Rpf蛋白结构域的生物学及免疫学特性的初步研究
发布时间：2018-07-20 13:38
【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F3D10G6C8G10D5;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A9C8D3A5B8G11。 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)(P0.01),但不及BCG免疫组(3.50±0.23)(P0.05)。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(p0.01),但不及BCG免疫组(p0.05)。 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)(p0.05),但不如BCG免疫组(差值为2.83 log10)(p0.05)。 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。
[Abstract]:Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB). It is also one of the most important pathogenic and fatal factors in the world today. According to WHO, approximately 1/3 population in the world is in the state of MTB infection, and the number of TB patients in China is living in the world. The number of second people has reached 400 million. Only 10% of these people may eventually develop into active TB, and the overwhelming majority of them are recessive. The infected bacteria exist in the form of dormant bacteria. The drug resistance of this kind of dormant bacteria is very strong, it can exist in the body for a long time, and the conventional method is difficult to separate and culture. Bacille Calmette-Guerin (BCG) is M The TB strain is the only vaccine used for the prevention of TB, but it is ineffective for the recessive infection, and has the defects of short protection period and weak immune response. Therefore, the study of the pathogenesis and immune mechanism of MTB, the development of the more effective diagnosis, the treatment and prevention of MTB infection, especially the new methods, new measures and new vaccines against the recessive infection Significance.
In recent years, it is found that in the recovery process of MTB dormant bacteria, 5 kinds of Resuscitation promoting factor (Rpf) secreted by MTB have played an important role, which are Rv0867c (RpfA), Rv1009 (RpfB), Rv1884c (RpfC). Homology analysis found that Rpf exists in a variety of Gram-positive bacteria rich in G+C, and their gene encoded proteins have Rpf like domains. In the study, the domain of Rpf protein has a consistent biological characteristic with the complete Rpf protein. The study of the Rpf family also found that the Rpf like protein is not only related to the proliferation of bacteria, but also may be The target antigen identified by the host immune system.
In this study, we cloned, expressed and purified Rpf protein, Rpf domain protein and RpfB domain protein, respectively, and prepared a monoclonal antibody (monoclonal antibody, MAb) against the Rpf domain and anti RpfB domain of Micrococcus gonorrhoeae, and studied the biological function of Rpf, Rpf domain and RpfB domain protein of rattan Micrococcus. And immunological characteristics. Evaluate its application for MTB isolation and culture additives, the establishment of related antigen detection methods and the possibility of developing new TB vaccines. Objective: to express and purify Rpf, Rpf domain and RpfB domain protein of Micrococcus lutea, to prepare Rpf domain of Micrococcus Garcinia and to resist MAb in RpfB domain by the expression system of Escherichia coli. Further study of its biological and immunological characteristics.
Experimental methods and results:
1. clones of Rpf, Rpf domain and RpfB domain of Micrococcus luptois, expression and purification were amplified by PCR method from the genome of gonorrhoeae micrococcus and MTB H37Rv respectively, which were respectively amplified from Rpf, Rpf domain and RpfB domain target gene fragment respectively, and were cloned into pUC-19 vector and sequenced respectively, and the results were reported to GenBank reports. The target genes were cloned in human pProEX HTb expression vector respectively. After the enzyme digestion was identified, the target protein was induced by IPTG to express the target protein in E.coli. The 3 target proteins expressed by SDS-PAGE were in accordance with the expected molecular weight. The Western-blot analysis showed that the 3 fusion 6 * His target proteins could react with the anti 6 x His MAb specific.3. The target proteins were expressed in inclusion bodies, and 3 target proteins were purified by affinity chromatography under the condition of denaturation.
