【作者】 樊爱琳；

【导师】 徐志凯； 郝晓柯； 师长宏；

【作者基本信息】 第四军医大学 ， 微生物学， 2008， 博士

【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,也是现今全世界范围内最重要的致病和致死因素之一。据WHO估计,目前全世界约有1/3人口处于MTB感染状态,我国TB患者数量居世界第二位,感染人数已达4亿。这部分人中只有10%可能最终发展成为活动性TB,而绝大多数为隐性感染,感染的细菌以休眠菌的形式存在。这种休眠菌耐药性极强,可在体内长期存在,常规方法难于分离培养。卡介苗(Bacille Calmette-Guerin,BCG)是MTB的减毒株,是目前唯一用于TB预防的疫苗,但其对隐性感染者无效,还存在保护期短,免疫应答较弱等缺陷。因此,研究MTB的致病及免疫机制,研制更为有效的诊断、治疗和预防MTB感染,特别是针对隐性感染的新方法、新措施及新疫苗等具有重要意义。近年来研究发现,在MTB休眠菌的复苏过程中,MTB分泌的5种促进复活因子(Resuscitation promoting factor,Rpf)起了重要作用,分别为Rv0867c(RpfA),Rv1009(RpfB),Rv1884c(RpfC),Rv2389c(RpfD)和Rv2450c(RpfE)。Rpf最早在藤黄微球菌(Micrococcus luteus,M.luteus)中发现。通过同源性分析发现,Rpf存在于多种富含G+C的革兰阳性细菌中,其基因编码的蛋白均具有Rpf样结构域。在研究中发现,Rpf蛋白的结构域与完整Rpf蛋白具有一致的生物学特性。对Rpf家族的研究还发现,Rpf样蛋白不仅与细菌的增殖有关,还可能是宿主免疫系统识别的靶抗原。在本研究中,我们分别克隆、表达和纯化了藤黄微球菌Rpf蛋白、Rpf结构域蛋白、RpfB结构域蛋白;制备了抗藤黄微球菌Rpf结构域和抗RpfB结构域的单克隆抗体(monoclonal antibody,MAb);研究了藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白的生物学功能和免疫学特性。评价其用于MTB分离培养添加剂、相关抗原检测方法建立及TB新型疫苗研制的可能性。实验目的:利用大肠杆菌表达系统表达并纯化藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白;制备抗藤黄微球菌Rpf结构域、抗RpfB结构域的MAb;进一步研究其生物学功能和免疫学特性。实验方法和结果:1.藤黄微球菌Rpf、Rpf结构域和RpfB结构域的克隆、表达与纯化采用PCR方法分别从藤黄微球菌和MTB H37Rv的基因组中分别扩增出相应大小的藤黄微球菌Rpf、Rpf结构域和RpfB结构域目的基因片段,并分别克隆入pUC-19载体中测序,结果与GenBank报道的完全一致。将目的基因分别克隆人pProEX HTb表达载体中,酶切鉴定后,分别在E.coli中由IPTG诱导表达目的蛋白。经SDS-PAGE分析,表达的3种目的蛋白与预期分子量一致;Western-blot分析表明3种融合6×His的目的蛋白均可与抗6×His MAb特异反应。3种目的蛋白均以包涵体的形式表达,用亲和色谱法在变性条件下分别纯化3种目的蛋白。2.藤黄微球菌Rpf、Rpf结构域和RpfB结构域的生物学功能研究2.1抗藤黄微球菌Rpf结构域、抗RpfB结构域MAb的制备及鉴定用藤黄微球菌Rpf结构域蛋白分别免疫BALB/c小鼠,进行细胞融合,获得了3株能稳定分泌抗藤黄微球菌Rpf结构域MAb的杂交瘤细胞系,分别命名为F3D10、G10D5、G6C8,其中F3D10、G10D5为IgG1亚类,G6C8为IgM亚类,其相对亲和力为F3D10>G6C8>G10D5;用RpfB结构域蛋白免疫BALB/c小鼠,进行细胞融合,获得了3株能稳定分泌抗RpfB结构域MAb的杂交瘤细胞系,分别命名为D3A5、B8G11、A9C8,其中D3A5、B8G11为IgG1亚类,A9C8为IgM亚类,其相对亲和力为A9C8>D3A5>B8G11。分别构建了pcDNA3.1(-)-Rpf结构域及pcDNA3.1(-)-RpfB结构域真核表达载体,转染COS-7细胞,用间接免疫荧光法检测表明藤黄微球菌Rpf结构域蛋白和RpfB结构域蛋白在COS-7细胞中有表达,也间接验证了MAb的特异性。2.2抗藤黄微球菌Rpf结构域及抗RpfB结构域MAb的交叉实验。分别用藤黄微球菌Rpf、Rpf结构域、RpfB结构域、RpfA(本实验室纯化的MTB RpfA蛋白,质粒为国外Mike Young教授馈赠)及H37Ra作为抗原与制备的两种MAb反应,结果显示:两种抗结构域MAb均可以与以上蛋白和H37Ra菌株发生反应。2.3藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白促藤黄微球菌及MTB H37Ra休眠菌的复苏和生长作用取藤黄微球菌和MTB H37Ra的休眠菌适当稀释后,随机分为3组。每组分别加入不同稀释浓度的纯化蛋白及相应的抗结构域MAb,取不同时间点的培养液测OD600值,绘制生长曲线。结果显示:当藤黄微球菌Rpf和Rpf结构域浓度均为100pmol/L时,刺激藤黄微球菌复苏和生长作用明显,当藤黄微球菌Rpf浓度为10pmol/L、Rpf结构域浓度为100pmol/L时,刺激MTB H37Ra复苏和生长作用明显,而且这种刺激作用在加入了1:600的抗藤黄微球菌Rpf结构域MAb后明显被抑制;当RpfB结构域浓度为1000pmol/L时,刺激藤黄微球菌复苏和生长作用明显,当RpfB结构域浓度为500pmol/L时,刺激MTB H37Ra复苏和生长作用明显,而且这种刺激作用在加入了1:1000的抗RpfB结构域MAb后明显被抑制。3.藤黄微球菌Rpf、Rpf结构域和RpfB结构域的免疫学特性研究采用皮下包埋的方法,分别将藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白滴到硝酸纤维素膜小块上,免疫小鼠3次,每次间隔2周,同时设BCG免疫组和生理盐水对照组。用ELISA方法检测免疫小鼠血清中的特异性抗体平均滴度。结果显示:藤黄微球菌Rpf蛋白免疫组最高抗体效价为1:12800,藤黄微球菌Rpf结构域蛋白免疫组最高抗体效价为1:4800,RpfB结构域蛋白免疫组最高抗体效价为1:6400。为了检测蛋白免疫小鼠引起的细胞免疫应答,最后一次免疫完成两周后,分离各免疫组小鼠脾淋巴细胞,在体外经PPD刺激后,MTT法检测淋巴细胞增殖反应,藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白免疫小鼠后的刺激指数分别为:2.86±0.12,2.10±0.09,2.40±0.11,明显高于生理盐水对照组(0.90±0.21)(P<0.01),但不及BCG免疫组(3.50±0.23)(P<0.05)。藤黄微球菌Rpf蛋白诱导产生的IFN-γ,IL-10和IL-12平均水平分别为1528±36ng/L,485±13ng/L和302±14ng/L;藤黄微球菌Rpf结构域蛋白诱导产生的IFN-γ,IL-10和IL-12平均水平分别为1126±36ng/L,368±13ng/L和289±14ng/L;RpfB结构域蛋白诱导产生的IFN-γ,IL-10和IL-12平均水平分别为1432±30ng/L,503±11ng/L和311±11ng/L;BCG免疫组诱导产生的IFN-γ,IL-10和IL-12平均水平分别为2022±38ng/L,578±13ng/L和400±10ng/L;生理盐水对照组IFN-γ,IL-10和IL-12水平分别为256±6ng/L,76±3ng/L和56±4ng/L。