【摘要】 目的:在大肠杆菌BL21（DE3）中重组表达寨卡病毒（ZIKV）囊膜（E）蛋白的200个氨基酸（138～338）截短片段ZIKV-E²⁰⁰,制备其多克隆抗体,为寨卡病毒亚单位疫苗后续研究提供检测抗体。方法:用全基因序列合成方法合成寨卡病毒E蛋白全长基因,以该基因为模板,用PCR方法克隆ZIKV-E²⁰⁰基因片段,经EcoRⅠ/XhoⅠ双酶切后连接到pET22b载体并转化大肠杆菌DH5α感受态细胞,获得重组质粒pET22b-ZIKV-E²⁰⁰,将其转化大肠杆菌BL21（DE3）感受态细胞得到重组表达菌株pET22b-ZIKV-E²⁰⁰。将37℃诱导表达后的菌液超声波破碎处理后制备ZIKV-E²⁰⁰蛋白,将制备的抗原ZIKV-E²⁰⁰免疫BALB/c小鼠,经3次免疫后,采集血清制备其相应的多克隆抗体,采用Western印迹检测多克隆抗体的特异性。结果:获得ZIKV-E²⁰⁰基因片段,并构建了其相应的原核表达载体pET22b-ZIKV-E²⁰⁰,在大肠杆菌中表达了重组ZIKV-E²⁰⁰蛋白,其相对分子质量约为25 000,与理论值一致;用分离的蛋白ZIKV-E²⁰⁰免疫小鼠后获得了抗ZIKV-E蛋白的多克隆抗体,该多克隆抗体检测到了酵母表达的寨卡病毒E蛋白。结论:ZIKV-E²⁰⁰蛋白的多克隆抗体可用于酵母表达的寨卡病毒E蛋白的检测等研究。更多还原

【Abstract】 Objective: To express a 200 amino acid fragment of Zika virus（ZIKV） envelope（E） protein in Escherichia coli BL21（DE3） and to produce its polyclonal antibodies used as the detection antibody for subunit vaccine for ZIKV E protein. Methods: The ZIKV-E²⁰⁰ gene was amplified by PCR from the template of full ZIKV E gene, and was cloned into prokaryotic expression vector pET22b. The recombinant plasmid pET22b-ZIKV-E²⁰⁰ was transformed into E.coli BL21（DE3）. The female mice were immunized with the obtained ZIKV-E²⁰⁰ protein to prepare the polyclonal antibodies against ZIKV E protein. Its specificity was detected by the method of Western blot.Results: The ZIKV-E²⁰⁰ gene has been cloned and expressed in E.coli BL21（DE3）. According to SDS-PAGE, the mass of the recombinant protein was consistent with the theoretical value（25 000）. The polyclonal antibodies obtained from the immunized mice, detected the recombinant expression protein ZIKV E by yeast in Western blot.Conclusion: The polyclonal antibodies can be used in the detection of the ZIKV E recombinant protein expressed by yeast.更多还原