【摘要】： 結(jié)核病(Tuberculosis,TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)所致的以呼吸系統(tǒng)感染為主的慢性傳染病,也是現(xiàn)今全世界范圍內(nèi)最重要的致病和致死因素之一。據(jù)WHO估計(jì),目前全世界約有1/3人口處于MTB感染狀態(tài),我國TB患者數(shù)量居世界第二位,感染人數(shù)已達(dá)4億。這部分人中只有10%可能最終發(fā)展成為活動(dòng)性TB,而絕大多數(shù)為隱性感染,感染的細(xì)菌以休眠菌的形式存在。這種休眠菌耐藥性極強(qiáng),可在體內(nèi)長期存在,常規(guī)方法難于分離培養(yǎng)�？ń槊�(Bacille Calmette-Guerin,BCG)是MTB的減毒株,是目前唯一用于TB預(yù)防的疫苗,但其對(duì)隱性感染者無效,還存在保護(hù)期短,免疫應(yīng)答較弱等缺陷。因此,研究MTB的致病及免疫機(jī)制,研制更為有效的診斷、治療和預(yù)防MTB感染,特別是針對(duì)隱性感染的新方法、新措施及新疫苗等具有重要意義。 近年來研究發(fā)現(xiàn),在MTB休眠菌的復(fù)蘇過程中,MTB分泌的5種促進(jìn)復(fù)活因子(Resuscitation promoting factor,Rpf)起了重要作用,分別為Rv0867c(RpfA),Rv1009(RpfB),Rv1884c(RpfC),Rv2389c(RpfD)和Rv2450c(RpfE)。Rpf最早在藤黃微球菌(Micrococcus luteus,M.luteus)中發(fā)現(xiàn)。通過同源性分析發(fā)現(xiàn),Rpf存在于多種富含G+C的革蘭陽性細(xì)菌中,其基因編碼的蛋白均具有Rpf樣結(jié)構(gòu)域。在研究中發(fā)現(xiàn),Rpf蛋白的結(jié)構(gòu)域與完整Rpf蛋白具有一致的生物學(xué)特性。對(duì)Rpf家族的研究還發(fā)現(xiàn),Rpf樣蛋白不僅與細(xì)菌的增殖有關(guān),還可能是宿主免疫系統(tǒng)識(shí)別的靶抗原。 在本研究中,我們分別克隆、表達(dá)和純化了藤黃微球菌Rpf蛋白、Rpf結(jié)構(gòu)域蛋白、RpfB結(jié)構(gòu)域蛋白;制備了抗藤黃微球菌Rpf結(jié)構(gòu)域和抗RpfB結(jié)構(gòu)域的單克隆抗體(monoclonal antibody,MAb);研究了藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白的生物學(xué)功能和免疫學(xué)特性。評(píng)價(jià)其用于MTB分離培養(yǎng)添加劑、相關(guān)抗原檢測(cè)方法建立及TB新型疫苗研制的可能性。實(shí)驗(yàn)?zāi)康?利用大腸桿菌表達(dá)系統(tǒng)表達(dá)并純化藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白;制備抗藤黃微球菌Rpf結(jié)構(gòu)域、抗RpfB結(jié)構(gòu)域的MAb;進(jìn)一步研究其生物學(xué)功能和免疫學(xué)特性。 實(shí)驗(yàn)方法和結(jié)果: 1.藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域的克隆、表達(dá)與純化采用PCR方法分別從藤黃微球菌和MTB H37Rv的基因組中分別擴(kuò)增出相應(yīng)大小的藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域目的基因片段,并分別克隆入pUC-19載體中測(cè)序,結(jié)果與GenBank報(bào)道的完全一致。將目的基因分別克隆人pProEX HTb表達(dá)載體中,酶切鑒定后,分別在E.coli中由IPTG誘導(dǎo)表達(dá)目的蛋白。