抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。

Expression and Characterization of Fusion Protein of Single-chain Variable Fragment of Alpha Fetoprotein and Allophycocyanin Alpha Subunit

Objective:By using multiple gene expressing plasmids, fusion protein of single-chain variable fragment of alpha fetoprotein and allophycocyanin alpha subunit, phycobiliprotein lyase (cpcS), and enzymes (Ho1 and pebS) for conversion of heme to phycoerythrobilin were co-expressed in E. coli, with an aim to prepare fluorescent fusion protein which is covalently bound with phycoerythrobilin. Method:fragments of alpha fetoprotein and allophycocyanin alpha subunit were fused by fusion PCR. Then the fused gene, together with cpcS gene, were ligated into vector pCDFDuet-1. Ho1 and pebS genes were ligated into another vector pRSFDuet-1. The two vectors were then transformed into E. coli. Recombinant proteinwere expressed efficiently in E. coli by addition of IPTG and purified by affinity chromatography.The activities of the recombinant protein were evaluated by spectral analysis and competitive ELISA.Results:The recombinant protein, with a molecular weight approximately to 45.0kDa, has an absorbance maximum at 549.5nm and an emission maximum at 660nm. Competitive ELISA results showed that inhibition rate reached 48% when the concentration ratio of scFv and parent monoclonal antibodies was 16:1.Conclusion:A recombinant protein, which was fluorescent and immunologic active, was successfully expressed in E. coli.