衔接蛋白p66^(Shc) CH2结构域分泌表达载体的构建及其表达

Construction of the lentiviral vector for the expression of adaptor protein p66^(Shc) CH2 domain

目的:构建可以产生分泌p66ShcCH2结构域重组蛋白的质粒,期望进一步研究p66Shc的功能。方法:利用定点诱变的方法在pDisplay中去除HA和Myc表达序列,但保留小鼠Igκ信号肽和PDGFR跨膜结构域。在小鼠Igκ信号肽后是外源CH2结构域、凝血因子Xa识别位点和Flag序列,与PDGFR跨膜结构域形成融合基因。PCR扩增得到完整表达片段后用于慢病毒载体上的克隆,之后在包装细胞Fx293中产生重组慢病毒用于后续实验。结果:经测序证实所构建的质粒序列与预期序列一致,酶切和PCR验证质粒构建正确,体外感染细胞后产生重组蛋白。结论:成功构建体外可以产生p66ShcCH2结构域的慢病毒载体,为体外产生重组分泌的CH2多肽和体外鉴定与p66ShcCH2结构域结合的蛋白分子实验奠定基础。

Objective： To construct the plasmid which expresses the secretory p66^ShcCH2 domain peptide in vitro. Methods： Site direct- ed mutagenesis was used to delete HA and Myc sequence on pDisplay plasmid which still contains the mouse Ig kappa-chain signal pep- tide directing the protein to the secretory pathway and the platelet derived growth factor receptor （PDGFR） transmembrane domain which anchors the protein on the extracellular side. Then the p66^ShcCH2 domain was fused at the N-terminus to the murine Ig K-chain signal se- quence followed by the factor Xa cleavage site and Flag sequence and at the C-terminus to PDGFR transmembrane domain. The fusion fragment was subcloned into the lentiviral vector to produce recombinant virus particles by packaging Fx293 cells. Results：The p66^ShcCH2 domain recombinant viral vector was created for its secretory expression in vitro. Conclusion：The construction of the secretory viral vector for overexpression of p66^ShcCH2 domain paves the road to its functional investigation such as the binding partners identification.