【作者】 刘双全；

【导师】 汪世平；

【作者基本信息】 中南大学 ， 微生物学， 2010， 博士

【摘要】 明确梅毒螺旋体(Treponema pallidum, TP)的致病因子及进行TP致病机制的研究,将为研制TP抗感染疫苗和开发TP感染治疗新药提供科学依据,是防治本病的关键环节。然而,迄今为止,由于TP体外人工培养尚未成功,抗原获取困难,限制了对TP的深入研究,以致TP的致病机制目前尚未被阐明。炎症和持续的获得性免疫应答被认为是造成TP感染后机体病理损伤的主要原因。研究表明,TP膜蛋白是介导炎症反应的主要成分,可能为TP的主要致病因子,因此对TP膜蛋白的研究是认识其对宿主的致病性和进行致病机制研究的关键。但是对于具体是哪些TP膜蛋白在发挥这方面的作用报道甚少,因而有必要进行TP膜蛋白的致病性研究和筛选,这对深入研究TP的致病机制至关重要。Tp0751为近年来新发现的一种TP主要的黏附膜蛋白,其主要介导TP与宿主细胞的结合而与TP入侵宿主有关,是机体较早接触的TP膜蛋白之一。那么其在TP致病过程中是否还具有其他的作用呢?譬如,其是否具有细胞毒性?其是否能够诱导宿主免疫细胞产生前炎症反应而与介导机体的炎症反应有关呢?这些均值得深入探索和研究。本研究试图通过探讨Tp0751黏附蛋白的免疫活性、细胞毒性及是否能诱导THP-1细胞产生前炎症细胞因子(CKs)TNF-α、IL-1β和IL-6,并初步研究其诱导CKs产生的信号传导通路,为进一步探索Tp0751黏附蛋白在TP感染免疫中的作用及进行梅毒的致病机制研究奠定重要的基础。通过生物信息学分析,去除Tp0751信号肽序列,以TP Nichols株的基因组DNA为模板,PCR扩增Tp0751基因；通过BamHI和EcoRI酶切位点将Tp0751基因克隆进pET-28a(+)质粒中构建重组质粒,筛选阳性质粒,酶切鉴定和PCR鉴定,经测序鉴定的重组质粒转化至表达宿主菌ER2566中构建原核重组表达体；进行诱导表达,Ni亲合层析柱纯化重组蛋白,BCA法测定蛋白浓度。Western blot检测其免疫反应性；用重组蛋白免疫新西兰兔,检测兔免疫血清中多克隆抗体的效价,评价其免疫原性。利用Detoxi-Gel内毒素去除胶去除重组蛋白中的内毒素；将重组蛋白经皮下注入新西兰兔大腿内侧,观察注射部位皮肤变化；佛波酯(Phorbol 12-myristate 13-acetate, PMA)诱导THP-I细胞转化为巨噬细胞,将经内毒素去除处理后的Tp0751重组蛋白分别刺激巨噬细胞,通过检测细胞的乳酸脱氢酶(lactate dehydrogenase, LDH)漏出率和NO释放量研究其细胞毒性。为研究Tp0751诱导炎症反应作用,以去内毒素的Tp0751蛋白刺激THP-1细胞,检测其产生前炎症细胞因子(CKs)TNF-α、IL-1β和IL-6的情况,同时用LPS做阳性对照和PBS为阴性对照组。为了探讨诱导CKs产生的信号传导通路是否与TLR2、CD14、MAPKs和NF-κB有关,分别用TLR2抗体、CD14抗体、PD98059(ERK1/2特异性抑制剂)、SP600125 (SAPK/JNK特异性抑制剂)SB203580(p38特异性抑制剂)和PDTC (NF-κB特异性抑制剂)预处理THP-1细胞,然后再用Tp0751刺激细胞,检测细胞因子的产生情况；Western blot检测Tp0751刺激THP-1细胞后细胞MAPKs磷酸化水平和NF-κB的活化情况。PCR扩增得到一大小约为600 bp的目的基因片断(Tp0751去信号肽片段,Tp0751基因全长度为764 bp,编码255个氨基酸)；构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片断为Tp0751目的基因,测序结果与Genbank上登录序列完全一致；SDS-PAGE分析显示,在IPTG诱导下,重组工程菌表达了一相对分子量(Mr)约为26kDa的目的蛋白条带,目的蛋白在菌体细胞内主要以可溶性形式存在;经Ni-NTA亲和纯化获得了纯度在95%以上的重组蛋白,BCA法测得纯化蛋白浓度为1.2 mg/mLWestern blot检测其能与梅毒阳性血清发生特异性反应；利用纯化的Tp0751重组蛋白免疫新西兰兔,间接ELISA法测定兔免疫血清特异性抗体效价到第四次免疫时达到顶峰,其抗体滴度在1:10 240以上。重组蛋白能诱发新西兰兔产生迟发型超敏反应(delayed type hypersensitivity, DTH),注射部位出现明显红肿,红肿部位皮肤温度比对侧相应部位高,于注射后6-1Od红肿消退,生理盐水对照部位未见反应；经Tp0751重组蛋白刺激后的巨噬细胞LDH漏出率和NO释放量较低,和6-His-Tag蛋白作用组比较,差异无统计学意义,与蛋白刺激浓度无关。Tp0751重组蛋白刺激THP-1细胞后,其能以剂量和时间依赖方式诱导THP-1细胞产生TNF-α、IL-1β和IL-6,诱导THP-1细胞产生TNF-α、IL-1β和IL-6的最适Tp0751刺激浓度为5μg/mL,Tp0751刺激细胞6h后即可从培养基中检测到TNF-α、IL-1β和IL-6,随着刺激时间的延长炎症因子的产生量逐渐增加,而刺激48h(TNF-α、IL-1β)和24 h(IL-6)产生的量则达到高峰。TLR2抗体、CD14抗体能明显抑制TNF-α、IL-1β和IL-6的产生,并且TLR2和CD14抗体联合处理组对于CKs产生的抑制率明显高于单独处理组；SAPK/JNK特异性抑制剂SP600125对TNF-α、IL-1β和IL-6的产生无明显影响；ERK1/2特异性抑制剂PD98059能较低程度抑制TNF-a的产生,但对IL-1β和IL-6的产生无明显影响；而p38特异性抑制剂SB203580和NF-κB特异性抑制剂PDTC能明显抑制TNF-a, IL-1β和IL-6的产生,且呈剂量依赖关系；Tp0751重组蛋白刺激THP-1细胞后,Western blot能检测到明显的磷酸化的SAPK/JNK、p38和ERK1/2条带,60 min达到高峰(P-JNK 45 min后达到高峰),随后逐渐下降,可持续到90 min以上(P-JNK 60 min后基本消失)；同时Western blot检测结果表明,NF-ΚB抑制剂PDTC能部分抑制NF-κB从胞浆转位至胞核,而未用PDTC处理的细胞核抽提物中发现Tp0751重组蛋白能明显激活NF-κB,在细胞核中能检测到明显的NF-κB蛋白条带结果。1)成功构建了pET-28a(+)-Tp0751原核表达载体,经表达、纯化后获得了高含量的相对分子量约为26 kDa的可溶性融合蛋白；2)Tp0751重组蛋白具有良好的免疫反应性和免疫原性,能刺激新西兰兔产生高效价的特异性抗体；3) Tp0751重组蛋白诱发新西兰兔产生明显的DTH反应；4) Tp0751重组蛋白对THP-1细胞无明显的细胞毒性作用；5) Tp0751重组蛋白可以诱导THP-1细胞以时间和剂量依赖方式表达CKs(IL-1β、TNF-α和IL-6),可能为TP的一个新的重要的致病因子；6)Tp0751重组蛋白诱导THP-1表达CKs的信号传导可能与TLR2和CD14途径有关；7)Tp0751重组蛋白能激活THP-1细胞内的MAPKs和NF-κB,从而表达CKs。更多还原

【Abstract】 Syphilis, caused by Treponema pallidum subsp. Pallidum, is a serious, multistage sexually transmitted infection with diverse and wide-ranging manifestations. Although effective drug treatment regimes exist, syphilis remains a pandemic disease. As this disease also contributes to the spread of human immunodeficiency virus (HIV), the development of an effective vaccine against syphilis has remained an important research and public health goal. Understanding of the pathogenesis of this disease and how host-pathogen interactions influence the course of disease