结核分枝杆菌保护性抗原的表达、纯化及单克隆抗体的制备

发布时间：2019-04-11 15:22

【摘要】： 结核病(Tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)所致的、以呼吸系统感染为主的慢性传染病。全球目前约有20亿人感染了MTB,每年死于TB的人数高达200万。TB已成为人类的头号传染病杀手。我国目前至少有5亿人感染了MTB,每年死于TB的人数约有25万,是我国其他传染病和寄生虫病死亡人数总和的两倍多。卡介苗(Bacille Calmette-Guerin,BCG)接种目前仍然是预防TB的主要手段,但总体保护率不稳定,介于0%-80%。TB目前仍没有十分有效的早期诊断方法。随着MTB耐药株的出现与人类免疫缺陷病毒(Human immunodeficiency virus HIV)合并感染及人口流动增加等因素的出现,进一步加剧了TB对人类的威胁。因此,积极研究MTB致病机制及免疫机制,研制更加有效的诊断及治疗MTB感染的新方法、新技术、新疫苗就具有重大意义。分泌蛋白是MTB在对数生长早期分泌到菌体外的一类蛋白,也是目前确认的MTB蛋白中能够诱发保护性免疫的一类蛋白,Ag85B和ESAT6是其中重要的组成成分,广泛用于治疗TB的预防和诊断研究中。复活促进因子(Resuscitation promoting factor,RPF)最早是在藤黄微球菌(Micrococcus luteus)中发现的,它可以促进休眠期MTB的生长。RPFD以分泌形式表达,抗原性较强,因此,RPFD在TB的疫苗研制和快速诊断试剂方面有较大的应用潜力。实验目的: 构建表达MTB H37RV保护性抗原Ag85B、Ag85B-ESAT6和ESAT6-RPFD的原核表达载体,表达并纯化三种蛋白,并制备抗Ag85B蛋白的单克隆抗体(mAb)。实验方法和结果: 1.以MTB H37RV株基因组为模版,采用PCR方法分别扩增ag85B, esat6,rpfD基因,经测序证实与Genebank公布的序列完全一致。将目的基因片段亚克隆入pProEXHTb原核表达载体,酶切鉴定阳性重组质粒pPro-ag85B、pPro-ag85B-esat6和pPro-esat6-rpfD。用IPTG诱导表达Ag85B、Ag85B-ESAT6和ESAT6-RPFD三种蛋白。SDS-PAGE分析和Western Blot表明,成功地表达了Ag85B、Ag85B-ESAT6和ESAT6-RPFD蛋白。相对分子量分别为32 kD,43 kD和30 kD,与预计蛋白大小相符。用Ni-NTA亲和色谱柱变性条件下纯化获得三种重组蛋白,200 mL培养物可分别获得25.52 mg、17.16 mg和23.76 mg的纯化蛋白,得率分别为4.4%、3%和4.1%。 2.取纯化的Ag85B蛋白与弗氏不完全佐剂等体积混合,乳化。6周龄雌性BAL/c小鼠100μg/只背部皮下多点注射。间隔2周,共免疫三次。末次免疫两周后,选择血清效价高的小鼠再经腹腔加强免疫50μg/只。3天后取脾细胞与小鼠骨髓瘤细胞(Sp2/0)融合,加入铺有饲养细胞的96孔板。用HAT培养液培养1周左右,换成HT培养液用以筛选阳性融合细胞。获得杂交瘤细胞系1C11,2D5,3H3。经测定,这三株杂交瘤细胞系均为IgG1亚类。取效价最高的1C11杂交瘤细胞系注射BAL/c小鼠腹腔,抽取腹水,用硫酸铵法纯化腹水。采用Western Blot、间接免疫荧光法和ELISA法测定mAb为抗Ag85B的单克隆抗体,特异性检测显示mAb可与MTB发生特异性反应。
[Abstract]:Tuberculosis (Tuberculosis, TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), mainly respiratory infection. About 2 billion people around the world are infected with MTB, and up to 20 million people die from TB every year. TB has become the number one killer of infectious diseases in humans. At present, there are at least 500 million people infected with MTB, in China, about 250000 of them die from TB every year, which is more than double the total number of deaths of other infectious and parasitic diseases in China. BCG vaccination (Bacille Calmette-Guerin,BCG) is still the main method to prevent TB, but the overall protection rate is not stable. There is still no effective early diagnosis method for 0%-80%.TB. With the emergence of MTB-resistant strains and the co-infection of human immunodeficiency virus (Human immunodeficiency virus HIV) and the increase of population mobility, the threat of TB to human beings is further aggravated. Therefore, it is of great significance to study the pathogenesis and immune mechanism of MTB, to develop a more effective method for diagnosis and treatment of MTB infection, and to develop a new vaccine. Secretory protein is a class of proteins secreted by MTB in the early logarithmic growth phase, and it is also a class of proteins that can induce protective immunity in MTB proteins. Ag85B and ESAT6 are important components of these proteins. It is widely used in the prevention and diagnosis of TB. The resurrection promoter (Resuscitation promoting factor,RPF was first found in the (Micrococcus luteus) of Micrococcus vinifera. It can promote the growth of MTB during dormancy. RPFD is expressed in secretory form and has a strong antigenicity, therefore, the antigenicity of RPFD is stronger. RPFD has great potential in vaccine development and rapid diagnosis of TB. Objective: to construct prokaryotic expression vector expressing MTB H37RV protective antigen Ag85B,Ag85B-ESAT6 and ESAT6-RPFD, express and purify three proteins, and prepare monoclonal antibody (mAb). Against Ag85B protein. Experimental methods and results: 1. Using the genome of MTB H37RV strain as template, the ag85B, esat6,rpfD gene was amplified by PCR and confirmed to be identical with the published sequence of Genebank by sequencing. The target gene fragment was subcloned into the prokaryotic expression vector pProEXHTb, and the positive recombinant plasmids pPro-ag85B,pPro-ag85B-esat6 and pPro-esat6-rpfD. were identified by enzyme digestion. Three proteins, Ag85B,Ag85B-ESAT6 and ESAT6-RPFD, were induced by IPTG. SDS-PAGE analysis and Western Blot showed that Ag85B,Ag85B-ESAT6 and ESAT6-RPFD proteins were successfully expressed. The relative molecular weight was 32 kD,43 kD and 30 kD, respectively, which were consistent with the predicted protein size. Three recombinant proteins were purified by Ni-NTA affinity chromatography column denaturation. The purified proteins of 25.52 mg,17.16 mg and 23.76 mg were obtained by 200 mL culture, and the yields were 4.4%, 3% and 4.1%, respectively. 2. The purified Ag85B protein was mixed with Freund's incomplete adjuvant and emulsified. 6-week-old female BAL/c mice were injected subcutaneously with 100 渭 g / mouse. The mice were immunized three times at a interval of 2 weeks. Two weeks after the last immunization, mice with high serum titer were selected and then immunized with 50 渭 g / mouse intraperitoneally. After 3 days, spleen cells were fused with mouse myeloma cells (Sp2/0), and 96-well plates with feeder cells were added. The cells were cultured in HAT medium for one week or so, then replaced with HT medium for screening positive fusion cells. Hybridoma cell lines 1C11, 2D5, 3H3were obtained. The three hybridoma cell lines were identified as IgG1 subclass. The highest titer 1C11 hybridoma cell line was injected into the abdominal cavity of BAL/c mice, ascites was extracted and purified by ammonium sulfate method. Western Blot, indirect immunofluorescence assay and ELISA method were used to detect mAb as a monoclonal antibody against Ag85B. Specific detection showed that mAb could react specifically with MTB.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】