-1 cell had the characteristics of resistance to infection of 1×10 TCID₅₀ ALV - J.The pcDNA-env-DF-1 cell developed in this paper provides a good tool for further research of the mechanisms of interaction between ALV-J Env protein and the host cell receptor.更多还原