Site-direct Mutation And Eukaryotic Expression Of The Human Growth Hormone Receptor

Objects: In earlier stage, Dr.chen screened and identified two novel mutations from 47 children with non-growth hormone deficiency and marked short stature, including a girl with Laron Syndrome and 46 children with idiopathic short stature. According to these primary results, here, we performed some works related to functional research of mutation. We not only completed site-directed mutation of hGHR, but also constructed two stable cell lines that expressed the wild-type hGHR and hGHR-muta. These works are the basis of the further functional analysis of these two mutant hGHR, including the binding affinity of GHR-muta toGH ,JAK-STAT signal transduction pathway.Methods: The PUC119 vector containing the wild-type GHR complementary DNA(cDNA; PUC-GHR)was kindly provided by Dr.W.I.Wood. Our studies was as follows:1.Firstly,using QuickChange site-directed mutagenesis kit, we got two mutant plasmids (hGHR-E42K,hGHR-H56R).Then, with restriction enzymes BamH I and Pst I, Two mutant plasmids( PUC-hGHR-E42K,PUC- hGHR-H56R) were digested and inserted into the pcDNA3.1(zeo) vector(pcDNA3.1-hGHR-wt,pcDNA3.1-hGHR-E42K, pcDNA3.1-hGHR-H56R) . 3.CHO cells were successfully transfected with wild-type and mutant GHR expression vectors (pcDNA3.1-hGHR-wt,pcDNA3.1-hGHR-E42K) using Lipofectamine 2000, stable expression cell lines were screened by medium which contains a measured amount of antibiotic. We tested expression product by RT-PCR and Western-Blot.Results: 1.Two mutant plasmids (PUC-hGHR-E42K. PUC-hGHR-H56R)and three eukaryotic expression vectors(pcDNA3.1-hGHR-wt,pcDNA3.1-hGHR-E42K pcDNA3.1-hGHR-H56R) were confirmed by sequencing analysis.2.The two cell lines which stably expressed hGHR and hGHR-E42K were successfully selected. This result was identified by RT-PCR and total proteins Western-Blot.Conclusion: In this study, we obtained two mutant plasmids and two stable expression cell lines. The effect of the mutant hGHR on JAK-STAT signal transduction pathway and its binding affinity to GH should be further studied.