【作者】 赵文忠；

【导师】 富宁；

【作者基本信息】 第一军医大学 ， 免疫学， 2004， 博士

【摘要】 哺乳动物Toll like receptors(TLRs)是果蝇Toll的同系物，它们构成一个新的蛋白家族，参与天然免疫的介导和获得性免疫的激活。自1997年发现第一个哺乳动物TLRs以来，现已发现了小鼠的TLRs有9个(即mTLR1-9)；人的TLRs有10个(即TLR1-10)中。研究表明，TLRs家族的作用并不仅限于抗感染免疫，还可参与抗原递呈、诱导免疫耐受、细胞凋亡，并以此在自身免疫病、超敏反应、移植排斥及肿瘤免疫中发挥作用。在过去几年中，TLR-2作为脊椎动物蛋白TLR家族的一员，得到广泛的研究。TLR-2可介导多种不同的病原微生物(尤其是革兰氏阳性菌、分枝杆菌及螺旋体等)及其产物引起的、性质相似的细胞信号转导活动及细胞因子产生。因此,TLR-2被认为是具中心作用的或中枢型识别(central pattern recognition)受体。尽管有许多体外实验证实了TLR-2参与免疫相关事件，但是来自体内实验关于TLR-2功能的数据很少。 小鼠和人的TLR-2都是果蝇TLR-2的同源蛋白，两者在氨基酸水平上有83％的同源性。基于mTLR-2与hTLR-2结构的高度同源性，及在分布、生物学活性方面的诸多相似，小鼠无疑是研究TLR-2结构、功能及其结合肽特性的良好模型。本研究拟对小鼠TLR-2进行克隆、表达及其抗体的生物活性研究。首先对小鼠TLR-2的全基因及部分基因进行克隆、表达；制备抗mTLR-2胞外段B细胞识别表位抗体；在获得了抗mTLR-2抗体的基础上，进行抗mTLR-2抗体抗过敏和肿瘤抑制实验，从整体水平上来研究TLR-2的生物学功能。 第一部分 mTLR-2基因的克隆及在不同体系中的表达 根据已发表的mTLR-2的全基因序列，采用Primer premier 5.0软件，设计并合成以下两对引物。以P2,P4为引物，以Balb／c小鼠肝脏eDNA为模板的PCR反应，扩增出约1500bp特异性扩增带，而以P1,P3为引物的PCR反应，扩增出约1400bp特异性扩增带通过蓝白斑筛选挑选，并经PCR和测序鉴定，分别获得上游阳性克隆U3和下游阳性克隆D4。以u3、04为模板，以PZ，P4为引物的pCR反应，扩增出约2 soobp特异性扩增带，将其克隆至puCm一T载体中，经PcR和测序鉴定，获得puc一mTLR一2重组质粒。经测序鉴定，表明获得mTLR一2基因，该序列已被GenBank收录(AY179346)，与已发表的mTLR一2的同源性为99.84%。在线运行BLASTZ，AY179346与标准mTLR一2 eDNA序列AF124741之间有4个碱基的差别。分别是515位由t变为c，963位由t变为c位，2476位由a变为g，2500位由t变为。由AY179346推导而来的氨基酸与AF 124741所编码的氨基酸序列比较，有3个氨基酸的差异，分别是F266L，Q770R，L778P。 从体外获取mTLR一2蛋白，对于从细胞模型及动物模型进行TLR一2的研究具有重要意义。选用哺乳动物CHO细胞表达系统、毕赤酵母表达系统及大肠杆菌原核表达系统对全长及部分mTLR一2基因进行表达研究。(l)将目的基因克隆到真核表达载体peDNA3 .1中，成功构建了PcDNA一mTLR一2重组质粒，并将其转染到CHo细胞，用G418筛选阳性克隆。PCR检测表明外源基因得到稳定整合，Westem blot分析显示在分子量约95 000处可见清晰的阳性反应条带。免疫组织化学和流式细胞术检测显示转染细胞表达mTLR一2。(2)将目的基因编码序列插入毕赤酵母表达载体pPICZac中，获得重组质粒pPIcz一mTLR一2，采用电穿孔法转化毕赤酵母。重组酵母以PeR、RT一PCR验证，sDs一AGE和Westem blot分析显示，在相对分子量为约97 000处有1特异蛋白带，且能与抗mTLR一2多抗发生反应。(3)采用PcR技术，扩增编码mTLR一ZN端153氨基酸的基因，并将其克隆到pET32a载体中，成功构建了融合表达载体pET一，融合蛋白在大肠杆菌中高效表达，采用Probond树脂柱纯化融合蛋白。第二部分抗mTLR一2胞外段抗体的制备与鉴定 制备两种抗mTLR一2胞外段抗体。(1)在对小鼠Toll样受体2(mTLR一2)B细胞优势表位预测的基础上，合成B细胞识别表位20mer短肤，将其与载体蛋白偶联，制备免疫原。经ELISA方法检测抗体滴度为1:25 600，采用spG柱纯化法获得兔抗mTLR一2胞外段B细胞识别表位多克隆抗体TSP一1;采用抗原肤亲和层析纯化法获得兔抗mTLR一2胞外段B细胞识别表位抗体TSP一2。Westem blot分析显示在相对分子量约95 000处可见清晰的反应条带;免疫组织化学和流式细胞术检测显示抗体可与表达天然mTLR一2的J774A.1细胞反应。(2)将已获得的N端融合蛋白免疫动物，获取抗mTLR一2多表位抗体。经ELISA方法检测抗体滴度为1:16 000，采用spG柱纯化法获得兔抗mTLR一2胞外段B细胞识别表位多克隆抗体TsP一3。流式细胞术检测显示TsP一3可与25.5%的CHO一mTLR一2细胞反应。第三部分抗mTLR一2胞外段B细胞识别表位抗体抑制肉瘤生长的研究 以小鼠肉瘤株5180细胞注射小鼠左后肢，其中TSP一l组混合有100雌纯化抗体TSP一1，RlgG组混合有100雌正常兔lgG，Sahne组仅接种5180。小鼠经麻醉分别于10d、14d、18d处死，TSP一l组瘤重在10d、14d、18d与Saline组相比差异显著(p<0.02、p<0.05、P<0.05);抑瘤率分别为77%、51%和53%。局部应用抗mTLR一2胞外段B细胞识别表位抗体可抑制小鼠肉瘤5180生长，且主要表现在肿瘤生长的早期。更多还原

