本文選題：梅毒螺旋體　切入點(diǎn)：Tp0751　出處：《中南大學(xué)》2010年博士論文　論文類型：學(xué)位論文

【摘要】： 明確梅毒螺旋體(Treponema pallidum, TP)的致病因子及進(jìn)行TP致病機(jī)制的研究,將為研制TP抗感染疫苗和開發(fā)TP感染治療新藥提供科學(xué)依據(jù),是防治本病的關(guān)鍵環(huán)節(jié)。然而,迄今為止,由于TP體外人工培養(yǎng)尚未成功,抗原獲取困難,限制了對TP的深入研究,以致TP的致病機(jī)制目前尚未被闡明。 炎癥和持續(xù)的獲得性免疫應(yīng)答被認(rèn)為是造成TP感染后機(jī)體病理損傷的主要原因。研究表明,TP膜蛋白是介導(dǎo)炎癥反應(yīng)的主要成分,可能為TP的主要致病因子,因此對TP膜蛋白的研究是認(rèn)識其對宿主的致病性和進(jìn)行致病機(jī)制研究的關(guān)鍵。但是對于具體是哪些TP膜蛋白在發(fā)揮這方面的作用報道甚少,因而有必要進(jìn)行TP膜蛋白的致病性研究和篩選,這對深入研究TP的致病機(jī)制至關(guān)重要。 Tp0751為近年來新發(fā)現(xiàn)的一種TP主要的黏附膜蛋白,其主要介導(dǎo)TP與宿主細(xì)胞的結(jié)合而與TP入侵宿主有關(guān),是機(jī)體較早接觸的TP膜蛋白之一。那么其在TP致病過程中是否還具有其他的作用呢?譬如,其是否具有細(xì)胞毒性?其是否能夠誘導(dǎo)宿主免疫細(xì)胞產(chǎn)生前炎癥反應(yīng)而與介導(dǎo)機(jī)體的炎癥反應(yīng)有關(guān)呢?這些均值得深入探索和研究。 本研究試圖通過探討Tp0751黏附蛋白的免疫活性、細(xì)胞毒性及是否能誘導(dǎo)THP-1細(xì)胞產(chǎn)生前炎癥細(xì)胞因子(CKs)TNF-α、IL-1β和IL-6,并初步研究其誘導(dǎo)CKs產(chǎn)生的信號傳導(dǎo)通路,為進(jìn)一步探索Tp0751黏附蛋白在TP感染免疫中的作用及進(jìn)行梅毒的致病機(jī)制研究奠定重要的基礎(chǔ)。 通過生物信息學(xué)分析,去除Tp0751信號肽序列,以TP Nichols株的基因組DNA為模板,PCR擴(kuò)增Tp0751基因；通過BamHI和EcoRI酶切位點(diǎn)將Tp0751基因克隆進(jìn)pET-28a(+)質(zhì)粒中構(gòu)建重組質(zhì)粒,篩選陽性質(zhì)粒,酶切鑒定和PCR鑒定,經(jīng)測序鑒定的重組質(zhì)粒轉(zhuǎn)化至表達(dá)宿主菌ER2566中構(gòu)建原核重組表達(dá)體；進(jìn)行誘導(dǎo)表達(dá),Ni親合層析柱純化重組蛋白,BCA法測定蛋白濃度。 Western blot檢測其免疫反應(yīng)性；用重組蛋白免疫新西蘭兔,檢測兔免疫血清中多克隆抗體的效價,評價其免疫原性。 利用Detoxi-Gel內(nèi)毒素去除膠去除重組蛋白中的內(nèi)毒素；將重組蛋白經(jīng)皮下注入新西蘭兔大腿內(nèi)側(cè),觀察注射部位皮膚變化；佛波酯(Phorbol 12-myristate 13-acetate, PMA)誘導(dǎo)THP-I細(xì)胞轉(zhuǎn)化為巨噬細(xì)胞,將經(jīng)內(nèi)毒素去除處理后的Tp0751重組蛋白分別刺激巨噬細(xì)胞,通過檢測細(xì)胞的乳酸脫氫酶(lactate dehydrogenase, LDH)漏出率和NO釋放量研究其細(xì)胞毒性。 為研究Tp0751誘導(dǎo)炎癥反應(yīng)作用,以去內(nèi)毒素的Tp0751蛋白刺激THP-1細(xì)胞,檢測其產(chǎn)生前炎癥細(xì)胞因子(CKs)TNF-α、IL-1β和IL-6的情況,同時用LPS做陽性對照和PBS為陰性對照組。 為了探討誘導(dǎo)CKs產(chǎn)生的信號傳導(dǎo)通路是否與TLR2、CD14、MAPKs和NF-κB有關(guān),分別用TLR2抗體、CD14抗體、PD98059(ERK1/2特異性抑制劑)、SP600125 (SAPK/JNK特異性抑制劑)SB203580(p38特異性抑制劑)和PDTC (NF-κB特異性抑制劑)預(yù)處理THP-1細(xì)胞,然后再用Tp0751刺激細(xì)胞,檢測細(xì)胞因子的產(chǎn)生情況；Western blot檢測Tp0751刺激THP-1細(xì)胞后細(xì)胞MAPKs磷酸化水平和NF-κB的活化情況。 