脊髓灰质炎病毒Sabin2株结构蛋白在昆虫杆状病毒表达系统中的表达

发布时间：2018-04-19 14:08

本文选题：脊髓灰质炎病毒 + 结构蛋白　；参考：《中国药品生物制品检定所》2011年硕士论文

【摘要】：脊髓灰质炎是由脊髓灰质炎病毒感染引起的,没有特效治疗方法,只能接种疫苗预防。口服脊髓灰质炎疫苗已大幅度降低了全球脊髓灰质炎的发病率,然而,由于疫苗相关麻痹型脊灰和疫苗衍生脊髓灰质炎病毒风险,今后必须停止使用口服脊髓灰质炎疫苗,以彻底根除脊髓灰质炎。消灭脊髓灰质炎野病毒之后,将会严格管理来源于野毒株的传统脊髓灰质炎灭活疫苗生产,而用Sabin株研制的脊髓灰质炎灭活疫苗抗原性及免疫原性与传统脊髓灰质炎灭活疫苗不同。新型脊髓灰质炎疫苗形式有待开发。本研究利用Bac-to-Bac杆状病毒表达系统表达脊髓灰质炎病毒Sabin2结构蛋白,由于脊髓灰质炎病毒的主要抗原位点位于结构蛋白VP1上,其功能蛋白3C及其前体3CD具有蛋白酶的功能,能将多聚蛋白P1剪切加工成结构蛋白VP0、VP3及VP1,因此,首先利用RT-PCR方法扩增得到脊髓灰质炎病毒Sabin2株基因VP1、P1、3C及3CD,分别构建以上基因的重组杆状病毒,然后将重组杆状病毒rBac-VP1、rBac-P1和rBac-3C、rBac-P1和rBac-3CD三组分别感染Sf9细胞进行表达。结果显示:分别克隆有Sabin2株基因VP1、P1、3C及3CD的重组杆状病毒表达质粒转染Sf9细胞后24小时出现细胞病变,72小时细胞病变明显;间接免疫荧光检测显示目的蛋白为细胞内表达;间接免疫荧光及Western-Blot结果显示三组均有VP1结构蛋白的生成,且均具有抗原性;rBac-P1和rBac-3C、rBac-P1和rBac-3CD分别共感染Sf9细胞两组有结构蛋白多聚体的生成,提示可能有病毒样颗粒的生成。脊髓灰质炎病毒Sabin2株结构蛋白在昆虫杆状病毒表达系统中的成功表达,为脊髓灰灰质炎病毒分子生物学特性研究提供了新的科研思路,为新型脊髓灰质炎疫苗形式的开发奠定了基础,提供了新的技术路线。
[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.
【学位授予单位】：中国药品生物制品检定所
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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