CAJ | 학술논문

目的：构建小鼠热休克蛋白GP96（GP96）基因真核表达载体并在真核细胞293T中表达。方法用RT-PCR法从小鼠脾脏扩增出GP96的成熟肽基因片段，先克隆于pMD19-T载体，再亚克隆到真核表达载体pcDNA3.1中。以脂质体法将重组质粒转入293T细胞，免疫印迹法鉴定GP96的蛋白表达。结果限制性内切酶酶切和测序结果表明，扩增出GP96基因完全正确；免疫印迹法检测表明，转染的重组质粒能在293T细胞中高效表达。结论构建的pcDNA3.1-GP96重组载体在293T细胞系中高效表达，为深入研究GP96作为DNA疫苗在病毒性心肌炎中的作用及机制打下基础。
목적：구건소서열휴극단백GP96（GP96）기인진핵표체재체병재진핵세포293T중표체。방법용RT-PCR법종소서비장확증출GP96적성숙태기인편단，선극륭우pMD19-T재체，재아극륭도진핵표체재체pcDNA3.1중。이지질체법장중조질립전입293T세포，면역인적법감정GP96적단백표체。결과한제성내절매매절화측서결과표명，확증출GP96기인완전정학；면역인적법검측표명，전염적중조질립능재293T세포중고효표체。결론구건적pcDNA3.1-GP96중조재체재293T세포계중고효표체，위심입연구GP96작위DNA역묘재병독성심기염중적작용급궤제타하기출。
Objective To construct an eukaryotic vector expressing heat shock protein GP96 (GP96) gene and detect its expression in human embryonic kidney 293T cells. Methods The GP96 full length gene was amplified by RT-PCR using spleen cDNA from BALB/c mice. It was then cloned into the pMD19-T vector before subclone into an eukaryotic vector pcDNA3.1. The recombinant vectors were transfected into HEK 293T cells by lipofectamine 2000. The quantity of GP96 was determined by Western blotting. Results The resultant GP96 gene was correct according to restriction enzyme digestion and sequencing. The recombinant vector was highly expressed in 293T cells. Conclusion The high expression of GP96 protein in 293T cells provide experimental basis for investigating the role of GP96 in viral myocarditis as a DNA adjuvant and potential mechanism.