18型人乳头瘤病毒E6蛋白突变体的制备及其对肿瘤细胞生长的影响

发布时间：2018-05-05 08:01

本文选题：人乳头瘤病毒(HPV) + E6　；参考：《华东理工大学》2014年硕士论文

【摘要】：宫颈癌是最常见的妇科恶性肿瘤之一。人乳头瘤病毒(human papillomavirus, HPV)感染是诱发宫颈癌的根本原因,而90%以上的宫颈癌与高危型HPV(尤其16、18型HPV)感染有关。HPV表达的早期蛋白E5/E6/E7可通过与宿主细胞内PDZ结构域相互作用,营造适合其转化、增殖的体内环境,引起一系列的疾病,甚至诱导肿瘤的发生。近年研究发现HPV16-E6单一氨基酸突变体(F47R)能抑转HPV-16的效应,是一种潜在的新型抗肿瘤药物。高危型HPV18-E6蛋白与HPV16-E6相似,其上类似位点的突变是否也具有相似的抑癌效果呢?本论文就此进行了探索,并获得如下结果： (1)以HeLa细胞基因组为模板,成功扩增了HPV18-E6蛋白的编码基因,构建了其真核表达载体pcDNA3.1-E6与pEGFP-Cl-E6;利用重叠PCR与常规重组相结合法扩增并构建了HPV18-E6单突变体蛋白(F49R与F127R)真核表达载体pcDNA3.1-E6F49R, pcDNA3.1-E6F127R、pEGFP-Cl-E6F49R、pEGFP-Cl-E6F127R,利用定点突变试剂盒构建了双突变体蛋白(F49R/F127R)的真核表达载体pcDN A3.1-E6F49R-F127R与pEGFP-Cl-E6F49R-F127R。 (2)将HPV18-E6及其突变体蛋白的真核载体转染细胞,RT-PCR首次显示HeLa细胞内HPV18-E6的转录、表达水平有差异。借助MTT、吖啶橙/溴化乙啶双染法、稳转细胞株生长曲线差异分析等首次证实了HPV18-E6蛋白有促癌细胞增殖的效果；而HPV18-E6突变体蛋白均能有效抑制感染HPV-18的癌细胞增殖,诱导癌细胞凋亡；HPV18-E6突变体对差异表达HPV18-E6的Hela细胞株作用效果不同；三种突变体比较而言,单突变体HPV18-E6F49R和双突变体HPV18-E6F49R-F127R的抑癌效果更好。 (3)人工合成了针对大肠杆菌密码子优化的HPV18-E6突变体F49R的编码基因,成功构建该突变体与穿膜肽(TAT或R9)的融合蛋白原核表达载体,实现重组蛋白在大肠杆菌中的表达。经优化,在15。C和180rpm转速下,添加0.1mmol/LZn2+,利用终浓度为0.06mmol/L的IPTG诱导20h,重组HPV18-E6(F49R)突变体-穿膜肽(TAT或R9)融合蛋白的可溶性表达量最高。虽然重组蛋白含有His标签,但常规的Ni柱亲和层析不能有效纯化目标蛋白,通过阴离子交换层析与锌柱亲和层析相结合,获得纯度85%以上的重组HPV18-E6(F49R)突变体-穿膜肽(TAT或R9)融合蛋白,细胞实验证实重组蛋白也能有效抑制Hela细胞增殖,HPV18E6F49R-TAT和HPV18E6F49R-R9蛋白对表达HPV18-E6全长的HeLa细胞的半数抑制浓度分别约在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.

【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.33

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