【作者】 曹炬；

【导师】 尹一兵；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2007， 硕士

【摘要】 目的肺炎链球菌(streptococcus pneumoniae, S.pn)是肺炎,脑膜炎和中耳炎的主要病因,引起全世界范围内婴幼儿,老年人和免疫缺陷个体疾病的主要病原菌。随着耐药菌株的不断发现,疫苗在其感染防治中的作用越来越重要。目前主要所用的是23价肺炎多糖疫苗和7价结合疫苗,但前者覆盖的血清型有限而且不是T细胞依赖性的,对免疫系统发育不完善的2岁以下小孩和老年人的保护性弱;后者存在荚膜血清型转换和载体携带的荚膜多糖血清型数量有限以及在肺炎高发人群中的保护作用小等问题,并且生产成本很高,不适合在发展中国家大量使用。因此发展肺炎链球菌蛋白疫苗迫在眉睫。肺炎链球菌表面蛋白A( pneumococcal surface protein A,PspA)是S.pn主要的毒力因子之一,在肺炎链球菌侵袭性感染时起着重要的作用,它是最早作为S.pn蛋白质疫苗的候选抗原之一;S.pn胆碱结合蛋白A(choline binding protein A,CbpA)作为S.pn的最为重要的表面蛋白成分之一,在S.pn引起脑膜炎时功能独特,是最关键的毒力因子。而S.pn热休克蛋白ClpP( caseinolytic protease )在S.pn从呼吸道(大约30到34℃)转移到血液和(或)脑膜(37℃),帮助S.pn适应宿主环境从而致病的过程中发挥着重要的功能。因此,本研究拟对上述三种保护性抗原进行克隆表达,通过三种抗原间的不同组合方式来探索联合多肽疫苗相对于单一多肽成分的优势性,并利用抗体被动保护实验来验证保护作用。方法1,用引物修饰法将S.pn基因组中扩增PspA,CbpA和ClpP片段并构建在原核表达载体PET-32a中。应用生物信息学软件DNAssist和GenBank数据库对已公布的TIGR4型S.pn全基因组序列进行相似性分析。将含目的基因片段的重组载体转化大肠杆菌BL21(DE3)中,经IPTG诱导表达,镍柱亲和层析纯化蛋白。应用SDS-PAGE对表达和纯化产物进行分析,透析除盐,并用抗组氨酸单克隆抗体Western blot鉴定。纯化的重组抗原分别免疫BALB/C小鼠,制备相应多克隆抗体血清。2,主动免疫保护实验,将96只BALB/C小鼠随机分为8组(每组12只):对照组,PspA组, CbpA组, ClpP组, PspA+ CbpA混合组, PspA+ ClpP混合组, CbpA+ ClpP混合组, PspA+ CbpA+ ClpP混合组。分别于0.14.28天经腹腔注射免疫,剂量为10 ug/只/次/种抗原。末次免疫后7天,每只小鼠经眼眶取血,ELISA检测免疫后血清特异IgG以证实高效价抗体的产生。7天后,每只小鼠经腹腔感染TIGR4型肺炎链球菌,连续监测21天小鼠的生存时间,计算各组的生存率。3,抗体被动免疫保护实验,将96只BALB/C小鼠随机分为8组(每组12只):对照组,Anti-PspA血清组, Anti-CbpA血清组, Anti-ClpP血清组, Anti-PspA + Anti-CbpA血清混合组,Anti-PspA+Anti-ClpP血清混合组, Anti-CbpA+Anti-ClpP血清混合组, Anti-PspA+ Anti-CbpA+ Anti-ClpP血清混合组。分别经腹腔注射免疫,剂量为100 ul抗血清/只/种抗血清。免疫24小时后,每只小鼠经腹腔感染TIGR4型肺炎链球菌,连续监测21天小鼠的生存时间,计算各组的生存率。结果1,经酶切鉴定和基因测序结果显示,S.pn PspA基因片段被正确克隆到PET-32a原核表达载体中。PspA基因片段的核苷酸序列长度为1329bp,与GeneBank中序列比较,无碱基突变。重组工程菌IPTG诱导,在BL21(DE3)菌中表达出了分子量约为66kD的Trx-PspA融合蛋白。重组蛋白以可溶形式表达,表达量约占全菌蛋白的40%;经Ni-NTA树脂纯化后得到了纯度>90%的重组蛋白。PspA重组蛋白免疫BALB/C小鼠制备多克隆抗体,Western blot结果显示抗体能够与S.pn菌体蛋白起反应。2,经酶切鉴定和基因测序结果显示,成功构建出原核表达载体pET32a(+)/CbpA。将重组子转化至大肠杆菌BL21(DE3)后,IPTG诱导,表达出了分子量约为80kD的Trx-CbpA融合蛋白。重组蛋白以可溶形式表达,表达量约占全菌蛋白的20%;经Ni-NTA树脂纯化后得到了纯度>90%的重组蛋白。CbpA重组蛋白免疫BALB/C小鼠制备多克隆抗体,Western blot结果显示抗体能够与S.pn菌体蛋白起反应。3,重组工程菌pET32a(+)/ClpP经IPTG诱导,表达重组工程菌IPTG诱导,在BL21(DE3)菌中表达出了分子量约为42kD的Trx-PspA融合蛋白。重组蛋白以包涵体形式表达,表达量约占全菌蛋白的35%;经Ni-NTA树脂纯化后得到了纯度>90%的重组蛋白。ClpP重组蛋白免疫BALB/C小鼠制备多克隆抗体,Western blot结果显示抗体能够与S.pn菌体蛋白起反应。4,重组蛋白的主动免疫保护实验,单一重组蛋白及混合组份经腹腔免疫BALB/C小鼠产生高滴度的抗体后,致死量的TIGR4腹腔感染。与对照组相比,抗原免疫组小鼠半数生存时间明显延长(P<0.05)。与其他抗原免疫组小鼠相比,三种亚单位重组蛋白免疫组半数生存时间最长(P<0.05)。PspA免疫只能延长小鼠的生存时间而不能防止其死亡,三种亚单位重组蛋白免疫组保护率为100%,与其他组小鼠(ClpP和PspA混合免疫组除外)相比,具有统计学意义(P<0.05)。5,抗体的被动免疫保护实验,与对照组相比,抗体免疫组小鼠生存时间明显延长(P<0.001)。三种抗体(Anti-PspA+Anti-CbpA+Anti-ClpP)混合免疫组与单一抗体免疫组相比,半数生存时间明显延长(P<0.025)。PspA多克隆抗体血清同样只能延长小鼠生存时间而不能防止其死亡。与单一抗体免疫组小鼠相比,三种抗体混合免疫组保护效率最高(P<0.05)。结论1,成功构建了pET32a(+)/PspA, pET32a(+)/CbpA, pET32a(+)/ClpP原核表达载体。纯化的重组蛋白PspA,CbpA和ClpP,具有良好的免疫原性和免疫反应性,可以作为S.pn基因工程疫苗的候选抗原。2,成功制备了重组蛋白PspA,CbpA和ClpP的多克隆抗血清,它们都能和肺炎链球菌菌体蛋白发生特异性的反应。3,亚单位疫苗主动免疫动物实验结果显示它们能够保护BALB/C小鼠抵抗致死量TIGR4型S.pn的攻击,其中三种疫苗蛋白混合免疫能够产生很好的保护效果,为进一步的S.pn多肽联合疫苗的研制奠定了基础。4,抗血清的被动免疫实验结果显示重组蛋白的多克窿抗体能够能够保护BALB/C小鼠抵抗致死量TIGR4型S.pn的攻击,其中三种抗体联合免疫能够得到最佳的保护效果,进一步证实主动免疫保护实验所取得的结果。更多还原

【Abstract】 ObjectiveStreptococcus pneumoniae is the major pathogen causing invasive disease in infants, the elderly, and the immunocompromised individuals, including sepsis, meningitis and pneumoniae.The 23 valent capsular polysacchride vaccine is useful in adults but fails to protect those at highest risk of disease at the youngest and oldest ages, and although the current 7-valent conjugate vaccines are effective against invasive disease caused by the vaccine-type strains, yet streptococcus pneumoniae can be divided into more than 90 serotypes according to the immunochemistry of their capsular polysaccharide, so vaccine coverage is limited, the cost of production is ver high and this conjugate vaccines are not applicable in the developing countries. Futhermore, replacement by invasive nonvaccine-serotypes in vaccinated individuals is a grim problem confronting us. Therefore, development of effective protein-based vaccines