【作者】 张宗德；

【导师】 王甦民； 李琦；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 免疫学， 2007， 博士

【摘要】 结核病是由结核分枝杆菌引起的传染病,全世界约有三分之一的人口被结核杆菌感染,每年约有300万人因此而死亡。结核病是单一致病菌引起死亡最主要原因。近年来耐多药结核菌株的快速出现以及卡介苗保护的不确定性,使得控制结核病的两条途径——治疗和预防捉襟见肘,因此,另辟蹊径来寻找新的抗结核药物和疫苗来摆脱目前的困境迫在眉睫。结核杆菌侵入人体后,为了适应体内的环境,会启动一系列基因的表达,这些基因有可能是结核杆菌逃避免疫系统攻击、侵入并在吞噬细胞内繁殖,进而引起疾病的关键基因。而体内诱导抗原技术(in vivo induced antigen technology,IVIAT)可以不通过动物模型直接筛选出病原体进入人体后才特异表达的基因,这些基因有可能作为候选的免疫及药物分子新靶位。本实验首先提取结核分枝杆菌(H37Rv)的基因组DNA,用Sau3A I酶切后回收片段插入到表达载体pET30a(+)、pET30b(+)和pET30c(+)中,构建H37Rv基因组表达文库。应用IVIAT的原理,先用IPTG诱导文库表达蛋白,然后用经体外培养的H37Rv菌株和大肠杆菌BL21(DE3)吸收过的结核病人血清对该文库进行初筛和两轮复筛,对筛选得到的阳性克隆提取质粒用T7启动子引物测序,测得的序列与结核杆菌基因组数据库比对后确定开放阅读框。选定PPE15基因为研究对象构建基因敲除载体,然后将PPE15基因敲除载体电转入H37Rv菌株,用卡那霉素和蔗糖进行筛选,用PCR初步鉴定PPE15基因敲除的菌株。本实验构建了H37Rv基因组表达文库,文库大小为4.3×104,文库重组质粒含有率大于80%,达到了理论要求。经过筛选、测序和比对本实验最终确定了51个开放阅读框。按照功能分类分为8类基因:毒力、解毒及调节相关基因1个,细胞壁与细胞处理相关基因13个,中间代谢和呼吸相关基因11个,脂类代谢基因7个,信号通路基因2个,PE/PPE蛋白家族基因3个,保守假设蛋白基因12个,与牛型分枝杆菌同源的保守假设蛋白基因2个。本实验成功构建了结核杆菌H37Rv PPE15基因的敲除载体(pJQ200-XylE∷ΔPPE15∷Km),并将该载体电转入到H37Rv菌株中,初步的PCR鉴定尚未得到PPE15基因突变菌株,目前该工作仍正在进行。总之,本研究应用体内诱导抗原技术(IVIAT)筛选到了一些结核杆菌进入人体后诱导表达的基因,并对基因的功能作了初步探讨,在找寻结核杆菌在人体内生存以及致病关键基因的道路上前进了一步,为结核病的预防和治疗分子靶位的研究积累了有价值的资料。更多还原

【Abstract】 Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, infects one-third of the world’s population and kills 3 million people each year. Tuberculosis is the largest cause of death caused by a single infectious organism. In recent years, the rapid development of multidrug resistant strains and the dubious protective efficacy of BCG made the two important strategies to control tuberculosis become ineffective. So it is very imminent to find new drugs and vaccines to deal with the crisis.After M.tb invades the human body, a series of bacterial genes will start up and express to adapt the internal environment. These genes may be the key ones to escape the immune attack, enter and multiply in the phagocyte, and cause the disease. In vivo induced antigen technology (IVIAT) provides a simple method for identifing genes specifically expressed in vivo, not depending on the animal model of infection. These genes are possible molecular targets of the immunological and therapeutic research.Genomic DNA from M.tb strain H37Rv was extracted. The DNA was partially digested with Sau3A I and the purified fragments were inserted into the pET30a(+), pET30b(+) and pET30c(+) expression vectors to construct the genomic library. The library was induced with IPTG and then was screened with pooled tuberculosis patient sera preabsorbed with in vitro grown M.tb strain H37Rv and Escherichia coli BL21(DE3). The inserts of positive clones were sequenced with primer T7 promoter. The sequences were aligned in the genomic database of M.tb strain H37Rv (http://genolist.pasteur.fr/Tuberculist) to identify the open reading frames. PPE15 gene knockout vector was constructed and electrotransformed into strain H37Rv. Transformants were selected with kanamycin and sucrose. The PPE15 mutagenesis strains were preliminarily identified using PCR.We constructed the genomic expression library of strain H37Rv. The library included 4.3×104 clones, and more than eighty percent were recombinated plasmids. The library had reached the theoretic requirement.Fifty-one open reading frames were obtained in this study including one virulence gene, thirteen cell wall and cell processes genes, eleven intermediary metabolism and respiration genes, seven lipid metabolism genes, two information pathways genes, three PE/PPE genes, twelve conserved hypotheticals and two conserved hypotheticals with an orthologue in M. bovis.We successfully constructed PPE15 gene knockout vector(pJQ200-XylE∷ΔPPE15∷Km)and electrotransformed it into strain H37Rv. Yet we haven’t obtained PPE15 mutagenesis strains, and this work is underway.In conclusion, this study identified some in vivo induced genes of M.tb by IVIAT approach and did some initial work about the gene function. We marched one step on the road of exploring the key genes which help M.tb survive in the body and result in tuberculosis. And the study also provided some new molecular targets data of prophylaxis and therapy of tuberculosis.更多还原