本文選題：人乳頭瘤病毒(HPV) + E6　； 參考：《華東理工大學(xué)》2014年碩士論文

【摘要】：宮頸癌是最常見的婦科惡性腫瘤之一。人乳頭瘤病毒(human papillomavirus, HPV)感染是誘發(fā)宮頸癌的根本原因,而90%以上的宮頸癌與高危型HPV(尤其16、18型HPV)感染有關(guān)。HPV表達(dá)的早期蛋白E5/E6/E7可通過與宿主細(xì)胞內(nèi)PDZ結(jié)構(gòu)域相互作用,營造適合其轉(zhuǎn)化、增殖的體內(nèi)環(huán)境,引起一系列的疾病,甚至誘導(dǎo)腫瘤的發(fā)生。近年研究發(fā)現(xiàn)HPV16-E6單一氨基酸突變體(F47R)能抑轉(zhuǎn)HPV-16的效應(yīng),是一種潛在的新型抗腫瘤藥物。高危型HPV18-E6蛋白與HPV16-E6相似,其上類似位點(diǎn)的突變是否也具有相似的抑癌效果呢?本論文就此進(jìn)行了探索,并獲得如下結(jié)果： (1)以HeLa細(xì)胞基因組為模板,成功擴(kuò)增了HPV18-E6蛋白的編碼基因,構(gòu)建了其真核表達(dá)載體pcDNA3.1-E6與pEGFP-Cl-E6;利用重疊PCR與常規(guī)重組相結(jié)合法擴(kuò)增并構(gòu)建了HPV18-E6單突變體蛋白(F49R與F127R)真核表達(dá)載體pcDNA3.1-E6F49R, pcDNA3.1-E6F127R、pEGFP-Cl-E6F49R、pEGFP-Cl-E6F127R,利用定點(diǎn)突變試劑盒構(gòu)建了雙突變體蛋白(F49R/F127R)的真核表達(dá)載體pcDN A3.1-E6F49R-F127R與pEGFP-Cl-E6F49R-F127R。 (2)將HPV18-E6及其突變體蛋白的真核載體轉(zhuǎn)染細(xì)胞,RT-PCR首次顯示HeLa細(xì)胞內(nèi)HPV18-E6的轉(zhuǎn)錄、表達(dá)水平有差異。借助MTT、吖啶橙/溴化乙啶雙染法、穩(wěn)轉(zhuǎn)細(xì)胞株生長曲線差異分析等首次證實(shí)了HPV18-E6蛋白有促癌細(xì)胞增殖的效果；而HPV18-E6突變體蛋白均能有效抑制感染HPV-18的癌細(xì)胞增殖,誘導(dǎo)癌細(xì)胞凋亡；HPV18-E6突變體對差異表達(dá)HPV18-E6的Hela細(xì)胞株作用效果不同；三種突變體比較而言,單突變體HPV18-E6F49R和雙突變體HPV18-E6F49R-F127R的抑癌效果更好。 (3)人工合成了針對大腸桿菌密碼子優(yōu)化的HPV18-E6突變體F49R的編碼基因,成功構(gòu)建該突變體與穿膜肽(TAT或R9)的融合蛋白原核表達(dá)載體,實(shí)現(xiàn)重組蛋白在大腸桿菌中的表達(dá)。經(jīng)優(yōu)化,在15。C和180rpm轉(zhuǎn)速下,添加0.1mmol/LZn2+,利用終濃度為0.06mmol/L的IPTG誘導(dǎo)20h,重組HPV18-E6(F49R)突變體-穿膜肽(TAT或R9)融合蛋白的可溶性表達(dá)量最高。雖然重組蛋白含有His標(biāo)簽,但常規(guī)的Ni柱親和層析不能有效純化目標(biāo)蛋白,通過陰離子交換層析與鋅柱親和層析相結(jié)合,獲得純度85%以上的重組HPV18-E6(F49R)突變體-穿膜肽(TAT或R9)融合蛋白,細(xì)胞實(shí)驗(yàn)證實(shí)重組蛋白也能有效抑制Hela細(xì)胞增殖,HPV18E6F49R-TAT和HPV18E6F49R-R9蛋白對表達(dá)HPV18-E6全長的HeLa細(xì)胞的半數(shù)抑制濃度分別約在280μg/m1和260μg/ml。
[Abstract]:Cervical cancer is one of the most common gynecologic malignancies. Human papillomavirus (human papillomavirus, HPV) infection is the fundamental cause of cervical cancer, while more than 90% of the early protein E5/E6/E7 associated with high-risk HPV (especially 16,18 HPV) infection of.HPV can interact with the PDZ domain in the host cell to create a suitable one. In recent years, the HPV16-E6 single amino acid mutant (F47R) can inhibit the effect of HPV-16, which is a potential new antitumor drug. The high risk HPV18-E6 protein is similar to HPV16-E6, and the mutation of the similar loci is similar. In this paper, we explored the results and obtained the following results:
(1) the encoding gene of HPV18-E6 protein was amplified successfully with the HeLa cell genome as a template, and its eukaryotic expression vector pcDNA3.1-E6 and pEGFP-Cl-E6 were constructed. The eukaryotic expression vector of HPV18-E6 single mutant protein (F49R and F127R), pcDNA3.1-E6F49R, pcDNA3.1-E6F127R, pEGFP-Cl-E6, was amplified and constructed by overlapping PCR with conventional recombination. F49R, pEGFP-Cl-E6F127R, using site directed mutagenesis kit to construct a eukaryotic expression vector of double mutant protein (F49R/F127R), pcDN A3.1-E6F49R-F127R and pEGFP-Cl-E6F49R-F127R.
(2) transfection of the eukaryotic vector of HPV18-E6 and its mutant protein to the cells, RT-PCR first showed the transcription of HPV18-E6 in HeLa cells for the first time, and the expression level was different. With the aid of MTT, acridine orange / ethidium double staining and the difference analysis of the growth curve of the stable cell line confirmed the effect of HPV18-E6 protein to promote the proliferation of cancer cells for the first time; and HPV18-E6 mutation. The body protein can effectively inhibit the proliferation of HPV-18 infected cancer cells and induce the apoptosis of cancer cells. The HPV18-E6 mutant has different effects on the Hela cell lines with differential expression of HPV18-E6. Compared with the three mutants, the single mutant HPV18-E6F49R and the double mutant HPV18-E6F49R-F127R have better effect on inhibiting cancer.
(3) the encoding gene of the HPV18-E6 mutant F49R was artificially synthesized for the optimization of the Escherichia coli codon. The fusion protein prokaryotic expression vector of the mutant and the membrane peptide (TAT or R9) was successfully constructed to realize the expression of the recombinant protein in Escherichia coli. After optimization, 0.1mmol/LZn2+ was added at the speed of 15.C and 180rpm, and the final concentration was 0.06mmol. IPTG induced 20h in /L, and the soluble expression of recombinant HPV18-E6 (F49R) mutant - membrane peptide (TAT or R9) fusion protein was the highest. Although the recombinant protein contained His label, the conventional Ni column affinity chromatography could not effectively purify the target protein. The recombinant HPV18-E6 was obtained by anionic exchange chromatography with the affinity chromatography of zinc column and chromatography, and the purity of the recombinant HPV18-E6 was more than 85%. F49R) mutants - transmembrane peptide (TAT or R9) fusion protein. Cell experiments confirmed that the recombinant protein could also effectively inhibit the proliferation of Hela cells. The median inhibitory concentration of HPV18E6F49R-TAT and HPV18E6F49R-R9 protein on HeLa cells expressing full length of HPV18-E6 was about 280 u g/m1 and 260 micron g/ml. respectively.

