抗2型登革病毒M蛋白和NS1蛋白单克隆抗体的制备及鉴定

发布时间：2018-01-13 06:29

本文关键词：抗2型登革病毒M蛋白和NS1蛋白单克隆抗体的制备及鉴定　出处：《重庆医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:近年来,登革病毒(DV)在世界许多国家和地区,尤其是东南亚流行趋势加重,已成为全球严重的公共卫生问题。由于目前尚无有效预防登革病毒感染的疫苗上市,而登革出血热和登革热休克综合征的死亡率较高,因此早期快速准确的诊断登革病毒感染就显得尤为重要。目前新问市的登革病毒感染诊断试剂主要是以全病毒或重组E蛋白为检测抗原,其特异性和灵敏性有待进一步深入研究。本研究拟利用2型登革病毒New Guinea C株(NGC株)重组质粒克隆表达重组M蛋白和NS1蛋白,制备抗2型登革病毒M蛋白和NS1蛋白的单克隆抗体,并对其生物学特性进行了鉴定,为下一步制备快速检测登革病毒感染的胶体金试纸条奠定基础。方法:诱导表达含DV2 prm、ns1基因的原核表达重组载体pET-32a(+)/prM、pET-32a(+)/M、pQE-30/NS1,并且采用了Ni~(2+)-NTA树脂亲和层析纯化重组蛋白,以SDS-PAGE进行初步鉴定,并以兔抗登革病毒免疫血清鉴定其免疫活性。以重组蛋白免疫Balb/c小鼠,采用杂交瘤技术制备抗登革病毒重组蛋白的McAb,采用间接ELISA方法和Western blot进行McAb特异性鉴定;同时采用间接ELISA方法鉴定McAb的Ig亚类、效价及亲和力进行分析。结果:重组蛋白pET-32a(+)/M、pQE-30/NS1以上清和包涵体表达,用兔抗登革病毒血清检测发现上清中蛋白抗原活性强。利用Ni~(2+)-NTA树脂亲和层析方法,从上清中纯化蛋白获得成功。通过杂交瘤技术成功获得3株可分泌特异性McAb的杂交瘤细胞Ⅲ1A6,Ⅲ3D2和Ⅲ4C3。其中Ⅲ1A6,Ⅲ4C3的抗体亚类均为IgG1;Ⅲ3D2的抗体亚类为IgG2a。细胞培养上清的效价分别为1:10~6 , 1:10~5和1:10~6;3株单抗的亲和常数均在10~7以上。Western bolt检测证明3株杂交瘤细胞所分泌的McAb特异性良好。结论:成功地制备出抗M蛋白的2株McAb和抗NS1蛋白的1株McAb,为进一步建立快速特异检测登革病毒感染的实验方法提供了有力的工具。
[Abstract]:Objective: in recent years, dengue virus (DV) in many countries and regions in the world, especially in Southeast Asia increased trend, has become a serious public health problem in the world. Due to lack of effective prevention of listed dengue virus infection and vaccine, dengue fever and dengue shock syndrome with high mortality, early diagnosis fast and accurate dengue virus infection is particularly important. At present, the new asked the city of dengue virus infection is the main diagnostic reagent of whole virus or recombinant E protein as antigen specificity and sensitivity needs further study.
This study intends to use the 2 dengue virus type New Guinea strain C (NGC strain) expression of recombinant M protein and NS1 protein in recombinant plasmid cloning and preparation of anti dengue virus type 2 monoclonal antibody of M protein and NS1 protein, and its biological characteristics were identified, and laid the foundation for the next step of preparation for rapid detection the colloidal gold test strip of dengue virus infection.
Methods: expression containing DV2 PRM, Recombinant Prokaryotic expression vector of pET-32a gene of NS1 (+) /prM (+) pET-32a, /M, pQE-30/NS1, and the use of Ni~ (2+) affinity purification of recombinant protein -NTA chromatography resin, were identified by SDS-PAGE, and the Rabbit anti dengue virus immune serum immune activity identification with the recombinant Balb/c protein immune mice were prepared by hybridoma technique of anti dengue virus recombinant protein McAb by indirect ELISA method and Western blot McAb specific Ig subclass identification; the indirect ELISA method of McAb identification, and the titer of the affinity analysis.
Results: recombinant protein pET-32a (+) /M and pQE-30/NS1 were expressed in supernatant and inclusion body. The detection of Rabbit anti dengue virus sera showed that the protein antigen in the supernatant was highly active. Ni~ (2+) -NTA resin affinity chromatography was applied to purify the protein from the supernatant.
By using the hybridoma technique successfully obtained hybridoma cell lines can secrete 3 III 1A6 specific McAb, 3D2 and 4C3. III III 1A6 III, III 4C3 antibody subclasses were IgG1; III 3D2 antibody subclasses supernatant for IgG2a. cell titer were 1:10~6, 1:10~5 and 1:10~6; the specificity of McAb 3 the affinity constants were single strains of anti.Western in 10~7 or bolt proved that the secretion of 3 hybridoma cells.
Conclusion: 2 McAb and 1 NS1 McAb resistant to M protein were successfully prepared, which provided a powerful tool for further establishment of a rapid and specific detection method for dengue virus infection.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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