本文關(guān)鍵詞：重組人膜型Klotho蛋白在CHO細(xì)胞中的穩(wěn)定表達(dá)　出處：《吉林大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人klotho基因 人Klotho蛋白 膜型 CHO細(xì)胞 穩(wěn)定轉(zhuǎn)染

【摘要】： 本實(shí)驗(yàn)以人基因組DNA為模板,用基因拼接的方式構(gòu)建克隆質(zhì)粒pEGFP-C1-hmkl:①利用PCR方法,從pcDNA3.1/zeo(+)-hskl上獲得前三個(gè)外顯子,將其克隆到載體pEGFP-C1中,并引入SalI位點(diǎn)。②以人基因組DNA為模板采用PCR方法獲得hmkl基因ex4,與載體pEGFP-C1-hmkl ex(1+2+3)連接構(gòu)建成pEGFP-C1-hmkl ex(1+2+3+4),同時(shí)引入EcoRI和AflII酶切位點(diǎn)。③以人基因組DNA為模板采用PCR方法獲得hmkl基因ex5,連接載體pEGFP-C1-hmkl ex(1+2+3+4)最終構(gòu)建成克隆質(zhì)粒pEGFP-C1-hmkl。BamHI和XhoI雙酶切克隆質(zhì)粒pEGFP-C1-hmkl,獲得完整hmkl基因,構(gòu)建真核表達(dá)質(zhì)粒pcDNA3.1/zeo(+)-hmkl。測(cè)序確認(rèn)后用Qiagen公司的無(wú)內(nèi)毒素質(zhì)粒提取純化試劑盒純化質(zhì)粒。采用陽(yáng)離子聚合物轉(zhuǎn)染試劑將表達(dá)質(zhì)粒pcDNA3.1/zeo(+)-hmkl轉(zhuǎn)入CHO細(xì)胞;含600μg/ml zeocin的DMEM完全培養(yǎng)液(含10%小牛血清)篩選陽(yáng)性細(xì)胞克隆;含300μg/ml zeocin的DMEM完全培養(yǎng)液維持?jǐn)U大培養(yǎng)陽(yáng)性克隆細(xì)胞;建立穩(wěn)定表達(dá)hmKL蛋白的細(xì)胞株CHO-hmKL。結(jié)果表明,本實(shí)驗(yàn)首次獲得穩(wěn)定表達(dá)天然結(jié)構(gòu)的hmKL蛋白的細(xì)胞株CHO-hmKL,從而使具有天然結(jié)構(gòu)的hmKL蛋白的重組表達(dá)得以實(shí)現(xiàn)。重組hmKL蛋白天然結(jié)構(gòu)的成功獲得,為探討KL蛋白的確切生物學(xué)功能提供了前提條件。
[Abstract]:The cloned plasmid pEGFP-C1-hmkl:1 was constructed by gene splicing using human genomic DNA as template and PCR method. The first three exons were obtained from pcDNA3.1% Zeo (-hskl) and cloned into the vector pEGFP-C1. SalI locus .2 was introduced to obtain hmkl gene ex4 using human genomic DNA as template and PCR method. PEGFP-C1-hmkl ex(1 234) was constructed by ligating with the carrier pEGFP-C1-hmkl ex(1 23). At the same time, EcoRI and AflII restriction sites were introduced. 3. Hmkl gene ex5 was obtained by PCR method using human genomic DNA as template. PEGFP-C1-hmkl ex(1 2 3 4). Finally, the cloned plasmid pEGFP-C1-hmkl.BamHI and XhoI double enzyme digested clone plasmid pEGFP-C1-hmkl were constructed. The complete hmkl gene was obtained. Construction of eukaryotic expression plasmid pcDNA3.1% zeo (). After confirmed by sequencing, the plasmid was purified by Qiagen's non-endotoxin plasmid extraction kit. The expression plasmid pcDNA3.1 / zeo() was transfected with cationic polymer transfection reagent. CHO cells were transfected with hmkl. Positive cell clones were screened in the DMEM complete culture medium containing 600 渭 g / ml zeocin (containing 10% calf serum). DMEM complete culture medium containing 300 渭 g / ml zeocin maintained expanded positive clone cells. A cell line CHO-hmKLexpressing hmKL protein stably was established. The results showed that the cell line CHO-hmKL which stably expressed hmKL protein with natural structure was obtained for the first time in this experiment. Thus, the recombinant expression of hmKL protein with natural structure can be realized. The success of the natural structure of recombinant hmKL protein provides a prerequisite for the study of the exact biological function of KL protein.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R346

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