發(fā)布時間：2018-01-08 18:29

  本文關(guān)鍵詞：融合蛋白CTP-SOD在畢赤酵母中的表達(dá)、純化及抗氧化能力　出處：《西南大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 超氧化物歧化酶 細(xì)胞質(zhì)轉(zhuǎn)導(dǎo)肽 畢赤酵母 分泌表達(dá) 氧化脅迫

【摘要】： 超氧化物歧化酶(Superoxide dismutase SOD)是一種在生物界廣泛存在的抗氧化酶,在抗衰老、抗腫瘤、抗免疫疾病和電磁輻射上都起著重要的作用。SOD主要在細(xì)胞質(zhì)中發(fā)揮作用,但是由于SOD是生物大分子,沒有特異性的受體和通道,不容易跨過細(xì)胞膜進(jìn)入細(xì)胞質(zhì),大大降低了生物利用度。 細(xì)胞質(zhì)轉(zhuǎn)導(dǎo)肽(Cytoplasmic transduction peptide CTP);是在轉(zhuǎn)導(dǎo)肽TAT基礎(chǔ)上人工設(shè)計的一種新型轉(zhuǎn)導(dǎo)肽,能夠攜帶外源大分子穿過細(xì)胞膜,定位于細(xì)胞質(zhì)。本實(shí)驗(yàn)利用含有細(xì)胞質(zhì)轉(zhuǎn)導(dǎo)肽基因序列的SOD特異引物,以人總RNA反轉(zhuǎn)錄產(chǎn)物為模板,擴(kuò)增出了CTP和銅鋅超氧化物歧化酶(Cu／Zn SOD)的融合基因。將其克隆到畢赤酵母分泌表達(dá)載體pPIC9K中,成功地構(gòu)建重組表達(dá)載體CTP-SOD／pPIC9K。通過電激法將重組載體轉(zhuǎn)化巴斯德畢赤酵母GS115,經(jīng)酵母菌落PCR和SDS-PAGE篩選得到可表達(dá)重組蛋白的陽性重組子。大量誘導(dǎo)融合蛋白CTP-SOD表達(dá),收集發(fā)酵上清經(jīng)鹽析、透析后,采用鎳親和層析介質(zhì)純化重組蛋白,蛋白純度達(dá)到90%以上,比活為676.5U／mg。經(jīng)Western-Blotting分析,進(jìn)一步驗(yàn)證了重組蛋白的正確表達(dá)。 為了分析CTP-SOD的生物功能,我們利用鄰苯三酚(pyrogallol)誘導(dǎo)HeLa氧化損傷,研究了重組蛋白對氧化脅迫下HeLa細(xì)胞的保護(hù)作用。60μmol／L的鄰苯三酚處理細(xì)胞48h后只有42.2%細(xì)胞存活下來；而CTP-SOD預(yù)處理HeLa細(xì)胞,明顯提高了HeLa細(xì)胞在氧化脅迫下的存活率,隨著CTP-SOD劑量的從1μmol／L增加到4μmol／L,HeLa細(xì)胞的存活率比對照組上升了10%到30%。CTP-SOD預(yù)處理HeLa細(xì)胞,明顯提高了HeLa細(xì)胞的抗氧化能力,和對照組野生型SOD相比具有顯著性差異。結(jié)果表明,CTP-SOD融合蛋白比野生型SOD更容易進(jìn)入細(xì)胞,具有更高效的抗氧化功能。
[Abstract]:Superoxide dismutase (Superoxide dismutase SOD) is a kind of widely existed in biological antioxidant enzymes, anti-aging, anti-tumor, anti immune diseases and radiation plays an important role of.SOD play an important role in the cytosol, but because SOD is the biological macromolecules, no specific receptors and channels that is not easy to cross the cell membrane into the cytoplasm, greatly reduces the bioavailability.
Cytoplasmic transduction peptide (Cytoplasmic transduction; peptide CTP) is a new type of transduction peptide transduction peptide in artificial design based on TAT, capable of carrying exogenous macromolecules across the cell membrane, in the cytoplasm of cells. This experiment using the cytoplasmic transduction peptide gene sequence containing SOD specific primers, with total RNA reverse transcription products as template. Amplified CTP and copper zinc superoxide dismutase (Cu / Zn SOD). The fusion gene was cloned into Pichia pastoris expression vector pPIC9K, successfully constructed the recombinant expression vector CTP-SOD / pPIC9K. by electroporation to Pasteur recombinant plasmid was transformed to Pichia pastoris GS115, PCR and SDS-PAGE by yeast colony expression of recombinant protein screened positive recombinant fusion protein. A large number of induced CTP-SOD expression, collecting supernatant by salting out, dialysis, the recombinant protein was purified by nickel affinity chromatography, the purity of protein It was more than 90%, and the specific activity was 676.5U / mg. by Western-Blotting analysis, which further verified the correct expression of the recombinant protein.
In order to study the biological function of CTP-SOD, we use the adjacent benzene three phenol (pyrogallol) induced oxidative damage to HeLa, the research of recombinant protein on oxidative stress of HeLa cells under the protective effect of.60 on mol / L of the adjacent benzene three phenol treated cells 48h after only 42.2% cells to survive; and the pretreatment of CTP-SOD HeLa cells significantly increased HeLa cells under oxidative stress in the survival rate, with the dose of CTP-SOD from 1 mol / L increased to 4 mol / L, the survival rate of HeLa cells than the control group rose 10% to 30%.CTP-SOD pretreated HeLa cells significantly increased the antioxidant capacity of HeLa cells, compared with wild-type controls SOD with a significant difference. The results showed that CTP-SOD fusion protein than the wild type SOD are more likely to enter the cell, with a more efficient antioxidant function.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R346

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 王多寧,趙雁武,田芙蓉;考馬斯亮藍(lán)微盤比色法測定蛋白質(zhì)含量[J];第四軍醫(yī)大學(xué)學(xué)報;2001年06期

2 陳鴻鵬;譚曉風(fēng);;超氧化物歧化酶(SOD)研究綜述[J];經(jīng)濟(jì)林研究;2007年01期

3 張?zhí)m馨;張書祥;;穿膜肽的內(nèi)化機(jī)制及其應(yīng)用[J];中國生物化學(xué)與分子生物學(xué)報;2008年12期

4 崔慧斐,張?zhí)烀?超氧化物歧化酶在食品和化妝品中的應(yīng)用及其發(fā)酵法生產(chǎn)進(jìn)展[J];藥物生物技術(shù);2000年03期

5 王鳳山,張?zhí)烀?姬勝利;超氧化物歧化酶的臨床應(yīng)用及開發(fā)[J];中國醫(yī)藥工業(yè)雜志;1998年10期

6 李文杰;;超氧化物歧化酶在治療超氧陰離子自由基所引起的疾病及抗衰老上的應(yīng)用[J];中國藥學(xué)雜志;1989年07期

，

本文編號：1398227