【摘要】：金黃色葡萄球菌(Staphylococcus aureus, S.aureus)為革蘭氏陽性致病菌，常引起人和動物感染。隨著抗生素使用，耐藥性菌株不斷出現(xiàn)，使S.aureus感染難以治療。因此，人們對S.aureus感染的研究重點(diǎn)從治療轉(zhuǎn)向免疫預(yù)防，S.aureus許多菌體蛋白便成為疫苗研制的主要候選對象。TRAP蛋白為葡萄球菌所特有，并且是一種免疫保護(hù)作用較為理想的疫苗候選蛋白，但其在細(xì)胞的定位和具體功能還不十分清楚。本研究制備了抗TRAP單克隆抗體，并用單克隆抗體研究了其對應(yīng)表位，為表位疫苗研制、TRAP蛋白定位及其生物功能研究提供依據(jù)。 用重組TRAP蛋白免疫BALB/c小鼠，以雜交瘤技術(shù)制備并篩選出11株能夠穩(wěn)定分泌抗TRAP抗體的細(xì)胞株，分別命名為2A1、3A6、3B1、1B4、2C5、4C7、5C3、2D8、2A7、3A1、1C4；經(jīng)McAbs亞類檢測，其中2A1、3A6、3B1、1B4、2C5、4C7、5C3、2D8的重鏈為IgG1型，2A7、3A1、1C4的重鏈為IgM型；所有McAbs的輕鏈均為κ鏈；用Western blot檢測8株IgG1型雜交瘤細(xì)胞上清，都能與TRAP蛋白有特異性結(jié)合；通過交叉性和親和性ELISA試驗證明McAbs特異性良好且親和能力較強(qiáng)。 分別以8株McAbs為配基，經(jīng)噬菌體展示技術(shù)篩選和間接ELISA檢測，得到與對應(yīng)單抗親和性高的噬菌體克隆，經(jīng)測序及生物信息學(xué)分析后共得到3個表位，其中2A1、2C5、4C7針對同一表位，氨基酸序列為YLYP；3A6、3B1、5C3、2D8針對同一表位，氨基酸序列為SYFE；1B4與TRAP同源性不高，氨基酸序列為不連續(xù)的L-G-L，但該單抗與TRAP蛋白的結(jié)合力很強(qiáng)，可能是模擬表位；經(jīng)調(diào)理吞噬實(shí)驗證實(shí)，2A1、3A6、1B4三株代表單抗都有很強(qiáng)的調(diào)理活性；用制備的單抗對S.aureus株Wood46進(jìn)行全菌體ELISA檢測證實(shí)，3個表位部分均暴露在菌體表面。
[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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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