日本血吸虫SIEA26-28kDa单链抗体与人白介素18融合蛋白质粒的构建及表达

发布时间：2018-02-24 08:23

本文关键词： 日本血吸虫未成熟虫卵可溶性抗原(SIEA) 单链抗体(scFv) 人白介素18(IL-18)　出处：《中南大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的: 本研究旨在构建日本血吸虫未成熟虫卵可溶性抗原26-28kDa单链抗体与人白介素18融合蛋白原核表达质粒,并通过大肠杆菌BL21菌株表达该融合蛋白,确定该融合蛋白的表达水平,探讨利用单链抗体联合白介素18进行日本血吸虫病靶向免疫治疗的可能性。方法: 以pET32a/SIEA26-28kDa-EGFP-scFv和pGEM-T-CMV/IL18质粒为基础,利用PCR技术从pGEM-T-CMV/IL18质粒中扩增得到带有特定酶切位点的IL-18全长基因片段,经相应的限制性内切酶酶切该片段和pET32a/SIEA26-28kDa-EGFP-scFv质粒,分离并纯化相应目的片段后,利用连接酶连接,转化至感受态细胞,经固体LB细菌培养基培养,挑取单克隆,纯化质粒、鉴定克隆,获得pET32a/SIEA26-28kDa-IL18-scFv质粒;将该质粒转染至大肠杆菌BL21菌株,通过IPTG诱导表达融合蛋白IL18-scFv,同时表达EGFP-scFv融合蛋白,观察该融合蛋白的表达水平;并用Western-blot鉴定该融合蛋白的表达。结果: 1.经酶切鉴定,原有保存的质粒证实为pET32a/SIEA26-28kDa-EGFP-scFv; 2.经PCR成功扩增IL-18基因片段; 3.经限制性内切酶酶切,连接酶连接,转化至感受态细胞,鉴定克隆,获得pET32a/SIEA26-28kDa-IL18-scFv质粒; 4.经考马斯亮蓝染色鉴定,在IPTG诱导不同时间后,融合蛋白IL18-scFv和EGFP-scFv得到成功的表达,并经Western-blot鉴定。结论: 1.成功构建pET32a/SIEA26-28kDa-IL18-scFv质粒; 2.经IPTG诱导,IL18-scFv融合蛋白可有效表达。
[Abstract]:Objective:. The aim of this study was to construct the prokaryotic expression plasmid of soluble single chain antibody 26-28kDa single chain antibody (scFv) of Schistosoma japonicum eggs and human interleukin-18 (IL-18) fusion protein, and to express the fusion protein by E. coli BL21 strain to determine the expression level of the fusion protein. To explore the possibility of targeted immunotherapy of schistosomiasis japonicum with single chain antibody (scFv) combined with interleukin 18 (IL 18). Methods:. Based on pET32a/SIEA26-28kDa-EGFP-scFv and pGEM-T-CMV/IL18 plasmids, the full-length IL-18 gene fragment with specific restriction endonuclease was amplified from pGEM-T-CMV/IL18 plasmid by PCR technique. The fragment and pET32a/SIEA26-28kDa-EGFP-scFv plasmid were digested by restriction endonuclease, and the corresponding target fragment was isolated and purified. By ligation of ligase, it was transformed into receptive cells, cultured in solid LB bacteria culture medium, selected monoclonal, purified plasmids, identified clones, obtained pET32a/SIEA26-28kDa-IL18-scFv plasmid, and transfected the plasmid into Escherichia coli BL21 strain. The fusion protein IL18-scFvwas induced by IPTG and the EGFP-scFv fusion protein was expressed at the same time. The expression level of the fusion protein was observed, and the expression of the fusion protein was identified by Western-blot. Results:. 1. The original plasmid was confirmed as pET32a / SIEA 26-28kDa-EGFP-scFv. 2. The IL-18 gene fragment was successfully amplified by PCR. 3. After restriction endonuclease digestion, ligase ligase was ligated and transformed into receptive cells. The clone was identified and the pET32a/SIEA26-28kDa-IL18-scFv plasmid was obtained. 4. By Coomassie brilliant blue staining, the fusion proteins IL18-scFv and EGFP-scFv were successfully expressed at different time after induction by IPTG, and identified by Western-blot. Conclusion:. 1. Construct pET32a/SIEA26-28kDa-IL18-scFv plasmid successfully; 2. The fusion protein of IL18-scFv was effectively expressed by IPTG.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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