抗S.aureus TRAP蛋白单克隆抗体的制备及其抗原表位分析
发布时间：2018-07-30 08:20
【摘要】：金黄色葡萄球菌(Staphylococcus aureus, S.aureus)为革兰氏阳性致病菌，常引起人和动物感染。随着抗生素使用，耐药性菌株不断出现，使S.aureus感染难以治疗。因此，人们对S.aureus感染的研究重点从治疗转向免疫预防，S.aureus许多菌体蛋白便成为疫苗研制的主要候选对象。TRAP蛋白为葡萄球菌所特有，并且是一种免疫保护作用较为理想的疫苗候选蛋白，但其在细胞的定位和具体功能还不十分清楚。本研究制备了抗TRAP单克隆抗体，并用单克隆抗体研究了其对应表位，为表位疫苗研制、TRAP蛋白定位及其生物功能研究提供依据。 用重组TRAP蛋白免疫BALB/c小鼠，以杂交瘤技术制备并筛选出11株能够稳定分泌抗TRAP抗体的细胞株，分别命名为2A1、3A6、3B1、1B4、2C5、4C7、5C3、2D8、2A7、3A1、1C4；经McAbs亚类检测，其中2A1、3A6、3B1、1B4、2C5、4C7、5C3、2D8的重链为IgG1型，2A7、3A1、1C4的重链为IgM型；所有McAbs的轻链均为κ链；用Western blot检测8株IgG1型杂交瘤细胞上清，都能与TRAP蛋白有特异性结合；通过交叉性和亲和性ELISA试验证明McAbs特异性良好且亲和能力较强。 分别以8株McAbs为配基，经噬菌体展示技术筛选和间接ELISA检测，得到与对应单抗亲和性高的噬菌体克隆，经测序及生物信息学分析后共得到3个表位，其中2A1、2C5、4C7针对同一表位，氨基酸序列为YLYP；3A6、3B1、5C3、2D8针对同一表位，氨基酸序列为SYFE；1B4与TRAP同源性不高，氨基酸序列为不连续的L-G-L，但该单抗与TRAP蛋白的结合力很强，可能是模拟表位；经调理吞噬实验证实，2A1、3A6、1B4三株代表单抗都有很强的调理活性；用制备的单抗对S.aureus株Wood46进行全菌体ELISA检测证实，3个表位部分均暴露在菌体表面。
[Abstract]:Staphylococcus aureus (Staphylococcus aureus, S.aureus) is a gram-positive pathogen that often causes human and animal infections. With the use of antibiotics, drug-resistant strains continue to appear, making S.aureus infection difficult to treat. Therefore, the focus of research on S.aureus infection has shifted from treatment to immune prevention. Many bacterial proteins have become the main candidate for vaccine development. Trap protein is unique to Staphylococcus. And it is an ideal vaccine candidate protein for immune protection, but its location and specific function in cells are not very clear. In this study, monoclonal antibodies against TRAP were prepared and their corresponding epitopes were studied with monoclonal antibodies, which provided the basis for the development of epitope vaccine for the localization of trap protein and its biological function. BALB/c mice were immunized with recombinant TRAP protein. Eleven cell lines which could stably secrete anti-TRAP antibody were prepared and screened by hybridoma technique. They were named as 2A1O3A6A6O3B1B1B4C5C7C7C7C7C7C7D8A7A7A1A1C4, and the heavy chain of 2A1O3A3A6A6B1B4B4C7C5C3D8 was IgG1 type, the heavy chain of 2A73A1C was IgM. The light chain of all McAbs was 魏 chain, the supernatant of 8 IgG1 hybridoma cells were detected by Western blot, all of them could bind to TRAP protein, and the cross and affinity ELISA test showed that McAbs had good specificity and strong affinity. The phage clones with high affinity to the corresponding monoclonal antibodies were obtained by phage display screening and indirect ELISA detection using 8 McAbs as ligand respectively. After sequencing and bioinformatics analysis, three epitopes were obtained, among which 2A1O2C5O4C7 was targeted at the same epitope. The amino acid sequence was YLYP3A6, 3B1O5C3O2D8, the amino acid sequence of which was not high homology with TRAP and the amino acid sequence was discontinuous L-G-L, but the McAb had strong binding force with TRAP protein, which might be a mimic epitope. The results of conditioning phagocytosis test showed that all three representative McAbs had strong conditioning activity, and that the three epitopes were exposed to the surface of S.aureus strain Wood46 by ELISA detection with the prepared McAbs.
【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】：2012
【分类号】：R392
本文编号：2154399
[Abstract]:Staphylococcus aureus (Staphylococcus aureus, S.aureus) is a gram-positive pathogen that often causes human and animal infections. With the use of antibiotics, drug-resistant strains continue to appear, making S.aureus infection difficult to treat. Therefore, the focus of research on S.aureus infection has shifted from treatment to immune prevention. Many bacterial proteins have become the main candidate for vaccine development. Trap protein is unique to Staphylococcus. And it is an ideal vaccine candidate protein for immune protection, but its location and specific function in cells are not very clear. In this study, monoclonal antibodies against TRAP were prepared and their corresponding epitopes were studied with monoclonal antibodies, which provided the basis for the development of epitope vaccine for the localization of trap protein and its biological function. BALB/c mice were immunized with recombinant TRAP protein. Eleven cell lines which could stably secrete anti-TRAP antibody were prepared and screened by hybridoma technique. They were named as 2A1O3A6A6O3B1B1B4C5C7C7C7C7C7C7D8A7A7A1A1C4, and the heavy chain of 2A1O3A3A6A6B1B4B4C7C5C3D8 was IgG1 type, the heavy chain of 2A73A1C was IgM. The light chain of all McAbs was 魏 chain, the supernatant of 8 IgG1 hybridoma cells were detected by Western blot, all of them could bind to TRAP protein, and the cross and affinity ELISA test showed that McAbs had good specificity and strong affinity. The phage clones with high affinity to the corresponding monoclonal antibodies were obtained by phage display screening and indirect ELISA detection using 8 McAbs as ligand respectively. After sequencing and bioinformatics analysis, three epitopes were obtained, among which 2A1O2C5O4C7 was targeted at the same epitope. The amino acid sequence was YLYP3A6, 3B1O5C3O2D8, the amino acid sequence of which was not high homology with TRAP and the amino acid sequence was discontinuous L-G-L, but the McAb had strong binding force with TRAP protein, which might be a mimic epitope. The results of conditioning phagocytosis test showed that all three representative McAbs had strong conditioning activity, and that the three epitopes were exposed to the surface of S.aureus strain Wood46 by ELISA detection with the prepared McAbs.
【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】：2012
【分类号】：R392
【参考文献】
相关期刊论文 前2条
1 孔繁德,黄印尧,赖清金;免疫胶体金技术及其发展前景[J];福建畜牧兽医;2002年S1期
2 朱锡华,吴玉章;对表位生物学研究的认识和体会[J];上海免疫学杂志;1998年01期
，本文编号：2154399
本文链接：https://www.wllwen.com/xiyixuelunwen/2154399.html
最近更新
教材专著
