人牙本质磷蛋白功能片段的真核表达

目的本研究旨在利用分子生物学技术真核表达包含功能区的人DPP片段。方法构建了人DPP蛋白5’端基因片段的杆状病毒表达质粒pFastBacHb—hDPP—N，经BH10BAC转座反应后感染昆虫细胞sf9，进行重组蛋白的表达。结果经2～3代后，重组病毒产生明显的CPE，人DSPP蛋白特异性多克隆抗体Western blot鉴定，结果显示重组病毒感染3代毒sf9细胞的裂解物中在约17KD位置上出现了特异性的反应条带。结论表明杆状病毒系统可表达人DPP基因片段。为进一步通过对人DPP功能片段的研究揭示DPP在牙齿发育和牙髓修复中的作用及其机制奠定基础。

Eukaryotic expression of human dentin phosphoprotein domain

Objective In this study, the human Dentin Phosphoprotein(DPP) domain containing DSS was cloned and expressed by using genetic engineering in eukaryotic cells with Bac-to-Bac bacular expression system.Methods The 504-bp fragment of human DPP gene was cloned into the the donor plasmid pFastBacHb. After transposition with BH10BAC, recombinant Bacmid DNAs were isolated and transfected in Sf9 cells.Results The typical cytopathologic effect caused by the recombinant baculovirus became obvious, after two or three passages. The expression of DPP-derived protein was screened by Western blot assays with a polyclonal antibody specific for human DSPP. It revealed a positive band mobilizing around 17 KD in the lysate of SF9 cells.Conclusion It suggests that the N-terminus DPP peptide could be expressed in the baculovirus expression system. It will lay a theoretical foundation for revealing the mechanism of DPP on tooth development and the effect on pulp restoration.