annexinA2基因片段的克隆、表达、纯化及多克隆抗体的制备

Prokaryotic expression and purification of the human annexinA2 protein and preparation of its polyclonal antibody

目的:构建人源annexinA2基因片段的原核表达载体,表达并纯化蛋白,制备多克隆抗体。方法:从GenBank(BC093056.1)中获得人源annexinA2基因的cDNA序列并根据不含信号肽的编码序列设计引物,用PCR的方法扩增编码an-nexinA2基因部分序列,将其克隆到原核表达载体pET28a(+)中表达annexinA2的蛋白。表达的蛋白产物利用切胶回收的方法纯化,用His抗体进行Western blot鉴定。用纯化的目的蛋白作为抗原免疫日本大耳白兔,获得annexinA2的抗血清,通过ELISA测定兔多克隆抗体的滴度。在CaSki细胞内采用细胞免疫荧光(CIF)和流式细胞术(FCM)检测内源annexinA2蛋白的表达,用于鉴定制备的多克隆抗体特异性。结果:通过双酶切及测序证实原核表达质粒pET28a(+)-annexinA2构建成功,可在大肠杆菌E.coli BL21(DE3)中诱导高表达,得到相对分子质量约28 000的annexinA2蛋白。纯化的蛋白免疫日本大白兔后,产生特异的高滴度的抗体(ELISA滴度达1∶640 000)。CIF和FCM结果显示抗血清与肿瘤细胞表达的annexinA2有高度特异的结合活性。结论:构建了annexinA2的原核表达质粒并获得高纯度的蛋白,免疫动物后,获得了特异、高效价的多克隆抗体,为人源annexinA2蛋白的生物学功能及肿瘤治疗的研究提供了实验基础。

Objective：To prepare annexinA2 polyclonal antibody,human annexinA2 prokaryotie expression vector was construc- ted, its protein was expressed and purified. Methods ：The primers were designed according to eDNA sequence of human annexinA2 gene without signal peptide sequence which was obtained from GenBank （ BC093056.1 ）. The part sequence of annexinA2 coding region was amplified by PCR, and then cloned into pET28a（ ＋ ） prokaryotic expressing vector. The expressed protein was purified by recycling pro- tein from cut gel, and then it was identified with SDS-PAGE and Western blot. The purified protein was used to immunize rabbits from Japan for 3 times to prepare polyelonal antibody. Rabbit anti-sernm titer was assayed by ELISA, and its specificity was detected by cell immunofluoreseence assay（CIF） and Flow Cytometry （FCM） in CaSki cells. Results：Confirmed by restriction enzyme digestion and se- quencing ,pET28a（ ＋ ） prokaryotie expressing vector was successfully constructed, and annexinA2 protein could be expressed in E. coli BI21 （ DE3 ） with high efficiency, abtained molecular weight protein of about 28 kD. Rabbit from Japan immunized with the purified protein produced high specificity and high titer of the antibody（ ELISA titer of 1 ： 640 000）. Immunofluoreseenee assay and Flow Cytometry displayed that the annexinA2 antibody was specifically combined with the protein highly expressed in CaSki cells. Conclusion：Human annexinA2 prokaryotie expression plasmid was constructed and the high purified protein was obtained successfully. After animals were immunized,the high specificity and high titer of the polyelonal antibody was obtained. The preparation of human annexinA2 polyelonal antibody laid the solid foundation for future research on annexinA2 protein function and its application for tumor treatment .