Construction and identification of eukaryotic expression vector of human full-length PLCγ1 gene

doi:10.3321/j.issn:1673-4254.2004.08.002

Abstract：Objective To construct the eukaryotic expression vector of human full-length PLCγl gene for further study of the role of PLCγl in cancer invasion. Methods Reverse transcription-polymerase chain reaction (RT-PCR) technique was used to amplify human full-length PLCγ1 gene from MG63 cells with a pair of specific primers containing the restriction sites for HindⅢ and Not I. After purification, the product of RT-PCR was digested with HindⅢ and Not I before insertion into the corresponding sites of eukaryotic expression vector pLNCX2, yielding the recombinant plasmid pLNCX2/PLCγ1. PCR,restriction endonuclease analysis and DNA sequencing were performed to identify the recombinant eukaryotic expression vector pLNCX2/PLCγ1. RT-PCR and Westem blotting were used to detect the expression of the PLCγ1 gene in LoVo cells after transient transfection via Lipofectamine TM 2000. Results A 3 878-bp full-length PLCγ1 gene fragment was successfully amplified by RT-PCR and inserted into eukaryotic expression vector pLNCX2. After digestion by Hind Ⅲ and Not I , the recombinant eukaryotic expression vector pLNCX2/PLCγ1 yielded a 3 878-bp fragment (PLCγ1 gene) and a 6 100 bp fragment (vector). Hind Ⅲ-Bgl Ⅱ digestion was also done to verify the correctness of the recombinant plasmid, resulting in the identification of the fragments as expected. Sequencing analysis further confirmed the results. In addition, RT-PCR and Western blotting verified that the PLCγ 1 could overexpress in LoVo cells after transfection with recombinant eukaryotic expression vector pLNCX2/PLCγ1. Conclusion The recombinant eukaryotic expression vector pLNCX2/PLCγ1 has been constructed successfully.