人4-1BBL胞外区/抗CD20融合蛋白的构建和表达

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

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150g/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4mg/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。

AIM： To construct and express human 4- 1 BBL./anti-CD20 bispecific fusion protein and identify its biological activity. METHODS： PCR and overlapping PCR were used to construct human 4-1BBL/anti-CD20 bispeciflc 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 A.549 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 cells and A549 cells.