本文選題：脊髓灰質(zhì)炎病毒 + 結(jié)構(gòu)蛋白��； 參考：《中國藥品生物制品檢定所》2011年碩士論文

【摘要】：脊髓灰質(zhì)炎是由脊髓灰質(zhì)炎病毒感染引起的,沒有特效治療方法,只能接種疫苗預防�？诜顾杌屹|(zhì)炎疫苗已大幅度降低了全球脊髓灰質(zhì)炎的發(fā)病率,然而,由于疫苗相關麻痹型脊灰和疫苗衍生脊髓灰質(zhì)炎病毒風險,今后必須停止使用口服脊髓灰質(zhì)炎疫苗,以徹底根除脊髓灰質(zhì)炎。消滅脊髓灰質(zhì)炎野病毒之后,將會嚴格管理來源于野毒株的傳統(tǒng)脊髓灰質(zhì)炎滅活疫苗生產(chǎn),而用Sabin株研制的脊髓灰質(zhì)炎滅活疫苗抗原性及免疫原性與傳統(tǒng)脊髓灰質(zhì)炎滅活疫苗不同。新型脊髓灰質(zhì)炎疫苗形式有待開發(fā)。 本研究利用Bac-to-Bac桿狀病毒表達系統(tǒng)表達脊髓灰質(zhì)炎病毒Sabin2結(jié)構(gòu)蛋白,由于脊髓灰質(zhì)炎病毒的主要抗原位點位于結(jié)構(gòu)蛋白VP1上,其功能蛋白3C及其前體3CD具有蛋白酶的功能,能將多聚蛋白P1剪切加工成結(jié)構(gòu)蛋白VP0、VP3及VP1,因此,首先利用RT-PCR方法擴增得到脊髓灰質(zhì)炎病毒Sabin2株基因VP1、P1、3C及3CD,分別構(gòu)建以上基因的重組桿狀病毒,然后將重組桿狀病毒rBac-VP1、rBac-P1和rBac-3C、rBac-P1和rBac-3CD三組分別感染Sf9細胞進行表達。結(jié)果顯示:分別克隆有Sabin2株基因VP1、P1、3C及3CD的重組桿狀病毒表達質(zhì)粒轉(zhuǎn)染Sf9細胞后24小時出現(xiàn)細胞病變,72小時細胞病變明顯;間接免疫熒光檢測顯示目的蛋白為細胞內(nèi)表達;間接免疫熒光及Western-Blot結(jié)果顯示三組均有VP1結(jié)構(gòu)蛋白的生成,且均具有抗原性;rBac-P1和rBac-3C、rBac-P1和rBac-3CD分別共感染Sf9細胞兩組有結(jié)構(gòu)蛋白多聚體的生成,提示可能有病毒樣顆粒的生成。 脊髓灰質(zhì)炎病毒Sabin2株結(jié)構(gòu)蛋白在昆蟲桿狀病毒表達系統(tǒng)中的成功表達,為脊髓灰灰質(zhì)炎病毒分子生物學特性研究提供了新的科研思路,為新型脊髓灰質(zhì)炎疫苗形式的開發(fā)奠定了基礎,提供了新的技術路線。
[Abstract]:Poliomyelitis is caused by polio virus infection, there is no special treatment, can only be vaccinated against.Oral polio vaccines have significantly reduced the global incidence of poliomyelitis. However, because of the vaccine-related paralytic polio and vaccine-derived poliovirus risks, the use of oral poliomyelitis vaccines must be discontinued in the future.To eradicate polio.After the eradication of wild poliovirus, the production of traditional poliovirus inactivated vaccine from wild strain will be strictly managed.The antigenicity and immunogenicity of inactivated poliomyelitis vaccine developed by Sabin strain were different from that of traditional inactivated poliomyelitis vaccine.New forms of polio vaccine need to be developed.In this study, Bac-to-Bac baculovirus expression system was used to express poliovirus Sabin2 structural protein. Because the main antigen site of poliovirus was located on the structural protein VP1, its functional protein 3C and its precursor 3CD had protease function.The poly-protein P1 can be cut into structural proteins VP0VP3 and VP1. therefore, firstly, the poliovirus Sabin2 strain VP1p1P13C and 3CD3 were amplified by RT-PCR method, and the recombinant baculoviruses were constructed, respectively.Then three groups of recombinant baculovirus rBac-VP1rBac-P1 and rBac-3CnrBac-P1 and rBac-3CD were infected with Sf9 cells respectively.The results showed that the recombinant baculovirus expression plasmids containing Sabin2 strain VP1p1P13C and 3CD showed obvious cytopathic effect at 24 hours after transfection into Sf9 cells, and indirect immunofluorescence assay showed that the target protein was expressed in cells.The results of indirect immunofluorescence and Western-Blot showed that all three groups had the production of VP1 structural proteins, and all of them had antigenicity of rBac-P1 and rBac-3CnrBac-P1 and rBac-3CD co-infected Sf9 cells, respectively, indicating the formation of viro-like particles.The successful expression of structural protein of poliovirus Sabin2 strain in insect baculovirus expression system provides a new scientific idea for the study of molecular biological characteristics of poliovirus.It lays a foundation for the development of new poliomyelitis vaccine forms and provides a new technical route.
【學位授予單位】：中國藥品生物制品檢定所
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R373

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