【作者】 步春玲；

【导师】 张林波；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 结核病（Tuberculosis,TB）为全球性传染病,呈逐年增高趋势,给人类带来极大的危害,人们称之为白色瘟疫。近年来,由于卡介苗（Bacillus Caimette Guerin,BCG）保护性不完善、耐药结核分枝杆菌（Mycobacterium tuberculosis,MTB）的流行、与人类免疫缺陷病毒（Hunan immunodeficiency virus,HIV）的协同感染以及人口流动等原因使结核病疫情再度出现新高。目前全球有超过20亿人感染过结核分枝杆菌,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。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。因此进行TB新型疫苗或增强BCG免疫效果以及特异、快速诊断试剂的研究具有重要意义。近十年来,人们对结核杆菌培养滤液中的蛋白进行了广泛的研究,并取得了令人鼓舞的结果。结核菌生长期间分泌的蛋白对结核病的保护性免疫是很重要的。国内外研究资料表明,结核分枝杆菌Rv3407基因含有300个碱基对的开放阅读框架（Open reading frame,ORF）,该ORF编码99个氨基酸,编码蛋白分子质量为10kDa,结核分枝杆菌Rv3407蛋白是高度保守的有保守序列的假定蛋白（Conserved hypothetical protein）,是结核杆菌生长期分泌的蛋白,具有细胞和体液免疫活性,国内还未见其结构和功能方面的研究,对其进行相关研究,可为结核疫苗的研制提供一定的实验依据。本研究在国内外学者研究的基础上,运用现代分子生物学技术克隆了结核分枝杆菌Rv3407基因序列,并在大肠杆菌中得到表达,目的在于研究结核分枝杆菌Rv3407蛋白基因的结构和功能,为研制有效的亚单位疫苗奠定基础。在本研究中,以结核分枝杆菌H₃₇Rv的染色体DNA为摸板,PCR扩增了Rv3407蛋白基因,选用pGEM-Tvector system成功克隆了分枝杆菌Rv3407蛋白基因,经酶切鉴定和PCR鉴定,证实了所克隆的基因与目的基因大小一致。通过序列测定及DNASTAR分析发现,克隆的Rv3407蛋白基因序列与Genbank上发表的结核分枝杆菌H₃₇Rv菌株Rv3407蛋白基因序列同源性为100%,氨基酸序列也完全一致,表明该基因在结核分枝杆菌中是高度保守的。进一步将克隆至pGEM-T中的Rv3407蛋白基因亚克隆至pGEX-4T-1表达系统中,构建了高效原核表达质粒,在与之相匹配的E.coliBL21（DE 3）中经IPTG诱导4h后,表达量达到了高峰。本研究结果将进一步研究结核分枝杆菌基因工程亚单位疫苗及核酸疫苗莫定一定的基础。更多还原

【Abstract】 Tuberculosis（TB）is a chronic respiratory infectious disease caused by pathogen Mycobacterium tuberculosis（MTB）.In recent years,factors such as the unsuccessful protection of BCG,increasing frequency of drug-resistance MTB,incidence of HIV-associated tuberculosis and increasement of population movement,have aggravated further the opportunity of people worldwide face to the threat of TB.Currently. MTB has infected approximately two billion individuals in total.TB remains one of the great killers,causing between 2 and 3 million deaths,and an estimated more than 8 million new infections a year.There are two main strategies for reducing the transmission rate:（1）rapid diagnosis and promptly treatment of active TB;（2）preventing TB occurrence with an effective vaccine.There fore,it is very important to develop new vaccine,or enhance BCG effection and diagnosis reagent for finding TB patient timely and specially.For the past ten years,people’s has conducted the extensive research to bacillus tuberculosis nutrient fluid proteinand has obtained the inspiring result.The tubercidin bacillus growth period secretion’s protein to tuberculosis’s protection immunity is very important.The domestic and foreign research material indicated that the tubercidin bacillus Rv3407 protein is highly conservative has the conservative sequence hypothesis protein（Conserved hypothetical protein）,is the bacillus tuberculosis vegetal period secretion protein,（99 aa） is composed of 300 bp,the code molecular is 10kDa,has the cell and humoral immunity activeness,domestic has not seen its structure and the function aspect research,research to it,may provide certain experiment basis for the tuberculosis vaccine development.This research aims to study foreign scholars on the basis of the introduction of modern molecular biology techniques to Mycobacterium Determination of Rv3407 gene sequence and expression in Escherichia coli as the main research objective is to study the molecular people Mycobacterium biological characteristics, In order to develop an effective set of DNA-based vaccines.In this study,Mycobacterium tuberculosis Rv3407 gene was amplified from M.tuberculosis H₃₇Rv genomic DNA using PCR and use pGEM-T vector system successfully cloned human Mycobacterium Rv3407 protein genes,the identification and digested PCR,confirmed the the cloned gene consistent with the size of the target gene.Sequencing and DNASTAR through analysis found that the cloned Rv3407 protein gene sequence with Genbank publishes of Human Mycobacterium H₃₇Rv strain Rv3407 protein gene sequence homology of 100 percent,exactly the same amino acid sequence also showed that the gene in Mycobacterium is highly conserved.Further cloned in the pGEM-T-Rv3407 protein gene was subcloned into pGEX-4T-1 expression system to construct a highly efficient prokaryotic expression plasmid,in which the expression plasmid match E.coliBL21（DE 3）by IPTG induction 4 h,the expression level reached a peak,so as to further research to it,human genetic engineering Mycobacterium subunit vaccine and DNA vaccine for a certain foundation.更多还原