經(jīng)SDS-PAGE分析,表達(dá)的3種目的蛋白與預(yù)期分子量一致;Western-blot分析表明3種融合6×His的目的蛋白均可與抗6×His MAb特異反應(yīng)。3種目的蛋白均以包涵體的形式表達(dá),用親和色譜法在變性條件下分別純化3種目的蛋白。 2.藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域的生物學(xué)功能研究 2.1抗藤黃微球菌Rpf結(jié)構(gòu)域、抗RpfB結(jié)構(gòu)域MAb的制備及鑒定 用藤黃微球菌Rpf結(jié)構(gòu)域蛋白分別免疫BALB/c小鼠,進(jìn)行細(xì)胞融合,獲得了3株能穩(wěn)定分泌抗藤黃微球菌Rpf結(jié)構(gòu)域MAb的雜交瘤細(xì)胞系,分別命名為F3D10、G10D5、G6C8,其中F3D10、G10D5為IgG1亞類,G6C8為IgM亞類,其相對(duì)親和力為F3D10G6C8G10D5;用RpfB結(jié)構(gòu)域蛋白免疫BALB/c小鼠,進(jìn)行細(xì)胞融合,獲得了3株能穩(wěn)定分泌抗RpfB結(jié)構(gòu)域MAb的雜交瘤細(xì)胞系,分別命名為D3A5、B8G11、A9C8,其中D3A5、B8G11為IgG1亞類,A9C8為IgM亞類,其相對(duì)親和力為A9C8D3A5B8G11。 分別構(gòu)建了pcDNA3.1(-)-Rpf結(jié)構(gòu)域及pcDNA3.1(-)-RpfB結(jié)構(gòu)域真核表達(dá)載體,轉(zhuǎn)染COS-7細(xì)胞,用間接免疫熒光法檢測(cè)表明藤黃微球菌Rpf結(jié)構(gòu)域蛋白和RpfB結(jié)構(gòu)域蛋白在COS-7細(xì)胞中有表達(dá),也間接驗(yàn)證了MAb的特異性。 2.2抗藤黃微球菌Rpf結(jié)構(gòu)域及抗RpfB結(jié)構(gòu)域MAb的交叉實(shí)驗(yàn)。分別用藤黃微球菌Rpf、Rpf結(jié)構(gòu)域、RpfB結(jié)構(gòu)域、RpfA(本實(shí)驗(yàn)室純化的MTB RpfA蛋白,質(zhì)粒為國外Mike Young教授饋贈(zèng))及H37Ra作為抗原與制備的兩種MAb反應(yīng),結(jié)果顯示:兩種抗結(jié)構(gòu)域MAb均可以與以上蛋白和H37Ra菌株發(fā)生反應(yīng)。 2.3藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白促藤黃微球菌及MTB H37Ra休眠菌的復(fù)蘇和生長作用 取藤黃微球菌和MTB H37Ra的休眠菌適當(dāng)稀釋后,隨機(jī)分為3組。每組分別加入不同稀釋濃度的純化蛋白及相應(yīng)的抗結(jié)構(gòu)域MAb,取不同時(shí)間點(diǎn)的培養(yǎng)液測(cè)OD600值,繪制生長曲線。結(jié)果顯示:當(dāng)藤黃微球菌Rpf和Rpf結(jié)構(gòu)域濃度均為100pmol/L時(shí),刺激藤黃微球菌復(fù)蘇和生長作用明顯,當(dāng)藤黃微球菌Rpf濃度為10pmol/L、Rpf結(jié)構(gòu)域濃度為100pmol/L時(shí),刺激MTB H37Ra復(fù)蘇和生長作用明顯,而且這種刺激作用在加入了1:600的抗藤黃微球菌Rpf結(jié)構(gòu)域MAb后明顯被抑制;當(dāng)RpfB結(jié)構(gòu)域濃度為1000pmol/L時(shí),刺激藤黃微球菌復(fù)蘇和生長作用明顯,當(dāng)RpfB結(jié)構(gòu)域濃度為500pmol/L時(shí),刺激MTB H37Ra復(fù)蘇和生長作用明顯,而且這種刺激作用在加入了1:1000的抗RpfB結(jié)構(gòu)域MAb后明顯被抑制。 3.藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域的免疫學(xué)特性研究 采用皮下包埋的方法,分別將藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白滴到硝酸纖維素膜小塊上,免疫小鼠3次,每次間隔2周,同時(shí)設(shè)BCG免疫組和生理鹽水對(duì)照組。