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;West-ern 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 titer 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.
出处
《中国畜牧兽医》
CAS
北大核心
2013年第4期18-22,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家现代农业(奶牛)产业技术体系建设专项经费(何洪彬)
国家自然科学基金(31272586)
国家转基因重大专项(2009ZX08007-006B
2011ZX08007-002
2011ZX08008-004)
济南市高校院所主创新计划(201004027)
山东省青年自然科学基金项目(ZR2010ZR029)
山东省自然科学基金(ZR2010CM012)
关键词
MXA
原核表达
抗血清
MxA gene
prokaryotic expression
antisera