从结果可以看出,3种蛋白免疫小鼠后诱导产生的细胞因子水平明显高于生理盐水对照组(p<0.01),但不及BCG免疫组(p<0.05)。免疫完成后第四周,用105CFU MTB H37Rv毒株经尾静脉攻击上述各免疫组小鼠,计数脾脏细菌负荷数。与生理盐水对照组相比,藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白免疫组小鼠对H37Rv毒株攻击后MTB在脾脏中的增殖均有显著抑制作用(差值分别为1.89 log10、1.61 log10和1.78 log10)(p<0.05),但不如BCG免疫组(差值为2.83 log10)(p<0.05)。结论:藤黄微球菌Rpf、Rpf结构域和RpfB结构域蛋白均具有促进藤黄微球菌和MTB休眠菌复苏和生长的作用,有望用于临床标本分离培养的添加剂,促进MTB休眠菌的复苏和生长,从而提高隐性感染者的检出率;抗两种结构域的MAb不仅可以明显抑制3种蛋白的促复苏和生长作用,而且可以特异性的识别多种Rpf蛋白及其结构域,据此,有可能建立MTB相关抗原的检测方法;3种蛋白免疫动物后诱导产生的特异性免疫应答,对MTB毒株的攻击具有一定的保护力,它们有可能用于TB新型疫苗的研制。更多还原

【Abstract】 Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). It has been one of the most important contributory and mortiferous factors in the world. It is estimated by WHO that one-third of the world population are infected with MTB. In China, the number of TB patients was the second in the world, approximately 400 million people infected. 10% of these people will develop to activity TB, and the most of infected MTB exist in condition of dormant infection. This dormant MTB has strong drug resistance, it can exist in vivo for a long time, and it’s difficult to isolate and cultivate. The current vaccine against MTB, M. bovis Bacillus Calmette-Guérin(BCG)has many problems such as ineffective to subclinical infection,short period of protect and low immune response. Current preventive measures and diagnosis methods have no effect on MTB dormant. So it is great significant for studying new diagnosis reagents, therapy methods and vaccines to prevent TB, especially for dormant infection.Recent studies showed that the resuscitation promoting factor(Rpf) which secreted by MTB played a important role in promoting the resuscitation and growth of dormant mycobacterium, Rv0867c(RpfA),Rv1009(RpfB),Rv1884c(RpfC),Rv2389c(RpfD)and Rv2450c(RpfE). Rpf was first discovered in Micrococcus luteus(M.luteus). Homology analysis suggests that Rpf-like protein widely distribute among high G+C Gram-positive bacteria. It has been found the domain of Rpf has the same biological characteristics as Rpf-like protein. It also has been demonstrated that Rpf-like protein can not only stimulate the resuscitation of stationary-phase mycobacterial, including M. bovis BCG and MTB, but also a target antigen of immune system of host. In this study, we cloned, expressed and purified M.luteus Rpf protein, M.luteus Rpf domain protein and RpfB domain protein, generated MAb of M.luteus Rpf domain and RpfB domain. Furthermore, we studied the biological function and immunological characteristics of them, in order to evaluate the possibility of using them to rapidly isolate and cultivate MTB, detect related antigen, and become candidate vaccines.AIM: To express and purify M.luteus Rpf protein, Rpf domain and RpfB domain.To generate MAb of M.luteus Rpf domain and RpfB domain .To study the biological and immunological properties of M.luteus Rpf, Rpf domain and RpfB domain.METHODS AND RESULTS:1. Cloning, expression and purification of M.luteus Rpf protein, Rpf domain and RpfB domainThe genes of M.luteus