【作者】 郑磊；

【导师】 程浩；

【作者基本信息】 浙江大学 ， 皮肤病与性病学， 2005， 硕士

【摘要】 淋病是由淋病奈瑟菌感染引起的世界上发病率最高的性传播疾病之一。近年来,我国淋病发病人数逐年上升,淋病的广泛传播已严重危害人类的身心健康和社会稳定。随着淋球菌耐药菌株不断增加,抗菌治疗日益困难。寻找有效的淋病治疗和预防手段就显得非常重要。 据文献报道,淋病奈瑟菌进入机体后首先需粘附于泌尿生殖道上皮细胞表面,然后才能进入机体引起一系列病变,粘附是淋球菌致病的先决条件,一旦失去粘附能力,就不能引起淋病。进一步研究发现,淋球菌的外膜蛋白在细菌粘附中起了至关重要的作用。 淋球菌的外膜蛋白不仅与淋球菌致病机制有关,同时也可能是研究淋病疫苗的可选免疫原之一。目前已知的淋球菌表面抗原有多种,其中Porin I(PI)是外膜蛋白中含量最高的蛋白成分,约占60%,许多资料表明蛋白PI在自然感染过程中抗原性质稳定,有较强的免疫性,且抗PI抗体具有杀菌作用,因而理论上PI抗原很有可能用于制备成理想的淋病疫苗。 我们已成功构建表达了重组淋球菌GST-PI融合蛋白并用其免疫新西兰兔,获得兔抗PI蛋白的多抗血清。作为研究工作的延续,我们进一步将GST-PI多抗血清纯化获得PI-IgG抗体。建立淋球菌感染BALB/c小鼠模型,并通过观察更多还原

【Abstract】 Gonorrhea is at the present time one of the most common sexually transmitted diseases in the world. In our country the prevallance of gonococcal is increasing rapidly in recent decades. Gonorrhea is a purulent inflammation of the mucous membrane surface caused by a sexually transmitted microorganism, Neisseria gonorrhoeae. Virtually any mucous membrane can be infected. Due to the extensive transmission, gonorrhea seriously endangers mental and physical health of the mankind and social stability as well. Since Neisseria gonorrhoeae has throughout its history developed resistance to traditional antibiotics, it might be very important to develop an effective vaccine to prevent the infection of Neisseria gonorrhoeae.The pathophysiology of N gonorrhea and the relative virulence of different subtypes depend on the antigenic characteristics of the respective surface proteins. Certain subtypes are able to evade serum immune responses and are more likely to lead to disseminated （systemic） infection.The outer membrane of Neisseria gonorrhoeae includes a variety of protein, the most abundant of which is known as protein I （PI）, occupying about 60% of total outer membrane protein. PI may function as a porin creating a hydrophilic diffusionchannel and allow uptake of essential nutrients. Unlike the other major surface antigens, pili, PII and LPS, PI dose not undergo antigenic variation during infection. PI may also play an important role in pathogenesis through its ability to interact with the cell membrane of eukaryotic cells, that means PI is related to the adherence of gonococcus to mucosa. PI is also a major target for immune attack on the gonococcus and can stimulate the production of opsonins. And anti-PI antibody can also activate complement-mediated bactericidal activity in mice. Thus, by virtue of its stability and antigenicity, PI is of considerable interest as a potential gonococcal vaccine candidate.The goal of our study is to investigate the blockness effects of purified polyclonal Anti- Porin I antibody on N.gonorrhoeae adherence to genitourinary tract epithelia of BALB/c mouse. Polyclonal Anti GST-PI antibody was generated by immunizing rabbit with GST-PI fusion protein which was constructed and expressed by ourselves. We established mice model of gonorrhea. In order to evaluate the effects of PI-IgG on gonococcus adhesion to vagina mucus, the macroscopic and pathological assessing as well as gonococcus culture was employed after gonococcus challenge on PI-IgG immunized mice. We hope our study may lead to a potential possibility for the further study of gonococcal vaccine.Methods1. Induction and purification of GST-PI fusion proteinThe expression recombinant （pGEX-4T-2/PI） was transformed to E.Coli P2392, then GST-PI fusion protein was induced by IPTG. The purification process was: SDS-PAGE, excision of 60 KDa protein band, and electroelution to collect GST-PI fusion protein.2. Preparation and detection of Polyclonal antibody seraFirst immunization: Each rabbit was inoculalted with emulsified immunizator containing GST-PI fusion protein and complet Fu’s immunoadjuvant. Enhancingimmunization: Each was inoculated for further three times with the emulsified immunizator containing GST-PI protein and incomplete Fu’s immunoadjuvant. On the fifth day, the antibodies potence value of rabbit sera was detected by doubleeagar immunodiffusion. Last immunization: Each was inoculated with PI protein without immunoadjuvant, and on the fifth day the blood of rabbit was collected and the sera were separated out.3. Purification of the polyclonal antibody of rabbit anti-PI seraThe purified immunolobulin G was obtained by ammonium sulphate deposition and DEAE cellulose chromatography. The titer of antibody were determined by doubleeagar immunodiffusion, and the purity were characterized by SDS-PAGE.4. Adhesion modelRoutinly degerm the cunnus of BALB/c mouse, centrifugate and callback the culture solution of Diplococcus gonorrhoeae, then inoculate the bacteria endovaginal (5×10⁸ bacteria per mouse). The vaginal mucosa and the secre更多还原