【摘要】： 實(shí)驗(yàn)?zāi)康模涸趯?shí)驗(yàn)室的前期工作中，通過(guò)對(duì)按一定入篩條件挑選的47名非生長(zhǎng)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(jìn)行一系列的突變篩查和定位獲得GHR上三個(gè)新的突變。本實(shí)驗(yàn)根據(jù)這三個(gè)新的突變位點(diǎn)對(duì)GHR進(jìn)行定點(diǎn)突變，并構(gòu)建穩(wěn)定表達(dá)野生hGHR及hGHR-muta的CHO細(xì)胞系，為后期研究GHR突變體與GH的結(jié)合能力及該突變對(duì)JAK-STAT信號(hào)轉(zhuǎn)導(dǎo)途徑的影響等功能性研究做基礎(chǔ)。 實(shí)驗(yàn)方法：由美國(guó)Gene公司Ph．D．Wood惠贈(zèng)獲得含GHR全長(zhǎng)cDNA的質(zhì)粒PUC-GHR。進(jìn)而開(kāi)展以下實(shí)驗(yàn)：1．使用stratagene公司的quick change試劑盒定點(diǎn)突變PUC-GHR質(zhì)粒，然后通過(guò)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(dá)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(xì)胞；通過(guò)抗生素篩選構(gòu)建穩(wěn)定表達(dá)野生hGHR和hGHR-muta的細(xì)胞系；3．采用細(xì)胞總RNA的RT-PCR和細(xì)胞總蛋白的Western-Blot方法鑒定hGHR的表達(dá)。 結(jié)果：1．通過(guò)對(duì)突變質(zhì)粒進(jìn)行測(cè)序驗(yàn)證，，成功獲得兩個(gè)PUC-hGHR-E42K及PUC-hGHR-H56R的PUC-GHR突變型質(zhì)粒以及真核表達(dá)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(xì)胞有野生hGHR和hGHR-E42K表達(dá)，說(shuō)明穩(wěn)定表達(dá)野生hGHR和hGHR-E42K蛋白的細(xì)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(gè)新突變位點(diǎn)的真核表達(dá)質(zhì)粒pcDNA3.1-hGHR-E42K、pcDNA3.1-hGHR-H56R并構(gòu)建了穩(wěn)定表達(dá)hGHR和hGHR-E42K蛋白的CHO細(xì)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(hào)轉(zhuǎn)導(dǎo)途徑做進(jìn)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(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R346

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