【作者】 郑山根；

【导师】 富宁；

【作者基本信息】 中国人民解放军第一军医大学 ， 免疫学， 2003， 博士

【摘要】 脂多糖是引起革兰氏阴性菌败血症休克的重要始动因素之一，目前尚无理想的拮抗LPS或类脂A制剂。虽然抗LPS抗体可拮抗LPS而具保护性，但临床上抗体的应用时机难以掌握。因此，建立针对内毒素的主动免疫是防治内毒素血症及败血症休克的重要途径之一。 LPS属TI-Ⅰ抗原，不能诱导有效的再次应答及高亲和力、调理杀菌力强的抗体的产生，加之LPS存在众多菌株及血清型特异性，因此研制有效的、具广谱保护性的LPS疫苗有一定困难。本文拟用对革兰氏阴性菌感染具交叉保护作用的抗LPS单克隆抗体和多克隆抗体从噬菌体肽库中筛选多个LPS表位模拟肽，进行人工合成及原核表达。具体目标如下：(1)获得LPS表位模拟肽，将LPS的多糖(TI)表位变为多肽(TD)表位，诱导有效的再次应答；(2)获得模拟LPS多个表位的多肽，诱导针对多种革兰氏阴性菌及其LPS的交叉保护力，即广谱预防作用；(3)基因表达模拟肽的融合蛋白，探索制备短肽疫苗更有效、经济的途径。 本研究分为以下四部分： 一、交叉保护性抗LPS多克隆抗体及单克隆抗体的制备和鉴定 经特殊处理的鼠伤寒沙门氏菌免疫大白兔，获得了既能与鼠伤寒沙门氏菌LPS反应，又能与大肠菌LPS(L2630)，尤其是具保守结构的LPS如明尼苏达沙门氏菌Re595株LPS(L9764)和Lipid A(L0744和L5399)有较好交叉反应的兔多克隆抗血清。 细菌攻击试验表明，兔抗血清对大肠杆菌和绿脓杆菌感染小鼠分别具有非常显著(P＜0.005，表4)和显著的(P＜0.05，表5)交叉保护性。亲和纯化制备的抗鼠伤寒沙门氏菌LPS单抗和多抗，约10ng／ml水平可检出1μg／mlLPS，具较好的抗体活性，可作为筛选噬菌体肽库的靶分子。 二、LPS多表位模拟肽的筛选与鉴定 用纯化的抗鼠伤寒沙门氏菌LPS单抗，对噬菌体随机环七肽库进行了筛选，经ELISA鉴定，所挑选的克隆中有34个克隆显示与单抗有较强结合；竞争抑制试验表明，阳性噬菌体克隆与多抗结合可被浓度低达。.spg/ml的鼠伤寒沙门氏菌LPS抑制。挑选其中10个克隆，经DNA测序获得了六种序列，相应的氨基酸序列分别为PSWASFW、PEWASSW、PAWAS柳、MSWFP户Y、QSWF[PY和PPGwPGF。 为获得模拟LPS多表位的噬菌体展示肤，用纯化的抗鼠伤寒沙门氏菌LPS多抗筛选噬菌体随机环七肤库，经鉴定，所挑选的克隆中有20个显示与多抗有较强结合。竞争抑制试验表明阳性克隆与多抗结合可被浓度低达。.5阳/m1的鼠伤寒沙门氏菌LPS抑制。挑选其中10个阳性克隆，经DNA测序获得了四种序列，相应的氨基酸序列分别为FQFYP从、LQFYPGA、LFTFA盯和YQ丫YP从。 以阳性噬菌体展示肤免疫动物鉴定其免疫原性，挑选四个含不同序列的阳性克隆(分别展示PSWASFW、QSWFLPY、FQFYP从和YQ丫YP从)分别免疫小鼠，以及将四个克隆混合免疫小鼠。其中QSWFLPY和FQFYPAA克隆及混合克隆免疫后，部分小鼠血清产生LPS抗体，效价为1:100一1:800，而对照组小鼠及经过长期观察的其它免疫原接种的小鼠均无抗LPS抗体产生。证明这两个噬菌体短肤为结合抗体的互补位(paratope)，即可模拟LPS表位结构，在体内能诱导产生抗LPS抗体。三、LPS表位模拟肤的合成与鉴定 挑选了用单抗筛选的阳性噬菌体克隆Kl展示序列(SACPSwASFwCGG)及用多抗筛选的阳性噬菌体克隆P4展示序列(SACFQFYPAACGG)进行人工合成，合成时将原序列两侧加入氨基酸臂SA及GG以稳定环七肤构象，两种肚分别命名为13a和13b。 竞争抑制试验表明，游离的13a单体能抑制单抗与LPS以及Kl克隆与单抗的结合，其ICS。分别为125肛岁ml和巧.6卜岁ml，且呈现浓度依赖效应，无关对照12肤不显示任何抑制效应，提示13a合成肤能特异地与LPS单抗IB6的抗原结合部位(即互补位)结合，可在空间结构上模拟LPS。游离的13b单体抑制阳性噬菌体克隆P4与LPs多抗结合，其IC50为31 .25林留ml，呈现浓度依赖效应，无关对照12肤不显示任何抑制效应，说明13b模拟噬菌体表面展示肤构象而与LPS多抗结合;13b在500协留ml时不能抑制多抗与LPS抗原的结合，推测可能是因为13b只能抑制作为其筛选靶抗体与LPS的结合，而不能抑制大部分针对其它表位的抗体分子与LPS的结合，并非不能模拟LPS抗原表位。 13a和13b与蓝载体的交联物能分别与单抗和多抗呈结合反应，其反应性随抗体浓度的增加而加强，而阴性对照肚则无此反应，说明交联后环七肤构象无变化，仍然能模拟LPS表位。将交联肤免疫小鼠，其中四只小鼠血清产生抗LPS，抗体效价随免疫次数的增加而提高，即产生了抗体再次应答，实现了多糖抗原(TI抗原)表位到多肤抗原(TD抗原)表位的转变。四、模拟肤的原核表达与鉴定 考虑合成肤的成本、环肤合成和抗原多价肤(MAPs)制备的技术难度与成本等因素，我们挑选两个噬菌体克隆展示序列在两种的原核载体中以不同的方法进行了表达，以期探索获得低成本、高活性模拟序列的途径。 第一种方法是将阳性噬菌体克隆Kl和KIO的环七肤原序列CPS从7ASFWC和cQS从TLPYC在谷肤甘肤硫转移酶(GST)原核表达系统中表达，经纯化获得GST融合蛋白:为使表达序?更多还原

