抗肝癌单链抗体融合PE38重组免疫毒素的构建、表达及纯化

Construction and purification of a recombinant immunotoxin composed of PE38 and humanized scFv against hepatocellular carcinoma

目的 :构建绿脓杆菌外毒素PE38融合人源化鼠抗肝癌单链抗体 (hscFv)原核表达载体 ,并对其产物作初步纯化 ,检测其对人肝癌细胞系SMMC 772 1的毒性作用 .方法 :采用基因工程原理 ,将PE38基因片段亚克隆入hscFv表达载体 pGEX 4T 1中 ,重组克隆载体经酶切及DNA序列测定证实两片段连接正确 ;转化大肠杆菌JM10 9后 ,受IPTG诱导表达GST融合蛋白 ,产物包涵体经变性、复性及重折叠并经GST亲合层析柱层析、凝血酶消化后 ,得到纯化的hscFv PE38融合蛋白 .采用MTT法检测hscFv PE38对SMMC772 1的杀伤作用 .结果 :实验成功构建了免疫毒素表达载体pGEX 4T 1 hscFv PE38(以下简称pGEXh PE38) ;诱导产物主要以包涵体形式存在 ,表达量为 11% ;纯化的hscFv PE38对SMMC 772 1细胞系具有较明显的杀伤作用 ,最大杀伤率为78% ,IC50 为 0 .386mg·L-1.结论 :人源化抗肝癌单链抗体与PE38重组免疫毒素的成功表达纯化及对人肝癌细胞系的试验结果 。

AIM: To construct a new recombinant immunotoxin expression vector composed of a truncated form of pseudomonas exotoxin A ( PE38 ) gene and a humanized rodent anti human hepatocellular carcinoma (HCC)single chain Fv fragment (hscFv) gene, and to examine the cytotoxicity of the purified product (hscFv PE38) on human HCC cell line SMMC 7721. METHODS: The PE38 gene fragment was subcloned into a hscFv inserted GST fusing protein expression plasmid. The recombinant vector was identified by restriction endonucleases digestion and DNA sequencing. After transformed into E.cole JM109 and induced by IPTG, the inclusion bodies were denatured, renatured and refolded, then resolvable protein was purified by GST affinity chromatography. Desired hsdFv PE38 was obtained after cleaving fusion protein with thrombin. The cytotoxity of hsdFv PE38 on SMMC 7721 was evaluated by MTT assay. RESULTS: The new recombinant immunotoxin expression vector pGEXh PE38 was constructed successfully. The main product was in inclusion bodies, amounting to 11% of the total host bacterial proteins. MTT assay showed that purified hscFv PE38 had obvious effects on SMMC 7721, the maximum killing rate being 78% and IC 50 being 0.386 mg·L -1 . CONCLUSION: The results of our study on hscFv PE38 and its effects on SMMC 7721show its great potentials for the targeted therapy of hepatocelluar carcinoma.