2. biological functions of Rpf, Rpf domain and RpfB domain of Micrococcus lutea
2.1 preparation and identification of RpfB domain MAb against Rpf domain of Micrococcus lutea
BALB/c mice were immunized with Rpf domain protein of Micrococcus Garcinia Micrococcus, and 3 hybridoma cell lines, which could secrete the Rpf domain MAb of anti Micrococcus, were obtained, named F3D10, G10D5, G6C8 respectively. F3D10, G10D5 were IgG1 subclasses and G6C8 were IgM subclasses. In white immunized BALB/c mice, 3 hybridoma cell lines, named D3A5, B8G11, A9C8, were named D3A5, B8G11, A9C8, which could stabilize the secretory anti RpfB domain MAb, which were D3A5, B8G11 as IgG1 subclass, A9C8 for IgM subclass, and its relative affinity was A9C8D3A5B8G11.
The pcDNA3.1 (-) -Rpf domain and the pcDNA3.1 (-) -RpfB domain eukaryotic expression vector were respectively constructed and transfected to COS-7 cells. The indirect immunofluorescence assay showed that the Rpf domain proteins and RpfB domain proteins were expressed in COS-7 cells, and the specificity of MAb was indirectly verified.
2.2 cross experiments against the Rpf domain and the anti RpfB domain MAb of the micrococcus aureus. Rpf, Rpf domain, RpfB domain, RpfA (the purified MTB RpfA protein in our laboratory, the plasmid is a foreign Mike Young Professor) and the two kinds of H37Ra as antigen and preparation, respectively. The results show that the two kinds of anti structural domains are all available. The above protein reacted with the H37Ra strain.
2.3 the recovery and growth of Micrococcus lutea Rpf, Rpf domain and RpfB domain protein to Micrococcus lutea and MTB H37Ra dormancy bacteria
After proper dilution of the dormant bacteria of Micrococcus Garcinia micrococcus and MTB H37Ra, they were randomly divided into 3 groups. Each group added the purified protein with different dilution concentration and the corresponding anti structural domain MAb to measure the OD600 value at different time points and draw the growth curve. The results showed that when the concentration of Rpf and Rpf in the Rpf and Rpf domains of the micrococcus Garcinia were 100pmol/L, the rattan was stimulated. The effect of Micrococcus aureus resuscitation and growth was obvious. When the concentration of Rpf was 10pmol/L and the concentration of Rpf domain was 100pmol/L, MTB H37Ra was stimulated to resuscitation and growth, and the stimulation was inhibited obviously after the MAb Rpf domain MAb was added to 1:600. When RpfB domain concentration was 1000pmol/L, the stimulus was stimulated. The effect of Micrococcus Luba Micrococcus resuscitation and growth was obvious. When the concentration of RpfB domain was 500pmol/L, the stimulation of MTB H37Ra resuscitation and growth was obvious, and the stimulation was obviously inhibited after 1:1000's RpfB domain MAb was added.
Immunological characteristics of Rpf, Rpf domain and RpfB domain of Micrococcus lutea 3.
Using the subcutaneous embedding method, Rpf, Rpf domain and RpfB domain protein were dripped to the micromass of the nitrocellulose membrane respectively. The mice were immunized for 3 times, each interval was 2 weeks, at the same time, the BCG immunization group and the normal saline control group were set up. The average titer of the specific antibody in the serum of the immune mice was detected by ELISA. The results showed: rattan yellow micro The titer of the highest antibody in the Rpf protein immunization group was 1:12800, the highest antibody titer of the Rpf domain protein immune group was 1:4800, and the highest antibody titer of the RpfB domain protein immune group was 1:6400..
