【作者】 杨志伟；

【导师】 陈建平；

【作者基本信息】 四川大学 ， 病原生物学， 2007， 博士

【摘要】 嗜肺军团菌是引起军团病的主要病原体，可导致流行、散在和非典型肺炎。嗜肺军团菌是一种兼性胞内寄生菌，广泛存在于各种自然水源及人工管道水源中，污染空调冷凝器、浴室、淋浴喷头等处后，人因吸入含有军团菌的气溶胶而感染，并在肺泡上皮细胞和单核巨噬细胞内增殖。嗜肺军团菌侵入肺泡上皮细胞和单核巨噬细胞与外膜蛋白、Ⅳ型菌毛蛋白等有关。由嗜肺军团菌主要免疫原基因ip编码产生的29kDa外膜蛋白（29 kDa immunogenic protein，IP）在机体防御军团菌感染方面具有重要作用，是机体免疫应答反应的主要免疫原，也是军团菌致病因子，而其具体的机理仍不清楚。军团菌黏附于宿主细胞表面是其致病的先决条件，菌毛是细菌的黏附结构。嗜肺军团菌的pilE基因编码的Ⅳ型菌毛蛋白参与细菌多种功能活动：如黏附、活动力、特异靶细胞的识别及噬菌体的感染等。此外，29kDa外膜蛋白和Ⅳ型菌毛蛋白还是一种重要的保护性抗原。本研究在查阅分析国内外相关文献及结合本室研究进展的基础上，选择ip基因、pilE基因为疫苗候选基因，采用重叠延伸PCR法将嗜肺军团菌ip和pilE基因融合，构建ip基因、pilE基因及ip／pilE融合基因的重组真核表达载体作为DNA疫苗，检测其免疫原性和免疫保护性，为研制嗜肺军团菌DNA疫苗提供一定的实验依据。本研究共分六部分进行：第一部分，以嗜肺军团菌血清1型（L1）菌株基因组DNA为模板，采用常规PCR法扩增出792bp的ip基因，将其定向克隆到原核表达载体pET-32a（+），构建原核表达重组质粒pET-ip，经IPTG诱导有效表达出Trx-IP融合蛋白质。第二部分，采用PCR法从嗜肺军团菌基因组DNA中扩增出pilE基因，将其定向克隆入原核表达载体pET-32a（+），构建原核表达重组质粒pET-pilE,并诱导Trx-PILE融合蛋白在原核系统中高效表达。第三部分，采用重叠延伸PCR法将嗜肺军团菌ip和pilE基因融合，融合基因ip／pilE被定向克隆入原核表达载体pET-32a（+）中，构建原核表达重组质粒pET-ip／pilE，经IPTG诱导表达出Trx-IP／PILE融合蛋白。第四部分，将ip基因、pilE基因和ip／pilE融合基因分别克隆入pcDNA3.1（+）中，得到真核表达重组质粒pcDNA3.1-ip、pcDUA3.1-pilE和pcDNA3.1-ip／pilE。将这三种真核表达重组质粒分别用脂质体介导转染NIH3T3细胞，用免疫荧光法检测各克隆基因蛋白的瞬时表达，随后用Western-blot分别鉴定稳定转染的阳性细胞，均表达出相应大小的蛋白。第五部分，将构建好的真核重组质粒作为DNA疫苗，肌肉注射免疫BALB／c小鼠两次，并设pcDNA3.1（+）、PBS组，检测免疫小鼠体内的抗体水平、脾淋巴细胞增殖活性、IFN-γ、IL-2产生水平、CTL杀伤活性等指标，评价疫苗的免疫原性。结果发现，与空质粒对照组相比，各实验组均检测到不同程度的免疫原性，差异有显著性（P＜0.01）：pcDNA3.1-ip／pilE免疫组抗体的滴度高于pcDNA3.1-pilE免疫组（P＜0.05）；pcDNA3.1-ip／pilE免疫组细胞免疫各项指标均高于pcDNA3.1-ip、pcDNA3.1-pilE免疫组。pcDNA3.1-ip免疫组和pcDNA3.1-pilE免疫组相比，pcDNA3.1-ip的免疫原性强于DcDNA3.1-pilE。第六部分，将构建好的真核表达重组质粒作为DNA疫苗，免疫BALB／c小鼠，加强免疫后，用10倍LD₅₀嗜肺军团菌L1型菌株对免疫小鼠进行攻击，并设空白和阴性对照组，对照组小鼠全部死亡后，检测肺组织带菌量和肺组织病理形态学变化，评价疫苗对动物的免疫保护性。小鼠肺组织保护效率的比较发现，各实验组保护效率均明显高于pcDNA3.1（+）空质粒对照组，pcDNA3.1-ip／pilE免疫组保护效率比pcDNA3.1-ip、pcDNA3.1-pilE免疫组高。病理形态学检测发现：空质粒pcDNA3.1（+）免疫组主要表现为炎性病变，镜下可见肺泡隔明显增宽，局部毛细血管扩张充血，并可见中性粒细胞及单核细胞浸润，肺泡腔内可见渗出物；而DNA疫苗实验组炎性病变较轻，所构建的DNA疫苗免疫组均能诱导小鼠产生一定的免疫保护效应。本研究结果表明：所构建的嗜肺军团菌DNA疫苗具有较强的免疫原性和免疫保护作用；pcDNA3.1-ip／pilE的免疫原性和免疫保护作用均高于其它免疫组。因此，pcDNA3.1-ip／pilE作为嗜肺军团菌DNA疫苗在今后的相关研究中值得作进一步探索。更多还原

【Abstract】 Legionella pneumophila, the most common cause of Legionaires’ disease, is a facultative intracellular parasite bacterium that causes epidemic, sporadic, and nosocomial pneumonia. Epidemiological evidence suggests that the environmental source of theses infection is natural freshwater systems and man-made aquatic environments. Human infection occurs by the inhalation of aerosolized bacteria from L. pneumophila-contaminated water sources. L. pneumophila invades and multiplies within alveolar macrophages. The major outer membrane protein and typeⅣpili are associated with L. pneurnophila infection of macrophages have been reported. In the outer membrane of L. pneumophila, there is a protein with molecular weight of 29 kDa, which is not only an specific antigen of the species but also the major outer membrane protein; it also be factor ofpathogenecity in the bacterium. The 29 kDa protein (29 kDa immunogenic protein, IP) was encoded by the ip gene. The mechanisms still remain unclear. The presence of typeⅣpilin gene (pilE) and its expression by L. pneumophila provide an advantage for colonization of lung tissues and invasion of macrophage during Legionnaires’Disease. According to the current study, an astonishing number of cellular functions have been attributed to the typeⅣpili, including surface motility, microcolony and biofilm formation, adhesion, immune evasion, cell signaling, transformation of DNA and phage attachment. These observations suggest that the 29 kDa protein and typeⅣpili may play a vital role in the host immune response, therefore develope some safe and effective vaccines for the disease will be of practical significance.This study selected Legionella pneumophila DNA vaccine as research direction. According to the results of indexing and analysis in the relevant literature, this study selected ip gene, pilE gene of Legionella pneumophila as candidate genes of the DNA vaccines. We have used the technique of splicing by overlap extension by the polymerase chain resction (SOE by PCR) to produce the ip and pilE fusion gene of Legionella pneumophila. The eukaryofic expression recombinant plasmid containing the ip gene, pilE gene and ip/pilE fusion gene were constructed successfully. And then study the immunity characteristics of these DNA vaccines, which provided certain experimental basis for the study of the new generation of safe and efficient DNA vaccines.The study was divided into six parts.In the first part, the ip gene of 792 bp long was amplified from the total cell DNA of Legionella pneumophila with PCR, and then was inserted it into the prokaryotic expression vector pET-32a(+). After the recombinant plasmid pET-ip was identified. And also the Trx-IP fusion protein of approximately 49 kDa in size was expressed in prokaryotic cell efficiently as expected.In the second part, the pilE gene of Legionella pneumophila was amplified from the genomic DNA ofLegionella pneumophila with PCR, and then was inserted into the prokaryotic expression vector pET-32a(+). The prokaryotic expression recombinant plasmid pET- pilE was constructed. After identified with restriction enzyme analysis and polymerase chain reaction and nucleotide sequencing analysis, the Trx-PILE fusion protein of approximately 35.7 kDa in size was expressed efficiently as expected.In the third part, the fragments of the ip and pilE gene having matching sequences at their ends to be fused were amplified from the genomic DNA of Legionella pneumophila by PCR respectively, and then these PCR products were mixed, denatured, reannealed, the strands with matching sequences at their 3’ ends can overlap and serve as primers for each other. Extension of this overlap by DNA polymerase produces recombinant products. After amplification by using outer primers, the ip/pilE fusion gene of about 1218 bp long was amplified and then it was inserted into the prokaryotic expression vector pET-32a(+), the recombinant plasmid pET- ip/pilE was constructed successfully and also the approximately 64.7 kDa Trx-IP-PILE fusion recombinant protein