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目的构建以大鼠WAP启动子调控的人血小板生成素(TPO)真核表达质粒并进行瞬时表达,探讨TPO基因组中不同内含子对TPO表达影响。方法通过基因工程方法构建以大鼠WAP启动子调控的6种TPO真核表达质粒,并通过酶切和测序进行鉴定,将上述重组质粒分别在HC-11和COS-1细胞上转染48 h后,通过双抗体夹心ELISA定量分析转染上清中TPO表达。结果酶切及测序分析表明构建与设计相符,6种重组质粒在COS-1细胞和HC-11细胞中均获得表达,其表达水平从高到低依次为pTPOWE>pTPOWA>pTPOWD>pTPOWB>pTPOWC>pTPOWF,其中含有TPO IntronⅤ的重组质粒pTPOWE表达量最高,而含有TPO全基因组的重组质粒pTPOWF表达量最低。结论 IntronⅤ可能含有特殊增强TPO表达序列,而TPO基因组中可能存在抑制TPO高水平表达结构元件。
Objective To construct eukaryotic expression plasmids of human thrombopoietin (TPO) regulated by WAP promoter and to investigate the effect of different introns on TPO expression in TPO genome. Methods Six TPO eukaryotic expression plasmids regulated by WAP promoter were constructed by genetic engineering and identified by restriction enzyme digestion and sequencing. The recombinant plasmids were transfected into HC-11 and COS-1 cells for 48 h The TPO expression in the transfected supernatant was quantified by double antibody sandwich ELISA. Results The results of enzyme digestion and sequencing showed that the constructs were consistent with the designed ones. The six recombinant plasmids were expressed in COS-1 cells and HC-11 cells. The expression levels of the six recombinant plasmids were pTPOWE> pTPOWA> pTPOWD> pTPOWB> pTPOWC> pTPOWF, in which the expression plasmid pTPOWE containing the TPO IntronⅤ was the highest, while the pTPOWF containing the TPO genome was the lowest. Conclusion IntronⅤ may contain specially enhanced TPO expression sequences, while TPO may inhibit the expression of TPO at high levels.