抗Aβ人源融合抗体在毕赤酵母中的高效表达纯化及性质研究

High Level Expression Purification and Character Research of Human Fusion Antibody for Anti-Aβ in the Yeast Pichia pastoris

抗β-淀粉样多肽(Aβ)的抗体对老年性痴呆(AD)病变有清除作用,人源融合抗体由可识别结合Aβ的多肽序列A与人抗体Fc段连接重组构成。构建的单拷贝表达载体PA0815-αAFc,可以在毕赤酵母中高效表达。此表达载体含有一个AFc表达单元,并在受严格调控的醇氧化酶启动子的控制下由甲醇诱导表达,并利用α因子前导肽引导融合抗体分泌。载体线性化后整合入甲醇营养型酵母Pichia pastoris(Pp)GS115基因组中,经鉴定表达单位具有良好的遗传稳定性。对筛选得到的高分泌表达融合抗体的细胞株进行表达和培养条件的优化,使诱导上清液中融合抗体的产量选到100mg/L。利用Protein A层析柱分离、纯化,获得纯度较高的融合抗体。经鉴定分析其具有正确的分子量和N末端,且融合抗体可与Aβ抗原结合并能吸引巨噬细胞吞噬和清除抗原抗体复合物,具有抗体生物学活性。这是首次利用甲醇营养型酵母表达系统成功实现了抗Aβ融合抗体的高效分泌表达及产物纯化鉴定,为进一步研究AD及其治疗提供了新的途径。

Immunological treatment is a new therapeutic approach for treatment of Alzheimer's Diseases(AD) . Now a cell line of expressing fusion antibody in yeast system was established, this antibody is composed by two parts. One part(A) is designed to recongnize Aβ and conjugate it (the sequence has been patent) . The others comes from human Fc. The fusion antibody acts as natural antibodies against amyloid β-peptide triggered microglialcell to clear plaques through Fc receptor-mediated phagocytosis. The pAO815-αAFc expression vector can be under the control of tightly regulated alcohol oxidase(AOX1) promoter and the gene encoding the fusion antibody was fused in fame to the S.cerevisiae α-mating factor leader sequence,that is, the secretion of fusion antibody depends on the secretion signal sequence of α-factor. The vector can be linearized by enzyme and transformed into Pp. Therefore the expression strains would show good genetic stability during growth even under non-selective conditions. To increase the yield of fusion antibody and minimize the proteolytic process of fusion antibody secreted in the culture medium, the conditions of expressing and culturing were optimized .The yield of fusion antibody in superatant of 1L flake pine culture is at the level of 100mg/L. Through Protein A affinity chromatography, the fusion antibody with high purity and biological activity was obtained. It owns correct MW, N-terminal amino acid sequence and shows the biological activity of combining Aβ and stimulating microphage to clear Ag+Ab complex. It may provide a new potential approach to treat and study Alzheimer's disease.