二硫键稳定的人源化抗肝癌单链抗体与人嗜酸性细胞神经毒素重组免疫毒素融合基因的构建及表达

目的：构建二硫键稳定的人源化抗肝癌单链抗体(hdsFv)与人嗜酸性细胞神经毒素(hEnS)融合基因原核表达载体，并在大肠杆菌中表达。方法：利用RCR的方法扩增hEDN基因，克隆人含抗肝癌hdsFv基因的TIH原核表达载体，转化大肠杆菌BL21(DE3)plyS后，以IPTG诱导融合蛋白表达。表达产物用SDS-PACE及Westen-blot分析及鉴定。结果：成功构建了抗肝癌HdsFv-hEDN融合基因原核表达载体；SDS-PACE和wbsten—blot分析显示，诱导表达产物出现一条Mr约为45．0KD的新生蛋白带，表达量占菌体总蛋白量的x％，主要以包涵体形式存在。结论：抗肝癌hdsFv—hEDN重组免疫毒素融合基因的成功构建及表达，为其进一步进行肝癌的导向治疗研究奠定了基础。

Construction and expression of disulfide-stabilized humanized scFv and hEDN recombinant immunotoxin fusion gene against hepatocellular carcinoma

Objective:To study the construction and expression of a novel recombinant immunotoxin fusion gene prokaryotic expression vector composed of disulfide-stabilized humanized scFv (hdsFv) and hEDN gene against hepatocellular carcinoma (HCC) in E.coli. WT5HZ]Methods: PCR was used to amplify the hEDN gene fragment encoding the mature protein. Recombinant immunotoxin fusion gene of hdsFv- hEDN prokaryotic expression vector was constructed by linkage hdsFv gene against HCC with hEDN gene and introduced into E.coli cell BL21(DE3)plyS. The expression of recombinant immunotoxin hdsFv-hEDN against HCC was induced by IPTG in E.coli. The expression product was analyzed and identified by SDS-PAGE and Western-blot.WT5HZ] Results:A 470 bp long gene fragment was amplified by PCR, and the size and sequence were accorded with those of hEDN reported in Genebank. PCR identification and nucleotide sequencing confirmed that the construction of recombinant immunotoxin fusion gene prokaryotic expression vector was correct. After induced by IPTG, the recombinant immunotoxin hdsFv-hEDN against HCC was successfully expressed in E.coli. A new anticipated Mr 45 KD protein band appeared on SDS-PAGE gel and amounted to 22% of total bacterial protein. The expressed product existed in a form of inclusion body that was confirmed by Western-blot. WT5HZ]Conclusion:The prokaryotic expression vector of recombinant immunotoxin fusion gene hdsFv-hEDN against HCC is successfully constructed and induced to express recombinant immunotoxin, which lay the foundation for the further study of targeting therapy of HCC. WT5HZ]