摘要
目的:构建抗人肝细胞癌单链抗体基因,并在大肠杆菌中表达.方法:采用RTPCR法扩增抗体重链和轻链可变区基因,用(Gly4Ser)3肽段连接VH基因和VL基因,构建克隆载体,将测序完全正确的ScFv基因克隆到分泌性表达载体pCANTAB5E中诱导表达转化的TG1和HB2151细胞.用SDSPAGE和免疫组化方法分析检测表达产物.结果:DNA测序证实ScFv基因结构正确,可在大肠杆菌中成功表达,游离ScFv相对分子质量大约为30ku,与HC108,HepG2和SMMC7721肝癌细胞有特异性的结合反应.结论:重组制备的抗人HCD78分子单链抗体在人肝癌的基础和临床研究中具有潜在的应用价值.
AIM: To generate and express a single-chain Fv (ScFv) fragment from the HCD78 hybridoma producing monoclonal antibodies against hepatocellular carcinoma (HCC). METHODS: VH and VL gene segments were generated from the HCD78 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). The ScFv was arranged in VH-VL orientation, joined together with a 15-amino-acid (Gly4Ser) 3 linker. The ScFv encoding sequence was amplified and cloned into pMD18-T vector. Amplification of the resultant ScFv gene was performed before it was introduced into E.coli TG1 and HB 2151 via a phagemid vector pCANTAB5E. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunohistochemistry analysis were used to determine the characteristics of the soluble ScFv. RESULTS: DNA sequencing proved that the HCD78 ScFv gene was correctly cloned into the expression vector. SDS-PAGE analysis of the recombinant antibody revealed that a protein with apparent molecular weight of approximately 3×10 4 was successfully expressed in E.coli HB2151. The ScFv antibody showed a specific reaction with HC-108, HepG-2 and SMMC-7721 hepatocellular carcinoma cells by immunohistochemical assay. CONCLUSION: The HCD78 ScFv is successfully constructed and expressed, which has the potential to be used as an agent in the research and clinical management of hepatocellular carcinoma.
出处
《第四军医大学学报》
CAS
北大核心
2005年第14期1268-1271,共4页
Journal of the Fourth Military Medical University
基金
陕西省自然科学基金(1999SM52)
关键词
癌
肝细胞
单链抗体
碱基序列
carcinoma, hepatocellular
single-chain antibody
base sequence
