重组人血管抑素表达载体的构建及在人肝癌细胞系中的表达

Construction of recombinant human angiostatin expression vector and stable expression on human hepatocelluar cancer cells

目的:构建重组人血管抑素(hANG)的表达载体并检测其在肝癌细胞系中的稳定表达.方法:利用RTPCR从人胚胎肝组织中扩增出hANGcDNA片段,将其克隆到pCRBluntIITOPO载体和pcDNA3.1(+)表达载体,利用克隆PCR、限制性内切酶消化以及序列测定对获得的hANG基因片段及重组载体进行验证.将重组pcDNA3.1hANG真核表达载体转染到人肝癌细胞SMMC7721对其表达状况进行检测.结果:所获得的hANG片段(1.1kb)序列与报道的序列完全一致.酶切鉴定的结果表明含血管抑素基因的重组pcDNA3.1hANG表达载体构建成功.转染重组pcDNA3.1hANG的人肝癌细胞SMMC7721表达了血管抑素.另外,从生长曲线结果来看,转染pcDNA3.1hANG真核表达载体和空载体的人肝癌细胞SSMC7721和未转染的SSMC7721细胞的生长速度无明显差别.结论:成功地构建了重组人血管抑素真核表达载体并获得能稳定表达人血管抑素的SSMC7721肝癌细胞,为开展下一步的实验奠定了基础.

AIM： To construct the recombinant human angiostatin （hANG） expression vector and to detect its expression in hepatocellular carcinoma cells. METHODS： hANG cDNA fragment was obtained by RT-PCR from human embryonic tissue and the fragment was cloned into pCR-Blunt Ⅱ-TOPO vector and into pcDNA3.1 （ ＋ ） expression vector. Colony PCR, restriction enzyme digestion and sequencing were used to verify the obtained hANG cDNA fragment. The recombinant pcDNA3. 1-hANG plasmid was then transfected into hepatocellular carcinoma cells to detect the expression of hANG in these cells. RESULTS： The obtained hANG fragment （ 1, 1 kb） was identical to the sequence of reported human angiostatin. Restriction enzyme digestion demonstrated that the recombinant pcDNA3. 1-hANG expression vector was successfully constructed. The transfected SMMC-7221 cells successfully expressed hANG. In addition, no difference in the growth speed was observed between the untransfected SMMC-7221 cells, the SMMC-7221 cells transfected with pcDNA3. 1 （ ＋ ） vector and the SMMC-7211 cells transfected with recombinant pcDNA3.1-hANG. CONCLUSION： We have successfully constructed the recombinant pcDNA3.1-hANG expression vector and obtained SMMC-7221 cells that can stable express hANG.