Construction,Identification and Expression of Recombinant Eukaryotic Expression Vector of Human KiSS-1 Gene

Objective To construct a recombinant eukaryotic expression vector containing the functional region of human KiSS-1 gene and then to transfect this vector into human hepatocellular carcinoma cell line MHCC97-H with high metastasis potential.Transient-expression cell lines are to be obtained so as to establish a foundation for a further study on the impact of KiSS-1 on hepatoma carcinoma cell invasion and metastasis.Methods Total RNA was extracted by using Trizol one-step method from fresh placenta tissues.cDNA was amplified via reverse transcription polymerase chain reaction(RT-PCR).The product of PCR,amplified by being transformed into E.coli Dh5α,was inserted into the eukaryotic expression vector after digestion and ligation using restriction endonucleases and ligase.Liposome was used to transfect this KiSS-1 vector into MHCC97-H cell 1ines and the result was detected by RT-PCR,immunocytochemistry(ICC)and Western blot.Results The correct KiSS-1 cDNA clone was obtained.It was confirmed that KiSS-1 cDNA was inserted into the eukaryotic expression vector correctly by employing PCR,double digestion identification and sequencing.RT-PCR,ICC and Western blot showed that the KiSS-1 gene had been transfected into MHCC97-H cell.Conclusions If recombinant eukaryotic expression vector pcDNA3.1/HisC/KiSS-1 and transient-expression KiSS-1 cell lines are successfully obtained,a foundation will be laid for a further study on how to establish KiSS-1 cell lines of stable transfection and how to observe the impact of KiSS-1 on hepatoma carcinoma cell proliferation,invasion and metastasis through in vivo and in vitro experiment.