is pretty urgent.PspA is one of the most important virulence factors playing some crtical roles in the pathogenesis of pneumococcal infection, and it is one of the earlist pneumococcal candidate vaccines.CbpA is a surface protein is necessary for pneumococcal meningitis. Streptococcus pneumoniae encounters heat stress after penetration from the nasal mucosa(30℃to 34℃) into the blood and/or meninges (37℃) during the pathogenic process, and ClpP plays some important roles in helping S.pn adapt to the host environment and cause invasive diseases.In this study, we attempte to express those proteins and compare abilities of PspA, PspC and ClpP or serums containing their specific polyclonal antibodies to elicit protection against pneumococcal infection and examined the possibility that an immunization combining these proteins or their polyclonal antibodies might elicit better protection against pneumococcal infection.Methods1 The production of recombinant PspA, CbpA, and ClpP was achieved by PCR amplification of pneumococcal genes, with subsequent cloning into pET32a(+) plasmid. The plasmids were transformed into E.coli BL21. With IPTG induction the proteins were expressed. The recombinant proteins were identified by SDS-PAGE and purified with Ni-NTA column. The antigenicity of proteins was confirmed with anti-His monoclonal antibodies by Western-blot. Hyperimmune mouse sera specific for PspA (anti-PspA), CbpA (anti-CbpA),and ClpP (anti-ClpP) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 were to be identified with these serums by Western blot.2 Active immunization protection assays.96 BALB/C mice were divided 8 groups randomly.For this experiment, BALB/c mice (12 per group) were primed with either PspA, PspC, ClpP, PspA plus PspC, PspC plus ClpP, ClpP plus PspA, or a combination of PspA, PspC, and ClpP, each in CFA(1:1 ratio [vol/vol]) and each mouse received 10 ug of each protein on day zero ,and boosted with the same concentration of each protein in IFA (1:1 ratio [vol/vol]) on day 14,and on day 28,mice received the last dose of each protein in PBS. Mice injected with respective adjuvants in PBS served as negative controls. All vaccines were administered i.p., and sera were collected from all mice by retro-orbital bleeding 1 week after the third immunization. The sera were pooled on a group-by-group basis and assayed for anti-PspA, anti- PspC and anti-ClpP specific antibodies by ELISA prior to challenge with live pneumococci. After challenging with TIGR4, The mice were then monitored for death over 21 days, and the median survival time of each mouse were recorded.3 Active immunization protection assays, BALB/C mice were divided 8 groups randomly.For this experiment, the groups of 12 BALB/c mice to be challenged were passively immunized with 100ul of hyperimmune serums specific for PspA, PspC, and ClpP by i.p. injection and followed by challenging with TIGR4 24 hours later, the survival time of mice were monitored for 21 days.Results1 The results of didestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene PspA had been cloned into the plasmid pET32a(+). The length of the PspA gene was 1329bp. Compared to the sequence of GeneBank, no mutation was found.A recombinant protein about 66kD was expressed in BL21(DE3) after induction of IPTG. SDS-PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 50% of total baterial protein and the purity of target protein was up to 90%. Western-blot suggested that the rP30 could be recognized by anti-His monoclonal antibodies. Hyperimmune mouse sera specific for PspA(anti-PspA) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 could be identified with these serums by Western blot.2 The results of didestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene CbpA had been cloned into the plasmid pET32a(+). The length of the CbpA gene was 1369bp. Compared to the sequence of GeneBank, no mutation was found.A recombinant protein