抗CD20融合蛋白在CHO细胞中的表达及其纯化与鉴定

Expression of Anti-CD20 Fusion Protein in CHO Cells and its Purification and Identification

目的:构建CD20胞外区与Igβ胞外区和人IgG1 Fc融合基因的表达载体,并在CHO细胞中表达。方法与结果:根据已知的IgM的CH2区域和Igβ胞外区序列,分别设计PCR引物并进行PCR扩增,然后用重叠PCR法扩增得到900 bp的Igβ-CH2序列,插入本实验室构建的pIRIS-EGFP-Fc载体,转化大肠杆菌,得到pIRIS-Igβ-CH2-Fc重组质粒,将其转染CHO-K1细胞,在G418抗性培养基中培养,荧光显微镜观察结合ELISA法筛选高表达细胞系,细胞系扩大培养后,通过亲和层析rProtein A柱纯化得到纯度融合蛋白,SDS-PAGE显示目的蛋白去糖基化后,相对分子质量约为42×103。结论:得到了Igβ-CH2-Fc重链抗体样分子,并鉴定为糖蛋白;须进一步对所得蛋白的生物学活性进行检测,验证其是否能够通过胞外区蛋白定位于B细胞淋巴瘤细胞表面对B淋巴瘤细胞进行杀伤。

Objective： To construct an expression vector of antibody like molecule targeting B-cell lymphoma surface biomarker CD20 and express it in CHO cells. Methods and Results： According to the known sequences, PCR primers were designed and used to amplify CH2 region of IgM and Igβ extracellular region respectively, then they were fused by overlapping PCR. The resulting Igβ-CH2 sequence about 900 bp was inserted into pIRIS-EGFP-Fc vector followed by transformed into E.coli to get the pIRIS-Igβ-CH2-Fc recombinant plasmid. The expression plasmid was transfected to CHO-K1 cells and cultured in G41g-resistant medium, then with fluo- rescence microscopy observation combined with ELISA method to screen out the high-expressing cell line. After larger scale culture of this cell line, the protein of interest was purified by affinity chromatography rProtein A col- umn. SDS-PAGE showed that deglycosylated target protein with molecular weight about 42kD. Conclusion： We ob- tained the recombinant Igβ-CH2-Fc, and it was characterized as glycoprotein. Its biological activity should be further examined, and whether the protein can locate on surface of B-cell lymphoma cells through the extracellular domain to kill B-cell lymphoma ceils should also be verified in the future studies.