【Abstract】 Cloning and Expression of murine TLR-2 and Research of Anti-mTLR-2 AntibodiesThe mammalian Toll-like receptors (TLRs) are homologues of Drosophila Toll and constitute a novel protein family involved in mediating innate immunity and linking to acquired immunity. Nine members in murine TLR family and ten members in human TLR family have been found since the first TLR molecular was identified in 1997. TLRs participate in both infection and non-infection immunity, such as antigen presenting, immune tolerance, and cell apoptosis. TLRs play an important role in autoimmune diseases, allergic diseases, transplantation rejection and tumor. Toll-like receptor 2 (TLR-2) has been studied in substantial detail over the last years. TLR-2 can recognize a wide variety of pathogen associated molecular patterns (PAMPs) such as gram-positive bacteria, mycobacterium tuberculosis and their component, and mediate similar signal transduction and pro-inflammatory cytokine production. Therefore TLR-2 was considered as a central pattern recognition receptor in innate immunity. There have been a lot of research in vitro regarding the importance of TLR-2 and their ligands in inducing immune related events. But few work has been done in vivo regarding the exploration of TLR-2 in innate immune and induction of acquired immunity.Both mTLR-2 and hTLR-2 are Drosophila Toll homologues, and amino acid of mTLR-2 shared 83% homologous with hTLR-2. Based on high homologous structure and similar distribution and biological activitities, mouse is undoubtedly a good model for us to study the structure and functions of hTLR-2. In this research, complete gene of murine TLR-2 was cloned and expressed; antibody against B cell epitope on extracellular domain and a fragment of N terminal were prepared; the activities of antibody on anti-allergy and anti-tumor experiment were performed.-6-Part I Cloning and expression of murine TLR-2Based on the complete sequence of murine TLR-2 gene published in GenBank, four primers from P1 to P4 were designed and synthesized. The downstream gene and the upstream gene of murine TLR-2 was amplified using P2 and P4, P1 and P3 respectively, P1 and P4 were used to amplify the complete gene. One fragment of 1 500bp was amplified using cDNA from mouse liver as template, another fragment of 1 400bp was obtained by RT-PCR using the same template. Two amplified products were cloned into pUC-T vector respectively. The upstream clone U3 and the downstream clone D4 were obtained through blue/white color selection, PCR and sequence analysis. Then U3 and D4 as template, the fragment of over 2 500 bp was amplified and cloned into pUC-T vector using P1 and P4. The recombinant plasmid pUC-mTLR-2 was obtained, and identified by sequence analysis. It showed the complete gene of mTLR-2 was acquired. The gene had been accepted as accession number AY179346, which has 99.84% homology with the standard sequence of mTLR-2. Using BLAST2 online, there are substitutions of four bases between AY179346 and the standard sequence AF124741, among which t substitute c at nucleotide position 515, t substitute c at 963, a substitute g at 2476, and t substitute c at 2500. Compared the deduced sequence of amino acid from AY179346 with that from AF124741, there are three amino acid substitutions, F266L, Q770R and L778P.It is significant for us to obtain mTLR-2 protein, which is helpful to study the biological functions of mTLR-2 in vitro and in vivo. Now the expression system of mammalian cell, the methylotrophic yeast Pichia pastoris, Escherichia coli were chosen to express the complete or partial gene of mTLR-2. In briefly, (l)The mTLR-2 gene was cloned into pcDNA3.1 of mammalian expression vector, and the recombinant plasmid pcDNA-mTLR-2 was transfected into CHO cell by electroporation , the positive cell clones were obtained by screening with G418, and insertion of-7-exogenous gene was confirmed by PCR; One clear band was shown in relative molecular weight(Mr) of 95 000 using Western blot analysis, and mTLR-2 was identified to be expresse更多还原