【作者】 佟丹丹；

【导师】 崔玉东；

【作者基本信息】 黑龙江八一农垦大学 ， 生化与分子生物学， 2010， 硕士

【摘要】 由于金黄色葡萄球菌(Staphyloccocus aureus, S.aureus)耐药性菌株的出现及传播,使S.aureus感染的治疗无计可施,因此S.aureus疫苗研究备受关注。但研究表明,全菌体灭活苗、荚膜多糖苗或荚膜多糖结合苗、类毒素等免疫效果不理想,而菌体表面蛋白的免疫保护作用,成为近年S.aureus疫苗研究中较优的候选者。铁调节表面决定簇B(IsdB)是镶嵌在S.aureus表面的蛋白,并证明具有良好的免疫原性,但对具有免疫保护作用的其抗原表位尚不了解。本研究用IsdB免疫优势片段(129-361aa)——重组IsdB3免疫BALB/c小鼠,以杂交瘤技术制备筛选出6株能够稳定分泌抗IsdB3蛋白抗体的细胞株,分别命名为2E1、2E3、3A7、3B6、3H5和4D3。通过Giemsa染色法观察,所得杂交瘤细胞染色体数约在80-96之间,符合融合细胞的染色体变化规律;对McAbs亚类进行检测,其中2E1、2E3和3H5的重链为IgGl型,3B6和4D3的重链为IgG2b型,而3A7的重链为IgG2a型,所有McAbs的轻链均为K链；在交义试验中,IsdB3与S.aureus的TRAP蛋白之间无交叉反应,说明所得的McAbs特异性良好;用Western blot检测6株杂交瘤细胞上清都能与IsdB3蛋白有特异性结合；以S.aureus全菌体作为包被抗原,用间接ELISA方法检测所得McAbs的反应原性,结果3A7、3B6和4D3可与S.aureus结合,而2E1、2E3和3H5与S.aureus不结合。根据IsdB3的结构设计5对引物,以S.aureus Newman株DNA为模板,经PCR扩增出isdB3a、isdB3b、isdB3c、isdB3d和isdB3e基因片段。这些基因片段编码的多肽表达后,分别与3A7、3B6和4D3McAbs进行Western blot分析,结果发现5种重组蛋白多肽均可以与这3株McAbs结合，初步表明重组IsdB3蛋白片段暴露于菌体表面的有效免疫抗原表位为模拟表位。以3B6株McAb为配基,从噬菌体随机七肽库中筛选抗体识别的模拟肽,对获得的与3B6株McAb反应的阳性噬菌体克隆,并对其进行测序及序列分析,结果也显示IsdB3蛋白的抗原表位为模拟表位。该研究为进一步研究S.aureus多抗原嵌合表位疫苗提供重要参考。更多还原

【Abstract】 Staphyloccocus aureus infection has become a big problem worldwide, and the harm to human is more serious due to the appearance and spread of resistant strains. Vaccine against S.aureus is urgently needed. Wide studies showed traditional vaccines, such as attenuated live vaccine and inactivated vaccine, capsule polysaccharides vaccine, and toxoid vaccine were not available for S.aureus, some surface proteins of S.aureus were ideal potential vaccine. Iron-regulated surface determinant B (IsdB), demonstrated a good immunogenicity, but IsdB epitopes inducing immuno-protection are not yet clear.Recombinant IsdB3 (129-361aa) was purified and used as an immunogen for BALB/c mice in this experiment. Six cell strains, which could stably secret monoclonal antibodies (McAbs) against IsdB3, were obtained by hybridoma technique, and named 2E1,2E3,3A7,3B6,3H5 and 4D3. The chromosome number of the cell strains were between 80-96 detected by Giemsa’s stain method, consistent with fusion cell genetic algorithm. The heavy chain subtype of 2E1,2E3 and 3H5 McAbs were IgG1,3B6 and 4D3 were IgG2b, and 3A7 was IgG2a. All light chains of the McAbs wereκ. There was no cross reaction between IsdB3 and TraP detected by the McAbs. This indicated the McAbs were of good specificity. All 6 McAbs could specifically bind to IsdB3 by Western blot, but 3A7,3B6 and 4D3 could bind to S.aureus by indirect ELISA based on coating with whole S.aureus.Five pairs of primer were designed according to the sequence of IsdB3. Chromosome DNA of S.aureus Newman strain was used as the templet to amplify isdB3a, isdB3b, isdB3c, isdB3d and isdB3e fragments. The expressed peptides coded by these fragments were analyzed with McAbs 3A7,3B6 and 4D3 respectively by Western blot. All expressed peptides could bind to the 3 McAbs. This result indicated that the IsdB3 antigen epitopes exposed on bacterium surface were mimic epitopes. Using phage display technique, the phages interacting with 3B6 McAbs were screened in heptapeptide library and the peptide sequence of the phages were analyzed. The result further confirmed that the IsdB3 antigen epitope exposed on bacterium surface was a mimic epitope. This research would provide important reference for further research on anti-S. aureus poly antigen chimeric epitope vaccine.更多还原