【摘要】：目的慢性粒細(xì)胞白血�。╟hronic myelogenous leukemia, CML）是一種造血祖細(xì)胞惡性增殖性血液系統(tǒng)腫瘤，目前耐藥和復(fù)發(fā)仍然是CML的兩大難題。近年來(lái)，隨著DNA重組技術(shù)的發(fā)展，免疫毒素尤其是重組免疫毒素(recombinant immunotoxin, rIT)迅猛發(fā)展，已經(jīng)成為腫瘤免疫治療研究中的重點(diǎn)和熱點(diǎn)。到目前為止，國(guó)內(nèi)外已經(jīng)有許多種免疫毒素試劑進(jìn)入臨床試驗(yàn)。在本研究中，我們制備了抗CML細(xì)胞人源化單鏈抗體（humanized single chain variable region fragment,hscFv)，并將其與截短了的假單胞菌外毒素基因（truncated pseudomonasexotoxin A, ETA′)融合來(lái)制備免疫毒素，，對(duì)其進(jìn)行了原核表達(dá)、純化，并研究了其生物學(xué)活性，旨在為CML的靶向治療提供一種高效的選擇性的方法。 方法1.將抗CML細(xì)胞hscFv片段插入到pET32a(+)原核表達(dá)載體中。將構(gòu)建正確的pET32a-hscFv重組質(zhì)粒轉(zhuǎn)化大腸桿菌BL21(DE3)菌株，在IPTG誘導(dǎo)下表達(dá)融合蛋白，表達(dá)產(chǎn)物用SDS-PAGE和WesternBlot進(jìn)行鑒定；蛋白純化后，采用細(xì)胞膜酶聯(lián)免疫吸附試驗(yàn)（cellmembrane-enzyme linked immunosorbent asssay, CM-ELISA)和流式細(xì)胞術(shù)（flow cytometer, FCM)鑒定hscFv融合蛋白的抗原結(jié)合特性。 2.將hscFv與ETA′基因通過(guò)酶切與連接反應(yīng)，在pWW20載體上構(gòu)建hscFv-ETA'免疫毒素基因；再將其亞克隆入pET32a(+)中，轉(zhuǎn)化BL21（DH3）宿主菌，IPTG誘導(dǎo)融合蛋白表達(dá)；SDS-PAGE和WesternBlot實(shí)驗(yàn)觀察目的蛋白的表達(dá)情況并對(duì)純化后的目的蛋白進(jìn)行鑒定。CM-ELISA和FCM鑒定融合蛋白的抗原結(jié)合特性。通過(guò)細(xì)胞增殖實(shí)驗(yàn)（MTT)、細(xì)胞凋亡實(shí)驗(yàn)等來(lái)探討hscFv-ETA'融合蛋白對(duì)CML細(xì)胞株和患者細(xì)胞的靶向殺傷作用。 結(jié)果1.本研究成功構(gòu)建了hscFv的重組原核表達(dá)載體；該蛋白在25℃、l mM IPTG誘導(dǎo)4h的條件下獲得高效、可溶性的表達(dá)；經(jīng)純化獲得了高純度的hscFv融合蛋白; CM-ELISA、FCM實(shí)驗(yàn)證實(shí)該蛋白具有CML細(xì)胞表面抗原結(jié)合的特性。 2.成功構(gòu)建了hscFv-ETA'重組原核表達(dá)載體；hscFv-ETA'融合蛋白在23℃、l mM IPTG誘導(dǎo)6h的條件下獲得可溶性表達(dá)；經(jīng)Ni2+-NTA親和純化獲得了hscFv-ETA'融合蛋白；經(jīng)CM-ELISA、FCM等實(shí)驗(yàn)證實(shí)了該融合蛋白具有CML細(xì)胞表面抗原結(jié)合的特性；凋亡實(shí)驗(yàn)證明了該融合蛋白對(duì)CML細(xì)胞株或者患者細(xì)胞具有明顯的靶向殺傷能力。 結(jié)論本研究成功制備、表達(dá)了hscFv蛋白和hscFv-ETA'重組免疫毒素，通過(guò)一系列的試驗(yàn)證明了制備的hscFv和hscFv-ETA'具有與CML細(xì)胞結(jié)合的活性并且hscFv-ETA'能特異性地靶向殺傷CML細(xì)胞，為慢性粒細(xì)胞白血病的靶向治療提供了新的免疫學(xué)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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