含结核分枝杆菌Ag85A基因的2型重组腺相关病毒的构建及其免疫原性研究

发布时间：2018-04-12 08:07

本文选题：结核分枝杆菌 + Ag85A　；参考：《第一军医大学》2007年硕士论文

【摘要】：近年来随着流动人口的增加、人类免疫缺陷病毒与结核分枝杆菌(Mycobacterium tuberculosis，Mtb)并发感染，以及Mtb多重耐药菌株的出现等原因，全球结核病(tuberculosis，TB)疫情再度出现新高。目前全球有超过20亿人感染过Mtb，每年新发TB病例超过800万，同时每年约有200～300万人死于TB，TB已成为世界上最严重的公共卫生问题之一。我国是世界上TB疫情非常严重的国家，发病率排名世界第二，占全球所有TB病例的15％左右，流行病学调查结果显示我国TB疫情相当严重，主要表现为结核菌感染人数多、现患肺结核病人多、结核病死亡人数多、农村结核病人多和传染性肺结核病疫情居高不下等特征。显然，要降低TB的发病率的就必须从根本上降低TB的传播速度，目前有效控制TB蔓延的主要策略有两方面：(1)活动性TB的快速诊断和及时治疗；(2)使用有效的疫苗阻止疾病的发生。因此，研制新型Mtb疫苗和寻求有效的治疗活动性TB的手段迫在眉睫。 Ag85A作为一种能激活机体产生细胞和体液免疫反应能力的Mtb分泌蛋白、2型重组腺相关病毒(recombinant adeno-associated virus type 2，rAAV-2)载体作为一种能有效转导多种组织和细胞的基因转移工具，均在各自的领域引起了人们的高度重视。为此，本研究以研制新型Mtb疫苗为目的，构建了表达Ag85A的rAAV-2(rAAV-2-Ag85A)，初步研究了其免疫原性，旨在为研制Mtb疫苗提供较为理想的候选株。采用PCR法，以结核杆菌H37Rv株基因组DNA为模板扩增Ag85A基因；将PCR扩增产物插入rAAV-2表达质粒pSNAV-2中，构建重组质粒pSNAV-Ag85A；用脂质体转染的方法将重组质粒转入BHK—21细胞中，G418筛选得到转入重组质粒并能表达目的基因细胞系BHK-Ag85A；用具有rAAV包装功能的重组单纯疱疹病毒感染BHK-Ag85A，纯化后得到rAAV-2-Ag85A；用高效液相色谱仪(HPLC)检测rAAV-2-Ag85A的纯度；以点杂交方法检测重组病毒的物理滴度；Western blot检测重组病毒在体外的表达。结果显示，，PCR扩增的序列与Gene Bank公布的Ag85A序列(AY732095)完全一致；分离纯化后得到的rAAV-2-Ag85A HPLC分析显示主峰为总面积大于99％；点杂交检测rAAV-2-Ag85A的物理滴度为2×10~(12) virusparticles／ml(vp／ml)；Western blot检测rAAV-2-Ag85A在体外感染的BHK-21细胞显示出一条32kD的杂交带，该杂交带的位置与DNAStar软件分析的结果相符。提示已经获得了在体外能有效地感染培养细胞的、能满足疫苗使用需要的高滴度、高纯度的rAAV-2-Ag85A，为下一步工作奠定了基础。本研究首次报道了rAAV-2载体介导Mtb抗原基因用于Mtb疫苗的研究。为观察rAAV-2-Ag85A在体内的转导效率，通过滴鼻(intranasal，in)的途径给予Balb／c小鼠5×10~(11) vp／ml的rAAV-2-Ag85A三周后，用RT-PCR扩增肺组织中Ag85A基因cDNA、Western blot检测肺组织中Ag85A的表达。分别通过肌肉注射和滴鼻途径用rAAV-2-Ag85A免疫Balb／C小鼠，ELISA法检测血清中抗—Ag85A抗体的滴度和mIFN-γ的含量，~(51)Cr释放分析检测细胞毒性T淋巴细胞(CTL)活性，以比较两种不同免疫途径的免疫效能。结果显示，RT-PCR可从小鼠肺组织扩增出一条约1Kb的条带，其长度与预期结果一致；Western Blotting检测到rAAV-2-Ag85A在肺组织的表达产物；无论是通过肌肉注射途径还是经鼻粘膜免疫，在加强免疫后的第10天就能刺激小鼠体内产生特异性抗体、激发抗原特异性CTL的出现、还可诱导机体产生Th1型细胞因子IFN-γ；除在个别时相点肌注免疫组CTL活性、IFN-γ的含量(均为加强免疫后第10天)明显高于滴鼻免疫组外，其它时相点两组的抗体滴度、CTL活性和IFN-γ的含量均无明显差异，且它们在两组中的变化趋势基本一致。表明rAAV-2不仅能介导外源性Ag85A基因转移到小鼠肺细胞，还能有效地转录和翻译。通过肌肉注射途径和经鼻粘膜免疫均可以诱导体液和细胞免疫反应的同时出现、刺激机体产生IFN-γ。说明除了通过刺激机体产生保护性抗体而具有预防Mtb感染的功能之外，rAAV-2-Ag85A也能通过增强宿主Th1型免疫反应而对TB有一定免疫治疗作用，因而作为TB的治疗性疫苗可能也同样具有潜在的应用价值。rAAV-2-Ag85A通过鼻粘膜免疫可以取得与通过肌肉注射免疫基本相当的免疫效果，有成为粘膜免疫候选疫苗的可能。研究的结果提示rAAV-2-Ag85A有可能作为初免疫苗或初免—加强疫苗以防止结核分枝杆菌感染，甚至是作为结核病的治疗性疫苗均具有潜在的应用价值，值得进一步深入研究。
[Abstract]:In recent years, with the increase of floating population, human immunodeficiency virus and Mycobacterium tuberculosis (Mycobacterium tuberculosis, Mtb) and Mtb infection, the emergence of multi drug resistant strains and other reasons, the global TB epidemic (tuberculosis, TB) again high. At present there are more than 2 billion people worldwide infected with Mtb, TB more than 8 million new cases every year. At the same time every year there are about 200~300 million people died of TB, TB has become one of the most serious public health problems in the world. China is the world's TB epidemic severely, the incidence rate ranked second in the world, accounting for about 15% of all TB cases worldwide, epidemiological survey results show that China's TB epidemic is very serious, mainly for the number of patients infected with Mycobacterium tuberculosis, pulmonary tuberculosis, TB deaths, rural tuberculosis patients and the infectious tuberculosis epidemic characteristics such as high. Obviously, to To reduce the incidence of TB must be reduced TB transmission speed fundamentally, the effective control strategy of TB spread mainly has two aspects: (1) the activity of the TB rapid diagnosis and timely treatment; (2) the use of effective vaccines to prevent the disease. Therefore, the development of new vaccines for TB and Mtb treatment of active and effective means is imminent.
Ag85A is a kind of organism can activate cellular and humoral immune response ability of Mtb protein, recombinant adeno-associated virus serotype 2 (recombinant adeno-associated virus type 2, rAAV-2) as a carrier can efficiently transduce a variety of tissues and cells of the gene transfer tools have attracted the attention of people in their respective fields. Therefore, this study to develop a novel Mtb vaccine for the purpose of constructing the expression of Ag85A rAAV-2 (rAAV-2-Ag85A), a preliminary study on the immunogenicity of Mtb vaccine, so as to provide the ideal candidate strains.
