人CD24分子成熟多肽的原核表达及抗体制备

Prokaryotic Expression of Human CD24 and Its Polyclonal Antibody Preparation

目的:为获得抗人CD24分子成熟多肽核心蛋白(hCD24N)的多克隆抗体。方法:制备CD24表达阳性的人肿瘤细胞cDNA,采用PCR法扩增hCD24N的编码基因,构建pGEX-KGV-hCD24N原核表达质粒;转化大肠杆菌BL21(DE3),乳糖诱导表达;经GST亲和柱层析、SDS-PAGE和Western blotting制备并鉴定纯化的GST-StraptagII-hCD24N融合蛋白;免疫新西兰大白兔制备抗血清并用rProtein A亲和柱层析纯化多克隆IgG抗体;用间接ELISA法测定抗体效价,Western blotting鉴定抗体特异性,同时采用细胞免疫荧光检测技术对抗体的特异性和应用可行性作进一步评价。结果:实现了hCD24N基因的克隆以及在原核细胞中的可溶性重组融合表达,得到了纯化后的目的融合蛋白,并以其为免疫原获得了效价高于1∶100 000的抗hCD24N多克隆抗体,Western blotting及细胞免疫荧光检测证明该抗体与当前市售的抗人CD24抗体具有相似的免疫反应特异性,并且能够与CD24阳性人肿瘤细胞表达并加工的高度糖基化CD24天然分子发生特异性抗原-抗体反应。结论:抗hCD24N多克隆抗体的成功制备为进一步以CD24分子为靶点的肿瘤生物学基础研究以及相关癌症的诊断试剂开发奠定基础。

Human CD24 is a candidate biomarker specifically expresses in many types of malignant tumor cells. In order to obtain its polyclonal antibody for following-up studies, the encoding sequence of mature human CD24 polypeptides without glycosylation （hCD24N） was amplified and cloned into pGEX-KGV, which is a modified prokaryotic expression vector containing a N-terminus GST fusion tag and another internal Strap tag II. Prokaryotic expression plasmid pGEX-KGV-hCD24N was constructed and expressed in the strain of E. coli BL21 （DE3） using lactose induction. Fusion protein, named GST-StraptagII-hCD24N, mainly expressed in its soluble forms, and existed in the supernatant after cell lysis. Using GST affinity chromatographic separation, this target protein was purified and used to immunize rabbit to produce rabbit anti-hCD24N polyclonal antibody. An indirect ELISA assay was further used to measure the anti-hCD24N titer of prepared antibody. Moreover, the specificity against native glycosylated CD24 molecular which is expressed in the positive human cancer cells was also confirmed by Western blot and immunofluorescence, and compared with commercial antibody. The results showed that the prepared polyclonal antibody had a high titer to recombinant hCD24N, and could react with native human CD24 and its positive human malignant cancer cells specifically, which provides a favorable tool for further studies on application of CD24 as a candidate antigen for cancer prevention and treatment. Moreover, the GST- StraptagII-hCD24N fusion protein could also be used as a target for screening novel specific ligand against CD24 and associated positive cancer cells. In conclusion, the prepared anti-CD24N polyclonal antibody provides the basis for tumor biology research and related diagnostic reagents development which are targeting CD24 molecular.