【作者】 曾燚华；

【导师】 吴移谋；

【作者基本信息】 南华大学 ， 病原生物学， 2012， 博士

【摘要】 研究背景生殖支原体（Mycoplasma genitalium, Mg）是近年新明确的一种性病病原体，研究表明，Mg可能引起泌尿生殖道感染，与盆腔炎、呼吸道感染、关节炎等疾病有关，并可导致不育。另外，Mg还是引起艾滋病患者发生机会感染的主要病原体之一，被称为AIDS相关支原体。对于Mg的感染，目前仍然没有满足临床需要的疫苗可供使用，临床上急需安全、有效的疫苗以用于预防Mg的感染。生殖支原体粘附素蛋白（MgPa）是位于其尖形结构的一种主要的粘附素，其C端具有很强的免疫原性（最强区位于第1248～1364aa）。因此，MgPa是一种重要的中和抗原，在Mg的诊断和预防中起着重要的作用。本研究拟利用噬菌体展示随机肽库技术筛选MgPa的模拟表位，采用多抗原肽（MAP）的设计方案，制备并纯化含有模拟表位的八分枝MAP，并对其免疫原性进行研究。研究目的表达并纯化含生殖支原体MgPa优势表位（1075～1364aa的重组蛋白（rMgPa），制备并纯化rMgPa的多克隆抗体（pAb），以此pAb为靶分子，利用噬菌体展示随机12肽库筛选并鉴定MgPa的模拟表位，为研究MgPa的抗原表位结构提供实验依据；制备含有多个模拟表位的多抗原肽（MAP），检测其诱导小鼠产生特异性体液免疫和细胞免疫应答水平，为研制安全、有效的基于多个抗原表位的Mg表位肽疫苗奠定实验基础。研究方法（1）利用构建好的原核表达载体PET-30a(+)/MgPa在大肠杆菌中诱导表达MgPa的重组蛋白，并用ELISA、SDA-PAGE和Western blot等方法对其进行鉴定，再用Ni-NAT亲和层析柱纯化重组蛋白，用BCA法测定重组蛋白的浓度。（2）将经纯化的重组蛋白免疫新西兰兔以制备其相应的pAb，用饱和硫酸铵法和溴化氰活化的琼脂糖4B亲和层析柱纯化pAb，用间接ELISA法检测免疫兔血清中pAb的效价，再用Western blot鉴定pAb的特异性和免疫原性。（3）以此pAb为靶分子对噬菌体展示随机12肽库进行4轮生物淘洗，随机挑取经淘洗后的噬菌体克隆进行扩增培养，提取并纯化其单链DNA进行DNA测序分析，推导出噬菌体表面展示的外源性氨基酸序列，并用MIMOX工具进行生物信息学分析。用ELISA、竞争性结合试验和Western blot等方法检测噬菌体与pAb结合的特异性。（4）以多聚赖氨酸为核心基质，人工合成含有筛选到的模拟表位的八分枝MAP，用反向高效液相色谱（RP-HPLC）分析MAP的纯度，再用质谱分析仪测定MAP的分子量以期对其进行鉴定。（5）将30只6～8周龄雌性BALB/c小鼠随机分为PBS组、WP组、AS组、KH组和混合组，将100L PBS或100g各种合成的MAP或其混合物分别经皮下多点注射免疫小鼠，共免疫4次，每间隔2w免疫1次。每次于免疫前1天剪尾取血，末次免疫后第14d摘眼球放血收集小鼠血清，无菌制备脾细胞悬液。（6）间接ELISA测定小鼠血清中的特异性IgG抗体及其亚类的水平；ELISA检测脾细胞培养上清中的IL-4和IFN-γ水平；MTT比色法检测小鼠脾淋巴细胞的增殖反应。研究结果（1）SDS-PAGE显示，IPTG诱导转化有PET-30a(+)／MgPa的大肠杆菌成功表达了一分子量约为37kD的含6×His的融合蛋白rMgPa，经Ni-NAT亲和纯化获得了较高纯度的目的蛋白，目的蛋白在菌体内主要以包涵体形式存在，BCA法测定经纯化的目的蛋白浓度达到1160μg/mL。（2）利用纯化的经复性后的rMgPa免疫新西兰兔后获得了相应的pAb，间接ELISA结果显示血清中特异性pAb的效价为1∶25600；Western blot的结果表明rMgPa能与免疫血清发生特异性结合；免疫血清经饱和硫酸铵法初步纯化，随后经亲和层析法纯化后得到具有较高纯度的抗rMgPa的pAb，SDS-PAGE分析的结果表明所制备的pAb的轻链分子量约为25kD，重链分子量约为50kD，因此，该pAb的分子量约为150kD。（3）以经纯化的抗rMgPa的pAb抗体为靶分子，对噬菌体展示随机12肽库进行了4轮生物淘洗，第一轮和第四轮生物淘洗的产率分别为1.45×10-6和9.25×10-4，说明特异性噬菌体克隆得到了明显富集；经对45种不同的肽序列进行比较分析以及用MIMOX进行生物信息学分析，结果表明45个肽序列中的3组一致性的核心序列分别为： P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L和K-S-L-S-R-X-D-X-I。（4）ELISA实验的结果显示：45个含不同肽序列的噬菌体克隆中，共有36个为阳性噬菌体克隆，其中23个噬菌体克隆有较高的结合力，其A450值均高于1.5；竞争性结合试验的结果表明，这些噬菌体与pAb的特异性结合能被不同浓度的rMgPa部分抑制，且随着其浓度的增加其抑制效果也相应的增加；Western blot方法检测的结果表明A450值高于1.5的23个阳性噬菌体能与pAb发生特异性结合。（5）反向高效液相色谱分析的结果表明合成的3个MAP的纯度都在90%以上，质谱分析的结果说明所合成的MAP的分子量与预期的理论分子量基本一致，因此，成功地合成了较高纯度的3个模拟表位的MAP。（6）小鼠经4次免疫后，WP组、AS组、KH组和混合免疫组血清中IgG抗体的A450值分别为0.935±0.028、0.640±0.022、0.841±0.103和1.326±0.025，与PBS对照组（0.118±0.023）比较，均具有统计学意义（p<0.01）；与WP组、AS组和KH组相比，混合免疫组小鼠血清中IgG抗体的A450值升高更为明显（p<0.05）。（7）WP组、AS组、KH组和混合免疫组的IgG抗体效价分别为1∶2560、1∶640、1∶2560和1∶5120。WP组、AS组、KH组和混合免疫组小鼠血清中的IgG抗体以IgG2a型为主，其相应的IgG2a/IgG1分别为1.835±0.125、1.708±0.180、1.690±0.202和2.095±0.179，均显著高于PBS组（0.967±0.142）（p<0.01）。（8）WP组、AS组、KH组和混合组的IL-4含量分别为55.588±2.407、42.996±1.935、54.754±2.270和81.598±2.649pg/mL，与PBS组（16.673±1.662）相比，具有统计学意义（p<0.01）。与WP、AS、KH组相比，混合组产生的IL-4水平具有显著性差异（p＜0.01）。（9）WP组、AS组、KH组和混合免疫组的刺激指数（SI）分别为1.560±0.036、1.353±0.131、1.424±0.041和1.874±0.060，明显高于PBS组（1.107±0.032，p<0.01）；混合免疫组的刺激指数（SI）显著高于WP组、AS组和KH组（p<0.01）。（10）WP组、AS组、KH组和混合免疫组的IFN-γ含量分别为168.496±7.919、98.327±5.030、111.437±5.243和235.815±8.430pg/mL，显著高于PBS组（31.476±1.717，p<0.01）；且与WP组、AS组、KH组相比，混合免疫组的脾淋巴细胞产生了更多的IFN-γ（p<0.01），差异具有统计学意义。结论（1）成功表达并纯化了分子量为37kD的重组蛋白rMgPa；（2）成功制备并纯化了高效价、高特异性的兔抗rMgPa的pAb；（3）成功筛选到MgPa的3个可能的模拟表位：P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L和K-S-L-S-R-X-D-X-I；（4）成功制备并纯化了3个模拟表位相应的MAP——WP、AS和KH；（5）WP、AS和KH均能刺激小鼠产生较强的体液免疫和细胞免疫应答，且混合免疫组诱导的免疫应答效果比单个MAP组更好。更多还原

