人乳头瘤病毒11型E6基因的克

发布时间：2018-03-24 22:20

本文选题：人乳头瘤病毒　切入点：11　出处：《重庆医科大学》2005年硕士论文

【摘要】：人乳头瘤病毒(human papillomavirus,HPV)是一种无包膜的环状闭合双链 DNA 病毒,以特异感染人类皮肤和粘膜上皮为特征。迄今,经鉴定和测序的 HPV 基因型已超过 100 多种。根据 HPV 所致疾病的恶性程度,可将其分为高危型和低危型。高危型 HPV(如 HPV16、18、31)与生殖道、皮肤及喉部鳞状细胞癌密切相关,其基因组整合入宿主细胞染色体中,引起细胞恶性转化,致病机制已基本阐明;而低危型 HPV(如 HPV6、11)引起尖锐湿疣等良性增生,很少发展成为恶性,所以长期以来不被重视。由于缺乏对尖锐湿疣发病机制的了解,至今对尖锐湿疣只能进行对症治疗。近年来,随着尖锐湿疣的发病率大幅度上升,人们逐渐认识到“低危并不等于不重要”,从病因学入手,研究低危型 HPV,特别是最常见的 HPV11 的作用机制显得尤为重要与迫切。已知高危型 HPV E6 蛋白可以通过泛素蛋白水解途径特异性的降解细胞 p53 蛋白。由于 p53 能够通过 p21WAF1/CIP1间接导致 G1 期阻滞,使细胞赢得时间,在进入 S 期前修复损伤的 DNA。E6 引起的 p53 的降解,解除了 p53 对生长的负向调节功能。这就允许遗传改变的细胞继续增殖,导致基因组不稳定进而引发肿瘤。由于低危型 HPV 和高危型HPV 的基因组结构和核酸序列具有高度一致性,因此可以推断低危型HPV E6 蛋白也有相似的作用机制。本研究选用低危型 HPV 中有代表性的 HPV11 为研究对象,主要目的是通过构建带有增强型绿色荧光蛋白(EGFP)的 HPV11E6 基因重庆医科大学硕士研究生学位论文 4 的真核表达质粒,应用体外(in vitro)基因转染技术将该质粒转染入人真皮成纤维细胞,进而研究 HPV11E6 对该细胞 p53 蛋白稳定性的影响,借以了解 HPV11E6 的功能,为进一步全面阐明 HPV11 引起尖锐湿疣的机制奠定一定的基础。本研究主要由三部分组成: 一、HPV11E6 基因绿色荧光蛋白真核表达载体的构建和鉴定根据已知的 HPV11E6 序列,设计在目的片段两端分别带 BamHI 和 EcoRI 酶切位点的引物。用 PCR 方法以质粒 pBR322/HPV11 为模板扩增该基因片段,经 BamHI 和 EcoRI 双酶切后回收纯化,定向克隆至含有以增强型绿色荧光蛋白为报告基因的真核表达质粒 pIRES2-EGFP 中。重组质粒 pIRES2-HPV11E6-EGFP 经限制性酶切和 DNA 序列测定证明构建正确,为研究 HPV11E6 在细胞内的功能提供了一个方便而重要的工具。二、重组质粒 pIRES2-HPV11E6-EGFP 在人真皮成纤维细胞中的表达取临床包皮组织,用含氨苄青霉素和硫酸链霉素的 PBS 反复漂洗后,剪弃脂肪和结缔组织。将皮肤剪成 1mm3左右的小块,胶原酶和透明质酸酶消化,离心细胞加入 DMEM,在 37℃、5%CO2 培养箱中培养,形态学观察鉴定。收获处于指数生长期的成纤维细胞约 1×105个, 将细胞接种于 6 孔细胞培养板中培养 24h,使细胞达到 50-80%融合。用转染试剂 DOTAP,按照说明书将 pIRES2-HPV11E6-EGFP 质粒转染入成纤维细胞。转染后 12h,在荧光显微镜下可以观察到荧光蛋白的表达3,6-48h为荧光蛋白表达的高峰期;收集细胞提取RNA后,用RT-PCR 方法检测细胞总 RNA 中目的基因 mRNA 的表达,结果为阳性,说明在转录水平有目的基因的表达。三、HPV11E6 在人真皮成纤维细胞内功能的初步研究
[Abstract]:Human papilloma virus (human papillomavirus HPV) is a non enveloped closed circular double stranded DNA virus, with specific infection of human skin and mucosal epithelial features. So far, the HPV genotyping and sequencing has been more than 100. According to the degree of malignancy of HPV disease, which can be divided into high-risk and the low-risk type. The high-risk HPV (such as HPV16,18,31) and the reproductive tract, skin and laryngeal squamous cell carcinoma is closely related to the genome integration into the chromosome of the host cell, cause cell malignant transformation, the pathogenic mechanism has been clarified; and the low risk HPV (such as HPV6,11) caused by benign hyperplasia of condyloma acuminatum, rarely become for a long time not to be malignant, so seriously.
Due to the lack of understanding of the pathogenesis of condyloma acuminatum, since the symptomatic treatment of condyloma acuminatum. Only in recent years, with the incidence of condyloma acuminatum rate increased to a great extent, people gradually realize that "low risk is important, starting from etiology, of low risk HPV, especially the most common mechanism of HPV11 it is particularly important and urgent.
