胆囊癌hTR锤头状核酶基因真核表达载体的构建

Constructing the Hammerhead Ribozyme Gene’s Eukaryon Express Vectors for hTR Sequence of Gallbladder Carcin oma

目的 用反转录PCR法 ,调取胆囊癌细胞内的端粒酶RNA(hTR)基因 ,并针对其序列设计锤头状核酶切割基因序列 ,并将其构建入真核表达载体pTriEx 4内。方法 根据hTRcDNA序列 ,设计引物 ,经RT PCR法从体外培养的胆囊癌细胞内调取hTR模板区基因 ,根据其测序结果 ,合成锤头状核酶基因 ,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。结果 经RT PCR从细胞内调出 6 8bp序列 ,与hTRcDNA比较 ,与模板区域碱基序列一致。核酶基因重组子经酶切鉴定序列正确。结论 RT PCR法可调出胆囊癌hTR基因有效片段 ;成功构建了针对hTR模板区的核酶基因的真核表达载体 。

Objective To design the hammerhead ribozyme gene according to the hTR sequence in the gallbladder cancer cell, and build it into the eukaryon expression vector pTriEx 4.Methods According to the hTR cDNA sequence, the authors designed the primers and take the hTR template area gene from the gallbladder cancer cells by RT PCR.The hammerhead ribozyme gene was synthesize according to the result of sequencing, and combine them with eukaryon expressing vector. Identified the exactitude of recombine vector by digestion.Results ]The 68 bp sequence extracted from the cell through the RT PCR had the same template sequence comparing with the hTR cDNA. The recombinant plasmid with the hammerhead ribozyme gene was correct by digestion identification. Conclusion The RT PCR method can extract the gallbladder cancer cell’s hTR gene. We construct the eukaryon expression vector containing the hammerhead ribozyme gene successfully which is the foundation for gene therapy of gallbladder cancer.