人MxA基因的原核表达及其抗血清的制备

摘要

本试验旨在利用大肠杆菌BL21(DE3)表达MxA融合蛋白pET32a(+)-MxA,制备兔抗人MxA抗血清.采用PCR技术获得MxA基因,然后将MxA基因重组到pET32a(+)载体中,筛选阳性克隆,转化大肠杆菌,IPTG诱导表达,用超声波裂解重组菌BL21 (DE3)培养物,纯化蛋白质后免疫新西兰兔制备抗血清.SDS-PAGE分析结果显示,pET32a(+)-MxA融合蛋白在大肠杆菌BL21(DE3)菌株中以包涵体形式高效表达；获得的抗血清经间接ELISA法检测效价为1∶6400；Western blotting检测结果显示,抗血清可与真核表达的MxA蛋白进行特异性结合.试验结果表明,在大肠杆菌中成功表达了pET32a(+)-MxA融合蛋白,制备了具有高度特异性的兔抗人MxA抗血清,这样为采用酶标法测定病毒患者全血细胞或由干扰素诱导的细胞产生的MxA蛋白质含量及临床诊断试剂盒的开发和应用打下了基础.%This assay was aimed to express the fusion protein of the MxA in Escherichia coli and prepare the antisera of rabbit against MxA. The recombinant expression vector pET32a( + )-MxA was contrusted by combinating MxA fragment from PCR and prokaryotic expression vector pET32a( + ), recombinant protein was induced to express and be purified. We immuned New Zealand rabbits with purified recombinant protein to prepare MxA antisera. Then, ELISA results showed that antibody ti-ter was 1: 64000,results of Western blotting showed that antisera had well combination with MxA. Results indicated that we had succesfully prepared immunogenic MxA protein and antisera which laid a foundation for further study on the methods of clinical diagnosis.