人全长PLCγ1基因真核表达载体的构建及鉴定(英文)

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

目的构建人全长PLCγ1基因真核表达载体,以便进一步研究PLCγ1的作用及其机制。方法自行设计一对带有HindⅢ和NotⅠ酶切位点的引物,采用RT-PCR技术从MG63细胞中扩增人全长PLCγ1cDNA(3878bp),纯化后,经HindⅢ-NotⅠ酶切,插入真核表达载体pLNCX2中,构建重组质粒pLNCX2/PLCγ1。通过PCR,限制性酶切分析及DNA直接测序对所构建的重组质粒进行鉴定。同时经瞬时转染后,利用RT-PCR及Westernblotting转染后LoVo细胞中PLCγ1的表达。结果RT-PCR产物经琼脂糖电泳分析所得片段大小与预期相符(3878bp)。重组质粒经HindⅢ-NotⅠ酶切后,得到3878bp(PLCγ1基因)及6100bp(载体pLNCX2)两个片段,同时重组质粒经HindⅢ-BglⅡ酶切后,得到约1300bp及约8500bp两个片段,与预期一致。DNA测序也证实了重组质粒的正确。经RT-PCR和Westernblot分析证实转染pLNCX2/PLCγ1的LoVo细胞中PLCγ1的表达均高于转染空质粒pLNCX2的LoVo细胞及未转染的LoVo细胞。结论成功构建了人全长PLCγ1基因真核表达载体pLNCX2/PLCγ1,为进一步研究PLCγ1的作用奠定了基础。

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! and Not`!. After purification, the product of RT-PCR was digested with Hindb! and Not`! 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! and Not`!, the recombinant eukaryotic expression vector pLNCX2/PLCγ1 yielded a 3 878-bp fragment (PLCγ1 gene) and a 6 100 bp fragment (vector). Hindb!-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.