Rpf, Rpf domain and RpfB domain were amplified respectively from genome of M.luteus and MTB H37Rv strain by using polymerase chain reaction(PCR). Inserted the PCR product into pUC-19 vector accordingly and sequenced. The DNA sequence of M.luteus Rpf, Rpf domain and RpfB domain were identical with that published by GenBank. After sequencing was confirmed, Subcloned the three right sequenced genes into pPRO-EXHT prokaryotic expression vector . The recombinant clones were screened restriction enzyme analysis and transformed into E.coli DH5αstrain and induced with IPTG. The analysis of SDS-PAGE showed that there were specific protein expressions at the corresponding positions as we anticipated. The three proteins were further identified by Western-blot using anti-6×His MAb.Purified theproteins by Ni-NTA purification system under denaturing conditions.2. Biological Activity Of M.luteus Rpf protein, Rpf domain and RpfB domain2.1 Construction and identification of MAbs to M.luteus Rpf domain and RpfB domainWe obtained 3 hybridoma cell strains which could stablely secrete antibodies adainst M.luteus, named F3D10,G10D5 and G6C8 from BALB/c mice immunized with. The subclass of F3D10 and G10D5 is IgG1,while the subclass of G6C8 is IgM. The relative affinity of the MAbs are different and the sequence is A9C8>D3A5>B8G11.We also obtained 3 hybridoma cell strains which could stablely secrete antibodies adainst RpfB domain ,named D3A5,B8G11 and A9C8. The subclass of D3A5 and B8G11 is IgG1,while the subclass of A9C8 is IgM. The relative affinity sequence of the MAbs is A9C8>D3A5>B8G11.In order to identify the specificity of the MAb, we constructed eukaryotic expression vector pcDNA3.1(-)-Rpf domain and pcDNA3.1 (-)-RpfB domain, and expressed in COS-7 cells. The specificity of the MAb was confirmed with indirect immunofluorescence.2.2 Cross test of anti-M.luteus Rpf domain and anti-RpfB domain.Cross test of the two constructed MAbs were detected by ELISA, using Rpf, Rpf domain, RpfB domain, and other Rpf proteins of MTB as antigen to invest culture plate respectively and the two constructed MAbs to be the first antibody. The results showed both MAbs have reaction with all above proteins.2.3 The promotive action of resuscitation and growth of M.luteus Rpf, Rpf domain and RpfB domain on M.luteus and MTBH37Ra and M.luteus dormant strains were at a suitable dilution using culture medium, and then divided into 3 groups randomly. Different concentration proteins were added to each group respectively. Taken 200μl from each group at different time to detected the OD600. The results showed that Rpf protein and Rpf domain could significantly stimulate the growth of M.luteus at concentration of 100pmol/L, while they stimulated the growth of MTB H37Rv notably at concentration of 10pmol/L in Rpf protein and 100pmol/L in Rpf domain. This stimulation was significantly inhibited after adding 1:600 anti- M.luteus Rpf domain MAb. When the concentration of RpfB domain was 1000pmol/L, stimulation to M.luteus was significant, and When its concentration was 500pmol/L, stimulation to MTB H37Rv was also significant. The stimulation was inhibited after adding 1:1000 anti- RpfB