鸭肝炎病毒鸡胚化弱毒MY株VP1基因克隆、原核表达及抗原性分析

Cloning and Prokaryotic Expression of Chicken-embryo-adapted Attenuated Vaccine Strain MY of Duck Hepatitis Virus and Antigenicity of the Expressed Protein

应用巢氏PCR(nested-PCR)扩增出1型鸭肝炎病毒鸡胚化弱毒MY株结构蛋白VP1基因片段,将VP1克隆到pMD18-T载体上,测序结果为714 bp,GenBank登录号:GU363950。对MY株VP1基因编码蛋白的主要抗原位点进行预测,aa208-aa222氨基酸区段表现很高的亲水性、抗原指数和表面可及性。VP1经EcoRⅠ和XhoⅠ双酶切后克隆至pET32a(+)原核表达载体,获得重组质粒pET-VP1,转入BL21 PLyss(DE3)细胞中,IPTG诱导后SDS-PAGE检测结果表明,在大肠杆菌中表达了1个相对分子质量为47 ku的融合蛋白。Western blotting分析结果表明,该重组蛋白可与鸭肝炎标准阳性血清发生特异性反应,具有良好的反应原性。

The gene of structural protein VP1 was cloned from chicken-embryo-adapted attenuated vaccine strain MY of duck hepatitis type 1 virus by nested-PCR.Then,the VP1 gene was subcloned into the vector pMD18-T,and the gene fragment was 714 bp.In GenBank submitted for GU363950.The main antigen sites of VP1 gene from strain MY was predicted,and the segment containing 208-222 amino acid exhibited high hydrophilicity,antigenic index and surface probability.After being double digested by EcoR Ⅰand Xho Ⅰ,VP1 gene was subcloned into prokaryotic expression vector pET32a（＋） to construct expression plasmid pET-VP1.The pET-VP1 was transformed into BL21 PLyss（DE3） and induced by IPTG.SDS-PAGE analyses showed that a fusion protein with relative molecular mass of 47 ku was expressed in Escherichia coli.Western blotting analyses showed that specific immune response could occur with this protein and duck hepatitis virus positive serum,and this protein had good reactionogenicity.