山羊IL-9基因克隆表达与单克隆抗体的制备

Cloning, expression and monoclonal-antibody preparation of goat IL-9

为了研究捻转血矛线虫诱导山羊产生Th9型免疫应答的特点,制备了抗山羊IL-9单克隆抗体。采用基因克隆、表达和纯化得到重组蛋白IL-9,并以重组IL-9为免疫原,重组蛋白IL-9和His-tag为筛选抗原,通过杂交瘤细胞技术,筛出1株稳定分泌抗山羊IL-9单克隆抗体细胞株,命名为6A11A8。将该细胞株接种小鼠腹腔,制备腹水,并将纯化后的抗体与生物素耦合,用流式细胞术检测山羊外周血单核细胞(PBMCs)中表达IL-9的细胞亚类。结果显示:融合蛋白大小约为16 ku,主要分布于包涵体中,筛选出的6A11A8细胞株产生的抗体效价高、特异性强,其细胞上清抗体效价可达1∶2430,其抗体亚型为IgG2bκ,层析柱纯化小鼠腹水后其效价可达1∶512 000,该单抗可识别山羊PBMCs中分泌IL-9的亚类细胞。结论:本研究获得了山羊IL-9单克隆抗体,为研究山羊Th9型免疫反应奠定了基础。

In this study,monoclonal antibodies against goat IL-9 were prepared for further investigating Th9 immune response in goats induced by Haemonchus contortus.The recombinant protein pET30a(+)/IL-9 was obtained by gene cloning,expression and purification,and was used as the immunogen.Then,the recombinant protein and His-tag were used as screening antigens,and the cells were selected by hybridoma.A cell strain stably secreting the anti-goat IL-9 monoclonal antibody was designated as 6A11A8 whose cell line was administrated to BALB/c mice intraperitoneally,and purified by column chromatography.The antibody was bound with Biotin and was used in flow cytometry to track the IL-9 expressing cells in PBMCs of goats.The results were as follows:SDS-PAGE showed that the fusion protein was of 16 ku in size,and was induced and mainly distributed in the inclusion body.The cell line 6A11A8 was found with high specificity.The titer of the cell-culture supernatant was 1:2430.Furthermore,the subtype of the monoclonal antibody expressed by 6A11A8 was IgG2bκ.To obtain sufficient antibody,cell line 6A11A8 was administrated to BALB/c mice intraperitoneally,and the antibody titer reached 1:512000 after purification by column chromatography.The IL-9 expressing cells in goat PBMCs identified by the monoclonal antibody.To sum up,in this study,a monoclonal antibody against goat IL-9 was obtained,which laid the foundation for further research on Th9 immune response.