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

Abstract

Abstract: Objective To construct the eukaryotic expression vector of human full-length PLCγ1 gene for further study of the role of PLCγ1 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 Hindb Ⅲ and Not Ⅰ. After purification, the product of RT-PCR was digested with Hindb Ⅲ 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 Western 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 Hindb 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). Hindb Ⅲ-Bgla! 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 PLCγ1 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.

CLC Number:

Q785

LI Xiu-mei, DENG Fan, ZENG Wei-sen, HUA Liang, LU Di, LI Ming, WANG Hong, LIU Zhong-ying, LUO Shen-qiu. Construction and identification of eukaryotic expression vector of human full-length PLCγ1 gene[J]. Journal of Southern Medical University, 2004, 24(08): 849-853.

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