用ELISA方法檢測(cè)免疫小鼠血清中的特異性抗體平均滴度。結(jié)果顯示:藤黃微球菌Rpf蛋白免疫組最高抗體效價(jià)為1:12800,藤黃微球菌Rpf結(jié)構(gòu)域蛋白免疫組最高抗體效價(jià)為1:4800,RpfB結(jié)構(gòu)域蛋白免疫組最高抗體效價(jià)為1:6400。 為了檢測(cè)蛋白免疫小鼠引起的細(xì)胞免疫應(yīng)答,最后一次免疫完成兩周后,分離各免疫組小鼠脾淋巴細(xì)胞,在體外經(jīng)PPD刺激后,MTT法檢測(cè)淋巴細(xì)胞增殖反應(yīng),藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白免疫小鼠后的刺激指數(shù)分別為:2.86±0.12,2.10±0.09,2.40±0.11,明顯高于生理鹽水對(duì)照組(0.90±0.21)(P0.01),但不及BCG免疫組(3.50±0.23)(P0.05)。藤黃微球菌Rpf蛋白誘導(dǎo)產(chǎn)生的IFN-γ,IL-10和IL-12平均水平分別為1528±36ng/L,485±13ng/L和302±14ng/L;藤黃微球菌Rpf結(jié)構(gòu)域蛋白誘導(dǎo)產(chǎn)生的IFN-γ,IL-10和IL-12平均水平分別為1126±36ng/L,368±13ng/L和289±14ng/L;RpfB結(jié)構(gòu)域蛋白誘導(dǎo)產(chǎn)生的IFN-γ,IL-10和IL-12平均水平分別為1432±30ng/L,503±11ng/L和311±11ng/L;BCG免疫組誘導(dǎo)產(chǎn)生的IFN-γ,IL-10和IL-12平均水平分別為2022±38ng/L,578±13ng/L和400±10ng/L;生理鹽水對(duì)照組IFN-γ,IL-10和IL-12水平分別為256±6ng/L,76±3ng/L和56±4ng/L。從結(jié)果可以看出,3種蛋白免疫小鼠后誘導(dǎo)產(chǎn)生的細(xì)胞因子水平明顯高于生理鹽水對(duì)照組(p0.01),但不及BCG免疫組(p0.05)。 免疫完成后第四周,用105CFU MTB H37Rv毒株經(jīng)尾靜脈攻擊上述各免疫組小鼠,計(jì)數(shù)脾臟細(xì)菌負(fù)荷數(shù)。與生理鹽水對(duì)照組相比,藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白免疫組小鼠對(duì)H37Rv毒株攻擊后MTB在脾臟中的增殖均有顯著抑制作用(差值分別為1.89 log10、1.61 log10和1.78 log10)(p0.05),但不如BCG免疫組(差值為2.83 log10)(p0.05)。 結(jié)論:藤黃微球菌Rpf、Rpf結(jié)構(gòu)域和RpfB結(jié)構(gòu)域蛋白均具有促進(jìn)藤黃微球菌和MTB休眠菌復(fù)蘇和生長的作用,有望用于臨床標(biāo)本分離培養(yǎng)的添加劑,促進(jìn)MTB休眠菌的復(fù)蘇和生長,從而提高隱性感染者的檢出率;抗兩種結(jié)構(gòu)域的MAb不僅可以明顯抑制3種蛋白的促復(fù)蘇和生長作用,而且可以特異性的識(shí)別多種Rpf蛋白及其結(jié)構(gòu)域,據(jù)此,有可能建立MTB相關(guān)抗原的檢測(cè)方法;3種蛋白免疫動(dòng)物后誘導(dǎo)產(chǎn)生的特異性免疫應(yīng)答,對(duì)MTB毒株的攻擊具有一定的保護(hù)力,它們有可能用于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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392

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