GPI锚定修饰的慢粒白血病bcr/abl和鼠IL-12真核双表达质粒的构建及表达

Construction and expression of eukaryotic co-expression plasmid of bcr/abl anchored GPI and murine IL-12

目的:构建由糖基化磷脂酰肌醇(GPI)锚定修饰的bcr/abl和鼠IL-12(mIL-12)真核双表达质粒,在COS-7细胞中检测其基因和膜蛋白表达.方法:以含有慢粒白血病(b3a2型)migP210全长序列的质粒为模板,通过PCR扩增获得654bp的bcr/abl融合基因片段,酶切后插入pBudCE4.1空载中,经鉴定获得重组质粒pBB.同时,RT-PCR扩增人外周血淋巴细胞CD24分子中的GPI锚定序列,酶切后克隆入pBB质粒中与bcr/abl基因片断羧基端相连,获得重组质粒pBBG并鉴定.然后扩增mIL-12基因片段并亚克隆入pBBG另一多克隆位点,构建重组质粒pBBGI.在多聚阳离子介导下将pBBGI质粒转染COS-7细胞,采用RT-PCR,Western Blot检测bcr/abl融合基因及其蛋白的表达,ELISA检测mIL-12的表达.结果:经酶切和测序鉴定证实,由GPI锚定连接的bcr/abl及mIL-12基因插入片段正确;转染细胞中有bcr/abl融合基因、转染细胞膜上有bcr/abl融合蛋白的表达,细胞培养上清中也有mIL-12的表达.结论:成功构建能在真核细胞中表达bcr/abl和mIL-12的重组质粒PBBGI,为进一步研究bcr/abl基因疫苗诱导肿瘤特异性细胞免疫奠定了基础.

AIM： To construct the eukaryotic co-expression plasmid of bcr/abl anchored GPI and murine IL-12 and to identify the expressed product in cos-7 cells. METItODS： The gene fragment encoding bcr/abl was amplified by PCR using the plasmlid containing the DNA sequence of P210 as template and was inserted into eukaryofic expression vector pBudCE4. 1. The constructed recombinant plasmid pBudCE4. 1-bcr/abl was identified by restriction analysis and DNA sequencing. The lymphocytes from human peripheral blood were separated and the total RNA was extracted. The gene fragment encoding GPI was amplified by RT-PCR using the RNA as template and was inserted into the constructed plasmid pBudCE4. 1-bcr/abl, and anchored GPI and bcr/abl fragment to construct recombinant plasmid pBudCE4. 1- bcr/abl-GPI. The raiL-12 gene fragment was also amplified by PCR using the mIL-12 plasmid as template and was subcloned in restriction enzyme sites of another promoter in the constructed plasmid pBudCE4. 1-bcr/abl-GPI to construct recombinant plasmid pBudCE4. 1-bcr/abl-GPI-mIL12. The constructed recombinant plasmid pBudCE4.1-bcr/abl-GPI-mIL12 was transfected into Cos-7 cells with lipesome. The expressed bcr/abl genes and bcr/ abl proteins were analyzed by RT-PCR and Western Blot, and the expressed mIL-12 was detected by ELISA. RESULTS： The results of restriction analysis, PCR and DNA sequencing proved that bcr/abl fragment anchored GPI and mIL-12 gene fragment were all correctly inserted into vector pBudCEA. 1. The expressions of bcr/abl gene and bcr/abl protein were identified in transfected cos-7 cells and their membranes, the expression of mIL-12 was also proved in culture supernatant of cos-7 cells. CONCLUSION： The reombinant vector pBudCE4. 1-bcr/abl- GPI-mIL12 is successessfully constructed and expressed in eukaryotic cells, which lays a basis for further study on tumour specific cellular immunity with bcr/abl fusional gene vaccine.