期刊论文

Construction of Human Recombinant Plasmid of pCMV-Tag-2B-SIK2 and its Expression in Eukaryotic Cells

中文

实用预防医学

1006-3110

2013

20

5

528-530

10.3969/j.issn.1006-3110.2013.05.005

2012AA063501:国家高技术研究发展计划（863计划） 81272994,31270894:国家自然科学基金

本校为第一机构

目的构建人SIK2基因真核表达载体。方法利用PCR扩增目的基因SIK2,PCR产物经纯化后与pC-MV-Tag-2B线性质粒进行连接,连接产物转化大肠杆菌DH5α,涂卡那抗性琼脂糖平板;菌落PCR、酶切及测序鉴定阳性克隆后,抽提质粒,利用Lipofectamnie 2000TM试剂瞬转293T细胞,24 h后,Western Blot方法检测其在真核细胞内的表达。结果成功构建pCMV-Tag-2B-SIK2重组质粒,并在真核细胞内成功表达。结论 SIK2外源表达载体的构建成功,为进一步研究其功能奠定了实验基础。

Objective To construct human pCMV-Tag-2B-SIK2 eukaryotic expression plasmid.Methods The PCR-amplified SIK2 cDNA was purified and successfully ligated to the linear eukaryotic expression vector pCMV-Tag-2B.The recombinant plasmid was further identified by PCR,restriction enzyme digestion and sequencing.Then it was transfected into 293T cells using Lipofectamine 2000TM.Its expression was detected by Western blot 24 hours after transfection.Results The eukaryotic expression vector,pCMV-Tag-2B-SIK2,was successfully constructed.It was expressed normally in eukaryotic cells.Conclusions The successfu...