摘要
目的构建小鼠FasL基因和屋尘螨主要抗原Der p2双基因共表达载体,并检测其在树突状细胞(DC)中的表达。方法以plambd-Der p2为模板扩增Der p2基因,双酶切pIRES2EGFP和Der p2基因,将Der p2基因克隆入pIRES2EGFP得pIRES2EGFP-Der p2。双酶切pMD18T-FasL质粒获得FasL片段,克隆入pIRES2EGFP-Der p2,获得FasL和Der p2共表达重组载体pIRES2EGFP-FasL-Der p2,采用酶切及测序鉴定重组质粒。将重组质粒转染DC,采用RT-PCR和Western Blot法检测FasL和Der p2基因的mRNA及蛋白水平的表达。结果测序证实FasL和Der p2基因序列完全正确。重组质粒转染DC后,能表达FasL和Der p2的mRNA和蛋白。结论成功构建FasL和Der p2双基因共表达重组载体pIRES2EGFP-FasL-Der p2,转染DC后能在体外正确表达FasL和Der p2基因。
Objective To construct eukaryotic expression vector co-expressing murine FasL and Der p2 gene,and to detect their expressions in bone marrow-derived dendritic cell（DC）.Methods Plasmid plambd-Der p2 as a template,Der p2 gene is amplified using PCR.The plasmid pIRES2EFGP and Der p2 gene were double digested using restriction endonuclease BstX1 and Not1,then Der p2 gene was ligated in pIRES2EGFP（pIRES2EGFP-Der p2）.The FasL gene was excised from the plasmid pMD18T-FasL as a BamH1-Sal1 fragment and cloned in pIRES2EGFP-Der p2.Plasmid pIRES2EGFP-FasL-Der p2 containing both FasL gene and Der p2 gene was obtained.After transfected into DC,the expressions of FasL and Der p2 mRNA were detected by RT-PCR,and the expressions of FasL and Der p2 proteins were assayed by Western Blot.Results The cloned full reading frame of FasL and Der p2 cDNA was in coincidence with the sequence registered in GenBank.In the DC after transfection of pIRES2EGFP-FasL-Der p2,the expressions of mRNA and protein of FasL and Der p2 were detected.Conclusion The eukaryotic expression vector pIRES2EGFP-FasL-Der p2 co-expressing FasL and Der p2 gene is constructed successfully,and FasL and Der p2 gene could be expressed correctly in vitro after transfecting DC.
出处
《重庆医学》
CAS
CSCD
北大核心
2010年第20期2697-2699,2703,共4页
Chongqing medicine
基金
国家自然科学基金资助项目(30470772)
