shRNA随机文库结合TK自杀基因筛选靶向HIV-1LTR相关宿主因子

构建shRNA随机文库与HIV-1 LTR启动胸苷激酶基因(TK基因)稳定表达的稳定细胞系HEK293/TK,将两者结合起来,筛选靶向HIV-1 LTR相关宿主因子.方法:通过化学合成含有19个随机脱氧核苷酸的发夹结构,将其退火补平后与合成的接头Linker连接进行PCR反应,将PCR产物酶切后置于慢病毒载体pLenti-U6启动子下游由此构建shRNA随机文库;利用重叠PCR将HIV-1 LTR片段和TK基因连接起来,连接产物经酶切后与pcDNA3.1载体连接;将连接正确的质粒转染HEK293细胞同时用G418加压筛选获得稳定细胞系HEK293/TK;将所获得的文库质粒包装成慢病毒后侵染所构建的HEK293/TK细胞系,通过加入药物GCV进行加压筛选获得存活细胞.结果:成功筛选到加药后存活下来的细胞,抽提细胞基因组,采用巢式PCR扩增目的干扰序列并用Western blot对干扰序列进行验证,鉴定获得一个克隆所表达的shRNA能对TK基因的表达起到抑制作用,通过测序分析获得其干扰序列,该序列很有可能针对HIV-1 LTR某宿主相关因子.结论:成功构建了一种筛选HIV-1 LTR相关宿主因子的方法,筛选所得序列可以定位到具体相关宿主因子,为靶向筛选抗HIV-1药物提供了重要手段.

Random shRNA Library Screen for shRNAs Targeting HIV-1 LTR Related Host Factors with TK Suicide Gene

To screen HIV-1 LTR related host factors by constructing random RNAi library based on the lentiviral vector and HIV-1 LTR reporter cell line in which TK suicide gene can express stably.Methods: Oligo deoxynucleotides were chemically synthesized which formed hairpin structure that contain 19 random bases and were then fused with a linker sequence.The joined product was amplified by PCR and the PCR product was cloned downstream of U6 promoter of a lentiviral vector pLenti-U6 to prepare random shRNA Library.HIV-1 LTR and HSV-TK gene were amplified by PCR and then joined by overlapping PCR.The joined product was cloned into pcDNA3.1 expression vector.The recombinant plasmid obtained was transfected into HEK293 cells,and stable cell lines were selected with G418.Lentiviral particles were pachaged in 293FT cells by transfection with random shRNA library plasmid along with pLP1,pLP2 and VSV-G,using Lipofectamine 2000.HEK293/TK cells were plated and infected with enriched random lentivirus shRNA library and then treated by GCV.Results: The surviving cells were collected after about two months,and genomic DNAs were extracted from the surviving cells to amplify integrated shRNAs by nested PCR.After verifying the validity of the interference sequence by Western blotting,a clone was isolated that expressed shRNA which inhibited the expression of TK gene.The shRNA sequence was determined by sequencing,and this sequence is likely to inhibit a host factor required for HIV-1 LTR function.Conclusion: By this strategy,it is possible to identify host factors assistant HIV-1 LTR,which could be potential host cellular targets for preparing novel anti-HIV drug.