结核分枝杆菌体内表达基因的筛选研究

发布时间：2018-05-28 19:07

本文选题：结核分枝杆菌 + 体内诱导抗原技术(IVIAT)　；参考：《北京市结核病胸部肿瘤研究所》2007年博士论文

【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。
[Abstract]:Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis. About 1/3 people in the world are infected with TB, and about 3 million people die every year. Tuberculosis is the leading cause of death caused by a single pathogen. In recent years, the rapid emergence of multidrug-resistant tuberculous bacilli and the uncertainty of the protection of BCG vaccine make the treatment and prevention of tuberculosis overstretched. It is urgent to find new anti-TB drugs and vaccines to get out of the current predicament. Mycobacterium tuberculosis invades the human body, in order to adapt to the internal environment, will start a series of genes expression, these genes may be the tuberculosis bacillus escape the immune system attack, invades and propagates in the phagocyte, then causes the disease the key gene. However, in vivo induced antigen technique, vivo induced antigen technology IVIATA, can directly screen genes specifically expressed after pathogens enter human body without animal model. These genes may be used as candidate immunological and drug molecular new targets. In this experiment, the genomic DNA of Mycobacterium tuberculosis H37Rv) was extracted and inserted into the expression vectors pET30a (pET30b () and pET30c() to construct the genomic expression library of H37Rv. Using the principle of IVIAT, we first used IPTG to induce the expression of protein in the library, then screened the library with the sera of TB patients absorbed by H37Rv strain and Escherichia coli BL21DE3 in vitro. The extracted plasmid was sequenced by T7 promoter primer. The sequence was compared with the genome database of Mycobacterium tuberculosis and the open reading frame was determined. The PPE15 gene was selected as the research object to construct the gene knockout vector. Then the PPE15 gene knockout vector was transferred into H37Rv strain and screened with kanamycin and sucrose. The PPE15 gene knockout strain was preliminarily identified by PCR. The H37Rv genomic expression library was constructed in this experiment. The size of the library was 4.3 脳 10 4. The recombinant plasmid contained more than 80% of the recombinant plasmid, which met the theoretical requirements. After screening, sequencing and comparison, 51 open reading boxes were determined. According to the functional classification, there were 8 kinds of genes: virulence, detoxification and regulation related genes, cell wall and cell treatment related genes, intermediate metabolism and respiration related genes, lipid metabolism genes. There were 2 PEP / PPE family genes, 12 conserved protein genes and 2 conserved protein genes homologous to Mycobacterium bovis. In this experiment, the knockout vector pJQ200-XylE: 螖 PPE15: KMN of Mycobacterium tuberculosis H37Rv PPE15 gene was successfully constructed, and the vector was electrotransferred into H37Rv strain. The preliminary PCR identification has not yet obtained the mutant strain of PPE15 gene, and the work is still under way. In conclusion, some genes induced by Mycobacterium tuberculosis after entering human body were screened by IVIAT-induced antigen technique in vivo, and the function of the genes was preliminarily discussed. It is a step forward to search for the key genes of TB in human body and to accumulate valuable data for the study of molecular targets for prevention and treatment of tuberculosis.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R378

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