人生長激素受體的定點突變及其真核表達
發(fā)布時間：2019-01-30 14:51
【摘要】： 實驗?zāi)康模涸趯嶒炇业那捌诠ぷ髦校ㄟ^對按一定入篩條件挑選的47名非生長激素缺失(GH)、顯著矮小(包括1例Laron綜合征、46例特發(fā)性矮小)的兒童進行一系列的突變篩查和定位獲得GHR上三個新的突變。本實驗根據(jù)這三個新的突變位點對GHR進行定點突變，并構(gòu)建穩(wěn)定表達野生hGHR及hGHR-muta的CHO細胞系，為后期研究GHR突變體與GH的結(jié)合能力及該突變對JAK-STAT信號轉(zhuǎn)導(dǎo)途徑的影響等功能性研究做基礎(chǔ)。 實驗方法：由美國Gene公司Ph．D．Wood惠贈獲得含GHR全長cDNA的質(zhì)粒PUC-GHR。進而開展以下實驗：1．使用stratagene公司的quick change試劑盒定點突變PUC-GHR質(zhì)粒，然后通過限制性內(nèi)切酶的雙酶切及酶切片斷連接等分子生物學(xué)方法構(gòu)建真核表達載體pcDNA3-hGHR-wt、pcDNA3-hGHR-E42K、pcDNA3-hGHR-H56R：2．選擇pcDNA3-hGHR-E42K及pcDNA3-hGHR-wt質(zhì)粒用脂質(zhì)體轉(zhuǎn)染法轉(zhuǎn)染CHO細胞；通過抗生素篩選構(gòu)建穩(wěn)定表達野生hGHR和hGHR-muta的細胞系；3．采用細胞總RNA的RT-PCR和細胞總蛋白的Western-Blot方法鑒定hGHR的表達。 結(jié)果：1．通過對突變質(zhì)粒進行測序驗證，，成功獲得兩個PUC-hGHR-E42K及PUC-hGHR-H56R的PUC-GHR突變型質(zhì)粒以及真核表達載體pcDNA3.1-hGHR-wt、pcDNA3.1-hGHR-E42K、pcDNA3.1-hGHR-H56R。2．Western-Blot和RT-PCR結(jié)果證明轉(zhuǎn)染后的細胞有野生hGHR和hGHR-E42K表達，說明穩(wěn)定表達野生hGHR和hGHR-E42K蛋白的細胞系構(gòu)建成功。 結(jié)論：本研究首次成功獲得了hGHR基因上兩個新突變位點的真核表達質(zhì)粒pcDNA3.1-hGHR-E42K、pcDNA3.1-hGHR-H56R并構(gòu)建了穩(wěn)定表達hGHR和hGHR-E42K蛋白的CHO細胞系。在今后的工作中我們將就該突變是否影響GHR與GH的結(jié)合能了以及JAK-STAT信號轉(zhuǎn)導(dǎo)途徑做進一步研究。
[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.
【學(xué)位授予單位】：江西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346
本文編號：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.
【學(xué)位授予單位】：江西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R346
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