摘要
[目的]探索棉铃虫核型多角体病毒G4株Ha99蛋白的最佳表达条件。[方法]采用PCR从其基因组中扩增出orf99编码区,将其克隆到pGEM-Teasy载体,测序后将其亚克隆到原核表达载体pGEX-KG,转化大肠杆菌BL21加入IPTG诱导融合表达。并在orf99缺失杆粒HaBac△99的基础上构建了orf99敲除子HaBac-KO99及回复子HaBac-Rep99。[结果]SDS-PAGE分析表明,GST-Ha99融合蛋白得到成功表达,Westernblot进一步分析表明融合蛋白已得到成功表达。此外,PCR及酶切验证各载体构建成功。[结论]为orf99功能的进一步研究奠定基础。
[Objective]The aim was to research the best expression condition of Ha99 of helicoverpa armigera single nucleocapsid nucleopoly-hedrovirus.[Method]The orf99＇s coding region was amplified from genome by PCR,and it was cloned into the pGEM-T Easy vector,then it was subcloned into the prokaryotic expression vector-pGEX-KG after sequencing.The custom-crafted vector was transformed into the E.coli BL21 cells and induced to fusion-express by IPTG,HaBac-KO99 and HaBac-Rep99 were constructed based on the orf99 deletion rod granule-HaBac△99.[Result]SDS-PAGE pattern indicated that the fusion protein GST-Ha99 was expressed successfully.Western blot analysis proved that the result was right.In addition,each vector was proved right by PCR and restriction endonuclease digestion.[Conclusion] The study established the foundation for orf99＇s further function researching.
出处
《安徽农业科学》
CAS
北大核心
2010年第20期10543-10546,10574,共5页
Journal of Anhui Agricultural Sciences