In order to detect the cellular immune response caused by protein immunization in mice, the spleen lymphocytes of mice were separated after the last two weeks of immunization. The lymphocyte proliferation reaction was detected by MTT method after PPD stimulation in vitro. The stimulation index of Rpf, Rpf domain and RpfB domain protein in mice were 2.86 + 0.12,2 respectively. .10 + 0.09,2.40 + 0.11 was significantly higher than that in the normal saline control group (0.90 + 0.21) (P0.01), but less than the BCG immunization group (3.50 + 0.23) (P0.05). The average level of IFN- gamma, IL-10 and IL-12 induced by Rpf protein of Micrococcus Lutus Micrococcus were 1528 + 36ng/L, 485 + and 302 + 14ng. The average level of -12 was 1126 + 36ng/L, 368 + 13ng/L and 289 + 14ng/L, and IFN- gamma induced by RpfB domain protein, the average level of IL-10 and IL-12 was 1432 + 30ng/L, 503 + 11ng/L and 311 + 11ng/L, and 2022 +, 578 and 400 +, respectively, induced by BCG immunization group, respectively, and 578 + and 400 +. The levels of IFN- gamma, IL-10 and IL-12 were 256 + 6ng/L, 76 + 3ng/L and 56 + 4ng/L., respectively. The results showed that the level of cytokines induced by 3 protein immunization mice was significantly higher than that of normal saline control group (P0.01), but it was not as good as BCG immune group (P0.05).
In the fourth week after immunization, 105CFU MTB H37Rv strains were used to attack the mice in the above immunization groups and count the number of spleen bacterial loads. Compared with the normal saline control group, the Rpf, Rpf domain and RpfB domain protein immunization group had significant inhibitory effect on the proliferation of MTB in the spleen after the attack of H37Rv strain (the difference value). They were 1.89 log10,1.61 log10 and 1.78 log10) (P0.05), but not as BCG immunization group (2.83 log10) (P0.05).
Conclusion: the Rpf, Rpf domain and RpfB domain proteins of Micrococcus luberus all have the effect on promoting the recovery and growth of Micrococcus lubera and MTB dormant bacteria, which are expected to be used in the isolation and culture of clinical specimens, promote the recovery and growth of MTB dormant bacteria and improve the detection rate of the recessive infection, and the MAb of the two domains is not only obvious. The inhibition of the recovery and growth of the 3 proteins and the identification of a variety of Rpf proteins and their domains can be specifically identified. According to this, it is possible to establish a detection method for MTB related antigens; the specific immune responses induced by the 3 kinds of protein immunized animals have certain protective effects on the attack of MTB strains, and they may be used for the new epidemic of TB. The development of the seedlings.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
本文编号：2133720
[Abstract]:Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (MTB). It is also one of the most important pathogenic and fatal factors in the world today. According to WHO, approximately 1/3 population in the world is in the state of MTB infection, and the number of TB patients in China is living in the world. The number of second people has reached 400 million. Only 10% of these people may eventually develop into active TB, and the overwhelming majority of them are recessive. The infected bacteria exist in the form of dormant bacteria. The drug resistance of this kind of dormant bacteria is very strong, it can exist in the body for a long time, and the conventional method is difficult to separate and culture. Bacille Calmette-Guerin (BCG) is M The TB strain is the only vaccine used for the prevention of TB, but it is ineffective for the recessive infection, and has the defects of short protection period and weak immune response. Therefore, the study of the pathogenesis and immune mechanism of MTB, the development of the more effective diagnosis, the treatment and prevention of MTB infection, especially the new methods, new measures and new vaccines against the recessive infection Significance.
In recent years, it is found that in the recovery process of MTB dormant bacteria, 5 kinds of Resuscitation promoting factor (Rpf) secreted by MTB have played an important role, which are Rv0867c (RpfA), Rv1009 (RpfB), Rv1884c (RpfC). Homology analysis found that Rpf exists in a variety of Gram-positive bacteria rich in G+C, and their gene encoded proteins have Rpf like domains. In the study, the domain of Rpf protein has a consistent biological characteristic with the complete Rpf protein. The study of the Rpf family also found that the Rpf like protein is not only related to the proliferation of bacteria, but also may be The target antigen identified by the host immune system.
