摘要
目的:构建并表达超抗原葡萄(SEA)和鼠抗人大肠癌单链抗体ND-1scFv的融合蛋白,以提高SEA的靶向杀伤作用。方法:构建超抗原SEA和鼠抗人大肠癌单链抗ND-1scFv的融合基因ND-1 scFv/SEA的表达载体,转化到大肠杆菌E.coli M15中进行诱导表达。Ni-NTA亲和层析对表达产物进行分离、纯化。间接免疫荧光法检测融合蛋白的靶向结合活性,MTT法检测靶向杀伤效率。结果:成功构建了融合基因ND-1scFv/SEA,实现功能性表达,纯化的ND-1scFv/SEA融合蛋白与表达有ND-1相应抗原的大肠癌细胞CCL-187有高度亲和活性,通过激活外周血单核细胞,可特异性杀伤靶细胞,在4μg/mL浓度下对CCL-187的杀伤率达到91%,明显优于SEA的杀伤活性。结论:融合蛋白ND-1 scFv/SEA对大肠癌细胞CCL-187具有靶向结合和杀伤活性,为SEA用于靶向性的大肠癌治疗奠定了基础。
AIM： To construct and express the recombinant ND-1-scFv/SEA, a fusion protein of superantigen （staphylococcal enterotoxinA, SEA） and single-chain variable fragment of monoclonal antibody ND-1 against human clolorectal carcinoma, and to enhance the targeted killing effect of SEA. METHODS： The expression of the fusion protein was induced in E. coli M15 by IPTG. Ni-NTA resin affinity chromatography was used to separate and purify the expressed product. The specific binding activity of the purified ND-1-scFv/SEA protein was examined by indirect immunofluorescence assay and the targeted-cytotoxicity was determined using MTT assay. RESULTS： The expressing vector of fusion gene ND-lscFv/SEA was constructed successfully. ND-1-scFv/SEA protein retained a high binding affinity to antigen-positive human colorectal cancer cell CCL-187 and had a stronger capability to activate PBMC and kill the target cells compared to SEA alone, with a killing rate of 91% at 4 pg/mL. CONCLUSION： ND-1-scFv/SEA fusion protein could specifically target colorectal cancer cell, enhance the activity of kill tumor cell and has potential applications in the targeted therapy of colorectal cancer.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2012年第4期400-403,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金资助项目(20875103)
关键词
ND-1单克隆抗体
单链抗体Fv
葡萄球菌肠毒素A
大肠癌
monoclonal antibody ND-1
single chain anti-body Fv
staphylococcal enterotoxin A
color-ectal cancer