Known high-risk HPV E6 protein through the ubiquitin proteolytic pathway specific degradation of cell p53 protein. Because p53 can cause G1 arrest by indirect p21WAF1/CIP1, the cell gain time, degradation caused by in the S period before the repair of DNA.E6 p53 p53, released on the growth of the negative regulation of this function. Allow the genetically altered cells continue to proliferate, leading to genomic instability and tumor initiation. The nucleic acid sequence of genome structure and low risk HPV and high-risk HPV has a high degree of consistency, it can be concluded that the low risk HPV E6 protein has a similar mechanism.
In this study, the representative HPV11 in low risk HPV was selected as the research object, and the main purpose was to construct HPV11E6 gene with enhanced green fluorescent protein (EGFP).
Master's degree thesis of Medical University Of Chongqing
Four
The eukaryotic expression plasmid was transfected into the plasmid by in vitro gene transfection technique.
Human dermal fibroblasts, and then study the effect of HPV11E6 on the stability of p53 protein in this cell
Ringing to understand the function of HPV11E6 and to sharpen the HPV11 for further clarifying
The mechanism of condyloma acuminata lays a certain foundation.
This study mainly consists of three parts:
Construction and identification of the eukaryotic expression vector of HPV11E6 gene green fluorescent protein
According to the known HPV11E6 sequence, BamHI is designed at both ends of the target segment.
Primers at the site of the EcoRI enzyme cutting site. Plasmid pBR322/HPV11 is used as a template by PCR
The gene fragment was amplified and purified by BamHI and EcoRI double enzyme digestion.
Eukaryotic expression plasmid pIRES2-EGFP containing enhanced green fluorescent protein as the reporter gene
Determination of recombinant plasmid pIRES2-HPV11E6-EGFP by restriction enzyme digestion and DNA sequencing
Proving that the construction is correct, it provides a convenient and heavy study for the function of HPV11E6 in cell.
The tools you want.
Two, the table of recombinant plasmid pIRES2-HPV11E6-EGFP in human dermal fibroblasts
reach
The clinical prepuce tissue, repeated rinse with ampicillin and streptomycin sulfate PBS
After that, cut off the fat and connective tissue. Cut the skin into small pieces around 1mm3, collagenase and dialysis
After digestion, the centrifugation cells were added to DMEM and were cultured at 37 C, 5%CO2 culture box.
1 * 105 fibroblasts were harvested at an exponential growth period.
The cells were inoculated into the 6 cell culture plate and cultured 24h to achieve 50-80% fusion.
Transfection of pIRES2-HPV11E6-EGFP plasmid with the transfection reagent DOTAP according to the instructions
Into fibroblasts. After transfection of 12h, a fluorescent protein table can be observed under the fluorescence microscope.
3,6-48h is the peak of the expression of the fluorescent protein; after the collection of RNA, RT-PCR
Methods the expression of the target gene mRNA in the total cell RNA was detected, and the results were positive, indicating that the expression of the target gene was positive.
The expression of the target gene at the transcriptional level.
A preliminary study of the function of three, HPV11E6 in human dermal fibroblasts

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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