摘要
目的:构建和表达含BCR/ABL融合基因和葡萄球菌肠毒素A(SEA)的真核双表达质粒。方法:利用RT-PCR技术从K562细胞中扩增出含BCR/ABL融合位点基因片段,提取金黄色葡萄球菌基因组DNA扩增出SEA基因,分别将两基因片段连在pIRES载体的多克隆位点A和B上,构建BCR/ABL-pIRES-SEA、SEA-pIRES-BCR/ABL重组质粒。将重组质粒转染K293细胞,RT-PCR鉴定重组质粒在真核细胞的转录情况,SDS-PAGE电泳鉴定目的蛋白在真核细胞的表达。结果:成功扩增出BCR/ABL和SEA基因片段;双酶切鉴定BCR/ABL-pIRES-SEA、SEA-pIRES-BCR/ABL重组质粒中含有BCR/ABL和SEA基因,测序证实完全正确;将重组质粒转染K293细胞后,经RT-PCR扩增鉴定,插入到重组质粒的BCR/ABL和SEA基因能在真核细胞正常转录,经SDS-PAGE电泳鉴定重组质粒能够在真核细胞中表达BCR/ABL和SEA蛋白。结论:成功构建BCR/ABL-pIRES-SEA、SEA-pIRES-BCR/ABL真核双表达质粒,可在真核细胞中正常转录并表达BCR/ABL和SEA蛋白。
Aim：To construct and express of eukaryotic coexpression plasmid containing BCR/ABL gene and Staphyloccucal enterotoxins A（SEA）. Methods： BCR/ABL fusion gene was amplified from K562 cell line by RT-PCR,and the whole SEA gene was amplified from Staphylococcus aureus.Both PCR products were cloned into multi-clone site（MCS） A and B of pIRES plamid respectively to construct recombinant plasmids BCR/ABL-pIRES-SEA and SEA-pIRES-BCR/ABL.The plasmid was transfected into K293 cells to detect the expression of BCR/ABL and SEA by RT-PCR and SDS-PAGE electrophoresis respectively.Results： the length and the sequence of the fragments inserted into pIRES plamids were confirmed by double enzyme cutting and sequence analyzing.Both mRNA and protein expression of recombinant plasmids in K293 cell lines could be correctly detected.Conclusion：The recombinant eukaryotic plasmids BCR/ABL-pIRES-SEA and SEA-pIRES-BCR/ABL were successfully constructed,and expressed in eukaryotic cells.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2010年第4期336-340,共5页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
广东省自然科学基金项目(06025169)
广东省科技计划项目(2005B50301016)
广州市科技计划项目(2005Z1-E4015)
