淋病奈瑟菌表面蛋白A和孔蛋白B免疫保护性研究

发布时间：2018-01-26 05:32

本文关键词： 淋病奈瑟菌 NspA PorB 免疫保护　出处：《扬州大学》2008年硕士论文　论文类型：学位论文

【摘要】： 淋病是由淋病奈瑟菌(Neisseria gonorrhoeae)感染引起的一种常见性传播疾病(Sexual Transmitted Disease, STD),主要引起男性的急性尿道炎、前列腺炎、附睾炎;在女性主要表现为隐性感染,常因不能及时医治而导致病菌向深部扩散,引起盆腔炎、子宫内膜炎、输卵管炎,并能导致不孕症。淋病发病率高,是当前性病防治中的重点。由于淋病奈瑟菌容易对抗生素产生耐药性,给淋病的治疗带来了新的问题,因而加强疫苗研究对淋病防制工作具有重要意义。淋病奈瑟菌疫苗研制历经艰难,二十世纪七十年代全细胞疫苗的人体试验遭遇失败,研究的重点开始转向亚单位疫苗和基因疫苗等新型疫苗。菌毛是第一个被纯化的淋病奈瑟菌亚单位成分,尽管纯化后的菌毛能够刺激机体产生较高滴度的抗体,但是由于菌毛表面一些表位变异程度高,这些抗体并不具有显著的免疫保护作用。同样由于高度变异的原因,另一类重要粘附因子Opa蛋白在淋病疫苗研制中也受到了限制。奈瑟菌表面蛋白A (Neisseria surface protein A, NspA)表达于所有淋病奈瑟菌菌株,高度保守,免疫原性强。本实验室的前期研究表明,重组真核表达载体pCNspA免疫小鼠产生了具有介导补体溶解淋病奈瑟菌作用的NspA特异性抗体。孔蛋白(Porin)是淋病奈瑟菌的重要外膜蛋白,但是从菌体中纯化孔蛋白极易污染Rmp蛋白,而Rmp蛋白的抗体能够阻遏孔蛋白抗体的抗菌作用。高度纯化的孔蛋白可以刺激动物产生高滴度的保护性抗体。本研究进一步探索了NspA和孔蛋白B(Porin B, PorB)在淋病疫苗研制中的应用前景。为了改进基因免疫效价不高的缺陷,在基因免疫后用重组蛋白进行加强免疫。构建了NspA原核表达载体pQE31NspA。SDS-PAGE和Western-blotting检测表明pQE31NspA可在大肠杆菌中高效表达重组蛋白NspA。rNspA经Ni+-NTAPurification System纯化,并通过透析复性。然后用本室前期构建的NspA真核表达载体pCNspA基因免疫BALB/c小鼠。4次基因免疫后在小鼠血清中仅能检测到1:200的NspA特异性IgG。用rNspA加强免疫1次后,NspA特异性IgG则提高到1:3200以上。但是仍然略低于完全以rNspA免疫小鼠血清中特异性抗体的效价。抗体介导的补体溶淋病奈瑟菌实验和抗体调理吞噬实验结果表明,两种方案获得的免疫血清均表现出良好的抗淋病奈瑟菌作用;然而,免疫保护作用的强弱并不是与血清中特异性IgG水平相对应,抗体水平略低的基因免疫蛋白加强组血清表现出更强的抗菌作用。为了避免Rmp蛋白的污染和提高基因免疫的效果,从Christopher博士惠赠的PorB原核表达载体pUNCH682中切出PorB基因,构建了PorB真核表达载体pcDNA3.1PorB,瞬时转染COS-1细胞后,经Western-blotting验证,该重组载体可以在COS-1细胞中表达PorB。大量提取pcDNA3.1PorB,4次基因免疫BALB/c小鼠后诱生的淋病奈瑟菌特异性抗体效价较低,仅为1:200。用原核表达的rPorB加强免疫一次后抗体水平可大幅度提高,可达1:3200以上。完全以PorB蛋白免疫小鼠能诱生出更高的特异性抗体水平。但是在免疫血清免疫保护性方面,两种免疫方案获得的血清均表现出良好的抗菌活性和调理吞噬作用;完全以PorB蛋白免疫的小鼠血清免疫保护作用略低。为了研究NspA和PorB的免疫保护作用是否具有协同性,将二者进行联合免疫。实验小鼠分为2组,一是rNspA和rPorB蛋白免疫组,二是先以基因免疫然后用重组蛋白加强组。每组小鼠接种的免疫原总含量不变,其中两种免疫原各占一半。ELISA检测结果表明蛋白免疫获得的血清IgG水平略高,但两种免疫方案均产生了较高水平的血清IgG,且两种抗原联合免疫后的血清IgG的水平比原来单个抗原免疫的血清IgG水平要高出许多;抗体介导的补体溶菌实验和调理吞噬实验结果均表明,联合免疫血清的淋病奈瑟菌特异性抗菌活性高于单一抗原免疫血清。提示两种抗原之间确实存在一定的协同作用。此外,先基因免疫后蛋白加强组小鼠血清的保护作用仍强于蛋白免疫组血清,表明该免疫方案具有一定的优越性。
[Abstract]:Gonorrhoea is a common sexually transmitted disease ( STD ) , which is caused by Neisseria Gonorrhoea infection , which mainly causes acute urinary tract inflammation , prostatitis and accessory orchitis in men . In women , it is mainly manifested as recessive infection , which can lead to infection of pelvic inflammatory disease , endometritis and salpingitis , which can lead to infertility . The incidence of gonococcal disease is high , which is the focus of prevention and treatment of chronic diseases . As a result of the high degree of variation , the other important adhesion factor Opa protein is also limited in the development of gonococcus vaccine . The recombinant eukaryotic expression vector pCNspA has produced NspA specific antibody which has the effect of mediating complement dissolution of Neisseria . The recombinant eukaryotic expression vector pCNspA has a very important outer membrane protein , but the purified pore protein in the cells can contaminate the Rmp protein . The highly purified porin can stimulate the animal to produce protective antibody with high titer . The recombinant protein NspA . rNspA was immunized with rNspA . The results showed that pQE31NspA could express recombinant protein NspA . rNspA . In order to avoid the pollution of Rmp protein and improve the effect of gene immunization , the PorB gene was excised from the prokaryotic expression vector pUNCH682 of PorB prokaryotic expression vector from Dr . Christopher . In order to study whether the immune protective effects of NspA and PorB were synergistic , the two groups were combined . The experimental mice were divided into 2 groups , one was rNspA and rPorB protein immune group , and the two kinds of immunity were higher than that of single antigen . The results of ELISA showed that the serum IgG level was higher than that of single antigen .

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R759.2;R392

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