【摘要】： 本課題的主要目的是以CHO細(xì)胞表達的重組乙肝病毒表面抗原為載體蛋白,制備A群腦膜炎球菌莢膜多糖與重組乙肝表面抗原的多糖蛋白結(jié)合物(GAMP-HBsAg),研究和評價該結(jié)合物的免疫原性和生物化學(xué)特性,并與以破傷風(fēng)類毒素(TT)為蛋白載體的GAMP-TT結(jié)合物進行比較,探討乙肝表面抗原作為多糖抗原的載體蛋白的可行性。 采用溴化氰活化、ADH衍生、EDAC縮合的方式,制備了GAMP與重組乙肝表面抗原(CHO)的多糖蛋白結(jié)合物(GAMP-HBsAg);陰離子交換層析純化GAMP-HBsAg;用免疫雙擴散和ELISA方法對結(jié)合物免疫反應(yīng)性進行評價;此外,對結(jié)合物的分子大小、衍生率、多糖含量、蛋白含量,以及CNBr和EDAC殘留量進行檢測;對結(jié)合物免疫小鼠后血清產(chǎn)生的特異性抗體滴度和抗體類型進行評價。 實驗結(jié)果表明,制備的四批GAMP-HBsAgm生物的m生率分別為2.04%、2.44%、1.67%和2.21%,對應(yīng)的多糖蛋白比分別為1.21、1.42、1.87和1.65,結(jié)合物KD值均小于0.2。以兩批GAMP-HBsAg結(jié)合物免疫小鼠,均可產(chǎn)生針對多糖的特異性IgG抗體,兩批結(jié)合物抗體滴度分別為10.76EU和27.43EU ,多糖對照組抗體滴度為1.03EU,結(jié)合物組血清陽轉(zhuǎn)率為100%。兩批結(jié)合物與多糖組抗體平均幾何滴度的比較有顯著性差異;GAMP-TT產(chǎn)生抗體滴度為57.94EU, GAMP-HBsAg產(chǎn)生HBsAg特異性抗體滴度為58.5EU。 綜上所述,HBsAg作為載體蛋白制備的結(jié)合物(GAMP-HBsAg)在小鼠體內(nèi)可產(chǎn)生針對多糖的特異性抗體,同時也能產(chǎn)生針對HBsAg的特異性抗體,因此,可作為候選載體蛋白。
[Abstract]:The main purpose of this study is to prepare the conjugate of group A meningococcal capsule polysaccharide (GAMP-HBsAg) with recombinant hepatitis B virus surface antigen (GAMP-HBsAg) expressed by CHO cells. The immunogenicity and biochemical characteristics of the conjugate were studied and evaluated, and compared with the GAMP-TT conjugate with tetanus toxoid (TT) as protein carrier, the feasibility of hepatitis B surface antigen as carrier protein of polysaccharide antigen was discussed. The polysaccharide protein conjugates (GAMP-HBsAg) of GAMP and recombinant hepatitis B surface antigen (CHO) were prepared by cyanide bromide activation, ADH derivatization and EDAC condensation. Purification of GAMP-HBsAg; by anion exchange chromatography In addition, the molecular size, derivatization rate, polysaccharide content, protein content, CNBr and EDAC residues of the conjugates were determined by double diffusion and ELISA methods, and the molecular size, derivatization rate, polysaccharide content and protein content of the conjugates were determined. The titers and types of specific antibodies produced in sera of mice immunized with conjugates were evaluated. The results showed that the m growth rate of the four batches of GAMP-HBsAgm was 2.04%, 2.44%, 1.67% and 2.21%, respectively, and the ratio of polysaccharide to protein was 1.21, 1.42, 1.87 and 1.65, respectively. The KD values of the conjugates were all less than 0.2. Mice immunized with two batches of GAMP-HBsAg conjugate could produce specific IgG antibody against polysaccharide. The titers of antibody against polysaccharide were 10.76EU and 27.43EU respectively, the antibody titer of polysaccharide control group was 1.03EU, and the seroconversion rate of binding group was 100%. The average geometric titers of antibodies produced by GAMP-TT and GAMP-HBsAg were 57.94 EUU and 58.5EU respectively, and the average geometric titers of antibodies in the two groups were significantly different from those in the polysaccharide group, and the antibody titers were 57.94 EU and 58.5EU respectively. In conclusion, the conjugate (GAMP-HBsAg) prepared by HBsAg as a carrier protein can produce specific antibodies against polysaccharides and HBsAg in mice, so it can be used as a candidate vector protein.
【學(xué)位授予單位】：武漢生物制品研究所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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