抗慢性粒细胞白血病细胞人源化单链抗体—免疫毒素的制备及其生物活性的鉴定

发布时间：2019-04-03 08:52

【摘要】：目的慢性粒细胞白血病（chronic myelogenous leukemia, CML）是一种造血祖细胞恶性增殖性血液系统肿瘤，目前耐药和复发仍然是CML的两大难题。近年来，随着DNA重组技术的发展，免疫毒素尤其是重组免疫毒素(recombinant immunotoxin, rIT)迅猛发展，已经成为肿瘤免疫治疗研究中的重点和热点。到目前为止，国内外已经有许多种免疫毒素试剂进入临床试验。在本研究中，我们制备了抗CML细胞人源化单链抗体（humanized single chain variable region fragment,hscFv)，并将其与截短了的假单胞菌外毒素基因（truncated pseudomonasexotoxin A, ETA′)融合来制备免疫毒素，，对其进行了原核表达、纯化，并研究了其生物学活性，旨在为CML的靶向治疗提供一种高效的选择性的方法。 方法1.将抗CML细胞hscFv片段插入到pET32a(+)原核表达载体中。将构建正确的pET32a-hscFv重组质粒转化大肠杆菌BL21(DE3)菌株，在IPTG诱导下表达融合蛋白，表达产物用SDS-PAGE和WesternBlot进行鉴定；蛋白纯化后，采用细胞膜酶联免疫吸附试验（cellmembrane-enzyme linked immunosorbent asssay, CM-ELISA)和流式细胞术（flow cytometer, FCM)鉴定hscFv融合蛋白的抗原结合特性。 2.将hscFv与ETA′基因通过酶切与连接反应，在pWW20载体上构建hscFv-ETA'免疫毒素基因；再将其亚克隆入pET32a(+)中，转化BL21（DH3）宿主菌，IPTG诱导融合蛋白表达；SDS-PAGE和WesternBlot实验观察目的蛋白的表达情况并对纯化后的目的蛋白进行鉴定。CM-ELISA和FCM鉴定融合蛋白的抗原结合特性。通过细胞增殖实验（MTT)、细胞凋亡实验等来探讨hscFv-ETA'融合蛋白对CML细胞株和患者细胞的靶向杀伤作用。 结果1.本研究成功构建了hscFv的重组原核表达载体；该蛋白在25℃、l mM IPTG诱导4h的条件下获得高效、可溶性的表达；经纯化获得了高纯度的hscFv融合蛋白; CM-ELISA、FCM实验证实该蛋白具有CML细胞表面抗原结合的特性。 2.成功构建了hscFv-ETA'重组原核表达载体；hscFv-ETA'融合蛋白在23℃、l mM IPTG诱导6h的条件下获得可溶性表达；经Ni2+-NTA亲和纯化获得了hscFv-ETA'融合蛋白；经CM-ELISA、FCM等实验证实了该融合蛋白具有CML细胞表面抗原结合的特性；凋亡实验证明了该融合蛋白对CML细胞株或者患者细胞具有明显的靶向杀伤能力。 结论本研究成功制备、表达了hscFv蛋白和hscFv-ETA'重组免疫毒素，通过一系列的试验证明了制备的hscFv和hscFv-ETA'具有与CML细胞结合的活性并且hscFv-ETA'能特异性地靶向杀伤CML细胞，为慢性粒细胞白血病的靶向治疗提供了新的免疫学方法。
[Abstract]:Objective chronic myeloid leukemia (chronic myelogenous leukemia, CML) is a malignant proliferative hematological tumor of hematopoietic progenitor cells. Drug resistance and relapse are still two difficult problems in CML. In recent years, with the development of DNA recombination technology, immunotoxins, especially recombinant immunotoxin (recombinant immunotoxin, rIT), have become the focus and focus of tumor immunotherapy. So far, there have been many kinds of immunotoxin reagents in clinical trials at home and abroad. In this study, we prepared anti-CML cell humanized scFv (humanized single chain variable region fragment,hscFv and fused it with truncated Pseudomonas exotoxin gene (truncated pseudomonasexotoxin A, ETA' to prepare immunotoxin. Its prokaryotic expression, purification and biological activity were studied in order to provide an efficient and selective method for targeted therapy of CML. Method 1. The hscFv fragment of anti-CML cells was inserted into the prokaryotic expression vector pET32a (). The recombinant plasmid of pET32a-hscFv was transformed into E. coli BL21 (DE3) strain, and the fusion protein was expressed under the induction of IPTG. The expressed product was identified by SDS-PAGE and WesternBlot. After purification, the antigen binding characteristics of hscFv fusion protein were identified by membrane enzyme-linked immunosorbent assay (cellmembrane-enzyme linked immunosorbent asssay, CM-ELISA) and flow cytometry (flow cytometer, FCM). 2. The hscFv and ETA' genes were digested and ligated to construct the hscFv-ETA' immunotoxin gene in pWW20 vector, and then subcloned into pET32a () to transform BL21 (DH3) host strain into BL21 (DH3) host strain. The fusion protein expression was induced by IPTG. The expression of the target protein was observed by SDS-PAGE and WesternBlot assay and the purified target protein was identified. The antigen binding characteristics of the fusion protein were identified by CM-ELISA and FCM. The target killing effect of hscFv-ETA' fusion protein on CML cell line and patient cell was investigated by cell proliferation test (MTT), cell apoptosis assay and so on. Outcome 1. In this study, the recombinant prokaryotic expression vector of hscFv was successfully constructed, and the protein was expressed efficiently and soluble under the condition of, l mM IPTG induction at 25 鈩

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