棉铃虫核型多角体病毒orf86基因的原核表达、抗体制备与免疫印迹检测

Prokaryotic Expression, Preparation of Polyclonal Antibody and Immunodetection of HearNPV orf86

摘要:

棉铃虫核型多角体病毒(Helicoverpa armigera nucleopolyhedrovirus, HearNPV) orf86是一个功能未知的基因。从HearNPV基因组中通过PCR得到orf86基因编码序列, 将其构建于原核表达载体pGEX-4T-2, 转化大肠杆菌BL21获得融合表达产物。融合表达蛋白经分离纯化后免疫新西兰大白兔, 制备其多克隆抗体。用ELISA测定结果表明, ORF86多克隆抗体效价为1 : 5.12 × 105。利用该抗体Western印迹检测HearNPV感染的HZAM1细胞, 检测到一个36 kD的目的蛋白条带, 与ORF86预期大小一致。结果表明该多抗可用于对orf86编码蛋白的检测和相关功能研究。

Abstract:

HearNPV orf86 is a function unknown gene of Helicoverpa armigera nucleopolyhedrovirus (HearNPV). The coding region of orf86 was amplified from the HearNPV genome by PCR and then was inserted to the prokaryotic expression vector pGEX-4T-2. The recombinant plasmid was transformed into E. coli BL21 to express the GST fusion protein in the bacteria. The purified fusion protein was injected into New Zealand rabbits to prepare polyclonal antibody. The titer of the anti-HearNPV ORF86 polyclonal antibodies reached to 1 : 5.12 × 105 determined by ELISA. A 36 kD protein was identified by Western blot on HZAM1 cells infected by HearNPV using the prepared polyclonal antibody, which the protein size was consistent with the predicted protein size encoded by orf86. The results indicated that the polyclonal antibodies against ORF86 was successful prepared and it would facilitate the detection and related study on ORF86.