【作者】 刘建利；

【导师】 靳亚平；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2009， 硕士

【摘要】 结核病(Tuberculosis,TB)是由结核分支杆菌(Mycobacterium tuberculosis,MTB)所致以呼吸系统感染为主的慢性传染病。唯一的预防疫苗卡介苗(bacille calmetteguerin, BCG)保护性不完善,需有效的疫苗来控制TB的传播与蔓延。结核分枝杆菌亚单位疫苗的研究有望解决卡介苗预防结核效果不稳定的问题。研究发现MTB培养上清滤液蛋白(culture filtrate protein, CFP)中Ag85B和ESAT6均能刺激机体产生保护性免疫反应,是机体抗结核感染的有效靶抗原。因此,本试验通过构建融合表达Ag85B-ESAT6的原核表达载体,获得Ag85B-ESAT6融合蛋白,测定分析该蛋白在小鼠体内的免疫学特性和抵抗MTB感染的保护力。1.采用PCR方法分别从MTB H37Rv株DNA基因组中扩增Ag85B和ESAT6基因,在ESAT6基因上游引入48bp的柔性linker结构,确保两蛋白的正确折叠。将各目的片段克隆至pMD18-T载体中进行测序。鉴定阳性重组质粒pMD18-T-Ag85B和pMD18-T-ESAT6,经测序证实与Genebank公布的基因序列完全一致。将Ag85B和ESAT6基因亚克隆入pPROEXHTa原核表达载体,酶切鉴定阳性重组质粒pPROEXHTa-Ag85B-ESAT6,并用IPTG进行诱导表达。SDS-PAGE检测和Western-blot鉴定表明,在相对分子量为45 kD的位置有特异性目的蛋白表达。采用Ni-NTA亲和色谱柱在变性条件下纯化得到了Ag85B-ESAT6融合蛋白。2.用Ag85B-ESAT6原核表达融合蛋白免疫BALB/c小鼠,用ELISA检测证明,免疫小鼠血清中的特异性抗体滴度达到1∶12 800。将BALB/c小鼠用MTB毒株H37Rv经尾静脉注射感染。4周后,脱颈处死小鼠,无菌条件下进行脾脏和肺脏匀浆,倍比稀释后接种于7H10平板,37℃培养3～4周,计数细菌菌落形成单位(cloning forming units,CFU),以观察免疫小鼠对MTB毒株攻击的抵抗作用。结果表明,与生理盐水组相比,试验组和BCG组对MTB在脾脏和肺脏中的增殖均有抵抗作用(P <0.05);但与BCG组相比,试验组对MTB在脾脏和肺脏中增殖的抵抗作用不如BCG组(P >0.05)。在大肠杆菌表达系统中成功表达并纯化出Ag85B-ESAT6融合蛋白,该蛋白能够分别与Ag85B和ESAT6的mAb发生特异性反应,表明具有一定的生物活性。小鼠免疫结果显示:Ag85B-ESAT6能诱发一定水平的细胞免疫应答和体液免疫应答,并产生一定的免疫保护力,可以抵御MTB的感染。为进一步研究Ag85B-ESAT6疫苗的免疫学特性及与其他疫苗的优势组合,用于TB新型疫苗的开发提供了实验数据和物质基础。更多还原

【Abstract】 Tuberculosis (TB) is a chronic infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). BCG is currently the only vaccine against TB, but it shows little efficacy on the pulmonary TB of adults. So, developing an efficient new kind of vaccine is important to against TB. The study about subunit vaccine of MTB may solve the problem that using BCG to prevent TB is unstable. Both Ag85B and ESAT6 in CFP of MTB were reported efficient targent antigen against TB, that could stimulate protective immune response. In our study, we have constructed prokaryotic recombinant plasmid expressing Ag85B and ESAT6 fusion protein of MTB and obtained pure protein. After immunize mice, the immunological properties and protective efficacy of fusion protein against MTB infection are evaluateed. The results shown as follows:1. Ag85B and ESAT6 genes were amplified by polymerase chain reaction(PCR) from DNA genome of Mycobacterium tuberculosis H37Rv strain of MTB, and a flexible chain with 48bp was linked between Ag85B and ESAT6 genes, in order to ensure the correct folding of the fusion protein. The two genes were further cloned into pMD18-T respectively to analysis the DNA sequence. Identified the positive recombinant plasmid pMD18-T-Ag85B and pMD18-T-ESAT6, then we analyzed the DNA sequence which were identical with that reported by GenBank. The results confirmed that we had inserted the right sequence of linker successfully. Subsequently, Ag85B and ESAT6 genes were subcloned into the prokaryotic expression vector pPRO-EXHTa. The positive recombinant plasmid pPROEXHTa -Ag85B-ESAT6 was digested with restrictive enzyme and induced with IPTG. The analysis of SDS-PAGE showed that there was a specific protein expression brand at 45kD molecular mass, and the fusion protein was further identified by Western-blot. It mainly existed in inclusion body, and the fusion protein was purified by affinity chromatograph of Ni-NTA column.2. BALB/c mice were immunized subcutaneously three times at 2-week interval, by embedded in fold groin with recombinant fused protein loaded to nitrocellulose filter. The control groups were given BCG and saline respectively. The specific antibody titers in immune groups were detected by ELISA. The results showed that the antibody titers of Ag85B-ESAT6 increased up to 1:12800. BALB/c mice were challenged i.v. with H37Rv of MTB strain. Four weeks later, separated the lung and spleens from the mice aseptically. The tissue were homogenated and multiple diluted, then inoculated into 7H10 plate. They were cultivated 3 to 4 weeks at 37℃. Subsequently, we counted the cloning forming units (CFU) to observe the resist of MTB strain for immuned mice. The results indicated that than experimental group and the BCG group resisted the proliferative of MTB in spleens and lungs compared with the saline group(P <0.05). Nevertheless, the BCG group was better the experimental group in resisting the proliferative of MTB in spleens and lungs(P >0.05).Ag85B-ESAT6 fusion protein was expressed and purified in coli successfully. This protein can develop specific reaction with mAb of Ag85B and ESAT6. It indicated that this protein had some biological activity. Ag85B-ESAT6 fusion protein could induce cellular immunologic response and humoral immunoresponse in the mice, and resisted MTB proliferation in the lungs and spleens effectively. It provides some new clues for researching prevention and treatment of tuberculosis.更多还原