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 reovirus σC protein and its effect on proliferation of hepatocellular carcinoma cells
黄莉 1周艾玲 1李雨桐 1王紫薇 1徐昊 1钱明明 1周崇1
作者信息
- 1. 广西中医药大学,广西南宁 530200
- 折叠
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ARV σC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.
Abstract
Objective To construct the recombinant adenovirus vector pAd-σC for the expression of avian reovirus(ARV)σC 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 ARV σC 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 TCID50,the expression of σC 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 107.5/0.1 mL,which inhibited the proliferation of hepatocellular carcinoma cells SMMC7721.Conclusion The recombinant adenovirus vector pAd-σC containing ARV σC 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Key words
Adenovirus/Avian reovirus(ARV)/σC protein/Tumor cells/Proliferation/Inhibition引用本文复制引用
基金项目
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(201910600018)
广西中医药大学国家级大学生创新创业训练计划(202010600017)
出版年
2024
NETL
NSTL
万方数据