金黃色葡萄球菌IsdB免疫優(yōu)勢(shì)片段單抗制備及抗原表位初步分析
發(fā)布時(shí)間：2018-11-28 08:16
【摘要】： 由于金黃色葡萄球菌(Staphyloccocus aureus, S.aureus)耐藥性菌株的出現(xiàn)及傳播,使S.aureus感染的治療無(wú)計(jì)可施,因此S.aureus疫苗研究備受關(guān)注。但研究表明,全菌體滅活苗、莢膜多糖苗或莢膜多糖結(jié)合苗、類(lèi)毒素等免疫效果不理想,而菌體表面蛋白的免疫保護(hù)作用,成為近年S.aureus疫苗研究中較優(yōu)的候選者。鐵調(diào)節(jié)表面決定簇B(IsdB)是鑲嵌在S.aureus表面的蛋白,并證明具有良好的免疫原性,但對(duì)具有免疫保護(hù)作用的其抗原表位尚不了解。 本研究用IsdB免疫優(yōu)勢(shì)片段(129-361aa)——重組IsdB3免疫BALB/c小鼠,以雜交瘤技術(shù)制備篩選出6株能夠穩(wěn)定分泌抗IsdB3蛋白抗體的細(xì)胞株,分別命名為2E1、2E3、3A7、3B6、3H5和4D3。通過(guò)Giemsa染色法觀察,所得雜交瘤細(xì)胞染色體數(shù)約在80-96之間,符合融合細(xì)胞的染色體變化規(guī)律;對(duì)McAbs亞類(lèi)進(jìn)行檢測(cè),其中2E1、2E3和3H5的重鏈為IgGl型,3B6和4D3的重鏈為IgG2b型,而3A7的重鏈為IgG2a型,所有McAbs的輕鏈均為K鏈；在交義試驗(yàn)中,IsdB3與S.aureus的TRAP蛋白之間無(wú)交叉反應(yīng),說(shuō)明所得的McAbs特異性良好;用Western blot檢測(cè)6株雜交瘤細(xì)胞上清都能與IsdB3蛋白有特異性結(jié)合；以S.aureus全菌體作為包被抗原,用間接ELISA方法檢測(cè)所得McAbs的反應(yīng)原性,結(jié)果3A7、3B6和4D3可與S.aureus結(jié)合,而2E1、2E3和3H5與S.aureus不結(jié)合。 根據(jù)IsdB3的結(jié)構(gòu)設(shè)計(jì)5對(duì)引物,以S.aureus Newman株DNA為模板,經(jīng)PCR擴(kuò)增出isdB3a、isdB3b、isdB3c、isdB3d和isdB3e基因片段。這些基因片段編碼的多肽表達(dá)后,分別與3A7、3B6和4D3McAbs進(jìn)行Western blot分析,結(jié)果發(fā)現(xiàn)5種重組蛋白多肽均可以與這3株McAbs結(jié)合，初步表明重組IsdB3蛋白片段暴露于菌體表面的有效免疫抗原表位為模擬表位。以3B6株McAb為配基,從噬菌體隨機(jī)七肽庫(kù)中篩選抗體識(shí)別的模擬肽,對(duì)獲得的與3B6株McAb反應(yīng)的陽(yáng)性噬菌體克隆,并對(duì)其進(jìn)行測(cè)序及序列分析,結(jié)果也顯示IsdB3蛋白的抗原表位為模擬表位。該研究為進(jìn)一步研究S.aureus多抗原嵌合表位疫苗提供重要參考。
[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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392
【引證文獻(xiàn)】
相關(guān)碩士學(xué)位論文 前1條
1 陳瑩;金黃色葡萄球菌抗原鐵調(diào)節(jié)表面決定蛋白B及疫苗研究[D];吉林大學(xué);2013年
，本文編號(hào)：2362320
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