BST2敲除的MDCK细胞构建及H9亚型禽流感病毒增殖效果的观察  

Observation of H9 subtype avian influenza virus production in BST2-deficient MDCK cells

作　　者：曹兴林 恽君雯 陈丽[1,3] 宋伟 侯继波[1] 冯磊 徐业芬[2] CAO Xing-lin;YUN Jun-wen;CHEN Li;SONG Wei;HOU Ji-bo;ENG Lei;XU Ye-fen(National Research Center cf Engineering and Technology for Veterinary Biologtcals,Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China.;Department of Animal Science,Provincial Key Laboratory of Tibet Plateau Animal Epidemic Disease Research,Tibet Agricultural and Animal Husbandry College,Nyingchi 860003,China;Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose,Yangzhou 225009,China)

机构地区：[1]江苏省农业科学院动物免疫工程研究所国家兽用生物制品工程技术研究中心,江苏南京210014 [2]西藏农牧学院动物科学学院/西藏高原动物疫病研究自治区高校重点实验室,西藏林芝860003 [3]江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2020年第6期696-703,共8页Chinese Veterinary Science

基　　金：国家重点科技研发计划项目(2017YFD0500706);江苏省自然科学基金面上项目(BK20151360)。

摘　　要：为提高细胞培养系统中禽流感病毒的增殖效率,通过构建缺失BST2编码序列的MDCK细胞系,探索BST2对H9亚型禽流感病毒在细胞系中增殖的影响。利用CRISPR/Cas9系统对MDCK细胞BST2主要功能区域进行基因编辑,获得MDCK-BST2-KO单细胞克隆,并检测细胞的增殖能力。通过慢病毒表达转染获得BST2表达细胞系,检测接毒后不同细胞系的BST2蛋白表达情况,并比较病毒的增殖情况。结果表明,利用CRISPR/Cas9系统成功获得MDCK-BST2-KO单细胞克隆,该细胞克隆生长能力与母细胞无差异但增毒能力增强,且BST2蛋白抑制H9亚型禽流感病毒在MDCK细胞中的增殖。To improve the productivity efficiency of avian influenza virus in the cell culture systems,the effect of tetherin on the propagation of H9 subtype avian influenza virus in the cell lines was explored by constructing a MDCK cell line with missing coding sequence of the protein.By using the CRISPR/Cas9 system to perform gene editing on the major functional regions of BST2 in MDCK cells,a single-cell clone of MDCK-BST2-KO was obtained and cells growth capacity was tested.BST2-expressing cell lines were obtained by lentiviral expression transfection experiments.Detection of BST2 protein expression in different cell lines after virus inoculation,while comparing the ability of these cell lines to amplify the virus.In results,MDCK-BST2-KO single cell line was successfully obtained using CRISPR/Cas9 system,the cell clone has the same growth capacity as the original cells,but the cell clone has an increased ability to amplify the virus.Tetherin inhibits the propagation of H9 subtype avian influenza virus in MDCK cells.

关 键 词：CRISPR/Cas9 慢病毒表达 犬肾细胞 骨髓基质细胞抗原2 禽流感病毒 

分 类 号：S852.659.5[农业科学—基础兽医学]