摘要
目的构建和原核表达抗人肝癌单链抗体HAb25scFv,为其进一步的临床应用研究奠定基础。方法采用(Gly4Ser)3为连接肽,在pUC19质粒上构建HAb25scFv单链抗体基因;序列分析证实后将HAb25scFv基因亚克隆入pGFX4T1GST融合表达载体上,SDSPAGE电泳鉴定表达产物并建立目的蛋白的纯化方法;免疫荧光竞争实验鉴定HAb25scFv单链抗体的活性。结果序列分析证实在pUC19质粒上以(Gly4Ser)3连接肽成功构建了HAb25scFv单链抗体基因;HAb25scFv单链抗体基因在pGEX4T1GST融合表达载体中获得高效表达,其表达量占菌体总蛋白的60.8%;经GST亲和层析柱,可获得纯度达95.2%的GSTHAb25scFv融合蛋白;免疫荧光竞争实验证实HAb25scFv单链抗体具有与其亲本抗体相同的抗原结合特性,并至少保留了亲本抗体39.12%的亲合力。结论获得了HAb25scFv单链抗体基因,并在原核GST融合表达系统中实现了HAb25scFv的高效表达,免疫荧光竞争实验证实HAb25scFv较好地保留了其亲本抗体的特异性和亲和力。
Objective To construct a prokaiyotie expression vector of single chain Fv （scFV） of HAb25 antibody against hepatocellular carcinoma （HCC）. Methods HAb25-cFsv gene was constructed in the pUC19 plasmid by using the （Gly4 Ser）3 as the polypeptide linker. After the sequence confirmation, the HAb25-seFv gene was subcloned into a GST expression vector pGEX-4T-1. The expression of the objective gene was identified by SDS-PAGE. After purification, the bioactivity of the recombinant HAb25-seFv was measured by immunofluorescent competitive assay. Results Sequencing analysis proved that the VH gene and the VL gene of HAb25 were linked to the 5＇ and 3＇ terminals of （Gly4Ser）3 gene in the pUC19, respectively. The HAb25-ScFv gene was highly expressed in the pGEX-4T-1 （60.8%）. After GST affinity chromatography, the purity of the recombinant protein was about 95.2%. Immunofluorescent competitive assay showed that the HAb25-seFv had specific binding ability to HCC cells and retained an affinity of 39.12% in comparison with its native HAb25 antibody. Conclusion The HAb25-scFv gene is successfully constructed and highly expressed in a GST fusion prokaIyotic system, and remains the specificity and affinity of its native antibody.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2005年第6期463-467,共5页
Immunological Journal
基金
重庆市科委自然科学基金重点项目资助(2004BA5017)
关键词
肝细胞肝癌
单链抗体
克隆
表达
Hepatocellular carcinoma
scFv
Cloning
Expression
