稳定表达犬瘟热病毒基质蛋白的细胞系Vero-SLAM-M的构建  

Establishment of a Vero-SLAM-M Cell Line Stably Expressing Matrix Protein of Canine Distemper Virus

作　　者：林佳旭 王介淞 杨雅明 黄娟[1] 单虎[1] LIN Jia-xu;WANG Jie-song;YANG Ya-ming;HUANG Juan;SHAN Hu(College of Veterinary Medicine,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学动物医学院,山东青岛266109

出　　处：《中国兽医杂志》2020年第5期20-24,I0002,共6页Chinese Journal of Veterinary Medicine

基　　金：国家自然科学基金(31472196);山东省高等学校优势学科“兽医生物技术”人才团队培育计划。

摘　　要：为了建立稳定表达犬瘟热病毒基质蛋白(M)的细胞系,从犬瘟热病毒狐狸源分离毒株SD16F中扩增出M基因,将其克隆至真核表达质粒pCI-neo的Xho I/Not I位点处。重组质粒经PCR、Xho I/Not I双酶切及测序鉴定后转染Vero-SLAM细胞,利用G418抗性压力筛选阳性细胞,并采用RT-PCR及间接免疫荧光试验(IFA)鉴定转染细胞中M蛋白的表达情况。结果表明,成功构建了重组质粒pCI-neo-M,瞬时及稳定转染Vero-SLAM细胞后,RT-PCR和IFA能够检测到M基因的转录和表达,且G418筛选后细胞与其亲本Vero-SLAM细胞生长特性基本一致,表明获得1株能稳定表达M蛋白的细胞系,命名为Vero-SLAM-M。The aim of this study was to establish a cell line stably expressing the matrix(M)protein of canine distemper virus(CDV).The M gene fragment was amplified from SD16F,a fox-origin wild-type strain of CDV,and cloned into Xho I/Not I sites of plasmid pCI-neo.The recombinant plasmid was identified by PCR,Xho I/Not I digestion and sequencing,and was then transfected into Vero-SLAM cells.The positive cells expressing M protein were screened by G418,and verified by indirect immunofluorescence assay(IFA)and RT-PCR.The results showed that the expression plasmid pCI-neo-M was successfully constructed,and the mRNA and protein expression of M gene in transfected cells were detected by RT-PCR and IFA,respectively.After G418 screening,the transfected cells showed similar growth characteristics to their parental Vero-SLAM cells.It is indicated that a cell line stably expressing M protein was successfully established as and named as Vero-SLAM-M.

关 键 词：犬瘟热病毒 基质蛋白 Vero-SLAM-M细胞系 

分 类 号：S858.292[农业科学—临床兽医学]