【Abstract】 BackgroundMycoplasma genitalium (M. genitalium) is a newly defined pathogen of causessexually transmitted diseases. M. genitalium has been well described as a pathogenwith acute and chronic nongonococcal urethritis (NGU), and may cause genital tractdiseases in women, such as bacterial vaginosis cervicitis, pelvic inflammatory disease(PID), endometritis and infertility. And M. genitalium can cause conjunctivitis andpneumonia for newborn by the mother`s genital tract infection during childbirth. M.genitalium is one of the main pathogens cause opportunistic infection for AIDSpatients, and a synergistic factor of human immunodeficiency virus (HIV), thus it isknown as the AIDS-associated mycoplasma.So far, there is still not available vaccine to meet the clinical needs. Thus clinicaldoctors are badly in need of safe, effective vaccines to prevent M. genitalium infection.M. genitalium adhesion protein (MgPa) is the major adhesion of M. genitalium andit`s C-terminal part (amino acid1248～1364) is the most immunogenic region. ThusMgPa plays an important role in the diagnosis and prevention of M. genitalium. Thisstudy aim to screen the mimic epitopes of MgPa by phage display random peptidelibrary, and prepare the eight branches MAP containing the mimic epitopes, and studythe immunogenicity of MAP.ObjectivesTo express and purify the recombinant adhesion protein of M. genitalium (rMgPa)containing the dominant epitope (1075~1364aa), and prepare and purify the rabbit anti-rMgPa polyclonal antibody (pAb). A12-mer phage display peptide library wasscreened by using the purified pAb for target molecular in order to obtain theantigenic mimic epitopes of MgPa, and thus facilitate the understanding of theantigenic structure of MgPa. The multiple antigen peptides (MAP) containing themimic epitope were prepared, and the humoral and cellular immune response levelswere analyzed in MAP-immunized BALB/c mice, in order to provide the theoreticalbasis for the development of the safe, effective multi-epitope-baseded markervaccines to prevent M. genitalium infection.Methods（1）The recombinant prokaryotic expression plasmid was transformed into E.co1iRosettaTM2(DE3) to express the adhesion protein of M.genitalium. Therecombinant protein (rMgPa) was identified by ELISA, SDS-PAGE andWestern blot, and then purified by Ni-NAT immunoaffinity chromatography.The concentration of MgPa was analyzed by bicinchoninic acid method.（2） New Zealand Whites rabbits were immunized with the purified MgPa togenerate the corresponding polyclonal antibody. Polyclonal antibody wasinitially purified by saturated ammonium sulfate, and then affinitychromatography with CNBr-activated Sepharose4B coupled with recombinantprotein. The immune sera were characterized by ELISA and Western blotanalysis.