Cloning Of Human ENO1Gene And Construction Of Recombinant Retroviral Vector ENO1-pBABE-puro

Objective:Our laboratory constructed the human enolase1(ENOI) gene recombinantMethods:Human cervical cancer cells was revived from liquid nitrogen and cultured with DMEM cell culture medium and extracted total RNA of the cell. In accordance with the instructions of the cDNA synthesis kit, cDNA was synthesized human gastric cancer cell total RNA as a template. Based on enolase ENO1gene in the GenBank designed upstream and downstream primers and were synthesized by the company. Then synthesized cDNA as a template, PCR amplified gene ENOI. The plasmid pBABE-Puro and PCR products of ENO1were recovered according to the instructions of DNA gel extraction kit. Plasmid pBABE-Puro and ENO1were purificated according to the instructions of purification kit after digested with sal1and BamH1enzyme. The target gene was inserted into the plasmid by ligase, and than transformed, selected positive clone, expanded and extracted. The recombinant plasmid of ENOl-pBABE-Puro digested with sall and BamH1restriction enzymes was saved and tested sequence by company.Results:The ENOI sequence of recombinant plasmid of ENOl-pBABE-Puro was in conformity with the sequence reported in GenBank. The ENOI gene was inserted into the retroviral vector exactly.Conclusions:Human ENOI gene is cloned correctly and the recombinant retroviral vector is constructed successfully, and all these lay a foundation for research correlation between expression of ENOl gene and chemosensitivity of cervical cancer cell line and its functions in tumors.