人体c-Mpl分子配体不同表达载体的构建及其优化表达策略

Different Construction for Human c-Mpl Ligand Expressed Plasmid and Their Opt imizing Strategy for Expressation in Escherichia coli

利用酵母双杂交系统从人体肝脏cDNA文库筛选到血小板生成素受体Mpl分子配体的xp1基因,构建pET＿28(b)＿xp1,pBV220＿xp1和pBAD gⅢA＿xp1表达质粒,分别进行IPTG、温度和果糖诱导,表达产物利用小鼠巨核细胞集落形成单位测定活性。表达分析显示BL21 pET＿28(b)＿xp1水溶性XP1目的蛋白表达量为最高,活性最大;BL21 pBV220＿xp1被短时、低温诱导后可增加表达目的蛋白的水溶性,但稳定性差,表达量和活性均不及BL21 pET＿28(b)＿xp1高;TOP10 pBAD gⅢA＿xp1不能分泌到细胞质周膜,以包涵体形式存在细胞内中,并且为无活性表达。

Thrombopoietin(TPO), the major cytokine affect ing the proliferation and differentiation of megakaryocytes through its interaction with the c_Mpl receptors, is believed to be the major physiological regulator of circulating platelet levels.c_Mpl was used as the bait protein to screen human liver cDNA library using a yeast two_hybird system. A clone, \%xp\%1 gene, was obta ined. The constructions of expression plasmid pET_28(b)_\%xp\%1,pBV220_\%xp\%1 an d pBAD/gⅢA_\%xp\%1 were selected by using different promote controlling \%xp\%1 gene ex pression, then by inducing with IPTG, temperature, arabinose. The biological activity of XP1 protein was measured by culturing colony forming units of murine megakaryocyte(CFU_MK). Comparison of these expressions indicates that the solution and the stability of pET_28(b)_\%xp\%1 expression were higher than the others, The sol ution and the stability of pBV220_\%xp\%1 expression were enhanced after induced a l ittle time in the low temperature, but its expression capacity was weaker than that of pET_28(b)_\%xp\%1,and its activity was decreased. The expression proteins of pBAD/gⅢA_\%xp\%1 were not secreted to the periplasmic space, which existed in incl usion body and presented no activity. 