1型鸭甲型肝炎病毒3D基因的原核表达与多抗制备

根据血清1型鸭甲型肝炎病毒(DHAV-1)SH株3D基因序列设计并合成1对特异性引物,通过RT-PCR方法扩增3D基因,将其克隆入原核表达载体p ET-32a,转化大肠杆菌BL21(DE3),在IPTG的诱导培养下重组蛋白获得了成功表达。SDS-PAGE显示重组蛋白的分子量约为68 k D,Western blot分析显示该重组蛋白能与多聚组氨酸标签单抗发生特异性反应。将切胶后纯化的目的蛋白免疫ICR小鼠,制备针对重组蛋白的多抗血清,Western blot结果显示制备的抗血清能够与DHAV-1感染的SPF鸡胚尿囊液总蛋白发生特异反应。以上结果表明,3D基因在大肠杆菌中获得了成功表达,且制备的多抗血清可以用于3D蛋白的检测,为3D蛋白的功能研究奠定了基础。

Prokaryotic expression and antiserum preparation of the 3D gene of Duck hepatitis A virus type-1

According to the genome sequences of duck hepatitis A virus type-1(DHAV-1), one pair of specific primers was designed to amplify the 3D gene using RT-PCR. The amplified fragment was cloned into prokaryotic expression vector pET-32a and the recombinant plasmid was transformed into BL21(DE3) E.coli. Recombinant proteins were successfully expressed following IPTG induction. SDS-PAGE showed that the recombinant expression protein was about 68 kD. Western blot assay revealed that the desired proteins could be recognized by the monoclonal antibody against histidine-tagged proteins. The antiserum against the recombinant protein was produced by the immunized ICR mouse with recombinant proteins. Western blot results showed that the antiserum could react specifically with DHAV-1 allantoic fluid. These results showed that the 3D gene was successfully expressed in E.coli and the antiserum could be used for detection of the 3D protein, which would lay a foundation for function study of the 3D protein.