I型鸭肝炎病毒VP1、3D基因克隆及其在大肠杆菌中的表达

Cloning of VP1 and 3D Gene of Duck Hepatitis Virus 1 (DHV1) and Its Expression in Escherichia coli

摘要:

根据GenBank中的I型鸭肝炎病毒全基因序列设计了扩增I型鸭肝炎病毒VP1、3D基因的引物, 用该特异性表达引物从I型鸭肝炎病毒cDNA模板中扩增得到目的基因VP1、3D, 用相同的限制性内切酶酶切目的基因和表达载体pET32a后构建重组表达载体, 转化宿主BL21(DE3), 用不同浓度的IPTG诱导VP1、3D基因的表达, 收集菌液进行SDS-PAGE电泳, Western-blotting分析蛋白免疫原性。结果表明, VP1、3D在大肠杆菌中表达量较高, 表达产物的分子量约为48 kD、68 kD, 并能被兔抗DHV-1血清所识别。I型鸭肝炎病毒VP1、3D蛋白在大肠杆菌中表达产物具有免疫原性。

Abstract:

In this study, two special primer pair containing EcoR V and Xho I according to complete genome of duck hepatitis virus 1 (DHV1) were designed to amplify VP1 and 3D genes from cDNA of DHV1. The target genes VP1 and 3D were subcloned into PET32a vector digested by EcoR V and Xho I respectively. Then the recombinant plasmids were transfected into Escherichia coli BL21(DE3) for VP1and 3D expression. The bacteria containing PET32a-VP1 and PET32a-3D were collected and examined by SDS-PAGE and western-blotting. Result showed that the VP1 and 3Dprotein were expressed in E. coli and the amount of expression was higher. Molecular weight of the protein was 48 kD, 68 kD. The protein can be recognized by DHV1antibody. This study showed that the protein VP1 and 3D have antigenicity.