人肺癌细胞A549λ噬菌体cDNA文库的构建与鉴定  

Construction and identification of a lambda phage cDNA library of human lung cancer cells A549λ

作　　者：张红英 郭嘉雯 员月明 徐志勇 邓长生 王琪 ZHANG Hong-ying;GUO Jia-wen;YUAN Yue-ming;XU Zhi-yong;DENG Chang-sheng;WANG Qi(Science and Technology Industry Park,Guangzhou University of T.C.M,Guangzhou 510405,China;Artemis-ia annua Research Center,Guangzhou University of T.C.M,Guangzhou 510405,China)

机构地区：[1]广州中医药大学科技产业园,广州510405 [2]广州中医药大学青蒿研究中心,广州510405

出　　处：《西南医科大学学报》2021年第4期333-337,共5页Journal of Southwest Medical University

基　　金：广东省科技计划项目(2020A0505090009),广东省教育厅项目(2020KZDZX1055),国家自然科学基金项目(81873218、82074301)。

摘　　要：目的构建人肺癌细胞A549λ噬菌体cDNA文库,为抗肺癌药物的筛选和药物作用分子机理的研究奠定基础。方法提取人肺癌细胞A549λ总RNA,用DnaseⅠ处理后,采用RNA 5’末端移动反转录(switching mechanism at 5′end of RNA transcript,SMART)技术合成双链cDNA,切胶回收去除短片段,依次用蛋白酶K和SfiⅠ进行酶切,并与经过酶切的λTriplEx2载体连接后进行体外包装,得到初级文库。初级文库扩增后,测定扩增文库滴度,并通过PCR鉴定插入片段。结果构建的人肺癌细胞A549λ噬菌体初级文库库容为1.88×10^(7)pfu/mL,扩增文库滴度为2.50×10^(9)pfu/mL,插入cDNA片段为500~3000 bp。结论成功构建了人肺癌细胞A549λ噬菌体cDNA文库,为进一步筛选治疗肺癌的药物作用分子机理奠定了基础。Objective To construct a lambda phage cDNA library of human lung cancer cells A549λ,and to lay a foundation for screening for lung cancer therapeutic drugs and study on the molecular mechanism of action of the drugs.Methods Total RNA of human lung cancer cells A549λwas extracted and digested with DNase I,and switching mechanism at 5’end RNA transcription(SMART)technology was used to synthesize double-stranded cDNA(ds-cDNA).The short fragments of ds-cDNA were removed via gel extraction,and the product was digested with proteinase K and SfiI successively and ligated into enzymatically digestedλTriplEx2 vector,followed by in vitro packaging to obtain the primary library.The primary library was then amplified and titrated,and polymerase chain reaction was used to identify the inserted fragments.Results The titer of the constructed primary library and amplified library of human lung cancer cells A549λwas 1.88×10^(7) pfu/mL and 2.50×10^(9) pfu/mL,respectively.The inserted fragments of cDNA ranged from 500 bp to 3000 bp.Conclusion A lambda phage cDNA library of human lung cancer cells A549λwas successfully constructed,which lays the groundwork for screening for potential drugs targeted at lung cancer and study on the molecular mechanism of action of the drugs.

关 键 词：肺癌 Λ噬菌体 CDNA文库 SMART 

分 类 号：G312[文化科学]