人4-1BBL胞外区/抗CD20融合蛋白的构建和表达

目的:构建和表达人4-1BBL胞外区/抗CD20双功能融合蛋白,并测定该融合蛋白的生物学活性.方法:采用PCR和ovelap PCR方法构建人4-1BBL胞外区/抗CD20双功能融合蛋白,并用双脱氧终止法测定DNA序列;采用亲和层析法纯化该产物,并用150 g/L SDS-PAGE和Western blot鉴定纯化产物;采用FACS法鉴定纯化产物与靶细胞的结合活性.结果:DNA序列测定结果表明:人4-1BBL胞外区/抗CD20双功能融合蛋白已构建成功,表达可溶性产物的产量达4 mg/L以上,具有与Raji细胞(CD20 )和A549(4-1BB )细胞结合的活性.结论:利用融合蛋白形式,首次成功地构建了人4-1BBL胞外区/抗CD20融合蛋白,并获得较高表达.表达产物具有与相应2个靶抗原结合的活性.

Construction and expression of human 4-1BBL/anti-CD20 fusion protein

AIM: To construct and express human 4-1BBL/anti-CD20 bispecific fusion protein and identify its biological activity. METHODS: PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispecific fusion protein. DNA sequencing was performed by the terminus of the fusion protein nucleotide.The product was purified by affinity chromatography and analyzed by Western blot and its antigen-binding activity was examined by FACS. RESULTS: The data of DNA sequence showed that human 4-1BBL/anti-CD20 bispecific fusion protein was correct. The fusion protein was recovered in high yield (up to 4 mg/L) after E-tag purification and predominantly(90%) as a dimer. The fusion protein could bind to Raji cells(CD20(+)) and A549 cells(4-1BB(+)), respectively. CONCLUSION: The human 4-1BBL/anti-CD20 bispecific fusion protein with high level expression was successfully obtained and could bind to Raji ceIls and A549 cells.