肝内胆管癌hTR反义RNA及锤头状核酶基因真核表达载体的构建

Building antisense RNA gene and hammerhead ribozyme gene sequence in the liver inside bile duct cancer cell into the real expression vector

用反转录PCR法 ,调取肝内胆管癌细胞内的端粒酶RNA(hTR)基因 ,并针对其序列设计反义RNA封闭基因及锤头状核酶切割基因序列 ,将其构建入真核表达载体pTriEx 4内。根据hTRcDNA序列设计引物 ,经RT PCR法从体外培养的肝内胆管癌细胞内调取hTR模板区基因 ,根据其测序结果 ,合成反义RNA基因及核酶基因 ,与经相应酶切的真核表达载体连接 ,酶切鉴定重组体的正确性。经RT PCR从细胞内调出 68bp序列 ,与hTRcDNA比较 ,与模板区域碱基序列一致 ,反义RNA与核酶基因重组子经酶切鉴定序列正确。肝内胆管癌细胞内端粒酶的活性明显增高 ,RT PCR法可调出其hTR基因有效片段 ;

We design the antisense RNA gene and hammerhead ribozyme gene according to the hTR sequence in the liver inside bile duct cancer cell, and build them into the real expression vector pTriEx 4 separately. According to the hTR cDNA sequence, we designed the primers and take the hTR template area gene from the liver inside bile duct cancer cells by RT PCR .We synthesize the antisense RNA and hammerhead ribozyme gene according to the result of sequencing, and combine them with true nucleus expressing vector. We identified the exactitude of recombine vector by digestion. The 68 bp sequence extracted from the cell through the RT PCR have the same template sequence comparing with the hTR cDNA. The recombinant plasmid with the antisense RNA gene and hammerhead ribozyme gene are correct by digestion identification. The telemerase activity of liver inside bile duct cancer cell also obviously increase high, the RT PCR method can extract its hTR gene valid part. We construct the real necleus expression vector containing the antisense RNA and hammerhead ribozyme gene successfully.