本文選題：乳源抗癌六肽　切入點(diǎn)：GST　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的利用pull-down技術(shù)和雙向電泳兩種技術(shù)來(lái)篩選乳源抗癌六肽(PGPIPN)在人卵巢癌細(xì)胞株(SKOV3)上的靶點(diǎn)蛋白(受體)并進(jìn)行質(zhì)譜分析鑒定。 方法以PGPIPN的序列為參照,設(shè)計(jì)出了PGPIPN基因,以BamHI/XhoI為酶切位點(diǎn),將PGPIPN基因構(gòu)建到表達(dá)質(zhì)粒載體pGEX-4T-l中,轉(zhuǎn)化到E.coli BL21中,用誘導(dǎo)劑異丙基-β-D-硫代吡喃半乳糖苷(IPTG)低溫誘導(dǎo)表達(dá)的谷胱甘肽S轉(zhuǎn)移酶(GST)標(biāo)簽融合蛋白作為誘餌蛋白；從SKOV3細(xì)胞中提取的總蛋白質(zhì)作為捕獲蛋白,運(yùn)用GST pull-down技術(shù),篩選出靶點(diǎn)蛋白質(zhì),運(yùn)用SDS-PAGE電泳進(jìn)行初步鑒定,并用異硫氰酸熒光素(FITC)標(biāo)記的PGPIPN孵育驗(yàn)證,最后切膠取目的條帶進(jìn)行電噴霧質(zhì)譜分析；為了確保篩選結(jié)果的準(zhǔn)確性,同時(shí)利用雙向電泳技術(shù)進(jìn)行鑒定,將從SKOV3細(xì)胞中提取的總蛋白作為樣品,進(jìn)行雙向電泳,并利用異硫氰酸熒光素(FITC)標(biāo)記的PGPIPN進(jìn)行孵育結(jié)合,取點(diǎn)進(jìn)行電噴霧質(zhì)譜分析。 結(jié)果pull-down實(shí)驗(yàn)結(jié)果進(jìn)行SDS-PAGE電泳后,硝酸銀染色下可見(jiàn)兩條清晰的條帶,經(jīng)對(duì)比分析后得到一條為誘餌蛋白,而另一條為目的條帶；通過(guò)熒光標(biāo)記肽孵育結(jié)合實(shí)驗(yàn),在免疫熒光顯微鏡下可觀察到熒光PGPIPN結(jié)合到該條帶上,驗(yàn)證了受體的特異性；雙向電泳實(shí)驗(yàn)中,在免疫熒光顯微鏡下觀察到兩個(gè)熒光結(jié)合點(diǎn)；利用電噴霧質(zhì)譜技術(shù),對(duì)pull-down實(shí)驗(yàn)結(jié)果的目的條帶和雙向電泳實(shí)驗(yàn)中的熒光點(diǎn)同時(shí)進(jìn)行初步分析,鑒定出五種功能、性質(zhì)相似的相關(guān)蛋白,分別為aldolase A、aldolase C、eukaryotic translation elongation factor1delta isoform2、60S ribosomal protein L5、similar to F-box only protein2。 結(jié)論篩選和鑒定了PGPIPN作用于卵巢癌細(xì)胞上的靶蛋白,為研究其抗癌的作用機(jī)制及其信號(hào)轉(zhuǎn)導(dǎo)通路奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R737.31

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