筛选和鉴定抗卵巢癌活性六肽的靶点蛋白

发布时间：2018-03-20 18:22

本文选题：乳源抗癌六肽　切入点：GST　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的利用pull-down技术和双向电泳两种技术来筛选乳源抗癌六肽(PGPIPN)在人卵巢癌细胞株(SKOV3)上的靶点蛋白(受体)并进行质谱分析鉴定。方法以PGPIPN的序列为参照,设计出了PGPIPN基因,以BamHI/XhoI为酶切位点,将PGPIPN基因构建到表达质粒载体pGEX-4T-l中,转化到E.coli BL21中,用诱导剂异丙基-β-D-硫代吡喃半乳糖苷(IPTG)低温诱导表达的谷胱甘肽S转移酶(GST)标签融合蛋白作为诱饵蛋白；从SKOV3细胞中提取的总蛋白质作为捕获蛋白,运用GST pull-down技术,筛选出靶点蛋白质,运用SDS-PAGE电泳进行初步鉴定,并用异硫氰酸荧光素(FITC)标记的PGPIPN孵育验证,最后切胶取目的条带进行电喷雾质谱分析；为了确保筛选结果的准确性,同时利用双向电泳技术进行鉴定,将从SKOV3细胞中提取的总蛋白作为样品,进行双向电泳,并利用异硫氰酸荧光素(FITC)标记的PGPIPN进行孵育结合,取点进行电喷雾质谱分析。结果pull-down实验结果进行SDS-PAGE电泳后,硝酸银染色下可见两条清晰的条带,经对比分析后得到一条为诱饵蛋白,而另一条为目的条带；通过荧光标记肽孵育结合实验,在免疫荧光显微镜下可观察到荧光PGPIPN结合到该条带上,验证了受体的特异性；双向电泳实验中,在免疫荧光显微镜下观察到两个荧光结合点；利用电喷雾质谱技术,对pull-down实验结果的目的条带和双向电泳实验中的荧光点同时进行初步分析,鉴定出五种功能、性质相似的相关蛋白,分别为aldolase A、aldolase C、eukaryotic translation elongation factor1delta isoform2、60S ribosomal protein L5、similar to F-box only protein2。结论筛选和鉴定了PGPIPN作用于卵巢癌细胞上的靶蛋白,为研究其抗癌的作用机制及其信号转导通路奠定了基础。
[Abstract]:Objective to screen the target protein (receptor) of breast cancer Hexapeptide PGPIPN on human ovarian cancer cell line SKOV3 by pull-down and two-dimensional electrophoresis and to identify it by mass spectrometry. Methods based on the sequence of PGPIPN, the PGPIPN gene was designed. The PGPIPN gene was constructed into the expression plasmid pGEX-4T-l and transformed into E. coli BL21 with BamHI/XhoI as the restriction site. The glutathione S-transferase (GST) labeled fusion protein induced by isopropyl- 尾 -Dthiopyranoside galactoside was used as bait protein, the total protein extracted from SKOV3 cells was used as trapping protein, and the GST pull-down technique was used. The target proteins were screened and identified by SDS-PAGE electrophoresis, then incubated with PGPIPN labeled with fluorescein isothiocyanate (FITC). Finally, the target band was cut and analyzed by electrospray mass spectrometry. In order to ensure the accuracy of the screening results, At the same time, the total protein extracted from SKOV3 cells was identified by two dimensional electrophoresis, and the PGPIPN labeled with fluorescein isothiocyanate was incubated and analyzed by electrospray mass spectrometry. Results after SDS-PAGE electrophoresis, two clear bands were found in silver nitrate staining, one was the decoy protein and the other was the target band, and the other was the target band, which was incubated with fluorescent labeled peptide. Fluorescent PGPIPN binding to the band was observed under immunofluorescence microscopy, which verified the specificity of the receptor; in two-dimensional electrophoresis, two fluorescent binding sites were observed under the immunofluorescence microscope; and electrospray ionization mass spectrometry was used. The target bands of pull-down and fluorescence spots in two-dimensional electrophoresis were analyzed at the same time, and five similar functional proteins were identified as aldolase aldolase translation elongation factor1delta isoform2o60 S ribosomal protein L 5similar to F-box only protein 2. Conclusion the target proteins of PGPIPN acting on ovarian cancer cells were screened and identified, which laid a foundation for the study of its anticancer mechanism and its signal transduction pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31

【参考文献】