禽白血病病毒衣壳蛋白P27慢病毒载体的构建及其在鸡肝癌细胞中的表达  

Construction of Avian Leukemia Virus Capsid Protein P27 Lentiviral Vector and its Expression in Chicken Liver Cancer Cells

作　　者：何倩 沈逵 秦爱建 钱琨[1,2,3,4] HE Qian;SHEN Kui;QIN Aijian;QIAN Kun(Key Laboratory for Avian Preventive Medicine,Ministry of Education,Yangzhou University,Yangzhou,Jiangsu 225009;Key Laboratory of Jiangsu Preventive Veterinary Medicine,Yangzhou University,Yangzhou,Jiangsu 225009;Jiangsu Co-innovation Centre for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou,Jiangsu 225009;Institute of Comparative Medicine,Yangzhou University,Yangzhou,Jiangsu 225009)

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 [4]扬州大学比较医学研究院,江苏扬州225009

出　　处：《中国家禽》2021年第3期16-21,共6页China Poultry

基　　金：国家重点研发计划(2017YFD0500700、2016YFD0500800);国家自然科学基金项目(31772734、32072839);江苏高校优势学科建设工程资助项目

摘　　要：为构建禽白血病病毒(ALV)衣壳蛋白P27慢病毒表达载体,并检测其在鸡肝癌细胞LMH中的表达,试验以实验室前期构建的pcDNA3.1-p27为模板,扩增p27基因,克隆入pLVX-IRES-ZsGreen1载体,构建的重组质粒命名为pLVX-p27,与辅助质粒psPAX2和pMD2.G共转染至HEK293T细胞,包装获得表达p27基因的重组慢病毒。将慢病毒上清感染293T和LMH细胞,检测细胞中p27基因的转录水平和蛋白表达水平。结果显示:重组质粒pLVX-p27构建正确,收集的慢病毒上清滴度为9×10^(4)TU/mL;实时荧光定量PCR、Western blot和共聚焦荧光试验结果显示P27蛋白主要定位于胞浆,p27基因的RNA和蛋白表达量在感染后96 h内随着感染时间延长逐渐升高。研究表明:表达p27基因的慢病毒包装成功,并且能感染293T及禽源LMH细胞,为进一步研究P27的生物学功能提供了工具。In order to construct the avian leukemia virus capsid protein P27 lentiviral expression vector and detectits expression in chicken liver cancer cells LMH, pcDNA3.1-p27 constructed in the laboratory was used as the template to amplify the p27 gene and then cloned into the p LVX-IRES-Zs Green1 vector. The recombinant plasmid was namedp LVX-p27, which was co-transfected with the helper plasmids ps PAX2 and p MD2. G into HEK293 T cells, and therecombinant lentivirus expressing p27 gene was packaged. The lentiviral supernatant was infected with 293 T and LMHcells, and the transcription and protein expression of p27 gene in the cells were detected. The results showed that therecombinant plasmid p LVX-p27 was constructed correctly by restriction enzyme digestion and sequencing, the titer of thecollected lentiviral supernatant reached 9×104 TU/m L;The results of real-time PCR, Western blot and confocal fluorescenceexperiments showed that the P27 protein was mainly localized in the cytoplasm, the expression of p27 gene RNA andprotein gradually increased with the infection time within 96 hours after infection. It’s suggested that the lentivirusexpressing the p27 gene was successfully packaged and could infect 293 T and avian-derived LMH cells, provide agood tool for further research on the biological functions of P27.

关 键 词：ALV P27 慢病毒载体构建 蛋白表达 

分 类 号：S855.3[农业科学—临床兽医学]