脑膜炎球菌蛋白抗原的表达及免疫效果评价

发布时间：2018-04-29 22:25

本文选题：脑膜炎球菌 + 蛋白保护性抗原　；参考：《西北农林科技大学》2010年硕士论文

【摘要】： 鉴于流脑多糖疫苗对婴儿保护性差,对成人的免疫持久性不够以及多糖与蛋白结合疫苗价格昂贵和工艺复杂的缺陷,新一代流脑疫苗的研制主要以蛋白保护性抗原为主。自2000年以来反向疫苗学、蛋白质组学及基因芯片等方法相互补充发现了近30多个流脑蛋白保护性抗原。本试验中选取了其中五个能够诱生杀菌抗体的蛋白保护性抗原来进行研究,为进一步筛选流脑蛋白保护性抗原奠定一定的基础。为了对这五个蛋白在不同流脑菌株中的保守性进行验证,本试验以7株脑膜炎球菌全基因组为模板,PCR扩增五个目的基因并进行测序。将测得的基因序列以及NCBI GenBank公布的其他菌株来源的目的基因翻译成氨基酸序列进行比对。结果表明这五个蛋白在包括A、B、C、W135、Y群菌株中的氨基酸同源性均在80%以上。将截去信号肽编码序列和终止密码子的目的基因克隆到原核表达载体pET-21b(+)上,3′端与6×His标签融合表达,除NhhA以包涵体形式表达,其余四个蛋白(GNA2132、GNA1870-4436、GNA1870-88020及NMBNMB0928)均为可溶性表达。通过Ni柱螯合层析法纯化获得目的蛋白,并用脑膜炎球菌的混合免疫血清对重组目的蛋白进行免疫印迹鉴定,结果均呈阳性反应。以AL(OH)_3和CpG作为重组目的蛋白的佐剂免疫Balb/c小鼠,二针免疫后采血进行ELISA检测,其血清抗体滴度高达1:100000～1:1000000。用脑膜炎球菌裂解物鉴定免疫血清的特异性,结果显示均为阳性。
[Abstract]:In view of the poor protection of infantile and adult immune persistence, the expensive and complicated technology of polysaccharide and protein-bound vaccine, the new generation of meningitis vaccine is mainly developed with protein-protective antigen. Since 2000, more than 30 protective antigens have been found in reverse vaccinology, proteomics and gene chip. In this experiment, five protein-protective antigens which can induce bactericidal antibodies were selected to study, which laid a foundation for further screening of protein-protective antigens. In order to verify the conserved nature of these five proteins in different meningitis strains, the five target genes were amplified by PCR and sequenced using the whole genome of 7 strains of meningococci as template. The obtained gene sequence was compared with the amino acid sequence of the target gene from other strains published by NCBI GenBank. The results showed that the amino acid homology of the five proteins in all the strains including Abibus C135Y was more than 80%. The target gene encoding the truncated signal peptide and the terminating codon were cloned into the prokaryotic expression vector pET-21b () and fused with 6 脳 His tag. The other four proteins, GNA2132GNA1870-4436, GNA1870-88020 and NMBNMB0928, were expressed in the form of inclusion body. The target protein was purified by Ni column chelate chromatography. The recombinant protein was identified by immunoblotting with mixed immune serum of meningococcus. Balb/c mice were immunized with AL(OH)_3 and CpG as adjuvant of recombinant protein. The serum antibody titers of Balb/c mice were 1: 1000 000 and 1: 1000 000 respectively. The specificity of the immune serum was identified with the meningitis cocci lysate, and the results showed that all of them were positive.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【引证文献】