Using PCR method to Mycobacterium tuberculosis H37Rv genomic DNA as template to amplify Ag85A gene; PCR gene fragment was inserted into rAAV-2 expression plasmid pSNAV- to construct recombinant plasmid pSNAV-Ag85A; 2, by transfecting the recombinant plasmid transfected into BHK 21 cells, G418 was screened into recombinant plasmid and gene expression of BHK-Ag85A cells. With rAAV; packaging function of recombinant herpes simplex virus infection BHK-Ag85A, purified by rAAV-2-Ag85A; using high-performance liquid chromatography (HPLC) to detect the purity of rAAV-2-Ag85A recombinant virus titer detection; physical by dot blot; detection of recombinant virus Western blot expression in vitro. The results showed that the Ag85A sequence of Gene was amplified by PCR and Bank the released (AY732095) completely consistent; purified rAAV-2-Ag85A HPLC analysis showed that the peak to the total area of more than 99% points; miscellaneous The detection of rAAV-2-Ag85A physical titer was 2 * 10~ (12) virusparticles / ml (VP / ml); Western blot in detection of rAAV-2-Ag85A infection in vitro BHK-21 cells showed a hybridization with 32kD, analysis of the hybrid zone position and DNAStar software results. That has obtained can effectively infect cultured cells in vitro, the vaccine can meet the need of high titer, high purity rAAV-2-Ag85A, which laid the foundation for the next work.
This is the first report of rAAV-2 vector mediated Mtb antigen gene for Mtb vaccine research. In order to observe the rAAV-2-Ag85A in vivo transduction efficiency by intranasal (intranasal, in) way of giving the Balb / c mice 5 * 10~ (11) after three weeks of VP / ml rAAV-2-Ag85A, increased Ag85A gene expression in pulmonary tissue cDNA with the RT-PCR expansion, Ag85A to detect the expression of Western in lung tissue of blot. Respectively by intramuscular injection and intranasal immunization with rAAV-2-Ag85A Balb / C mice. The content of anti Ag85A antibody titer and mIFN- gamma in serum were measured by ELISA, ~ (51) detection of cytotoxic T lymphocyte (CTL) activity, the release of Cr the immune efficacy of two different immune pathways. The results showed that the RT-PCR from the lung tissue of mice was amplified. A band of 1Kb, the result is consistent with the expected length; Western Blotting detected the expression of rAAV-2-Ag85A in lung tissue of both products; By intramuscular injection or nasal mucosal immunity in tenth days after immunization can stimulate mice to produce specific antibody, appear to stimulate antigen-specific CTL, also can induce the production of Th1 type cytokines IFN-; except in the individual time points by intramuscular injection of CTL activity, the content of IFN- (gamma for tenth days following immunization) was significantly higher than that of intranasal immunization group, other time point antibody titer was two, there were no significant difference between the content of CTL and activity of IFN- gamma, and their change trend in the two groups are basically the same. Results show that rAAV-2 not only can mediate exogenous Ag85A gene transfer to the lung cells in mice, but also effectively transcription and translation. By intramuscular injection and intranasal immunization could induce humoral and cellular immune responses and induce IFN- gamma. In addition to stimulate the body to produce protective antibody With the function of preventing Mtb infection, rAAV-2-Ag85A can also enhance the host immune response to Th1 and has a certain therapeutic effect on immune TB, as TB therapeutic vaccine may also have the application value of.RAAV-2-Ag85A potential by nasal mucosal immunity can be achieved by intramuscular injection of immune and very basic immune effect, has become the mucous membrane the immune vaccine candidates. The results of the study suggest that rAAV-2-Ag85A may be used as primary immunization vaccine or primary immunization - strengthening vaccine to prevent infection with Mycobacterium tuberculosis, tuberculosis or even as a therapeutic vaccine has a potential application value, worthy of further study.

【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

【参考文献】