1型鸭甲型肝炎病毒3D基因的原核表达与多抗制备剖析.PDF

安徽农业大学学报, 2015, 42(4): 536-539 Journal of Anhui Agricultural University [DOI] 10.13610/ki.1672-352x006 网络出版时间：2015-6-26 9:12:33 [URL] /kcms/detail/34.1162.S0912.006.html 1 型鸭甲型肝炎病毒 3D 基因的原核表达与多抗制备 * 王安平，朱善元 ，吴 双 (江苏农牧科技职业学院，江苏省兽用生物制药高技术研究重点实验室，泰州 225300) 摘 要：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pET-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显示重组蛋白的分子量约为68 kD ，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。 关键词：鸭甲型肝炎病毒；3D 基因；原核表达；抗血清 中图分类号：S855.3 文献标识码：A 文章编号：1672−352X (2015)04−0536−04 Prokaryotic expression and antiserum preparation of the 3D gene of Duck hepatitis A virus type-1 WANG Anping, ZHU Shanyuan, WU Shuang (Veterinary Bio-pharmaceutical, Jiangsu Province Key Laboratory of High-tech Research, Jiangsu Agri-animal Husbandry Vocational College, Taizhou 225300) Abstract: According to the genome sequences of duck hepatitis A virus type-1(DHAV-1), one pair of spe- cific 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. SDSshowed that the re- combinant 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 recombi- nant protein was produced