AFP启动子介导胸苷激酶表达载体的构建及其特异性表达

Recombined and Construction of Human Alpha-fetoprotein Eukaryotic Expression Vector Plasmid pEGFP-C1-AFP-TK

【目的】构建AFP启动子介导HSV-TK基因表达载体,并对其在肝癌细胞中特异性表达进行分析。【方法】采用PCR法扩增人AFP基因启动子。回收纯化后克隆入pBluescript Ⅱ KDR-TK相同克隆位点,切取AFP-TK片段,然后通过酶联反应插入到pEGFP-C1中,构建成pEGFP-C1-AFP-TK。对获得的片段进行鉴定、细胞靶向和外源基因表达的鉴定。【结果】序列分析表明,该启动子含300bp核苷酸,与已报道的序列比较,100%相符。限制性酶切分析,重组质粒pEGFP-C1-AFP-TK已经克隆AFP启动子。RT-PCR及Western blotting分析pEGFP-C1-AFP-TK转染后的HepG2细胞后,表明TK基因在mRNA水平上能有效的表达,裂解后的HepG2细胞膜蛋白中有相对分子质量约61～83ku大小的特异性条带。【结论】人肝细胞癌(HepG2)靶向性质粒pEGFP-C1-AFP-TK构建成功。

【Objective】 To construct the herpes simplex virus thymidine kinase type 1 （HSV-1-TK） expression vector with alpha- fetoprotein （AFP） expression gene as starter, and analyzing its specific expression. 【Method】 The AFP promoter was amplified by means of PCR. It was purified and cloned on the cloning site of pBluescript II KDR-TK. Then the AFP-TK, fragment was obtained and inserted into pEGFP-C1 with T4 DNA ligase. The obtained fragment was identified for its cell targeting and exogenous genes expression. HepG2 genome DNA was extracted as template to amplify the AFP sequence （300 bp）. Its PCR product was then conjugated with pMD-18, then amplified and extracted. The pMD-18-AFP was digested, and then the fragment was cloned into pBluescriptII KDR-TK. Then to produce the whole expressing fragment of AFP-TK which was inserted into the pEGFP-C1. Then the construction of pEGFP-C1-AFP-TK finished. The gene product was sent for sequencing evaluation. And the expression of pEGFP-C1-AFP-TK in HepG2 cells and ECV 304 cells was evaluated. And its mRNA expression was also evaluated. 【Result】 The sequence analysis proved that the promoter contained 300 bp nucleic acid which was 100% consistency with literatures. The result of restriction endonuclease analysis demonstrated that the recombined pEGFP-C1-AFP-TK was cloned within the AFP as an AFP promoter. The results of RT-PCR and Western blotting from the HepG2 cells transfected with pEGFP-C1-AFP- TK demonstrated that TK mRNA was expressed effectively. The lysis from the HepG2 cells was a specific band of 61-83 ku. The pEGFP-C1-AFP-TK was transcripted in HepG2 cells and expressed in HepG2 mRNA effectively. 【Conclusion】 The plasmid of pEGFP-C1-AFP-TK specific for HepG2 cells was successfully constructed.