本文選題：腦膜炎球菌 + 蛋白保護(hù)性抗原　； 參考：《西北農(nóng)林科技大學(xué)》2010年碩士論文

【摘要】： 鑒于流腦多糖疫苗對(duì)嬰兒保護(hù)性差,對(duì)成人的免疫持久性不夠以及多糖與蛋白結(jié)合疫苗價(jià)格昂貴和工藝復(fù)雜的缺陷,新一代流腦疫苗的研制主要以蛋白保護(hù)性抗原為主。自2000年以來(lái)反向疫苗學(xué)、蛋白質(zhì)組學(xué)及基因芯片等方法相互補(bǔ)充發(fā)現(xiàn)了近30多個(gè)流腦蛋白保護(hù)性抗原。本試驗(yàn)中選取了其中五個(gè)能夠誘生殺菌抗體的蛋白保護(hù)性抗原來(lái)進(jìn)行研究,為進(jìn)一步篩選流腦蛋白保護(hù)性抗原奠定一定的基礎(chǔ)。 為了對(duì)這五個(gè)蛋白在不同流腦菌株中的保守性進(jìn)行驗(yàn)證,本試驗(yàn)以7株腦膜炎球菌全基因組為模板,PCR擴(kuò)增五個(gè)目的基因并進(jìn)行測(cè)序。將測(cè)得的基因序列以及NCBI GenBank公布的其他菌株來(lái)源的目的基因翻譯成氨基酸序列進(jìn)行比對(duì)。結(jié)果表明這五個(gè)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。 將截去信號(hào)肽編碼序列和終止密碼子的目的基因克隆到原核表達(dá)載體pET-21b(+)上,3′端與6×His標(biāo)簽融合表達(dá),除NhhA以包涵體形式表達(dá),其余四個(gè)蛋白(GNA2132、GNA1870-4436、GNA1870-88020及NMBNMB0928)均為可溶性表達(dá)。通過(guò)Ni柱螯合層析法純化獲得目的蛋白,并用腦膜炎球菌的混合免疫血清對(duì)重組目的蛋白進(jìn)行免疫印跡鑒定,結(jié)果均呈陽(yáng)性反應(yīng)。 以AL(OH)_3和CpG作為重組目的蛋白的佐劑免疫Balb/c小鼠,二針免疫后采血進(jìn)行ELISA檢測(cè),其血清抗體滴度高達(dá)1:100000～1:1000000。用腦膜炎球菌裂解物鑒定免疫血清的特異性,結(jié)果顯示均為陽(yáng)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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 鄭鑫;腦膜炎奈瑟氏菌表面蛋白NspA基因的克隆與原核表達(dá)[D];長(zhǎng)春理工大學(xué);2011年

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本文編號(hào)：1821849