Anti-CD3 scFv-B7.1真核表达载体的构建及在COS-7细胞中的初步表达

目的　构建anti CD3scFv B7.1真核表达载体 ,并进行初步表达。方法　采用重叠延伸拼接法 (splicingbyoverlapextention ,SOE)将anti CD3scFv和B7.1(V +C)两个基因片段通过spacer序列连接 ,将融合基因克隆入T载体 ,并测序证实。在此基础上构建真核表达载体pcDNA/anti CD3scFv B7.1,并经脂质体法转染COS 7细胞 ,免疫组织化学法检测表达。结果　获得了序列与预期完全相同的融合基因 ;构建了抗CD3scFv B7.1融合基因真核表达载体 ;在COS 7细胞中获得初步表达。结论　成功构建及表达抗CD3scFv B7.1融合基因真核表达载体 ,为进一步研究抗CD3scFv B7.1双功能分子的生物学活性及用于肿瘤生物治疗奠定了基础

Construction of anti-CD3 scFv-B7.1 eukaryotic expression vector and preliminary expression in COS-7 cells

Objective To construct eukaryotic expression vector bearing anti-CD3 scFv-B7.1 fusion gene for the functional investigation of the fusion protein. Methods Two DNA fragments, anti-CD3 scFv and B7.1(V+C), were linked through a spacer, assembled into a fusion gene by SOE (splicing by overlap extention) method and cloned into T-vector. Based on this fusion, eukaryotic expression vector pcDNA/ anti-CD3 scFv-B7.1 was then constructed. Lipofection strategy was subsequently adopted for the COS-7 transfection and fusion protein was detected by immunohistochemistry.Results Recombinant vector pcDNA/ anti-CD3 scFv-B7.1 was obtained and its preliminary expression was observed in COS-7. Conclusion The successful construction and expression of pcDNA/ anti-CD3 scFv-B7.1 has laid foundation for further study on its biological activities, functional assays and even for tumor biotherapy.