【Abstract】 Lipopolysaccharide(LPS), ie, endotoxin, which is the component of the cell wall of gram negative bacteria, has been considered as an very important factor starting the pathologic cascade leading to multi-organ failure and death. Up to now, there is no ideal antagonists against LPS or lipid A for clinical prophylaxis and therapy . Although there were some evidences that the antibodies against LPS are protective , but the time phase of antibody application is hard to master. Therefore, establishment of active immune against LPS which means to develop a vaccine is very necessary.There are two major difficulties in preparing effective LPS vaccine with broad spectrum protection: (1) LPS is a kind of Thymus-independent antigen (TI antigen), which can not elicit secondary antibody response and high affinity antibodies with strong opsonization; (2) there are too many kinds of specificities of strains and serotypes in Gram negative bacteria with LPS. In this work , peptides multi-mimotopes of LPS displayed on phage were screened from phage display peptide library by using monoclonal and polyclonal anti-LPS antibodies with cross protection against G" bacteria infection, and the peptides mimicry to LPS epitope were synthesized with Fmac amino acide and expressed in E. coli ,which were estimated for a possibility to induce production of anti-LPS antibody. Our objectives are to change LPS as TI antigen to peptide mimotope of LPS as TD antigen, and induce the cross protection against LPS.This research can be divided into the following four parts.Part I Preparation and identification of monoclonal and polyclonal antibodies with cross protection against LPS from different bacteriaIn this part, rabbits were immunized with S. typhi. treated specially . By this way, we got polyclonal antisera with cross reactions with LPS from different strainsof bacteria, such as S. typhi. LPS(L7261) and E.coli. LPS(L2630), especially LPS with conservative structure, e.g. LPS from strain S. minne. Re595 (L9764) and LipidA(L0744&L5399).In protection test in mice attacked with bacteria, the result showed that the antiserum can protect mice from infection (LD90) either by E. coli. (P<0.005 vs control) or by P. pyocyanea (PO.05 vs control). Monoclonal and polyclonal antibodies with high activity were prepared by affinity chromatography , and can be used as targets for screening.Part II Screening and identification of the mimotopes for LPS multi-epitopes from phage displayed peptide librariesPeptide motopes were screened from The cyclic 7mer phage displayed peptide library was screened with purified mAb to S. Typhi. LPS. 34 of selected positive showed excellent binding to mAb 1B6 by identified using ELISA. The binding of positive clones to 1B6 can be inhibited by S. typhi. LPS at a concentration of 0.5ug/ml by competitive inhibition assay. The amino acid sequences of peptide deduced from the corresponding DNA sequences of 10 of the positive clones were PSWASFW(3/10), PEWASSW(3/10), PAWASMW(1/10), MSWFPPY(1/10), QSWFLPY(l/10)and PPGWPGF(1/10).In order to obtain peptides mimetic to multi- epitopes of LPS, the same peptide library was used for screening of mimotopes with purified polyclonal antibody to S. Typhi. LPS. 20 of positive clones were identified as positive clones. The binding of all positive phage clones to polyclonal antibody can be inhibited by S. typhi. LPS at a concentration of 0.5fJ,g/ml by competitive inhibition assay. The amino acid sequences of peptide deduced from the corresponding DNA sequences of 10 of the positive clones were FQFYPAA(4/10) , LQFYPGA(1/10) , LFTFAHY(3/10) YQYYPAA(2/10).Mice were immunized with four phage clones(PSWASF\V\ QSWFLPY, FQFYPAA and YQYYPAA) respectively and also with mixed phage clones of the above four. Only a part of mice immunized with clones QSWFLP Y , FQFYPAA and mixed clones produced anti-LPS with titer of 1:100-1:800 in serum. Mice of immunized with other antigens and control group did not produce anti-LPS. These results suggested that peptides QSWFLPY and FQFYPAA dis更多还原