血小板生成素双体分子的融合构建、原核表达及其分子结构特性预测

FUSION CONSTRUCTION, PROKARYOTICAL EXPRESSION AND STRUCTURE CHARACTERISTICS PREDICTION OF BIMOLECULAR THROMBOPOIETIN

目的为了研制更稳定、高效、低毒的血小板生成素(TPO),拟设计构建TPO的双体分子(TT)并在原核中表达,同时预测其融合蛋白的分子结构特性。方法采用分子克隆方法分别构建TPO单体分子与双体分子的原核表达载体pET32/TPO和pET32/TT,并在大肠杆菌中进行表达,以Westernblot鉴定表达产物;用生物信息学方法DSGene1.1和ProtScale软件,对该融合蛋白的结构特征如电点、柔性、抗原性及亲水性等进行模拟分析。结果成功构建TPO双体分子pET32/TT的原核表达载体,并经NcoI和NotI双酶切电泳及测序证实。通过转化origamiTM(DE3)感受态菌及IPTG诱导获得高效表达,其产量在40%以上;Westernblot显示表达产物能与抗TPO单克隆抗体特异性结合。对TT双体分子融合蛋白的结构特性预测表明,其融合蛋白中的两个TPO单体分子等电点、抗原性及亲水性均无显著改变,接头处具有高柔性。与TPO的原始序列相比,TT的一级结构中有2个氨基酸发生改变,在接头(L)后插入一段34个氨基酸的新序列(N)。此序列具有一定的抗原性和亲水性,呈现β片层结构。结论本研究所构建表达的TPO单体和TT双体分子均可在大肠杆菌中获高效表达,TT双体分子的结构预测符合设计要求,为进一步研究新型TT双体分子融合蛋白的生物学特性提供了基础。

Aim: Design, construction, expression in E coli and protein characteristics prediction of bimolecular thrombopoietin(T T) with more stability, efficiency, and lower toxicity. Methods: The expression vectors of TPO and T T, pET32 a(+)/TPO and pET32 a(+)/T T, had been constructed by molecular cloning methods. Then, they were expressed in host bacterium. Their products were identified by Western blot. The protein characteristics, such as second structure, antigenicity, hydrophilicity, flexibility and isoelectric point, were predicted by DS Gene and Protscale software. Results: The expressing vectors pET32a(+)/ TPO and T T were constituted correctly and expressed in origami TM (DE3), and their expression efficiency were more than 40 percent of total protein. T T was identified correctly by Western blot. DS Gene and Protscale software predict the protein characteristics of TPO sequences in T T molecule were no change, there was high flexibility in the linker domain. But two amino acids in T T molecule have been mutated, and an insert fragment with 34 amino acids following the linker had antigenicity, hydrophilicity, and β sheet structure. Conclusion:We have constructed correctly and expressed T T with high level in E Coli. Protein characteristics prediction of T T accords with our design.