稳定表达LamR的RK13细胞系的建立及RHDV吸附能力的初步研究  

Generation of a RK13 cell line stably expressing Lam R and its effect on absorption of RHDV

作　　者：黄萍 宋艳华[2] 胡波[2] 魏后军[2] 范志宇[2] 仇汝龙 陈萌萌[2] 薛家宾[2] 王芳[2] JUNG Yong-Sam HUANG Ping;SONG Yanhua;HU Bo;WEI Houjun;FAN Zhiyu;QIU Rulong;CHEN Mengmeng;XUE Jiabin;WANG Fang;JUNG Yong-Saml(MOE Joint International Research Laboratory of Animal Health and Food Safety,College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural sciences/Key Laboratory for Veterinary Bio-Product Engineering,Ministry of Agriculture,Nanjing 210014,China)

机构地区：[1]南京农业大学动物医学院/教育部动物健康与食品安全国际合作联合实验室,江苏南京210095 [2]江苏省农业科学院兽医研究所/农业部动物疫病诊断与免疫重点开放实验室,江苏南京210014

出　　处：《畜牧与兽医》2018年第8期44-48,共5页Animal Husbandry & Veterinary Medicine

基　　金：现代农业产业技术体系建设专项资金资助(CARS-43-C-1);中央高校基本业务费(KYHW201702);国家自然科学基金项目(31600130)

摘　　要：为建立稳定表达层黏连蛋白受体（LamR）RK13细胞系（RK13-LamR）,并研究LamR蛋白在兔出血症病毒（RHDV）与宿主细胞结合中的作用,将完整的LamR序列通过同源重组克隆入慢病毒表达载体pLVX-m Cherry,并将该重组质粒pLVX-m Cherry-LamR与慢病毒包装质粒pLP1、pLP2、pLP/VSVG共转染293T细胞,在293T细胞中包装成含目的基因的重组慢病毒。将收获的重组病毒感染RK13细胞,经过嘌呤霉素筛选和细胞有限稀释法,筛选出表达LamR蛋白的RK13细胞。通过间接免疫荧光试验（IFA）和Western blotting证明所获细胞株能够稳定表达LamR蛋白,将细胞株命名为RK13-LamR。以纯化的RHDV接种RK13-LamR细胞,IFA检测结果显示,LamR蛋白能促进RHDV对RK13细胞的吸附。本研究建立了RHDV相对敏感的细胞株,证实在RHDV吸附宿主细胞的过程中,LamR蛋白起到一定的作用。To establish a RK13 cell line （RK13-LamR） which stably expresses LamR protein and to examine its effect on absorption of rabbit hemorrhagic disease virus （RHDV） , a complete LamR sequence was cloned into the lentiviral vector pLVX-mCherry via homologous recombination. Then the recombinant plasmid pLVX-mCherry-LamR was co-transfected into 293T cells with lentivirus packaging plasmids pLP1, pLP2 and pLP/VSVG. The recombinant virus was harvested and used to infect RK13 cells. The cells expressing the LamR protein were screened using the puromycin selection and cell limiting dilution method. The expression of LamR in RK13 was identified by IFA and western blotting. A monoclonal cell line was obtained and named RK13-LamR. After infected with RHDV, the cell line was identified by IFA. The results showed that the LamR protein promoted adsorption of RHDV to RK13 cells. The cell line sensitive to RHDV infection was successfully established here, which suggested that LamR protein might play a role in adsorption of RHDV to host cells.

关 键 词：慢病毒载体 LAM R RK13细胞 兔出血症病毒 

分 类 号：S858.291[农业科学—临床兽医学]