about 66kD was expressed in BL21(DE3) after induction of IPTG. SDS-PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 20% of total baterial protein and the purity of target protein was up to 90%. Western-blot suggested that the rCbpA could be recognized by anti-His monoclonal antibodies. Hyperimmune mouse sera specific for CbpA(anti-CbpA) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 could be identified with these serums by Western blot.3 The results of didestion with restriction DNA enzymes and sequencing of recombinant plasmid showed that the gene ClpP had been cloned into the plasmid pET32a(+). The length of the ClpP gene was 1369bp. Compared to the sequence of GeneBank, no mutation was found.A recombinant protein about 66kD was expressed in BL21(DE3) after induction of IPTG. SDS-PAGE showed that the recombinant protein mainly expressed as soluble form. The expression product of recombinant protein accounted for 20% of total baterial protein and the purity of target protein was up to 90%. Western-blot suggested that the rClpP could be recognized by anti-His monoclonal antibodies. Hyperimmune mouse sera specific for ClpP (anti-ClpP) were generated by i.p immunization of mice with each protein. Whole-cell lysates prepared from S.pneumonie strain TIGR4 could be identified with these serums by Western blot.4 Active immunization protection assays, In this experiment, the median survival times for mice that were immunized with single antigen or their combinations were significantly longer than that for mice that received adjuvants alone (p<0.0001), the PspA only delayed the time to death but failed to protect mice from death. Mice that received a combination of three antigens survived significantly longer than those that received single antigen or the combinations of two antigens(p<0.05). The highest survival rate of the various groups of mice was observed with the combination of three antigens, this rate was significantly different from that for mice that received either single antigens or two antigens (p<0.025)except the combination of ClpP and PspA.5 Passive immunization protection assays, mice that received either single serum containing specific antibody or their combinations lived significantly longer than mice that received serum from nonimmunized mice(p<0.001), Similarly, the anti-PspA serum could only elongate the survival time of the mice challenged intraperitoneally by S.pneumoniae TIGR4 but failed to protect these mice from death. the highest protection rate was observed with the combination of three serums containing anti-PspA, anti- PspC, and anti-ClpP polyclonal antibodies, it was significantly higher than those for mice treated with single serum(p<0.05). Conclusion1 The S.pn PspA, CbpA and ClpP genes were clone successfully into the plasmid pET32a(+). Sequencing and sequencing analysis were done and no mutation was found. The expressed and purified recombinant proteins were proved with immunogenicity and comtetence to induce protective immune response. These recombinant proteins can be candidate protein vaccines against S.pn.2 We successfully got the anti-PspA, anti-CbpA and anti-ClpP polyclonal antibodies and they could reacted with whole-cell lysates prepared from S.pneumonie strain TIGR4 specifically.3 Our active immunization protection assays showed that thses antigens could elicit protection against S.pn, and the combination of PspA, CbpA, and ClpP could enhance protection against S.pneumoniae TIGR4 challenged intraperitoneally, suggesting combining different pneumococcal virulence proteins to achive better protection is an applicable way.4 Our passive immunization protection assays showed that thses antibodies could elicit protection against S.pn, and the combination of anti-PspA, anti-CbpA, and anti-ClpP could enhance protection against S.pneumoniae TIGR4 challenged intraperitoneally, which proved our active immunization protection results.更多还原