RESUMO
Objective To eonstruct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity, then check their expression in CHO cells. Methods With the available PUC-hGHR vector, two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into eukaryotic expression vectors pcDNA3.1/zeo(+) with restriction enzymatic reactions.Then with Lipofectamine2000, we trans-fected expression vectors to CHO cells and screened the stably expressed CHO cells by Zeocin. RT-PCR and/or Western blotting were used to examine hGHR and STAT5-P. Results After sequencing, two mutations were introduced to hGHR, three eukaryotic expression vectors were identified. The transfected CHO cells expressed vd-hGHR or its mutants. Compared with hGH-wt, two mutate cells (E42K and H56R) had decreased phosphorylated STAT5 levels. Conclusion Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established, E42K and H56R partly interfered the phosphorylation of STAT5.
RESUMO
Objective To construct three eukaryotic expression vectors containing wilde-type hGHR gene and its mutants(hGHR-E42K and hGHR-H56R) related to congenital growth hormone insensitivity,then check their expression in CHO cells. Methods With the available PUC-hGHR vector,two mutate hGHR genes (hGHR-E42K and hGHR-H56R) were obtained through mutagenesis. Then three recombinants were cloned into 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 examine hGHR and STAT5-P.Results After sequencing,two mutations were introduced to hGHR,three eukaryotic expression vectors were identified. The transfected CHO cells expressed wt-hGHR or its mutants. Compared with hGH-wt,two mutate cells (E42K and H56R) had decreased phosphorylated STAT5levels. Conclusion Three CHO cells which stably expresses wide type hGHR and its mutants were successfully established,E42K and H56R partly interfered the phosphorylation of STAT5.