本文關(guān)鍵詞： 日本血吸蟲 未成熟蟲卵可溶性抗原(SIEA) 單鏈抗體(scFv) 人白介素18(IL-18)　出處：《中南大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 本研究旨在構(gòu)建日本血吸蟲未成熟蟲卵可溶性抗原26-28kDa單鏈抗體與人白介素18融合蛋白原核表達(dá)質(zhì)粒,并通過(guò)大腸桿菌BL21菌株表達(dá)該融合蛋白,確定該融合蛋白的表達(dá)水平,探討利用單鏈抗體聯(lián)合白介素18進(jìn)行日本血吸蟲病靶向免疫治療的可能性。 方法: 以pET32a/SIEA26-28kDa-EGFP-scFv和pGEM-T-CMV/IL18質(zhì)粒為基礎(chǔ),利用PCR技術(shù)從pGEM-T-CMV/IL18質(zhì)粒中擴(kuò)增得到帶有特定酶切位點(diǎn)的IL-18全長(zhǎng)基因片段,經(jīng)相應(yīng)的限制性內(nèi)切酶酶切該片段和pET32a/SIEA26-28kDa-EGFP-scFv質(zhì)粒,分離并純化相應(yīng)目的片段后,利用連接酶連接,轉(zhuǎn)化至感受態(tài)細(xì)胞,經(jīng)固體LB細(xì)菌培養(yǎng)基培養(yǎng),挑取單克隆,純化質(zhì)粒、鑒定克隆,獲得pET32a/SIEA26-28kDa-IL18-scFv質(zhì)粒;將該質(zhì)粒轉(zhuǎn)染至大腸桿菌BL21菌株,通過(guò)IPTG誘導(dǎo)表達(dá)融合蛋白IL18-scFv,同時(shí)表達(dá)EGFP-scFv融合蛋白,觀察該融合蛋白的表達(dá)水平;并用Western-blot鑒定該融合蛋白的表達(dá)。 結(jié)果: 1.經(jīng)酶切鑒定,原有保存的質(zhì)粒證實(shí)為pET32a/SIEA26-28kDa-EGFP-scFv; 2.經(jīng)PCR成功擴(kuò)增IL-18基因片段; 3.經(jīng)限制性內(nèi)切酶酶切,連接酶連接,轉(zhuǎn)化至感受態(tài)細(xì)胞,鑒定克隆,獲得pET32a/SIEA26-28kDa-IL18-scFv質(zhì)粒; 4.經(jīng)考馬斯亮藍(lán)染色鑒定,在IPTG誘導(dǎo)不同時(shí)間后,融合蛋白IL18-scFv和EGFP-scFv得到成功的表達(dá),并經(jīng)Western-blot鑒定。 結(jié)論: 1.成功構(gòu)建pET32a/SIEA26-28kDa-IL18-scFv質(zhì)粒; 2.經(jīng)IPTG誘導(dǎo),IL18-scFv融合蛋白可有效表達(dá)。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

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