（3）The purified pAb was used to target molecular to screen the immunodominantmimic epitopes of MgPa by using a random12-peptide phage display library.Phage clone were randomly selected and then the single chain DNA wereextracted and purified, DNA sequence analysis and computer-basedbioinformatics analysis were performed to define the consensus amino acidresidues of the mimotopes by MIMOX. The binding specificities of the selected phage-displayed peptides to the purified pAb were confirmed byELISA, competitive ELISA and Western blot analysis.（4）The eight branches MAP containing the screened mimic epitopes of MgPa wereprepared using poly-lysine as the core matrix. The purity of MAP was analyzedby reverse phase high performance liquid chromatography (RP-HPLC), andthen the molecular weights of MAP were characterizated by MassSpectrometry.（5）A total of30female BALB/c mice were randomly divided into5groups: GroupPBS, Group WP, Group AS, Group KH and Group mixed MAP. The miceswere inoculated intramuscularly with100g of MAP or100L of PBS attwo-week interval for four times. The sera of mice were collected and stored at-20℃in the day before every immunization or the day before execution. Themice spleen lymphocytes were separated for preparing the spleens cellssuspensions.（6）The specific IgG antibody and the subtype of IgG antibody in serum of theimmunized mice and. IFN-γ or IL-4levels in the cultured supernatant of spleenlymphocytes were detected by indirected ELISA. The proliferation responsesof the spleen lymphocyte were detected using MTT assay and delegated bystimulation index (SI).Results（1）The results of SDS-PAGE showed the recombinant protein containing6×His,with molecular weight of about37kD, was successfully expressed in E.co1iRosettaTM2(DE3). And the target protein in bacteria was mainly in the form ofinclusion bodies, the target protein with high purity were obtained by Ni-NATaffinity chromatography. The protein concentration reaches1160μg/mL byBCA method.（2）The corresponding polyclonal antibody were obtained by immunizing New Zealand rabbits with the purified and refolded rMgPa. The results of ELISAdemonstrated that the titer of pAb in serum is1∶25600. The results ofWestern blot showed that the polyclonal antibody has high specificity.SDS-PAGE analysis proved that the pAb in sera has high purity and themolecular weight of light chain of the prepared pAb was about25kD, and thatof heavy chain was approximately50kD, therefore, the molecular weight ofpAb about150kD.（3）After four rounds of biopanning to phage display random12peptide library, theyield ratios of the first and fourth round biopanning were1.45×10-6and9.25×10-4, respectively, which demonstrated the specific phages weresignificant enriched. The single-stranded DNA were successfully extractedfrom74phage clones and sequenced. The exogenous inserts from74phageclones distinguished45peptides sequences that can be divided into three groupsaccording to the different amino acid sequence. The results of bioinformaticsanalysis by MIMOX and comparative analysis for the45different peptidesequences revealed three different consistent core sequence wereP-S-A-A/V-X-R-F/W-E/S-L-S-P, A-K-I/L-T/Q-X-T-L-X-L andK-S-L-S-R-X-D-X-I.