金黄色葡萄球菌IsdB免疫优势片段单抗制备及抗原表位初步分析
发布时间：2018-11-28 08:16
【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。
[Abstract]:Due to the emergence and spread of Staphylococcus aureus (Staphyloccocus aureus, S.aureus)-resistant strains, there is no cure for S.aureus infection. Therefore, the study of S.aureus vaccine has attracted much attention. However, the immune effects of inactivated whole cell vaccine, capsule polysaccharide vaccine or capsule polysaccharide conjugate vaccine and toxoid were not satisfactory. However, the immune protection of bacterial surface protein has become an excellent candidate in the research of S.aureus vaccine in recent years. The iron-regulated surface determinant (B (IsdB) is a protein embedded on the surface of S.aureus and has good immunogenicity, but the antigenic epitopes with immuno-protective effect are unknown. In this study, BALB/c mice were immunized with IsdB immune dominant fragment (129-361aa)-recombinant IsdB3. Six cell lines which could stably secrete anti-IsdB3 protein antibodies were prepared by hybridoma technique and named 2E1O2E3E3A7A7A7B6O3H5 and 4D3 respectively. Observed by Giemsa staining, the chromosome number of hybridoma cells was about 80-96, which was consistent with the rule of chromosome change of fusion cells. The results showed that the heavy chain of 2E1O2E3 and 3H5 was IgGl type, the heavy chain of 3B6 and 4D3 was IgG2b type, the heavy chain of 3A7 was IgG2a type, and the light chain of all McAbs was K chain. In the crossover test, there was no cross reaction between IsdB3 and TRAP protein of S.aureus, which indicated that the McAbs was specific, and the supernatant of 6 hybridoma cells could bind to IsdB3 protein by Western blot. The reactivity of McAbs was detected by indirect ELISA method using S.aureus whole cell as coating antigen. The results showed that 3A7O3B6 and 4D3 could bind to S.aureus, but 2E1O2E3 and 3H5 could not bind to S.aureus. According to the structure of IsdB3, five pairs of primers were designed. IsdB3a,isdB3b,isdB3c,isdB3d and isdB3e gene fragments were amplified by PCR using DNA of S.aureus Newman strain as template. After the expression of the peptides encoded by these gene fragments, Western blot analysis was carried out with 3A7, 3B6 and 4D3McAbs, respectively. The results showed that all of the five recombinant protein peptides could bind to the three strains of McAbs. The results showed that the epitopes of recombinant IsdB3 protein were mimic epitopes. Using 3B6 strain McAb as ligand, the mimic peptides recognized by antibody were screened from phage random heptapeptide library. The positive phage clones which reacted with 3B6 strain McAb were sequenced and sequenced. The results also showed that the epitopes of IsdB3 protein were mimic epitopes. This study provides an important reference for the further study of S.aureus multiantigen chimeric epitope vaccine.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
本文编号：2362320
[Abstract]:Due to the emergence and spread of Staphylococcus aureus (Staphyloccocus aureus, S.aureus)-resistant strains, there is no cure for S.aureus infection. Therefore, the study of S.aureus vaccine has attracted much attention. However, the immune effects of inactivated whole cell vaccine, capsule polysaccharide vaccine or capsule polysaccharide conjugate vaccine and toxoid were not satisfactory. However, the immune protection of bacterial surface protein has become an excellent candidate in the research of S.aureus vaccine in recent years. The iron-regulated surface determinant (B (IsdB) is a protein embedded on the surface of S.aureus and has good immunogenicity, but the antigenic epitopes with immuno-protective effect are unknown. In this study, BALB/c mice were immunized with IsdB immune dominant fragment (129-361aa)-recombinant IsdB3. Six cell lines which could stably secrete anti-IsdB3 protein antibodies were prepared by hybridoma technique and named 2E1O2E3E3A7A7A7B6O3H5 and 4D3 respectively. Observed by Giemsa staining, the chromosome number of hybridoma cells was about 80-96, which was consistent with the rule of chromosome change of fusion cells. The results showed that the heavy chain of 2E1O2E3 and 3H5 was IgGl type, the heavy chain of 3B6 and 4D3 was IgG2b type, the heavy chain of 3A7 was IgG2a type, and the light chain of all McAbs was K chain. In the crossover test, there was no cross reaction between IsdB3 and TRAP protein of S.aureus, which indicated that the McAbs was specific, and the supernatant of 6 hybridoma cells could bind to IsdB3 protein by Western blot. The reactivity of McAbs was detected by indirect ELISA method using S.aureus whole cell as coating antigen. The results showed that 3A7O3B6 and 4D3 could bind to S.aureus, but 2E1O2E3 and 3H5 could not bind to S.aureus. According to the structure of IsdB3, five pairs of primers were designed. IsdB3a,isdB3b,isdB3c,isdB3d and isdB3e gene fragments were amplified by PCR using DNA of S.aureus Newman strain as template. After the expression of the peptides encoded by these gene fragments, Western blot analysis was carried out with 3A7, 3B6 and 4D3McAbs, respectively. The results showed that all of the five recombinant protein peptides could bind to the three strains of McAbs. The results showed that the epitopes of recombinant IsdB3 protein were mimic epitopes. Using 3B6 strain McAb as ligand, the mimic peptides recognized by antibody were screened from phage random heptapeptide library. The positive phage clones which reacted with 3B6 strain McAb were sequenced and sequenced. The results also showed that the epitopes of IsdB3 protein were mimic epitopes. This study provides an important reference for the further study of S.aureus multiantigen chimeric epitope vaccine.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 陈莹;金黄色葡萄球菌抗原铁调节表面决定蛋白B及疫苗研究[D];吉林大学;2013年
，本文编号：2362320
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