家蚕浓核病毒BmDNV-1荧光假病毒粒子的制备及人博卡病毒VP2的纯化

发布时间：2018-04-11 07:39

本文选题：家蚕浓核病毒 + 人博卡病毒　；参考：《华中师范大学》2012年硕士论文

【摘要】：导致人类疾病的病原体在不断进化,人类生产方式和生活环境的巨大演变也加速着这一进化过程。新病毒以及新型疾病的出现给人类生命健康和传统疾病治疗手段带来了前所未有的挑战,如2003年在亚洲急剧流行的严重急性呼吸道综合症(SARS)、2009年席卷全球的甲型流感病毒(H1N1、H5N1)和2005年新发现的细小病毒科人博卡病毒(HBoV)等。在众多威胁人类健康的疾病之中,恶性肿瘤(又称癌症)依旧占据着主要地位,并且尚无切实可行的治疗方法。寻找可行的癌症治疗手段和方法,已经引起了医学界和科学界的密切关注,人们不断探索着一些新的治疗方法。相对于传统癌症治疗手段(化疗、放疗)所带来的严重副反应特性,近年来出现的生物纳米技术在癌症治疗方面展现出了巨大潜力和优势。纳米生物技术是运用一些纳米颗粒或类似于纳米颗粒的生物材料(如病毒样颗粒)作为载体平台,来实现对病变等非正常细胞的特异性进攻,以达到治疗疾病的目的。目前,有关病毒样颗粒在细胞内或生物机体内的转运途径与机制、病毒样颗粒体外去组装与重组装、生物效应分子的偶联与包被等特性尚不够清楚。本研究基于已有有关细小病毒的研究,体外构建家蚕浓核病毒伊那株(BmDNV-1)结构蛋白基因vp4与增强型绿色荧光蛋白egfp基因融合表达载体,利用重组杆状病毒真核表达系统,制备荧光病毒样颗粒(fluorescent virus-like particle, fVLP)。得到的荧光病毒样颗粒因携带有绿色荧光蛋白标记,可以方便地用于其入胞、转运、定位以及体外自组装等生物学特性研究,为有关癌症治疗药物的研发提供参考。另外,以人博卡病毒(Human Bocavirus, HBoV-1)结构蛋白VP2为研究对象,构建VP2原核表达载体,亲和纯化VP2蛋白,用于多克隆抗体制备,用于人博卡病毒的快速检测和病毒蛋白亚细胞定位研究。完成的工作包括以下几个方面： 1.制备家蚕浓核病毒结构蛋白VP4与EGFP融合表达的荧光病毒样颗粒 1)PCR分别体外扩增家蚕浓核病毒结构蛋白基因vp4和绿色荧光蛋白egfp基因,通过酶切、连接和转化等步骤筛选重组克隆。将重组质粒转化大肠杆菌DH10Bac,在协助质粒的帮助下,构建含结构基因vp4和egfp基因融合表达的重组Bacmid。 2)重组Bacmid转染Sf9昆虫细胞表达产生重组杆状病毒,获得重组杆状病毒病毒液。以最适感染复数的病毒液感染hi5细胞以表达荧光病毒样颗粒EGFP-VP4fVLP,然后利用蔗糖垫层及CsCl梯度离心获得较纯净的fVLP,电镜观察。 2.表达并纯化HBoV-1结构蛋白VP2 1)PCR扩增HBoV结构蛋白基因vp2,并将其分别连接到pET-28a(+)和pMAL-c2x原核表达载体上,获得原核表达重组质粒pET-28a(+)-VP2和pMAL-c2x-VP2,将重组质粒分别转入相应表达宿主菌,检测目的蛋白VP2表达情况。 2)将不同分子伴侣导入重组表达菌,检测目的蛋白的可溶性及表达情况,利用亲和树脂纯化VP2蛋白,纯化获得的VP2蛋白应用于多克隆抗体的制备。
[Abstract]:Lead to human pathogens in the evolution of the great evolution of human mode of production and living environment is also accelerating the process of evolution. The emergence of new viruses and new diseases brought hitherto unknown challenge to human life and health and the traditional means of treatment of diseases, such as the 2003 rapidly popular in Asia the severe acute respiratory syndrome (SARS), 2009 the global influenza A virus (H1N1, H5N1) and Boka discovered in 2005, human parvovirus virus (HBoV). In many diseases threatening human health, malignant tumor (also called cancer) still occupy the main position, and there is no feasible treatment method.
The search for cancer treatment and feasible method, pay close attention to the medical and scientific community, people have been exploring some new methods of treatment. Compared with traditional cancer treatments (chemotherapy, radiotherapy) with serious side effects caused by the bio nanotechnology emerged in recent years in the treatment of cancer has shown a great the potential and advantage. Nano biotechnology is the use of some nano particles or biological materials similar to nano particles (such as virus like particles) as the carrier platform, to realize the specificity of the abnormal cells attack, in order to achieve the purpose of treating disease. At present, the virus like particles in cells or biological pathway the machine body and the mechanism of virus like particles in vitro assembly and re assembly, coupling and package molecules are characteristic of biological effects are still not clear.
This study is based on the research on parvovirus has been constructed in vitro of Bombyx mori Densovirus Iran that strains (BmDNV-1) fusion structure protein gene VP4 and enhanced green fluorescent protein expression vector of EGFP gene, eukaryotic expression system using recombinant baculovirus, preparation of fluorescent virus like particles (fluorescent virus-like, particle, fVLP) fluorescent virus. Like particles derived from carrying green fluorescent protein, can be used conveniently for the entry, transit, self-assembled on the biological characteristics of localization and in vitro, which provide the reference for the research on cancer treatment drugs. In addition, the Boka virus (Human Bocavirus HBoV-1) structural protein VP2 as the research object, construct VP2 the prokaryotic expression vector for VP2 protein, affinity purification, polyclonal antibody preparation, research of rapid detection and virus protein subcellular localization for Boka virus.
The work completed includes the following aspects:
1. preparation of fluorescent virus like particles fused by VP4 and EGFP in silkworm Bombyx mori
1) PCR respectively in vitro amplification of Bombyx mori densonucleosis virus structural protein VP4 Gene and green fluorescent protein EGFP gene by restriction enzyme digestion, ligation and transformation steps of screening recombinant cloning. The recombinant plasmid was transformed into Escherichia coli DH10Bac, to assist with the help of the recombinant plasmid, the expression of Bacmid. fusion gene containing VP4 and EGFP gene structure
2) the expression of recombinant Bacmid transfected Sf9 insect cells to produce recombinant baculovirus recombinant baculovirus virus. In order to liquid fluorescence expression of virus like particles of EGFP-VP4fVLP cells infected with hi5 virus was the optimal multiplicity of infection, and then using sucrose gradient and CsCl gradient centrifugation to obtain pure fVLP and electron microscopy.
2. expression and purification of HBoV-1 structural protein VP2
1) PCR amplified the HBoV structural protein gene VP2 and connected it to pET-28a (+) and pMAL-c2x prokaryotic expression vector respectively. The prokaryotic expression plasmid pET-28a (+) -VP2 and pMAL-c2x-VP2 were obtained. The recombinant plasmids were transferred into the corresponding host bacteria respectively, and the target protein VP2 status was detected.
2) introduce different molecular chaperones into recombinant expression bacteria, detect the solubility and expression of target protein, purify VP2 protein with affinity resin, and purify VP2 protein, and prepare polyclonal antibody.

【学位授予单位】：华中师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

【相似文献】