禽呼肠孤病毒σC蛋白重组腺病毒载体的构建及其对肝癌细胞增殖的影响  

Construction of recombinant adenovirus expression vector of avian reovirusσC protein and its effect on proliferation of hepatocellular carcinoma cells

作　　者：黄莉 周艾玲 李雨桐 王紫薇 徐昊 钱明明 周崇 HUANG Li;ZHOU Ailing;LI Yutong;WANG Ziwei;XU hao;QIAN Mingming;ZHOU Chong(Guangxi University of Chinese Medicine,Nanning 530200,Guangxi Zhuang Autonomous Region,China)

机构地区：[1]广西中医药大学,广西南宁530200

出　　处：《中国生物制品学杂志》2024年第3期287-291,297,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(81860539);广西中医药大学国家级大学生创新创业训练计划项目(201910600018,202010600017)。

摘　　要：目的构建表达禽呼肠孤病毒(avian reovirus,ARV)σC蛋白的重组腺病毒载体pAd-σC,并检测其对肝癌细胞增殖的影响,为新型抗肿瘤疫苗的研发奠定基础。方法采用PCR法扩增ARVσC基因,构建重组穿梭载体pShuttle-σC,随后转化含有腺病毒载体pAdessy-1的BJ5183感受态细胞,通过同源重组获得重组腺病毒载体pAd-σC,在HEK293细胞内包装病毒,TCID50法检测病毒滴度,Western blot和ELISA法检测σC蛋白的表达,CCK-8法检测病毒对人肝癌细胞SMMC7721增殖的影响。结果重组穿梭载体pShuttle-σC经双酶切及测序鉴定证明构建正确,重组腺病毒载体pAd-σC经菌落PCR鉴定证明构建正确;σC蛋白在肝癌细胞SMMC7721中可成功表达;重组腺病毒Ad-σC效价为107.5/0.1 mL,可抑制肝癌细胞SMMC7721增殖。结论成功构建了含有ARVσC基因的重组腺病毒载体pAd-σC,初步分析其对肿瘤细胞增殖具有抑制作用,为揭示ARV溶瘤效应的分子机制及进一步研发新型抗肿瘤生物制剂奠定了基础。Objective To construct the recombinant adenovirus vector pAd-σC for the expression of avian reovirus(ARV)aC protein and to detect its effects on the proliferation of hepatocellular carcinoma cells,in order to build up a basis for the development of novel anti-tumor vaccines.Methods The recombinant shuttle vector pShuttle-σC was constructed by PCR amplification of ARVσC gene,and then transformed into competent BJ5183 cells containing the adenovirus vector pAdessy-1.The recombi-nant adenovirus vector pAd-σC was obtained by homologous recombination,and the virus was packaged in HEK293 cells.The virus titer was measured by TCID_(50),the expression ofσC protein was determined by Western blot and ELISA,and the effect of virus on the proliferation of human hepatocellular carcinoma cells SMMC7721 was detected by CCK-8 assay.Results The recombinant shuttle vector pShuttle-σC was confirmed to be constructed correctly by double enzyme digestion and sequen-cing,and the recombinant adenovirus vector pAd-σC was constructed correctly as identified by colony PCR.σC protein was successfully expressed in hepatocellular carcinoma cells SMMC7721.The recombinant adenovirus Ad-σC had a titer of 10~(7.5)/0.1 mL,which inhibited the proliferation of hepatocellular carcinoma cells SMMC7721.Conclusion The recombinant adenovirus vector pAd-trC containing ARVσC gene was successfully constructed,and its inhibitory effect on tumor cell proliferation was preliminarily analyzed,which lays a foundation for revealing the molecular mechanism of ARV oncolytic effect and further developing novel anti-tumor biological preparation.

关 键 词：腺病毒 禽呼肠孤病毒 σC蛋白 肿瘤细胞 增殖 抑制 

分 类 号：R392[医药卫生—免疫学]