论文部分内容阅读
目的探讨抗去唾液酸糖蛋白受体(ASGPR)单链抗体C1与蜂毒肽(Melittin)重组蛋白C1M靶向抑制肝癌细胞的效果。方法将含C1M/pGC的XL1-Blue转化菌通过IPTG诱导表达。表达产物用Ni2+螯合柱亲和纯化,免疫组织化学技术分析重组蛋白C1M的抗原结合能力,四氮唑盐(MTT)法检测C1M对肝癌细胞株HepG2的增殖抑制作用。结果原核表达质粒C1M/pGC在XL1-Blue中获得有效表达;表达产物以可溶性形式存在;纯化的C1M相对分子量为29.4×103;免疫组化结果表明C1M能有效识别去唾液酸糖蛋白受体,与HepG2(C1M)共培养3d,细胞生长抑制率达92.0%。结论在大肠埃希菌中成功表达C1M,位于C端的Melittin蜂毒肽能有效抑制肿瘤细胞的生长,为体内研究Melittin的靶向抗肝癌效果奠定了基础。
Objective To investigate the effect of anti-asialoglycoprotein receptor (ASGPR) scFv C1 and Melittin recombinant protein C1M on inhibiting hepatoma cells. Methods XL1-Blue transformed bacteria harboring C1M / pGC were induced to express by IPTG. The expressed product was affinity purified with Ni2 + chelating column, the antigen binding ability of recombinant protein C1M was analyzed by immunohistochemistry, and the inhibitory effect of C1M on the proliferation of HepG2 hepatoma cell line was detected by MTT assay. Results The prokaryotic expression plasmid C1M / pGC was efficiently expressed in XL1-Blue. The expressed product existed in soluble form. The relative molecular weight of purified C1M was 29.4 × 103. The results of immunohistochemistry showed that C1M could effectively recognize asialoglycoprotein receptor, After co-cultured with HepG2 (C1M) for 3 days, the cell growth inhibition rate reached 92.0%. Conclusion The successful expression of C1M in E. coli and Melittin melittin at the C-terminus can effectively inhibit the growth of tumor cells, which lays the foundation for the study of Melittin targeting anti-hepatocellular carcinoma in vivo.