肌肉特异性酪氨酸激酶—红色荧光蛋白融合荧光蛋白在HEK293细胞中的表达

发布时间：2018-05-24 03:59

本文选题：重症肌无力 + 肌肉特异性酪氨酸激酶　；参考：《延边大学》2014年硕士论文

【摘要】：目的：将构建好的肌肉特异性酪氨酸激酶(MuSK)-红色荧光蛋白(mCherry)融合蛋白(MuSK-mCherry)基因,转染在人胚肾HEK293细胞中进行表达,为后续检测重症肌无力患者(MG) MuSK抗体制备抗原。方法：将转化有pMT/BiP/V5-His (MuSK-mCherry)的E coli DH5a扩大培养,提取出质粒,电泳后置于凝胶成像系统中观察其位置确认是否含有pMT/BiP/V5-His (MuSK-mCherry)质粒。分别在放有爬片的6孔细胞培养板和普通6孔细胞培养板中培养人胚肾HEK392细胞,将提取出的质粒转染至人胚肾HEK293细胞中。转染后取放有爬片的6孔细胞培养板第24小时的细胞,通过抗荧光猝灭封片剂封片,凝固后应用共聚焦显微镜在激发波长为587nm、发射波长为610nm处观察荧光图像确认转染表达的情况。在转染后48小时内每6个小时为一时间将转染后的普通6孔细胞培养板中的细胞提取冻存为细胞团。将细胞团统一进行细胞裂解提取出蛋白后,应用免疫印迹检测技术检测表达的融合蛋白,观察条带确认细胞表达情况及转染后每个时间段的表达差异。结果：通过电泳检测于E coli DH5a中提取出的质粒溶液在凝胶成像系统中观察,发现5647bp大小条带,与理论条带位置符合。在HE293细胞中转染后通过免疫荧光图像观察到有红色荧光蛋白,于每6小时的细胞团中提取的蛋白进行免疫印迹试验,可观察到72.4KDa大小条带,与理论条带位置符合,并观察到转染后每个时间段的表达有差异。结论：含有MuSK-mCherry融合蛋白的载体质粒pMT/BiP/V5-His (MuSK-mCherry)可在HEK293细胞中进行表达,获得了MuSK-mCherry融合蛋白。这为后续检查MG患者MuSKAb准备了抗原。
[Abstract]:Aim: to transfect the muscle-specific tyrosine kinase mCherryprotein muSK-mCherry) gene into human embryonic kidney HEK293 cells, and to prepare the antigen for the subsequent detection of MuSK antibody in patients with myasthenia gravis. Methods: the E. coli DH5a transformed with pMT/BiP/V5-His mSK-mCherrywas expanded and the plasmids were extracted, and then the plasmids were extracted by gel imaging system. The position of the plasmids was detected by gel imaging system to confirm the existence of pMT/BiP/V5-His MuSK-mCherryplasmids. Human embryonic kidney HEK392 cells were cultured in 6-well cell culture plate and 6-well cell culture plate respectively. The extracted plasmids were transfected into human embryonic kidney HEK293 cells. After transfection, the cells of the 6-well cell culture plate with crawling tablets were removed for 24 hours, and the cells were sealed with anti-fluorescence quenching tablets. After solidification the transfection expression was confirmed by confocal microscope at the excitation wavelength of 587 nm and the emission wavelength of 610nm. After 48 hours of transfection, the cells from the normal 6-well cell culture plate were extracted and frozen into a cell mass every 6 hours after transfection. The fusion protein was detected by Western blotting technique after the cell mass was extracted by cell lysis. The expression of the fusion protein was confirmed by bands and the difference of expression in each period after transfection was observed. Results: the plasmids extracted from E coli DH5a were observed in gel imaging system by electrophoresis. The size bands of 5647bp were found to be consistent with the theoretical bands. After transfection in HE293 cells, red fluorescent protein was observed by immunofluorescence image, and the protein extracted from the cell mass every 6 hours was detected by Western blotting test. The size bands of 72.4KDa were observed, which coincided with the position of the theoretical bands. It was observed that there were differences in expression at each time after transfection. Conclusion: the vector plasmid pMT/BiP/V5-His MuSK-mCherrycontaining MuSK-mCherry fusion protein can be expressed in HEK293 cells and the MuSK-mCherry fusion protein is obtained. This prepares antigens for follow-up examination of MuSKAb in MG patients.
【学位授予单位】：延边大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R746.1

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