梅毒螺旋体Tp0751黏附蛋白的免疫活性及在梅毒致病中的作用研究

发布时间：2018-03-17 13:25

本文选题：梅毒螺旋体　切入点：Tp0751　出处：《中南大学》2010年博士论文　论文类型：学位论文

【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/mL Western 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。
[Abstract]:Clear of Treponema pallidum (Treponema pallidum, TP) of pathogenic factors and research the pathogenesis of TP, the TP for the development of vaccines against infection and provide scientific basis for the development of TP new drug for the treatment of infection, is a key link in the prevention and treatment of this disease. However, so far, because the TP in vitro cultured antigen has not yet been successful, difficult to obtain, limit the research of TP, so that the pathogenesis of TP has not yet been elucidated.
Persistent inflammation and acquired immune response is considered to be the main reason for body pathological damage caused by TP infection. The study showed that the membrane protein is the main component of TP mediated inflammatory reaction, may be the main pathogenic factor of TP, so the research on the TP membrane protein is the understanding of the pathogenicity and pathogenic mechanism of host the key of the study. But for which specific TP membrane proteins play a role in this area reported little research and screening of pathogenicity and thus it is necessary for the TP membrane protein, the pathogenic mechanism of in-depth study of TP is very important.
Tp0751 is a major TP adhesion membrane protein discovered in recent years, with the main TP mediated by host cells and associated with TP invasion of host, is one of the TP membrane protein. Then the contact body early in the pathogenesis of TP, it also has other function? For example, whether it has the cell about its toxicity? Whether inflammation can produce inflammatory host immune cells induced by NK and mediated? These are worthy of further exploration and study.
This study attempts to explore the Tp0751 adhesion protein immune activity, cell toxicity and whether can produce proinflammatory cytokine induced THP-1 cells (CKs) of TNF- alpha, IL-1 beta and IL-6 signal transduction pathway and to study the induced CKs production, laid an important foundation for further exploring the pathogenic mechanism of Tp0751 adhesion protein in syphilis the role of TP infection and immunity.
Through bioinformatics analysis, the removal of Tp0751 signal peptide sequence, genomic DNA TP strain Nichols as template, PCR amplification of Tp0751 gene; through BamHI and EcoRI restriction sites of Tp0751 gene was cloned into pET-28a (+) to construct recombinant plasmid plasmid, screening positive plasmid, enzyme digestion and PCR identification and sequencing of recombinant plasmid the identification of transformed into E.coli ER2566 to construct recombinant prokaryotic expression; induced expression, Ni affinity chromatography to purify the recombinant protein and the protein concentration was determined by BCA method.
Western blot was used to detect its immunoreactivity, and the recombinant protein was used to immunize New Zealand rabbits to detect the titer of the polyclonal antibody in the rabbit immune sera and to evaluate its immunogenicity.
Use Detoxi-Gel glue removing endotoxin removal of endotoxin in recombinant protein; recombinant protein was injected into New Zealand rabbit thigh subcutaneous injection site, observe the change of skin; phorbol-12-myristate-13-acetate (Phorbol 12-myristate 13-acetate, PMA) to induce THP-I cells into macrophages by endotoxin removal after treatment with recombinant Tp0751 protein respectively stimulated macrophages, cells were detected by lactate dehydrogenase the (lactate dehydrogenase LDH) on the cytotoxicity of the leakage rate and the release of NO.
To study the role of Tp0751 induced inflammatory responses to endotoxin, Tp0751 protein stimulated THP-1 cells, detect the production of proinflammatory cytokines (CKs) TNF- alpha, IL-1 beta and IL-6, with LPS as positive control and PBS as negative control group.
In order to investigate whether the signal transduction pathway of CD14 induced CKs production and TLR2, MAPKs, and NF- K B respectively with TLR2 antibody, CD14 antibody, PD98059 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor) and SB203580 (p38 inhibitor) and PDTC (NF- kappa B inhibitor) pretreatment THP-1 cells, then Tp0751 cells were stimulated by detection of cytokines; cell activation and phosphorylation of MAPKs NF- Western blot Tp0751 K B detection after stimulation of THP-1 cells.
