人源TNF-α抗体基因的克隆与表达(1)
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[摘要] 目的:在原核系统中高效表达抗TNF-α单克隆抗体VH/K21,以便进一步研究其生物学功能,预测临床应用前景。方法:以已筛选获得的TNF-α抗体基因VH/K21为模板进行PCR扩增,产物与其表达质粒进行双酶切,连接后克隆进入PET22b(+)/BL21(DE3)感受态中并在该原核系统中实现可溶性表达;对表达产物进行Western blotting、ELISA鉴定。结果:成功克隆与表达出可溶性的高效表达的人源TNF-α单克隆抗体。序列分析表明该克隆具有全新的人源TNF-α抗体基因,它可以特异识别人TNF-α。结论:人源TNF-α单克隆抗体的获得为进一步研制应用于临床的TNF-α人源抗体奠定了基础,也将为其他人源抗体的制备提供理论依据和技术基础。
[关键词] 抗体;TNF-α;变应性鼻炎
[中图分类号] R765.21[文献标识码]A [文章编号]1673-7210(2009)07(c)-009-03
Cloning and expression of human TNF-α antibody gene
TIAN Lufang1, ZHAO Xiaoling2, ZHANG Kejun1, YANG Zhihua2
(1.Department of Otolaryngology, the First Hospital of Shanxi Medical University, Taiyuan 030001, China; 2.Department of Stress Medicine, Research Institute of Hygiene and Environmental Medicine, the Academy of Military Medical Sciences, Tianjin 300050, China)
[Abstract] Objective: To express the TNF-α monoclonal antibody VH/K21 in the prokaryotic expression system, in order to further explore its biological functions and predict its clinical application prospect. Methods: The screened TNF-α antibody VH/K21 gene was amplified by PCR and cloned into the prokaryotic expression vector pET-22b(+)/BL21(DE3), and achieved soluble expression. The products were identified by Western blotting and ELISA. Results: The soluble and high expressed human TNF-α monoclonal antibody was successfully cloned and expressed. Sequence analysis suggested that this clone had fully new human TNF-α antibody gene, and it could specifically recognize human TNF-α. Conclusion: The obtain of human TNF-α monoclonal antibody lays a foundation for its clinical application, and would provide a theoretical and technical basis for the preparation of other human antibodies.
[Key words] Antibody; TNF-α; Allergic rhinitis
变应性鼻炎(allergic rhinitis,AR)是由变应原激发的、由IgE介导的鼻部炎性疾病,是耳鼻喉科最常见的一种疾病,也是全球最常见的慢性疾病之一。随着人类现代生活方式和生态环境的急剧变化,AR的发病率在全球呈增长趋势[1-2],且城市的发病率远高于农村[3],已逐渐成为全球性的健康问题,虽非重大疾病,但对人们的工作、学习和生活产生了严重的影响。AR治疗原则主要为避免接触变应原、药物控制和免疫调节。目前AR的治疗还没有特效的治疗措施,常规的治疗只是缓解症状,减轻炎症,提高患者的健康度和生活质量。所以,找到一种治疗AR的最佳方法已经成为耳鼻喉科医师的研究热点。
研究发现,TNF-α在AR的发生、发展过程中起着至关重要的作用,用TNF-α的拮抗剂治疗AR成为一个新的研究热点 ......
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