靶向去唾液酸糖蛋白受体多功能分子的构建及抗肝癌效应

Anti-ASGPR scFv-mediated multi-function targeting molecule construction and characterization

目的构建具有靶向去唾液酸糖蛋白受体、溶酶体逃逸、DNA结合的多功能融合蛋白，并对表达产物进行功能鉴定。方法合成编码Melittin的两条寡核苷酸单链（GenBank：X02007），退火形成寡核苷酸双链；双酶切含抗去唾液酸糖蛋白单链抗体（ASGPRseFv）c1基因的载体C1／plT2，0．7％低融点琼脂糖凝胶回收C1；以pSW50．Gal4为模板，通过聚合酶链反应（PCR）扩增Gal4基因；采用分子克隆技术将其定向克隆至载体pGC4C26H中，获得重组质粒C1MG／pGC4C26H；双酶切载体C1MG／pGCAC26H，胶回收片段C1MG定向克隆至载体pET-32c中，将含C1MG／pET-32c的B121单菌落1：100接种至1000mlLB肉汤培养基中培养，并通过IPTG诱导表达。表达产物用Ni“螯合柱亲和纯化，免疫印迹技术（Western blot）和免疫组织化学分析重组蛋白C1MG的抗原结合能力，溶血实验分析C1MG的生物学活性，肿瘤细胞生长抑制实验分析C1MG的DNA结合能力。结果成功构建原核表达质粒C1MG!pET．32e，并经测序证实：在大肠杆菌BL21中有效表达重组融合蛋白C1MG；表达产物以包涵体形式存在；纯化的C1MG大小为64．1kDa，浓度为0．6g／L；Westernblot结果说明C1MG能有效识别重组ASGPR，免疫组织化学结果证实C1MG能结合到鼠肝细胞表面；溶血实验显示C1MG具有裂解红细胞膜功能；肿瘤细胞生长抑制实验证实C1MG将pEBAF／tk-GAL4rec质粒有效地导入表达ASGPR的细胞中并表达TK基因，氯喹对肿瘤细胞生长抑制无明显影响。结论在大肠杆菌中成功表达和纯化得到单链抗体一蜂毒肽一酵母转录因子（C1MG）的融合蛋白，该融合蛋白至少具有以下功能：ASGPR靶向识别能力、溶酶体膜裂解功能、以及DNA特异性结合功能，提示对肝癌的靶向治疗有潜在的应用价值。

Objective To construct a multi-function fusion protein with the target desasialoglyeoprotein receptor （ASGPR〉, endosome escape and DNA-binding ability, and identify the function of the expression product. Methods Two single oligonucleotide chains of Melittin were synthesized and double oli- gonueleotide chains formed after annealing. The vector of anti-desasialoglycoprotein single chains receptor C1 gene （CI/pIT2） was digested, and C1 was purified by hypo-melting point agarose gel. By using pSW50-Gal4 as template, Gal4 gene was amplified by using polymerase chain reaction （PCR）. All they were inserted into plasmid pGC4C26 to construct the recombinant plasmid C1MG/pGC. Then fused gene C1MG/pGC4C26H was subcloned into plasmid pET-32c to construct the recombinant plasmid C1MG/pET-32e. A single colony of E. BL21 containing the plasmid C1M/ET-32c was inoculated in LB broth, then diluted 1/100 into 1000 ml LB broth and induced with 1 mmol/L IPTG. The recombinant C1MG was purified with Ni2＋ chelating HiTrap HP column. Its antigen-binding ability was evaluated by isomg Western blotting and immnunohistochemistry, its cytolytic activity was analyzed by hemolysis test and DNA-binding capacity was substantiated by growth inhibition rate. Results The prokaryotic expression plasmid C1MG/pET-32e validly expressed recombinant protein C1MG by sequencing. C1MG about 64 kD was expressed as includion body （0. 6 g/L）. The results of WB and immnunohistochemistry showed that C1MG could effectively discriminate recombinant desasialoglycoprotein receptor （ASGPR）. Hemolysis test showed that C1 MG kept the membrance-disrupted activity of hepatic cells, and C1 MG could lyse cellular membrane of akaryocyte. Tumour cell growth inhibition test indicated C1 MG could introduce the pEBAF/tk-GAIArec plasmid into the cells expressing ASGPR and TK gene expressed. Chloroquine had no influence on tumour cell growth inhibition. Conelusion Successful expression and purity of C1MG are achieved in E. coli. C1MG recom binant protein confers targeting desasialoglycoprotein receptor （ ASGPR）, eytolytie and DNA-binding capacity, suggesting its potential value of liver-specific DAN delivery efficacy in vivo.