CAJ | 학술논문

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pB-ABE-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。
목적：구건함인희순화매1（ENO1）목적기인적중조역전록병독재체。방법종인암세포중제취총RNA합성cDNA， PCR확증목적기인ENO1，용sal1화BamH1쌍매절ENO1급pbabe질립，연후용련접매장ENO1삽입pbabe중，최후장중조질립전화감수태E．coli DH 5α대장간균，제취질립진행매절감정급 DNA 측서。결과중조역전록병독재체ENO1-pBABE-Puro측서결과여GenBank상적서렬완전일치，극륭적목적기인이경정학삽입도역전록병독재체pB-ABE-Puro중。결론성공구건료함유ENO1목적기인적중조역전록병독재체，위진일보관찰ENO1재종류발생、발전중적작용급여궁경암세포화료민감성적상관성주호준비。
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 .