【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.33

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 殷廣潔;羅兵;王言奎;;靶向HPV18 E6基因不同鋅指結(jié)構(gòu)編碼序列的siRNA對宮頸癌HeLa細(xì)胞作用的比較[J];青島大學(xué)醫(yī)學(xué)院學(xué)報(bào);2008年03期

2 許潔;洪穎;;HPV致宮頸癌的機(jī)制研究及防治策略[J];實(shí)用醫(yī)學(xué)雜志;2011年04期

3 梅泉;李雙;劉萍;奚玲;王世宣;孟玉菡;劉杰;楊欣慰;盧運(yùn)萍;汪輝;;Construction and Identification of a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein[J];Journal of Huazhong University of Science and Technology(Medical Sciences);2010年01期

4 金宏林;朱立新;金春順;;Rho GTPases研究進(jìn)展及與腫瘤的關(guān)系[J];醫(yī)學(xué)綜述;2009年07期

5 袁月秀;王言奎;羅兵;殷廣潔;;化學(xué)合成siRNA對宮頸癌細(xì)胞SiHa中E6基因表達(dá)的抑制作用[J];中國癌癥雜志;2007年12期

6 胡斯奇;高友鶴;;高危型HPV E6蛋白與宿主PDZ蛋白相互作用的研究進(jìn)展[J];微生物學(xué)免疫學(xué)進(jìn)展;2009年02期

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