人生长激素受体的定点突变及其真核表达
发布时间：2019-01-30 14:51
【摘要】： 实验目的：在实验室的前期工作中，通过对按一定入筛条件挑选的47名非生长激素缺失(GH)、显著矮小(包括1例Laron综合征、46例特发性矮小)的儿童进行一系列的突变筛查和定位获得GHR上三个新的突变。本实验根据这三个新的突变位点对GHR进行定点突变，并构建稳定表达野生hGHR及hGHR-muta的CHO细胞系，为后期研究GHR突变体与GH的结合能力及该突变对JAK-STAT信号转导途径的影响等功能性研究做基础。 实验方法：由美国Gene公司Ph．D．Wood惠赠获得含GHR全长cDNA的质粒PUC-GHR。进而开展以下实验：1．使用stratagene公司的quick change试剂盒定点突变PUC-GHR质粒，然后通过限制性内切酶的双酶切及酶切片断连接等分子生物学方法构建真核表达载体pcDNA3-hGHR-wt、pcDNA3-hGHR-E42K、pcDNA3-hGHR-H56R：2．选择pcDNA3-hGHR-E42K及pcDNA3-hGHR-wt质粒用脂质体转染法转染CHO细胞；通过抗生素筛选构建稳定表达野生hGHR和hGHR-muta的细胞系；3．采用细胞总RNA的RT-PCR和细胞总蛋白的Western-Blot方法鉴定hGHR的表达。 结果：1．通过对突变质粒进行测序验证，，成功获得两个PUC-hGHR-E42K及PUC-hGHR-H56R的PUC-GHR突变型质粒以及真核表达载体pcDNA3.1-hGHR-wt、pcDNA3.1-hGHR-E42K、pcDNA3.1-hGHR-H56R。2．Western-Blot和RT-PCR结果证明转染后的细胞有野生hGHR和hGHR-E42K表达，说明稳定表达野生hGHR和hGHR-E42K蛋白的细胞系构建成功。 结论：本研究首次成功获得了hGHR基因上两个新突变位点的真核表达质粒pcDNA3.1-hGHR-E42K、pcDNA3.1-hGHR-H56R并构建了稳定表达hGHR和hGHR-E42K蛋白的CHO细胞系。在今后的工作中我们将就该突变是否影响GHR与GH的结合能了以及JAK-STAT信号转导途径做进一步研究。
[Abstract]:Objective: in the early stage of laboratory work, 47 cases of non-growth hormone deficient (GH), (including 1 case of Laron syndrome) selected according to certain screening conditions were significantly shorter than those in the control group (including 1 case of Laron syndrome). A series of mutations were screened and located in 46 children with idiopathic dwarfism to obtain three new mutations in GHR. According to the three new mutation sites, the GHR was mutated and the CHO cell lines stably expressing wild hGHR and hGHR-muta were constructed. It provides a basis for the functional study of the binding ability of GHR mutants to GH and the effect of the mutation on the signal transduction pathway of JAK-STAT. Methods: the plasmid PUC-GHR. containing GHR full-length cDNA was obtained from Ph.D.Wood of Gene Company in USA. Then the following experiments were carried out: 1. Using the quick change kit of stratagene Company to mutate the PUC-GHR plasmid, the eukaryotic expression vector pcDNA3-hGHR-wt, was constructed by double enzyme digestion and ligation of restriction endonuclease fragments. 2. PcDNA3-hGHR-E42K and pcDNA3-hGHR-wt plasmids were selected by pcDNA3-hGHR-E42K,pcDNA3-hGHR-H56R:2. and transfected into CHO cells by liposome transfection. A stable cell line expressing wild hGHR and hGHR-muta was constructed by antibiotic screening. 3. The expression of hGHR was identified by RT-PCR of total RNA and Western-Blot of total protein. Results: 1. Two PUC-GHR mutant plasmids of PUC-hGHR-E42K and PUC-hGHR-H56R and eukaryotic expression vector pcDNA3.1-hGHR-wt,pcDNA3.1-hGHR-E42K, were successfully obtained by sequencing the mutant plasmids. 2. The results of pcDNA3.1-hGHR-H56R.2.Western-Blot and RT-PCR showed that the transfected cells expressed wild hGHR and hGHR-E42K, indicating that the cell lines stably expressing wild hGHR and hGHR-E42K proteins were successfully constructed. Conclusion: the eukaryotic expression plasmid pcDNA3.1-hGHR-E42K,pcDNA3.1-hGHR-H56R of two new mutation sites of hGHR gene was successfully obtained for the first time and the CHO cell line stably expressing hGHR and hGHR-E42K proteins was constructed. In the future, we will further study whether the mutation affects the binding energy of GHR to GH and the signal transduction pathway of JAK-STAT.
【学位授予单位】：江西师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2418243
[Abstract]:Objective: in the early stage of laboratory work, 47 cases of non-growth hormone deficient (GH), (including 1 case of Laron syndrome) selected according to certain screening conditions were significantly shorter than those in the control group (including 1 case of Laron syndrome). A series of mutations were screened and located in 46 children with idiopathic dwarfism to obtain three new mutations in GHR. According to the three new mutation sites, the GHR was mutated and the CHO cell lines stably expressing wild hGHR and hGHR-muta were constructed. It provides a basis for the functional study of the binding ability of GHR mutants to GH and the effect of the mutation on the signal transduction pathway of JAK-STAT. Methods: the plasmid PUC-GHR. containing GHR full-length cDNA was obtained from Ph.D.Wood of Gene Company in USA. Then the following experiments were carried out: 1. Using the quick change kit of stratagene Company to mutate the PUC-GHR plasmid, the eukaryotic expression vector pcDNA3-hGHR-wt, was constructed by double enzyme digestion and ligation of restriction endonuclease fragments. 2. PcDNA3-hGHR-E42K and pcDNA3-hGHR-wt plasmids were selected by pcDNA3-hGHR-E42K,pcDNA3-hGHR-H56R:2. and transfected into CHO cells by liposome transfection. A stable cell line expressing wild hGHR and hGHR-muta was constructed by antibiotic screening. 3. The expression of hGHR was identified by RT-PCR of total RNA and Western-Blot of total protein. Results: 1. Two PUC-GHR mutant plasmids of PUC-hGHR-E42K and PUC-hGHR-H56R and eukaryotic expression vector pcDNA3.1-hGHR-wt,pcDNA3.1-hGHR-E42K, were successfully obtained by sequencing the mutant plasmids. 2. The results of pcDNA3.1-hGHR-H56R.2.Western-Blot and RT-PCR showed that the transfected cells expressed wild hGHR and hGHR-E42K, indicating that the cell lines stably expressing wild hGHR and hGHR-E42K proteins were successfully constructed. Conclusion: the eukaryotic expression plasmid pcDNA3.1-hGHR-E42K,pcDNA3.1-hGHR-H56R of two new mutation sites of hGHR gene was successfully obtained for the first time and the CHO cell line stably expressing hGHR and hGHR-E42K proteins was constructed. In the future, we will further study whether the mutation affects the binding energy of GHR to GH and the signal transduction pathway of JAK-STAT.
【学位授予单位】：江西师范大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
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