Objective: To construct the secretory vector which expresses the transcriptional regulator p8. Methods: Site directed muta- genesis was used to delete HA and Myc sequence on pDisplay plasmid which still contains the mouse Ig kappa-chain signal peptide 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 p8 was fused at the N-terminus to the murine Ig K-chain signal sequence followed by the factor Xa cleavage site and Flag sequence and at the C-terminus to PDGFR transmembrane domain. The fusion fragment was sub-cloned into the lentiviral vector to produce recombinant virus particles by packaging Fx293 cells. Results:The p8 recombinant viral vector was created for its secretory expression in vitro. Conclusion: The construction of the secretory viral vector for overexpression of p8 paves the road to its functional investigation such as the binding partners identification.%目的:构建可以产生分泌型转录调节因子p8重组蛋白的载体,期望进一步研究p8的功能.方法:利用定点诱变的方法在pDisplay表达载体中去除HA和Myc标记序列,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p8、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,体外转染Fx293细胞后产生重组蛋白.结论:成功构建体外可以产生p8的慢病毒载体,为体外产生重组分泌的p8多肽和体外鉴定与p8结合的蛋白分子实验奠定基础.
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