domain MAb.3. Immunological activity of M.luteus Rpf protein, Rpf domain and RpfB domainTo test immunological properties of the proteins, BALB/c mice were immunized three times at 2-week interval subcutaneously on their backs with Rpf , Rpf domain and RpfB domain proteins respectively, which were transferred to NC membranes beforehand. The titers of specific antibody in BALB/c mice were detected by ELISA. The results suggested average titers of anti-Rpf was 1:12800, that of Rpf domain was 1:4800, and that of RpfB domain was 1:6400.To evaluate cell-mediated immune response, stimulating index(SI) of the spleen lymphocytes in immunized mice were measured by MTT method, the SI of Rpf , Rpf domain and RpfB domain were significantly higher(which were 2.86±0.12 for Rpf, 2.10±0.09 for Rpf domain, 2.40±0.11 for RpfB domain than that of normal saline controls ( P<0.05 ) , but lower than that of BCG groups(3.50±0.23)(P<0.05).The levels of IFN-γ, IL-10 and IL-12 secretion stimulated by specific antigen were detected by indirect ELISA. IFN-γ, IL-10 and IL-12 concentrations in cultured supernatant of spleen lymphocytes from mice immunized with Rpf protein were 1528±36 ng/L, 485±13 ng/L and 302±14 ng/L respectively. The levels of three cytokines mentioned above stimulated by Rpf domain protein were 1126±36 ng/L, 368±13 g/L and 289±14 ng/L respectively, while thats of RpfB domain protein were 1432±30 ng/L, 503±11 ng/L, and 311±11 ng/L respectively. In group immunized by BCG, the average levels of IFN-γ,IL-10 and IL-12 were 2022±38 ng/L, 578±13 ng/L and 400±10 ng/L respectively; while in normal saline groups, the average levels of IFN-γ,IL-10 and IL-12 were 256±6 ng/L, 76±3 ng/L and 56±4 ng/L. From above results, we found the levels of three cytokines immunized by three proteins were significantly higher than that of normal saline groups(p<0.01), but lower than that of BCG groups. (p<0.05).In order to test our three proteins’efficacies against MTB H37Rv, BALB/c mice were intravenously infected with 105 CFU MTB H37Rv. four weeks after the final immunization, the numbers of MTB CFU in spleens were determined. Compared with the normal saline groups, Bacteria loads in the three protein groups were dramatic reduction(differences were 1.89 log10, 1.61 log10 and 1.78 log10 respectively,P<0.05). But the protective efficacies of mice immunized with proteins were lower than that of BCG vaccination group(2.83 Log10) (p<0.05).CONCLUSION:M.luteus Rpf, Rpf domain and RpfB domain proteins could stimulate the resuscitation and growth of M.luteus dormant and MTB H37Ra dormant. MAb of the two domain could significantly inhibited the effectiveness of growth promotion and specifically identified many kinds of Rpf and Rpf domain. They also could induce high level cell-mediated immune reponse and confer effective protection against TB. So they may be an appropriate candidate for culture medium additives which promote growth of dormant MTB in order to elevate detection rate of subclinical patients. We can establish detection methods on MTB related antigen according to our results. At the same time, they may be new candidate for vaccine to TB control and prophylaxis.更多还原