發(fā)布時間：2018-04-12 08:07

  本文選題：結核分枝桿菌 + Ag85A��； 參考：《第一軍醫(yī)大學》2007年碩士論文

【摘要】：近年來隨著流動人口的增加、人類免疫缺陷病毒與結核分枝桿菌(Mycobacterium tuberculosis，Mtb)并發(fā)感染，以及Mtb多重耐藥菌株的出現(xiàn)等原因，全球結核病(tuberculosis，TB)疫情再度出現(xiàn)新高。目前全球有超過20億人感染過Mtb，每年新發(fā)TB病例超過800萬，同時每年約有200～300萬人死于TB，TB已成為世界上最嚴重的公共衛(wèi)生問題之一。我國是世界上TB疫情非常嚴重的國家，發(fā)病率排名世界第二，占全球所有TB病例的15％左右，流行病學調查結果顯示我國TB疫情相當嚴重，主要表現(xiàn)為結核菌感染人數(shù)多、現(xiàn)患肺結核病人多、結核病死亡人數(shù)多、農村結核病人多和傳染性肺結核病疫情居高不下等特征。顯然，要降低TB的發(fā)病率的就必須從根本上降低TB的傳播速度，目前有效控制TB蔓延的主要策略有兩方面：(1)活動性TB的快速診斷和及時治療；(2)使用有效的疫苗阻止疾病的發(fā)生。因此，研制新型Mtb疫苗和尋求有效的治療活動性TB的手段迫在眉睫。 Ag85A作為一種能激活機體產生細胞和體液免疫反應能力的Mtb分泌蛋白、2型重組腺相關病毒(recombinant adeno-associated virus type 2，rAAV-2)載體作為一種能有效轉導多種組織和細胞的基因轉移工具，均在各自的領域引起了人們的高度重視。為此，本研究以研制新型Mtb疫苗為目的，構建了表達Ag85A的rAAV-2(rAAV-2-Ag85A)，初步研究了其免疫原性，旨在為研制Mtb疫苗提供較為理想的候選株。 采用PCR法，以結核桿菌H37Rv株基因組DNA為模板擴增Ag85A基因；將PCR擴增產物插入rAAV-2表達質粒pSNAV-2中，構建重組質粒pSNAV-Ag85A；用脂質體轉染的方法將重組質粒轉入BHK—21細胞中，G418篩選得到轉入重組質粒并能表達目的基因細胞系BHK-Ag85A；用具有rAAV包裝功能的重組單純皰疹病毒感染BHK-Ag85A，純化后得到rAAV-2-Ag85A；用高效液相色譜儀(HPLC)檢測rAAV-2-Ag85A的純度；以點雜交方法檢測重組病毒的物理滴度；Western blot檢測重組病毒在體外的表達。結果顯示，，PCR擴增的序列與Gene Bank公布的Ag85A序列(AY732095)完全一致；分離純化后得到的rAAV-2-Ag85A HPLC分析顯示主峰為總面積大于99％；點雜交檢測rAAV-2-Ag85A的物理滴度為2×10~(12) virusparticles／ml(vp／ml)；Western blot檢測rAAV-2-Ag85A在體外感染的BHK-21細胞顯示出一條32kD的雜交帶，該雜交帶的位置與DNAStar軟件分析的結果相符。提示已經獲得了在體外能有效地感染培養(yǎng)細胞的、能滿足疫苗使用需要的高滴度、高純度的rAAV-2-Ag85A，為下一步工作奠定了基礎。 本研究首次報道了rAAV-2載體介導Mtb抗原基因用于Mtb疫苗的研究。為觀察rAAV-2-Ag85A在體內的轉導效率，通過滴鼻(intranasal，in)的途徑給予Balb／c小鼠5×10~(11) vp／ml的rAAV-2-Ag85A三周后，用RT-PCR擴增肺組織中Ag85A基因cDNA、Western blot檢測肺組織中Ag85A的表達。分別通過肌肉注射和滴鼻途徑用rAAV-2-Ag85A免疫Balb／C小鼠，ELISA法檢測血清中抗—Ag85A抗體的滴度和mIFN-γ的含量，~(51)Cr釋放分析檢測細胞毒性T淋巴細胞(CTL)活性，以比較兩種不同免疫途徑的免疫效能。結果顯示，RT-PCR可從小鼠肺組織擴增出一條約1Kb的條帶，其長度與預期結果一致；Western Blotting檢測到rAAV-2-Ag85A在肺組織的表達產物；無論是通過肌肉注射途徑還是經鼻粘膜免疫，在加強免疫后的第10天就能刺激小鼠體內產生特異性抗體、激發(fā)抗原特異性CTL的出現(xiàn)、還可誘導機體產生Th1型細胞因子IFN-γ；除在個別時相點肌注免疫組CTL活性、IFN-γ的含量(均為加強免疫后第10天)明顯高于滴鼻免疫組外，其它時相點兩組的抗體滴度、CTL活性和IFN-γ的含量均無明顯差異，且它們在兩組中的變化趨勢基本一致。表明rAAV-2不僅能介導外源性Ag85A基因轉移到小鼠肺細胞，還能有效地轉錄和翻譯。通過肌肉注射途徑和經鼻粘膜免疫均可以誘導體液和細胞免疫反應的同時出現(xiàn)、刺激機體產生IFN-γ。說明除了通過刺激機體產生保護性抗體而具有預防Mtb感染的功能之外，rAAV-2-Ag85A也能通過增強宿主Th1型免疫反應而對TB有一定免疫治療作用，因而作為TB的治療性疫苗可能也同樣具有潛在的應用價值。rAAV-2-Ag85A通過鼻粘膜免疫可以取得與通過肌肉注射免疫基本相當?shù)拿庖咝Ч�，有成為粘膜免疫候選疫苗的可能。研究的結果提示rAAV-2-Ag85A有可能作為初免疫苗或初免—加強疫苗以防止結核分枝桿菌感染，甚至是作為結核病的治療性疫苗均具有潛在的應用價值，值得進一步深入研究。
[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.

【學位授予單位】：第一軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

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