禽白血病病毒衣壳蛋白p15基因慢病毒表达载体的构建及其在鸡肝癌细胞系中的表达  

Construction of a lentiviral expression vector of avian leukosis virus capsid protein p15 gene and its expression in chicken liver cancer cell line

作　　者：沈逵 何倩 秦爱建 钱琨[1,2,3] SHEN Kui;HE Qian;QIN Aijian;QIAN Kun(MOE Key Lab for Avian Preventive Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Province Key Laboratory of Preventive Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-innovation Centre for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225009,China)

机构地区：[1]扬州大学教育部禽类预防医学重点实验室,江苏扬州225009 [2]扬州大学江苏省动物预防医学重点实验室,江苏扬州225009 [3]江苏高校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：《畜牧与兽医》2021年第4期89-94,共6页Animal Husbandry & Veterinary Medicine

基　　金：国家重点研发计划(2017YFD0500700,2016YFD0500800);国家自然科学基金(31772734,32072839);江苏省优势学科项目。

摘　　要：构建禽白血病病毒(ALV)衣壳蛋白p15基因慢病毒表达载体,并检测其在鸡肝癌细胞系(LMH)中的表达情况,以探讨p15基因对病毒复制、免疫信号通路的影响。以真核表达质粒pCAGGS-p15为模板扩增p15全长基因,经双酶切后克隆到慢病毒载体plvx-IRES-ZsGreen1上,构建成plvx-p15-Flag慢病毒表达质粒,将该质粒与辅助质粒共转染293T细胞产生慢病毒,将细胞上清中的病毒感染LMH细胞,检测p15蛋白表达情况。慢病毒载体在293T细胞上包装完成后,病毒滴度为2.25×10^(5) TU/mL。慢病毒感染LMH细胞,基因和蛋白水平检测结果表明,p15蛋白表达良好。表达ALV p15基因的慢病毒包装成功,并且能够感染鸡源LMH细胞。该载体的构建为进一步研究p15蛋白的生物学功能提供工具。This study was to construct a lentiviral expression vector of the avian leukosis virus(ALV)capsid protein p15 gene and to detect the expression of the gene in a chicken liver cancer cell line(LMH),in order to explore the effect of the p15 gene on virus replication and immune signaling pathways.The full-length p15 gene was amplified from pCAGGS-p15 and cloned into the lentiviral vector plvx-IRES-ZsGreen1 by double restriction enzyme digestion,and a lentivirus was produced by co-transfection of the 293 T cells with skeleton plasmid and auxiliary plasmids.LMH cells were infected with the viruses in supernatant.After the lentiviral vector was packaged on the 293 T cells,the titer of the virus was 2.25×10^(5) TU/mL.The results of gene and protein analysis showed that the p15 protein was well expressed in the LMH cells infected with the lentivirus.The lentivirus expressing the p15 gene was successfully packaged and could infect chicken derived LMH cells,which provided a tool for further research on the biological function of the p15 protein.

关 键 词：P15蛋白 慢病毒载体构建 鸡肝癌细胞系细胞 鉴定 

分 类 号：S857.4[农业科学—临床兽医学]