重组乙肝病毒表面抗原为载体的流脑多糖蛋白结合疫苗特性研究
发布时间：2019-04-04 14:02
【摘要】： 本课题的主要目的是以CHO细胞表达的重组乙肝病毒表面抗原为载体蛋白,制备A群脑膜炎球菌荚膜多糖与重组乙肝表面抗原的多糖蛋白结合物(GAMP-HBsAg),研究和评价该结合物的免疫原性和生物化学特性,并与以破伤风类毒素(TT)为蛋白载体的GAMP-TT结合物进行比较,探讨乙肝表面抗原作为多糖抗原的载体蛋白的可行性。 采用溴化氰活化、ADH衍生、EDAC缩合的方式,制备了GAMP与重组乙肝表面抗原(CHO)的多糖蛋白结合物(GAMP-HBsAg);阴离子交换层析纯化GAMP-HBsAg;用免疫双扩散和ELISA方法对结合物免疫反应性进行评价;此外,对结合物的分子大小、衍生率、多糖含量、蛋白含量,以及CNBr和EDAC残留量进行检测;对结合物免疫小鼠后血清产生的特异性抗体滴度和抗体类型进行评价。 实验结果表明,制备的四批GAMP-HBsAgm生物的m生率分别为2.04%、2.44%、1.67%和2.21%,对应的多糖蛋白比分别为1.21、1.42、1.87和1.65,结合物KD值均小于0.2。以两批GAMP-HBsAg结合物免疫小鼠,均可产生针对多糖的特异性IgG抗体,两批结合物抗体滴度分别为10.76EU和27.43EU ,多糖对照组抗体滴度为1.03EU,结合物组血清阳转率为100%。两批结合物与多糖组抗体平均几何滴度的比较有显著性差异;GAMP-TT产生抗体滴度为57.94EU, GAMP-HBsAg产生HBsAg特异性抗体滴度为58.5EU。 综上所述,HBsAg作为载体蛋白制备的结合物(GAMP-HBsAg)在小鼠体内可产生针对多糖的特异性抗体,同时也能产生针对HBsAg的特异性抗体,因此,可作为候选载体蛋白。
[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.
【学位授予单位】：武汉生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
[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.
【学位授予单位】：武汉生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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