CAJ | 학술논문

目的构建载有小鼠CD40-IgG2aFc-IRS-GFP融合基因片段的腺病毒载体,并检测其转染293细胞后的融合蛋白表达及出毒情况.方法提取小鼠CD40、IgG2aFc基因片段构建PDC316-IgG2aFc-GFP质粒,测序成功后包装构建Ad5-PDC316-CD40-IgG2aFc-GFP,转染293细胞.出毒后大量复制病毒并纯化,测定病毒滴度,体外感染细胞观察病毒复制及分泌目的蛋白情况并检测目的蛋白表达量.结果成功构建了质粒及病毒,体外感染显示该病毒能有效的感染细胞并表达蛋白(荧光显示),检测目的蛋白表达量随时间点及浓度增加而增加.当病毒浓度增加到一定程度时目的蛋白表达趋于无明显差异性.结论构建小鼠融合基因片段并成功转入细胞内,分泌表达目的蛋白是可行的,为进一步研究该病毒在大鼠体内感染及表达CD40Ig、大鼠肝移植模型免疫耐受及其机制的研究奠定了基础.
목적 구건재유소서CD40-IgG2aFc-IRS-GFP융합기인편단적선병독재체,병검측기전염293세포후적융합단백표체급출독정황.방법 제취소서CD40、IgG2aFc기인편단구건PDC316-IgG2aFc-GFP질립,측서성공후포장구건Ad5-PDC316-CD40-IgG2aFc-GFP,전염293세포.출독후대량복제병독병순화,측정병독적도,체외감염세포관찰병독복제급분비목적 단백정황병검측목적 단백표체량.결과 성공구건료질립급병독,체외감염현시해병독능유효적감염세포병표체단백(형광현시),검측목적 단백표체량수시간점급농도증가이증가.당병독농도증가도일정정도시목적 단백표체추우무명현차이성.결론 구건소서융합기인편단병성공전입세포내,분비표체목적 단백시가행적,위진일보연구해병독재대서체내감염급표체CD40Ig、대서간이식모형면역내수급기궤제적연구전정료기출.
Objective To construct the adenovirus vector containing mice CD40- IgG2aFc-IRS-GFP fusion gene and detect the expression of the fusion protein in transfeete 293cells.Methods The fragments of CD40, IgG2aFc were obtained and inserted into plasmid PDC316-IgG2aFc-GFP.After being verified by sequencing, Ad5-PDC316-CD40-IgG2aFc-GFP was contransfored with the bckbone vector into 293 cells.The virus titer was detected after replicating and purifing and the expression of the fusion protein was analyzed.Results A virus and plasmid were constructed successfully.The vitro infection showed that the virus can infect cells of which the fusion protein was confirmed by fluo-rescence.The expression of the fusion protein increased with the increased time and virus concentra-tion.The protein expression stopped increasing after the virus concentration came to a certain level.Conclusion CD40, IgG2aFc fragments are correctly ligated with plasmid PDC316-IgG2aFc-GFP, and the fusion protein can be expressed in 293cells.This might lay a foundation for further studies of the expresstion of virus, immune tolerance and mechanism of liver transplantation model in rats.