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百日咳博德特氏菌丝状血凝素三个重叠免疫显性重组片段的表达、纯化及特性分析

Expression, Purification and Characterization of Three Overlapping Immunodominant Recombinant Fragments from Bordetella pertussis Filamentous Hemagglutinin.

作者信息

Asgarian-Omran Hossein, Amirzargar Ali Akbar, Arjmand Mohammad, Eshraghian Mohammadreza, Nikbin Behrooz, Eshraghi Saeid, Mahdavi Marzieh, Khoshnoodi Jalal, Jeddi-Tehrani Mahmood, Rabbani Hodjatallah, Shokri Fazel

机构信息

Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran ; Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.

出版信息

Avicenna J Med Biotechnol. 2013 Jan;5(1):20-8.

PMID:23626873
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3572703/
Abstract

BACKGROUND

Filamentous hemagglutinin (FHA) is one of the most important immunoprotective antigens of Bordetella pertussis (B. pertussis) and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated.

METHODS

Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli (E. coli) BL21(DE3) strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells (PBMC) proliferation and IFN-γ production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively.

RESULTS

Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21(DE3). SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN-γ production.

CONCLUSION

Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immuno-protective.

摘要

背景

丝状血凝素（FHA）是百日咳博德特氏菌（百日咳杆菌）最重要的免疫保护性抗原之一，也是无细胞百日咳疫苗的主要成分。在本研究中，制备了来自FHA免疫显性区域的三个重叠重组片段，并对其免疫原性进行了研究。

方法

通过PCR从百日咳杆菌基因组DNA中扩增FHA抗原的三个重叠编码序列。扩增片段在大肠杆菌BL21(DE3)菌株中表达，并使用镍基色谱通过His标签进行纯化。纯化片段通过SDS-PAGE和蛋白质印迹技术进行鉴定。分别通过³H-胸腺嘧啶核苷掺入法和ELISA，对有限数量接种商业无细胞百日咳疫苗的健康成年人外周血单个核细胞（PBMC）对所有纯化的FHA片段的体外增殖和IFN-γ产生进行了评估。

结果

重组FHA片段在大肠杆菌BL21(DE3)中成功克隆并高水平表达。SDS-PAGE和蛋白质印迹分析证实了它们的纯度和反应性。所有三个重组片段与商业天然FHA一起均能够诱导体外PBMC增殖和IFN-γ产生。

结论

我们的初步结果表明，这些重叠的重组FHA片段具有免疫原性，可能具有免疫保护作用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/0b5edf4b1ae9/AJMB-5-20-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/edfbf5741a0c/AJMB-5-20-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/2fcefce9d8b8/AJMB-5-20-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/d0306a2b83e7/AJMB-5-20-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/bdd4a7722cf2/AJMB-5-20-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/0b5edf4b1ae9/AJMB-5-20-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/edfbf5741a0c/AJMB-5-20-g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/2fcefce9d8b8/AJMB-5-20-g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/d0306a2b83e7/AJMB-5-20-g003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/bdd4a7722cf2/AJMB-5-20-g004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/9bb2/3572703/0b5edf4b1ae9/AJMB-5-20-g005.jpg

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