In this study, we cloned, expressed and purified Rpf protein, Rpf domain protein and RpfB domain protein, respectively, and prepared a monoclonal antibody (monoclonal antibody, MAb) against the Rpf domain and anti RpfB domain of Micrococcus gonorrhoeae, and studied the biological function of Rpf, Rpf domain and RpfB domain protein of rattan Micrococcus. And immunological characteristics. Evaluate its application for MTB isolation and culture additives, the establishment of related antigen detection methods and the possibility of developing new TB vaccines. Objective: to express and purify Rpf, Rpf domain and RpfB domain protein of Micrococcus lutea, to prepare Rpf domain of Micrococcus Garcinia and to resist MAb in RpfB domain by the expression system of Escherichia coli. Further study of its biological and immunological characteristics.
Experimental methods and results:
1. clones of Rpf, Rpf domain and RpfB domain of Micrococcus luptois, expression and purification were amplified by PCR method from the genome of gonorrhoeae micrococcus and MTB H37Rv respectively, which were respectively amplified from Rpf, Rpf domain and RpfB domain target gene fragment respectively, and were cloned into pUC-19 vector and sequenced respectively, and the results were reported to GenBank reports. The target genes were cloned in human pProEX HTb expression vector respectively. After the enzyme digestion was identified, the target protein was induced by IPTG to express the target protein in E.coli. The 3 target proteins expressed by SDS-PAGE were in accordance with the expected molecular weight. The Western-blot analysis showed that the 3 fusion 6 * His target proteins could react with the anti 6 x His MAb specific.3. The target proteins were expressed in inclusion bodies, and 3 target proteins were purified by affinity chromatography under the condition of denaturation.
2. biological functions of Rpf, Rpf domain and RpfB domain of Micrococcus lutea
2.1 preparation and identification of RpfB domain MAb against Rpf domain of Micrococcus lutea
BALB/c mice were immunized with Rpf domain protein of Micrococcus Garcinia Micrococcus, and 3 hybridoma cell lines, which could secrete the Rpf domain MAb of anti Micrococcus, were obtained, named F3D10, G10D5, G6C8 respectively. F3D10, G10D5 were IgG1 subclasses and G6C8 were IgM subclasses. In white immunized BALB/c mice, 3 hybridoma cell lines, named D3A5, B8G11, A9C8, were named D3A5, B8G11, A9C8, which could stabilize the secretory anti RpfB domain MAb, which were D3A5, B8G11 as IgG1 subclass, A9C8 for IgM subclass, and its relative affinity was A9C8D3A5B8G11.
The pcDNA3.1 (-) -Rpf domain and the pcDNA3.1 (-) -RpfB domain eukaryotic expression vector were respectively constructed and transfected to COS-7 cells. The indirect immunofluorescence assay showed that the Rpf domain proteins and RpfB domain proteins were expressed in COS-7 cells, and the specificity of MAb was indirectly verified.
2.2 cross experiments against the Rpf domain and the anti RpfB domain MAb of the micrococcus aureus. Rpf, Rpf domain, RpfB domain, RpfA (the purified MTB RpfA protein in our laboratory, the plasmid is a foreign Mike Young Professor) and the two kinds of H37Ra as antigen and preparation, respectively. The results show that the two kinds of anti structural domains are all available. The above protein reacted with the H37Ra strain.
2.3 the recovery and growth of Micrococcus lutea Rpf, Rpf domain and RpfB domain protein to Micrococcus lutea and MTB H37Ra dormancy bacteria
After proper dilution of the dormant bacteria of Micrococcus Garcinia micrococcus and MTB H37Ra, they were randomly divided into 3 groups. Each group added the purified protein with different dilution concentration and the corresponding anti structural domain MAb to measure the OD600 value at different time points and draw the growth curve. The results showed that when the concentration of Rpf and Rpf in the Rpf and Rpf domains of the micrococcus Garcinia were 100pmol/L, the rattan was stimulated. The effect of Micrococcus aureus resuscitation and growth was obvious. When the concentration of Rpf was 10pmol/L and the concentration of Rpf domain was 100pmol/L, MTB H37Ra was stimulated to resuscitation and growth, and the stimulation was inhibited obviously after the MAb Rpf domain MAb was added to 1:600. When RpfB domain concentration was 1000pmol/L, the stimulus was stimulated. The effect of Micrococcus Luba Micrococcus resuscitation and growth was obvious. When the concentration of RpfB domain was 500pmol/L, the stimulation of MTB H37Ra resuscitation and growth was obvious, and the stimulation was obviously inhibited after 1:1000's RpfB domain MAb was added.
