重组人膜型Klotho蛋白在CHO细胞中的稳定表达

发布时间：2018-01-14 20:21

  本文关键词：重组人膜型Klotho蛋白在CHO细胞中的稳定表达　出处：《吉林大学》2007年硕士论文　论文类型：学位论文

  更多相关文章： 人klotho基因 人Klotho蛋白 膜型 CHO细胞 稳定转染

【摘要】： 本实验以人基因组DNA为模板,用基因拼接的方式构建克隆质粒pEGFP-C1-hmkl:①利用PCR方法,从pcDNA3.1/zeo(+)-hskl上获得前三个外显子,将其克隆到载体pEGFP-C1中,并引入SalI位点。②以人基因组DNA为模板采用PCR方法获得hmkl基因ex4,与载体pEGFP-C1-hmkl ex(1+2+3)连接构建成pEGFP-C1-hmkl ex(1+2+3+4),同时引入EcoRI和AflII酶切位点。③以人基因组DNA为模板采用PCR方法获得hmkl基因ex5,连接载体pEGFP-C1-hmkl ex(1+2+3+4)最终构建成克隆质粒pEGFP-C1-hmkl。BamHI和XhoI双酶切克隆质粒pEGFP-C1-hmkl,获得完整hmkl基因,构建真核表达质粒pcDNA3.1/zeo(+)-hmkl。测序确认后用Qiagen公司的无内毒素质粒提取纯化试剂盒纯化质粒。采用阳离子聚合物转染试剂将表达质粒pcDNA3.1/zeo(+)-hmkl转入CHO细胞;含600μg/ml zeocin的DMEM完全培养液(含10%小牛血清)筛选阳性细胞克隆;含300μg/ml zeocin的DMEM完全培养液维持扩大培养阳性克隆细胞;建立稳定表达hmKL蛋白的细胞株CHO-hmKL。结果表明,本实验首次获得稳定表达天然结构的hmKL蛋白的细胞株CHO-hmKL,从而使具有天然结构的hmKL蛋白的重组表达得以实现。重组hmKL蛋白天然结构的成功获得,为探讨KL蛋白的确切生物学功能提供了前提条件。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

【参考文献】

相关期刊论文 前10条

1 夏丽君;杨致邦;;KLOTHO基因表达产物的研究进展[J];国外医学(老年医学分册);2006年01期

2 杨泽,于普林;衰老与长寿的遗传机制探讨[J];国外医学.遗传学分册;2000年04期

3 李开龙,张建国;Klotho基因与肾脏疾病关系研究进展[J];中国中西医结合肾病杂志;2005年01期

4 童坦君,张宗玉;衰老相关基因研究进展[J];现代实用医学;2002年03期

5 黄燕,赵寿元;泛议衰老[J];生命科学;2002年02期

6 申烨华,耿信笃;CHO细胞表达系统研究新进展[J];生物工程进展;2000年04期

7 赵长安,刘素彩;Klotho基因及其生物学效应[J];国外医学(生理、病理科学与临床分册);2001年05期

8 吕宝璋;对人类衰老生物学研究现状及其前景的几点看法[J];中国老年学杂志;2002年04期

9 焦平;马杰;王文加;王建秋;王丁丁;张赢予;范伟全;颜炜群;;人分泌型Klotho蛋白在CHO细胞中的稳定表达[J];中国老年学杂志;2006年11期

10 余伍忠;Klotho基因及其与衰老相关的研究现状[J];中国优生与遗传杂志;2003年06期

，

本文编号：1425149