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目的:使卵巢癌相关基因HE4在杆状病毒表达系统中获得表达。方法:将卵巢癌相关基因HE4克隆到pMagic-his-HE4中,通过转化E.coliDH10B筛选克隆,阳性克隆通过接合转移的方式与杆状病毒框架质粒体外重组,构建Bac-phes-HE4杆状病毒表达载体,后者经Lipofectamine2000介导转染Sf9细胞,获取重组病毒,扩增病毒并感染Sf9细胞进行表达,SDS-PAGE、Western blot分析鉴定表达蛋白。结果:经HE4抗体、6×His单抗鉴定,成功通过杆状病毒表达系统获得重组卵巢癌相关基因HE4。结论:卵巢癌相关基因HE4通过接合转移的方法在杆状病毒表达系统中成功表达。
Objective: To make ovarian cancer related gene HE4 express in baculovirus expression system. Methods: The ovarian cancer-associated gene HE4 was cloned into pMagic-his-HE4. The clones were screened by transformation with E.coli DH10B. The positive clones were recombined with the baculovirus framework plasmids in vitro by conjugation and transfer to construct Bac-phes-HE4 baculovirus The latter was transfected into Sf9 cells by Lipofectamine 2000, and the recombinant virus was obtained. The virus was amplified and infected with Sf9 cells for expression. The expressed proteins were identified by SDS-PAGE and Western blot. Results: The HE4 was identified by HE4 antibody and 6 × His monoclonal antibody, and the recombinant ovarian cancer related gene HE4 was successfully obtained by baculovirus expression system. Conclusion: Ovarian cancer related gene HE4 was successfully expressed in baculovirus expression system by conjugation and transfer.