in size was expressed as expected.In the fourth part, the ip gene, pilE gene and ip/pilE fusion gene was amplified by PCR. The products of PCR were inserted respectively into eukaryotic expression plasmid pcDNA3.1(+). The eukaryotic expression recombinant plasmid pcDNA3.1-ip, pcDNA3.1-pilE and pcDNA3.1-ip/pilE were also constructed respectively. NIH3T3 cells were transfected by the recombinant plasmid pcDNA3.1-ip, pcDNA3.1-pilE and pcDNA3.1-ip/pilE with lipofection strategy. Transient products were detected in cell membrane and inside the cell by immunofluorescence. And about 29 kDa protein of the IP, 15.7 kDa protein of the PILE, and IP-PILE fusion protein of approximately 44.7 kDa in size were detected in stable transfercted cells with Westem-blot.In the fifth part, BALB/c female mice were immunized intramuscularly with the naked DNA vaccine, and an blank group was immunized intramuscularly with PBS. Immunogenicity was evaluated through antigen specific antibodies, lymphocyte proliferative response, IFN-γproduction, IL-2 production and cytotoxic T-lymphocyte response of immunized mice. The results showed that immunogenicity of DNA vaccine immunized mice were higher than control group. Humoral immunity and cellular immunity of each group of DNA vaccines immunized mice was higher than pcDNA3.1(+) immunized mice. Antigen specific antibodies of pcDNA3.1-ip/pilE immunized mice were higher than peDNA3.1-pilE immunized mice. Cell mediated immune responses of pcDNA3.1-ip/pilE immunized mice were higher than pcDNA3.1-ip, pcDNA3.1-pilE immunized mice. Immunogenicities of pcDNA3.1-ip immunized mice were higher than pcDNA3.1-pilE immunized mice.In the sixth part, BALB/c female mice were immunized intramuscularly with the DNA vaccines. 2 weeks after the second immunization, the immunized mice were challenged with L.pneumaphila serogroup 1. On the day of infection, bacteria were diluted in normal saline deliver approximately 10 times the 50% lethal dose that consistently resulted in the death of all control mice to each animal. The bacterial counts and the pathological section of the lungs of the immunized mice were detected to evaluate the protective immunity. The changes results of lung showed that the bacterial counts of DNA vaccine immunized mice were lower than control, especially the pcDNA3.1-ip/pilE group show significantly lower counts than other DNA vaccine immunized groups. The pathologic changes and cell recruitment into the lung of each animal was assessed by light microscopy. In five independent experiments, mice immunized with pcDNA3.1-pilE or pcDNA3.1-ip or pcDNA3.1-ip/pilE decreased significantly infiltration of inflammatory cells in alveolar and interstitial spaces than controls to lethal challenged.This study showed that the three DNA vaccines have higher immugenecity and protective immunity in some degree. It is meaningful to construct fusion gene of candidate genes. This study provided experimental proofs to further study DNA vaccine against Legionella pneumophila.更多还原