基于沙堡蠕虫和贻贝黏蛋白的融合基因构建及其在昆虫细胞中的表达

Construction of a fusion gene based on Phragmatopoma californica and mussel adhesive proteins and its expression in insect cells

目的构建基于贻贝足蛋白(Mfp)和沙堡蠕虫分泌蛋白(Pcs)的融合基因,并实现其在昆虫细胞中的表达,为新型黏附材料研制奠定基础。方法理性设计基于Mfp和Pcs的融合分子,通过PCR扩增得到目的基因,再通过pFastBacHBM载体所介导的平末端克隆以及DH10Bac所介导的转座合成带有目的基因的重组杆状病毒质粒(Bacmid DNA),利用昆虫表达系统生成携带目的基因的杆状病毒,并通过病毒感染细胞实现融合蛋白Pc1⁃Mfp5的可溶性表达。结果测序结果显示,重组载体构建成功。病毒滴度测试显示,收获高滴度的杆状病毒。SDS⁃PAGE以及Western印迹分析表明,Pc1⁃Mfp5融合蛋白在昆虫细胞中实现了可溶性表达。结论成功构建pc1⁃mfp5融合基因,并实现了其在昆虫系统中的可溶性表达,为新型仿生黏附材料的研制奠定了基础。

Objective To construct a fusion protein and realize its expression in insect cells so as to contribute to the development of new adhesive materials.Method Based on mussel foot proteins(Mfps)and cement precursor proteins of the Phragmatopoma californica(Pcs),the sequence of the fusion protein was designed,according to which the target gene was obtained through PCR amplification.The recombinant Bacmid DNA with the target gene was synthesized via pFastBacHBM vector mediated flat terminal cloning and DH10Bac mediated transposition.The baculovirus carrying the target gene was through the insect expression system.Then,the baculovirus was infected to the insect cells to obtain the soluble expression of fusion protein Pc1⁃Mfp5.Results The sequencing results indicated that the recombinant vector was constructed.The virus titer test showed that a high⁃titer baculovirus stock was obtained.SDS⁃PAGE and Western blotting analysis indicated that the Pc1⁃Mfp5 fusion protein was solublely expressed in insect cells.Conclusion The fusion gene of pc1⁃mfp5 has been constructed,and the soluble expression of Pc1⁃Mfp5 fusion protein in insect systems has been made available,which can provide data for the development of new bionic adhesive materials.