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目的去唾液酸糖蛋白受体(ASGPR)H1亚单位的重组、表达、纯化及其单链抗体的筛选与鉴定。方法将ASGPRH1亚单位糖识别区基因(CRDH1)定向克隆至原核表达载体pET-32c经IPTG诱导表达,表达产物用Ni2+螯合柱亲和纯化,免疫印迹技术(WB)检测;用纯化的CRDH1对人源噬菌体抗体库进行4轮固相筛选,阳性克隆用表达标记(Trx-His-stag)进行差异筛选。取阳性噬菌体C3感染宿主菌HB2151,37℃振荡培养过夜,终浓度为1mmol/L的IPTG诱导表达,12%SDS-PAGE与Westernblot鉴定以及免疫组化鉴定纯化的单链抗体C3与肝细胞结合的特性。结果CRDH1/pET-32c在原核系统经诱导表达出相对分子质量(Mr)约35×103大小的融合蛋白,以包涵体的形式存在;通过Ni2+亲和柱纯化获得CRDH1融合蛋白;经4轮筛选和差异筛选后,有2株可分泌性表达与CRDH1特异性结合的单链抗体,DNA序列分析表明单链抗体重链基因和轻链基因分别属于人免疫球蛋白VH3家族和VKI家族。噬菌体C3经诱导表达出Mr约28×103大小的融合蛋白,可溶性分泌表达于培养基中;通过Ni2+亲和柱纯化获得单链抗体,ELISA、Westernblot表明单链抗体C3能较好结合rCRDH1,免疫组化显示该单链抗体可与肝癌细胞、肝细胞特异结合。结论成功表达ASGPR并筛选出其人源单链抗体。该单链抗体可与表达的rCRDH1以及肝细胞和肝癌细胞特异性结合,提示对肝脏疾病的靶向治疗有潜在的应用价值。
AIM: To study the recombinant, expression and purification of asialoglycoprotein receptor (ASGPR) H1 subunit and the screening and identification of its single chain antibody. Methods The CRDH1 gene of ASGPRH1 subunit was cloned into prokaryotic expression vector pET-32c and induced by IPTG. The expressed product was purified by Ni2 + chelate column and detected by Western blot (WB). The purified CRDH1 Human phage antibody library was subjected to 4 rounds of solid-phase screening. The positive clones were screened by differential expression using Trx-His-stag. The positive bacteriophage C3-infected host strain HB2151 was cultured overnight at 37 ° C with IPTG induction at a final concentration of 1 mmol / L, and identified by 12% SDS-PAGE and Western blotting as well as by immunohistochemistry to identify purified C3 antibody conjugated to hepatocytes characteristic. Results CRDH1 / pET-32c was induced in prokaryotic system to express fusion protein of 35 × 103 molecular weight (Mr), and existed as inclusion body. CRDH1 fusion protein was purified by Ni2 + affinity column. After 4 rounds of screening, After differential screening, there were 2 single chain antibodies secreting specifically to CRDH1. DNA sequence analysis showed that the single chain antibody heavy chain genes and light chain genes belong to human immunoglobulin VH3 family and VKI family respectively. The phage C3 induced the expression of Mr about 28 × 103 size fusion protein, soluble secreted expression in the medium; purified by Ni2 + affinity column to obtain single-chain antibody, ELISA, Western blot showed that the single-chain antibody C3 can better bind rCRDH1, immune Histochemistry shows that the single chain antibody can specifically bind to liver cancer cells and hepatocytes. Conclusion ASGPR was successfully expressed and its human scFv was screened out. The single-chain antibody can specifically bind to the expressed rCRDH1 and hepatocellular and hepatocellular carcinoma cells, suggesting potential therapeutic value for the targeted therapy of liver diseases.