About the size of a PCR amplified gene fragment of 600 BP (Tp0751 signal peptide and Tp0751 gene full length is 764 BP, encoding 255 amino acids); recombinant plasmid by enzyme digestion and sequencing proved that the inserted fragment was Tp0751 gene sequencing results and Genbank sequence of identical SDS-PAGE; analysis showed that under the induction of IPTG, a recombinant expression of relative molecular weight (Mr) is about to 26kDa protein bands, the protein in bacterial cells mainly existed in a soluble form by Ni-NTA affinity; the purity of the recombinant protein in more than 95% of the purified protein concentration was measured by BCA method 1.2 mg/mL
Western blot detected its specific reaction with syphilis positive serum. The purified Tp0751 recombinant protein was used to immunize New Zealand rabbits. The titer of specific antibody of rabbit immune serum reached the peak at fourth times by indirect ELISA, and the titer of antibody was above 240 at 1:10.
The recombinant protein can produce delayed hypersensitivity induced by New Zealand rabbits (delayed type hypersensitivity, DTH), the injection site appeared obvious swelling, redness of skin temperature on the corresponding parts of the high side, after injection of 6-1Od saline control swelling, no site reaction; recombinant protein by Tp0751 stimulated macrophages and the leakage rate of LDH NO the release amount is low, and the role of protein in 6-His-Tag group, the difference was not statistically significant, has nothing to do with the protein concentration.
The recombinant Tp0751 protein in THP-1 cells stimulated, it can be in a dose and time-dependent manner in TNF- cells induced by THP-1 alpha, IL-1 beta and IL-6 generated TNF- cells induced by THP-1, the optimum IL-1 beta and IL-6 Tp0751 concentration of 5 g/mL, Tp0751 6h to TNF- after the stimulation of cell culture medium can be detected from alpha IL-1, beta and IL-6, with the extension of time to stimulate the production of inflammatory factors increased gradually, and the stimulation of 48h (TNF- alpha, IL-1 beta) and 24 h (IL-6) production reached the peak of.TLR2 antibody, CD14 antibody can inhibit TNF- alpha, IL-1 beta and IL-6, and TLR2 and CD14 the combined treatment group for CKs antibody inhibited the rate was significantly higher than the treatment group alone; SAPK/JNK specific inhibitor SP600125 on TNF- alpha, beta and IL-1 had no significant effect on IL-6 generation; ERK1/2 specific inhibitor PD98059 can inhibit the production of TNF-a low level, but the IL-1 beta and IL-6 production Effect; and p38 specific inhibitor SB203580 and NF- kappa B inhibitor PDTC could inhibit TNF-a and IL-1 beta and IL-6 production in a dose-dependent manner; the recombinant protein Tp0751 in THP-1 cells stimulated with Western, blot can detect significant phosphorylation of SAPK/JNK, p38 and ERK1/2 bands, min reached 60 the peak (P-JNK reached a peak after 45 min), then decreased gradually, sustainable to more than 90 min (P-JNK disappeared after 60 min); and Western blot results showed that NF- kappa B inhibitor PDTC could partially inhibit NF- K translocation of B from cytoplasm to the nucleus, but not using nuclear extracts in PDTC treatment Tp0751 found that the recombinant protein could activate NF- kappa B, can detect the obvious NF- kappa B protein bands results in the nucleus.
1) pET-28a (+) -Tp0751 prokaryotic expression vector was successfully constructed. After expression, the high content of soluble fusion protein with a relative molecular weight of about 26 kDa was obtained.
2) the recombinant protein of Tp0751 has good immunoreactivity and immunogenicity, which can stimulate New Zealand rabbits to produce specific antibodies with high efficiency.
3) the recombinant protein of Tp0751 induced a significant DTH reaction in New Zealand rabbits.
4) the recombinant protein of Tp0751 had no obvious cytotoxic effect on THP-1 cells.
5) Tp0751 recombinant protein can induce THP-1 cells to express CKs (IL-1 beta, TNF- alpha and IL-6) in a time and dose dependent manner, which may be a new important pathogenic factor of TP.
6) the signal transduction of THP-1 expression CKs induced by Tp0751 recombinant protein may be related to TLR2 and CD14 pathway.
7) Tp0751 recombinant protein activates MAPKs and NF- kappa B in THP-1 cells, thus expressing CKs.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R373;R759.1

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