progression is crucial to controlling and preventing syphilis. However, study of the disease has been restricted by the limitations that the organism cannot be grown in vitro, and that, as an exclusively human pathogen, inferences made from animal models are of limited applicability.In the absence of cytotoxins and other known virulence factors, inflammation caused by T. pallidum and the ensuing adaptive immune response to T. pallidum may cause the tissue destruction characteristic of syphilis infection. T. pallidum membrane proteins are thought to be potent inducers of inflammation during early syphilis infection. There are a number of potential candidates among the T. pallidum membrane proteins. For example, TpN47 has been shown to induce the production of tumor necrosis factor-a (TNF-a), human interleukin-1β(IL-1β), and cytokine interleukin-6 (IL-6) by activating nuclear factor kappa-B (NF-κB) through a toll-like receptor 2 (TLR2) and CD 14 signaling pathway. However, no other T. pallidum proteins have yet been characterized that might also induce the inflammatory reaction. The molecular mechanisms by which they might trigger and sustain the inflammatory cascades also remain obscure. Therefore, screening a cohort of T. pallidum membrane proteins for inflammatory responses is crucial for clarifying the full progression of disease pathogenesis.Tp0751 is an adhesion protein from the T. pallidum membrane that has recently been related to pathogen invasion by virtue of its potential for attachment to the host cells. Tp0751 is also one of the first T. pallidum proteins to come into contact with host cells. Tp0751 may have other functions in T. pallidum pathogenesis, for example, in stimulating inflammatory reactions, but its full role in the host immunity has yet to be explored.This study was carried out to investigate the immunocompetence, cytotoxicity and induction of proinflammatory reactivity of Tp0751 laminin-binding adhesin of T. pallidum. This study was very important to further understand the pathogenicity of T. pallidum at the molecular level.The Tp0751 ORF without upstream non-coding region was chosen by computer analysis, was amplified from T. pallidum complete genome by polymerase chain reactions (PCR), was ligased into the expression vector pET-28a(+) to generate recombinant plasmid pET-28a(+)/Tp0751, expressed in E.coli R2566, and analyzed by SDS-PAGE and Western blot.The fusion protein was purified with Ni-NTA affinity chromatography, Purified protein was analyzed by SDS-PAGE, and protein concentration was determined by the BCA protein assay kit.The immunoreactivity of fusion protein was evaluated by Western-blot. Zealand rabbits were immunized with the fusion protein, antibodies to anti-Tp0751 in sera were detected with indirected ELISA.In order to study the cytotoxicity of Tp0751 in THP-1 cells, Endotoxin was removed by endotoxin removal gel. The skin changes in the injection site were observed by subcutaneous injection in New Zealand white rabbits. Lactate dehydrogenase leakage rate and NO release of macrophages after Tp0751 treatment were detected to study the cytotoxicity in cells.THP-1 cells were stimulated with different concentrations of Tp0751 for suitable periods, further cells were stimulated with suitable concentration of Tp0751 for different time. The cytokines’(TNF-a, IL-1βand IL-6) concentration was measured using the quantitative "sandwich" ELISA technique.For inhibitor experiments, the cell culture were treated respectively 30 min with mouse anti-TLR2 mAb, mouse anti-CD 14 mAb, PD98059, SP600125, SB203580(Three-MAPK specific inhibitors, SP600125, PD98059 and SB203580 can selectively