PCR擴(kuò)增得到一大小約為600 bp的目的基因片斷(Tp0751去信號肽片段,Tp0751基因全長度為764 bp,編碼255個氨基酸)；構(gòu)建的重組質(zhì)粒經(jīng)酶切鑒定和測序鑒定證明其中插入片斷為Tp0751目的基因,測序結(jié)果與Genbank上登錄序列完全一致；SDS-PAGE分析顯示,在IPTG誘導(dǎo)下,重組工程菌表達(dá)了一相對分子量(Mr)約為26kDa的目的蛋白條帶,目的蛋白在菌體細(xì)胞內(nèi)主要以可溶性形式存在;經(jīng)Ni-NTA親和純化獲得了純度在95%以上的重組蛋白,BCA法測得純化蛋白濃度為1.2 mg/mL Western blot檢測其能與梅毒陽性血清發(fā)生特異性反應(yīng)；利用純化的Tp0751重組蛋白免疫新西蘭兔,間接ELISA法測定兔免疫血清特異性抗體效價到第四次免疫時達(dá)到頂峰,其抗體滴度在1:10 240以上。 重組蛋白能誘發(fā)新西蘭兔產(chǎn)生遲發(fā)型超敏反應(yīng)(delayed type hypersensitivity, DTH),注射部位出現(xiàn)明顯紅腫,紅腫部位皮膚溫度比對側(cè)相應(yīng)部位高,于注射后6-1Od紅腫消退,生理鹽水對照部位未見反應(yīng)；經(jīng)Tp0751重組蛋白刺激后的巨噬細(xì)胞LDH漏出率和NO釋放量較低,和6-His-Tag蛋白作用組比較,差異無統(tǒng)計學(xué)意義,與蛋白刺激濃度無關(guān)。 Tp0751重組蛋白刺激THP-1細(xì)胞后,其能以劑量和時間依賴方式誘導(dǎo)THP-1細(xì)胞產(chǎn)生TNF-α、IL-1β和IL-6,誘導(dǎo)THP-1細(xì)胞產(chǎn)生TNF-α、IL-1β和IL-6的最適Tp0751刺激濃度為5μg/mL,Tp0751刺激細(xì)胞6h后即可從培養(yǎng)基中檢測到TNF-α、IL-1β和IL-6,隨著刺激時間的延長炎癥因子的產(chǎn)生量逐漸增加,而刺激48h(TNF-α、IL-1β)和24 h(IL-6)產(chǎn)生的量則達(dá)到高峰。TLR2抗體、CD14抗體能明顯抑制TNF-α、IL-1β和IL-6的產(chǎn)生,并且TLR2和CD14抗體聯(lián)合處理組對于CKs產(chǎn)生的抑制率明顯高于單獨(dú)處理組；SAPK/JNK特異性抑制劑SP600125對TNF-α、IL-1β和IL-6的產(chǎn)生無明顯影響；ERK1/2特異性抑制劑PD98059能較低程度抑制TNF-a的產(chǎn)生,但對IL-1β和IL-6的產(chǎn)生無明顯影響；而p38特異性抑制劑SB203580和NF-κB特異性抑制劑PDTC能明顯抑制TNF-a, IL-1β和IL-6的產(chǎn)生,且呈劑量依賴關(guān)系；Tp0751重組蛋白刺激THP-1細(xì)胞后,Western blot能檢測到明顯的磷酸化的SAPK/JNK、p38和ERK1/2條帶,60 min達(dá)到高峰(P-JNK 45 min后達(dá)到高峰),隨后逐漸下降,可持續(xù)到90 min以上(P-JNK 60 min后基本消失)；同時Western blot檢測結(jié)果表明,NF-ΚB抑制劑PDTC能部分抑制NF-κB從胞漿轉(zhuǎn)位至胞核,而未用PDTC處理的細(xì)胞核抽提物中發(fā)現(xiàn)Tp0751重組蛋白能明顯激活NF-κB,在細(xì)胞核中能檢測到明顯的NF-κB蛋白條帶結(jié)果。 1)成功構(gòu)建了pET-28a(+)-Tp0751原核表達(dá)載體,經(jīng)表達(dá)、純化后獲得了高含量的相對分子量約為26 kDa的可溶性融合蛋白； 2)Tp0751重組蛋白具有良好的免疫反應(yīng)性和免疫原性,能刺激新西蘭兔產(chǎn)生高效價的特異性抗體； 3) Tp0751重組蛋白誘發(fā)新西蘭兔產(chǎn)生明顯的DTH反應(yīng)； 4) Tp0751重組蛋白對THP-1細(xì)胞無明顯的細(xì)胞毒性作用； 5) Tp0751重組蛋白可以誘導(dǎo)THP-1細(xì)胞以時間和劑量依賴方式表達(dá)CKs(IL-1β、TNF-α和IL-6),可能為TP的一個新的重要的致病因子； 6)Tp0751重組蛋白誘導(dǎo)THP-1表達(dá)CKs的信號傳導(dǎo)可能與TLR2和CD14途徑有關(guān)； 7)Tp0751重組蛋白能激活THP-1細(xì)胞內(nèi)的MAPKs和NF-κB,從而表達(dá)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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R373;R759.1

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