（4）Amongst45peptides, a total of36peptides were ELISA positive and theabsorbance values of23phage clones were higher than1.5,which demonstratedthe high reactivities with pAb. Competitive binding assay showed that thespecific binding between these phages and pAb could be partially inhibited bydifferent concentration of rMgPa, and the inhibitory effect was correspondinglyincreased when the concentration of rMgPa increased.23phage clones that theabsorbance values were higher than1.5could specifically bind with pAb.（5）The purity of three MAP were higher than90%by reverse phase highperformance liquid chromatography analysis. The results of MS analysis showed the molecular weight of MAP was basically consistent with theexpected theoretical molecular weight, which three MAP containing the mimicepitopes were successfully prepared.（6）The A450value of IgG antibody in the last immunized sera of group WP, AS,KHand mixed group were respectively0.935±0.028、0.640±0.022、0.841±0.103and1.326±0.025, which were significantly higher than that of PBS group(0.118±0.023)(p<0.01). And the A450value from mixed group was obviouslyhigher than those of group WP, AS and KH (p<0.05).（7）The titers of specific IgG antibody were1∶2560、1∶640,1∶2560, and1∶5120for group WP, AS, KH and mixed group, respectively. The IgG2a antibodywas mainly antibody in the mice sera of Group WP, AS, KH and mixed group,and the corresponding ratios IgG2a/IgG1were respectively1.835±0.125,1.708±0.180,1.690±0.202and2.095±0.179, which were significantly higherthan that of PBS group (0.967±0.142，p<0.01).（8）The contents of IL-4in the supernatant of the cultured spleen lymphocyte ofGroup WP, AS, KH and mixed group were respectively55.588±2.407,42.996±1.935,54.754±2.270and81.598±2.649pg/mL, which were higher thanthat of Group PBS（16.673±1.662）(p<0.01). And the IL-4level of mixed grouphas significantly difference from those of group WP, AS and KH (p＜0.01).（9）The stimulation indexs (SI) of the spleen lymphocyte from Group WP, AS, KHand mixed group were higher than that of Group PBS (1.107±0.032，p<0.01).And the SI of the mixed group was higher than those of other groups (p<0.01).（10）The contents of IFN-γ in the supernatant of the cultured spleen lymphocyte ofGroup WP, AS, KH and mixed group were significantly higher than that ofGroup PBS (31.476±1.717),(p<0.01). Meanwhile, the contents of IFN-γ fromthe mixed group was obviously higher than that those of other groups (p<0.01). Conclusion（1）The rMgPa with molecular weight of about37kD was successfully expressed inE.co1i RosettaTM2(DE3) and purified by by Ni-NAT affinity chromatography.（2）The rabbit anti-rMgPa pAb that had high titer and specificity was successfullyprepared and purified by affinity chromatography.（3）The mimic epitopes of MgPa were successfully screened and identified by phagedisplay peptide library and the motif P-S-A-A/V-X-R-F/W-E/S-L-S-P,A-K-I/L-T/Q-X-T-L-X-L and K-S-L-S-R-X-D-X-I may represent theimmunodominant mimic epitopes of MgPa.（4）The three corresponding MAP (WP, AS and KH) containing the three mimicepitopes were successfully prepared, identificated and purified.（5）The WP, AS and KH could induce strong specific cellular immune and humoralimmune response. And the competence of mixed group was stronger than thatof the Group WP, AS and KH.更多还原