inhibit SAPK/JNK, ERK and p38 signaling pathways without affecting the others) and pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor) before Tp0751 stimulation. The activation of NF-κB and MAPKs after Tp0751 treatment in THP-1 cells was detected by Western blot.The size of PCR amplification product was about 600 bp. Restriction enzyme digestion analysis and sequencing showed that the inserted target gene were Tp0751, compared with gene reported by GenBank, it had 100% similarity. and the results of BLAST confirmed that the sequence was Tp0751. A fusion protein with molecular weight about 26 kDa was attained after expression and purification, the concentration of Tp0751 recombinant protein was 1.2 mg/mLWestern blot proved that the recombinant protein can specifically react with T. pallidum IgG positive sera. Specific humoral response were elicited by recombinant protein in zealand rabbit and the specific antibody titer was above 1:10 240 after immunization for 4 times.Tp0751 stimulated the New Zealand rabbit to show fever red skin in leg, disappeared after six days to ten days, and delayed-type hypersensitivity was positive. THP-I were stimulated into macrophages by PMA and the macrophage cells stimulated by the target protein were collected after 48 h and cell supernatant. LDH leakage rate and NO concentration compared with the control group were unsignificantly.Cytokine production by Tp0751 treated THP-1 cells was found to be dose-dependent in a concentration range from 0.5μg/mL to 10.0μg/mL The level of cytokines decreased above a concentration of 5μg/mL Cytokine production by Tp0751 treated THP-1 cells was also found to be time-dependent in a time range from 6 hours to 72 hours. The amount of TNF-a and IL-1βreached peak levels at 48 hours after stimulation, but The peak amount of IL-6 occurred at 24 hours after stimulation.The expression of Tp0751-induced proinflammatory cytokines was significantly decreased by pretreatment with anti-TLR2 Ab, anti-CD 14 antibodies, SB203580 (p38 signaling pathways specific inhibitor) and PDTC (NF-κB signaling pathways specific inhibitor). PD98059 (ERK signaling pathways specific inhibitor) slightly inhibited TNF-a production, but the amounts of IL-1p, IL-6 were not affected. SP600125 (SAPK/JNK signaling pathways specific inhibitor) could not inhibit the production of TNF-a, IL-1βand IL-6. The phosphorylated SAPK/JNK, ERK and p38 increased in a time dependent manner, the peak occurred at 45 min and then decreased. The duration of MAPK/P38 and SAPK/JNK pathways were longer than SAPK/JNK pathway, they could last 90 min post induction. Western blot found that NF-κB translocation was obviously inhibited after treatment with PDTC (25μmol/L).1. Prokaryotic expression vector pET-28a(+)/Tp0751 was constructed successfully and Tp0751 recombinant protein with molecular weight near 26 kDa was well attained.2. Tp0751 recombinant protein showed excellent immunongenicity and immunoreactivity, can specifically react with T. pallidum IgG positive sera, and also can induce the humoral responses in zealand rabbits efficiently.3. Tp0751 recombinant protein can induce delayed-type hypersensitivity in New zealand rabbits.4. The leakage rate of LDH and NO release were not increased after Tp0751 recombinant protein stimulated the macrophages, which indicated no cytotoxicity effect in cells.5. Tp0751 stimulated THP-1 cells’production of proinflammatory cytokines, and may be relative to the inflammatory reaction after T. pallidum infection.6. TLR2, CD 14 might play important roles in the the activation of monocytes/macrophages by Tp0751.7. Tp0751 induced MAPKs and NF-κB activation in THP-1 cells.更多还原