Immunological characteristics of Rpf, Rpf domain and RpfB domain of Micrococcus lutea 3.
Using the subcutaneous embedding method, Rpf, Rpf domain and RpfB domain protein were dripped to the micromass of the nitrocellulose membrane respectively. The mice were immunized for 3 times, each interval was 2 weeks, at the same time, the BCG immunization group and the normal saline control group were set up. The average titer of the specific antibody in the serum of the immune mice was detected by ELISA. The results showed: rattan yellow micro The titer of the highest antibody in the Rpf protein immunization group was 1:12800, the highest antibody titer of the Rpf domain protein immune group was 1:4800, and the highest antibody titer of the RpfB domain protein immune group was 1:6400..
In order to detect the cellular immune response caused by protein immunization in mice, the spleen lymphocytes of mice were separated after the last two weeks of immunization. The lymphocyte proliferation reaction was detected by MTT method after PPD stimulation in vitro. The stimulation index of Rpf, Rpf domain and RpfB domain protein in mice were 2.86 + 0.12,2 respectively. .10 + 0.09,2.40 + 0.11 was significantly higher than that in the normal saline control group (0.90 + 0.21) (P0.01), but less than the BCG immunization group (3.50 + 0.23) (P0.05). The average level of IFN- gamma, IL-10 and IL-12 induced by Rpf protein of Micrococcus Lutus Micrococcus were 1528 + 36ng/L, 485 + and 302 + 14ng. The average level of -12 was 1126 + 36ng/L, 368 + 13ng/L and 289 + 14ng/L, and IFN- gamma induced by RpfB domain protein, the average level of IL-10 and IL-12 was 1432 + 30ng/L, 503 + 11ng/L and 311 + 11ng/L, and 2022 +, 578 and 400 +, respectively, induced by BCG immunization group, respectively, and 578 + and 400 +. The levels of IFN- gamma, IL-10 and IL-12 were 256 + 6ng/L, 76 + 3ng/L and 56 + 4ng/L., respectively. The results showed that the level of cytokines induced by 3 protein immunization mice was significantly higher than that of normal saline control group (P0.01), but it was not as good as BCG immune group (P0.05).
In the fourth week after immunization, 105CFU MTB H37Rv strains were used to attack the mice in the above immunization groups and count the number of spleen bacterial loads. Compared with the normal saline control group, the Rpf, Rpf domain and RpfB domain protein immunization group had significant inhibitory effect on the proliferation of MTB in the spleen after the attack of H37Rv strain (the difference value). They were 1.89 log10,1.61 log10 and 1.78 log10) (P0.05), but not as BCG immunization group (2.83 log10) (P0.05).
Conclusion: the Rpf, Rpf domain and RpfB domain proteins of Micrococcus luberus all have the effect on promoting the recovery and growth of Micrococcus lubera and MTB dormant bacteria, which are expected to be used in the isolation and culture of clinical specimens, promote the recovery and growth of MTB dormant bacteria and improve the detection rate of the recessive infection, and the MAb of the two domains is not only obvious. The inhibition of the recovery and growth of the 3 proteins and the identification of a variety of Rpf proteins and their domains can be specifically identified. According to this, it is possible to establish a detection method for MTB related antigens; the specific immune responses induced by the 3 kinds of protein immunized animals have certain protective effects on the attack of MTB strains, and they may be used for the new epidemic of TB. The development of the seedlings.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R392
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