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目的通过昆虫杆状病毒表达系统表达SETD4(SET domain-containing 4)蛋白,并纯化SETD4蛋白,为深入探讨SETD4的功能奠定基础。方法提取小鼠正常肝组织的RNA,通过RT-PCR扩增SETD4基因,并克隆至p Fast Bac-HTB构建重组载体,再转座获得重组杆粒;通过脂质体介导将重组杆粒转染SF9细胞产生重组病毒,扩增病毒感染细胞并获得重组蛋白;利用Ni2+亲和柱来纯化蛋白,并通过Western Blot及考马斯亮蓝染色鉴定SETD4蛋白。结果经双酶切鉴定及测序证实SETD4基因插入了供体质粒;经PCR鉴定证实SETD4基因插入了穿梭载体;经考马斯亮蓝染色证实纯化得到重组蛋白,用His-Tag抗体和SETD4特异性抗体在50 k D处可检测到目的条带。结论成功利用昆虫杆状病毒表达系统够表达了SETD4,并纯化了SETD4。
Objective To express SETD4 (SET domain-containing 4) protein in insect baculovirus expression system and purify SETD4 protein, so as to lay the foundation for the further study on the function of SETD4. Methods RNA was extracted from the normal liver tissue of mice. The SETD4 gene was amplified by RT-PCR and cloned into p Fast Bac-HTB to construct the recombinant vector. The recombinant bacmid was transposed to obtain the recombinant bacmid. Sf9 cells were infected with recombinant virus to amplify virus-infected cells and obtain recombinant protein. The protein was purified by Ni2 + affinity column and SETD4 protein was identified by Western Blot and Coomassie blue staining. Results The SETD4 gene was inserted into the donor plasmid by double enzyme digestion and sequencing. The SETD4 gene was inserted into the shuttle vector by PCR. The recombinant protein was confirmed by Coomassie brilliant blue staining. His-Tag antibody and SETD4-specific antibody The target band can be detected at 50 kD. Conclusion SETD4 was successfully expressed in insect baculovirus expression system and SETD4 was purified.