APP亚片段真核细胞载体的构建及其在HEK-293T细胞中的表达

发布时间：2018-04-10 23:19

本文选题：淀粉样蛋白前体 + 亚片段　；参考：《山西医科大学》2012年硕士论文

【摘要】：研究目的：目前AD的疫苗研制大多都是以Aβ为靶点进行的。但通过内吞作用沉积于细胞内的Aβ同样会影响AD的发生发展。可大多数抗体不能进入细胞内,故对此显得束手无策。因此从源头减少Aβ的产生就显得尤为重要。本实验探索APP β剪切位点上游区域,欲从中寻找出对Aβ生成起到调控作用的结构,从而为寻找新的治疗靶位及下一步新型疫苗的研发打下基础。实验方法：构建加载有APPsw695亚片段基因与GFP融合基因的pcdna3.1+真核细胞载体,其中亚片段基因包括APP1-695、APP265-695、APP507-695及APP590-695等多核苷酸链。构建后经通过测序,符合预期要求。将重组质粒及空质粒利用脂质体转染法分别转染至HEK-293T细胞中,作为实验组。以未转染的HEK-293T细胞作为对照组。转染36小时后通过WB(Western blot)方法对Aβ产量进行半定量分析及MTT法的对细胞活性进行检测。所得计量数据采用SPSS17.0统计软件进行χ2检验,检验水准α=0.05,P0.05表示有显著性差异。实验结果： 1.经优化设计后的片段于大肠杆菌中扩增量较高且对宿主增殖无明显影响,经测序证实符合预期要求。 2.转染后分别于24h、36h、48h于倒置显微镜下对细胞形态进行观测,发现贴壁率逐渐下降,且各培养基颜色未见变化提示无消耗产酸迹象。至60h左右,各组细胞完全脱壁,聚集成团悬浮于培养液中。 3.对各组细胞转染36h后上清液中Aβ进行检测,于4.5KD位置无明显条带显影。结论： 1.将突变基因点对应基因(APPsw695:K595N、M596L)优化为符合细菌宿主的AATCTG与AACCTG,有助于细菌的转化及质粒的扩增。 2.利用脂质体转染法在对HEK-293T细胞进行APPSW1-695转染过程中发现,转染后细胞存活率逐渐下降直至死亡,提示此种细胞可能不适用于AD模型的制造。接触性抑制弱的细胞可能成为AD模型的首选。 3.虽然Aβ作为“瀑布级联学说”的起始致病因素,但APP所造成细胞间的相互交联,可能也对细胞的活性也产生负性影响,其在发病过程中的作用较以往认识更为重要。 4.WB技术在对小分子蛋白的检测中仍表现欠佳,因此目前研究Aβ应避免采取此种方法进行检测。 5.在体外Aβ可能进一步聚合成多聚体形式,从而造成WB的特异性一抗,对于此产物并不能识别,因此造成此次试验的假阴性结果。进一步探索Aβ体外多聚体的形成及所需条件、时间及可能性,不仅可解决利用WB技术对Aβ体外检测时条带选择及小分子不可分辨问题,可能也有助于脑内老年斑形成时间窗的判定与干预。
[Abstract]:Objective: at present, most of AD vaccines are based on A 尾.But A 尾 deposited in cells through endocytosis also affected the development of AD.But most antibodies can not enter the cell, so this seems helpless.Therefore, it is particularly important to reduce the production of A 尾 from the source.The aim of this study was to explore the upstream region of the APP 尾 shear site, and to find out the structure that regulates the A 尾 production, thus laying a foundation for the development of new therapeutic targets and new vaccines.Methods: the eukaryotic vector of pcdna3.1 containing APPsw695 subfragment gene and GFP fusion gene was constructed. The subfragment genes included APP1-695, APP265-695, APP507-695 and APP590-695.After construction by sequencing, according with the expected requirements.Recombinant plasmids and empty plasmids were transfected into HEK-293T cells by liposome transfection.Untransfected HEK-293T cells were used as control group.After 36 hours of transfection, A 尾 production was semi-quantitatively analyzed by WB(Western blot method and the activity of cells was detected by MTT method.The measured data were tested by 蠂 ~ 2 test with SPSS17.0 software, and the test level was 0.05% (P < 0.05).Experimental results:1.The optimized fragment was amplified in Escherichia coli and had no obvious effect on host proliferation, which was confirmed by sequencing to meet the expected requirements.2.After transfection, the cell morphology was observed under inverted microscope at 24 h, 36 h and 48 h, respectively. It was found that the adherence rate decreased gradually, and no change in the color of the culture medium indicated that there was no sign of acid production.At about 60 h, the cells in each group were completely detached and clustered and suspended in the culture medium.3.After transfection for 36 h, A 尾 in the supernatant of each group was detected, and there were no obvious bands in the 4.5KD position.Conclusion:1.The mutated gene corresponding gene (APPsw695: K595NN M596L) was optimized to fit the bacterial host AATCTG and AACCTG, which was helpful to the transformation of bacteria and the amplification of plasmids.2.In the process of APPSW1-695 transfection of HEK-293T cells by liposome transfection, it was found that the survival rate of the cells decreased gradually until death after transfection, which suggested that the cells might not be suitable for the manufacture of AD model.The cells with weak contact inhibition may be the first choice of AD model.3.Although A 尾 is the initial pathogenetic factor of "cascade waterfall theory", the cross-linking of cells caused by APP may also have a negative effect on cell activity, and its role in pathogenesis is more important than previous understanding.The detection of small molecule protein by 4.WB is still poor, so we should avoid using this method to detect A 尾 at present.5.In vitro, A 尾 may be further polymerized to form a polymer, resulting in a specific antibody to WB, which can not be recognized for this product, thus resulting in false negative results of this test.Further exploring the conditions, time and possibility of formation, time and possibility of A 尾 in vitro Polymer can not only solve the problems of band selection and small molecule indiscernibility when using WB technology to detect A 尾 in vitro.It may also contribute to the determination and intervention of time window formation of senile plaques in the brain.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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