人ENO1基因的克隆及其重组逆病毒载体ENO1-pBABE-Puro的构建

Cloning of Human ENO1 Gene and Construction of Recombinant Retroviral Vector ENO1-pBABE-Puro

目的构建含人烯醇化酶1(ENO1)目的基因的重组逆转录病毒载体。方法从人癌细胞中提取总RNA合成cDNA,PCR扩增目的基因ENO1,用sal1和BamH1双酶切ENO1及pbabe质粒,然后用连接酶将ENO1插入pbabe中,最后将重组质粒转化感受态E.coli DH 5α大肠杆菌,提取质粒进行酶切鉴定及DNA测序。结果重组逆转录病毒载体ENO1-pBABE-Puro测序结果与GenBank上的序列完全一致,克隆的目的基因已经正确插入到逆转录病毒载体pBABE-Puro中。结论成功构建了含有ENO1目的基因的重组逆转录病毒载体,为进一步观察ENO1在肿瘤发生、发展中的作用及与宫颈癌细胞化疗敏感性的相关性做好准备。

Objective To clone human ENO1 gene and construct the retroviral vector ENO 1-pBABE-Puro.Methods The total RNA was extracted from human cancer cells for the total RNA reverse transcription into cDNA .Full-length ENO1 gene was amplified by using PCR from human cancer cells reverse transcription of cDNA .Both the PCR product and vector pBABE-Pu-ro were digested by sal1 and BamH1.Then the PCR was cloned in the retroviral vector pBABE-Puro.The plasmids were construc-ted and transformed into E .coli competent DH5α.The positive clones were selected ,and tested by sequencing .Results A DNA fragment 1305 bp was obtained by PCR and its sequence was in conformity with the sequence reported in GenBank .The ENO1 gene was inserted into the retroviral vector exactly .Conclusion Human ENO1 gene is cloned correctly and the recombinant ret-roviral vector is constructed successfully ,and it is helpful for study correlation between expression of ENO 1 gene and chemosensi-tivity of cervical cancer cell line and its function in tumors .