关键词: 生长激素受体基因, 稳定表达, CHO细胞, 突变体

Abstract: Objective To construct three eukaryotic expression vectors containing wide-type hGHR gene and its mutants (hGHR-E42K、hGHR-H56R) related to Congenital Growth Hormone Insensitivity, then make sure these vectors are expressed stably in CHO cells. After hGH stimulation, we measured the expression of phosphorylated STAT5 in those cells. Methods With the available PUC-hGHR vector, other two mutate hGHR genes (hGHR-E42K,hGHR-H56R) were achieved by quick site-directed mutagenesis. Then three recombinants were cloned to eukaryotic expression vectors, pcDNA3.1/zeo(+) with restriction enzymatic reactions. Then with Lipofectamine2000, we transfected expression vectors to CHO cells and screened the stably expressed CHO cells by Zeocin. RT-PCR and/or Western Blotting were used to testify levels of hGHR and STAT5-P. Results After sequencing, two mutations were introduced to hGHR smoothly, three eukaryotic expression vectors identified. The transfected CHO cells expressed wt-hGHR or its mutants. Compared with hGH-wt, two mutate cells (E42K, H56R) had decreased phosphorylated STAT5 levels. Conclusion：Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established, E42K, H56R partly interfered the phosphorylation of STAT5.

Key words: Human Growth Hormone Receptor Gene, stable Expression, CHO cells, Mutant