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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 the eukaryotic expression vector of human SIK2 gene. Methods The target gene SIK2 was amplified by PCR. The PCR product was purified and ligated with pC-MV-Tag-2B linear plasmid. The product was transformed into Escherichia coli DH5α and smecta-resistant agarose plates. PCR, restriction enzyme digestion and sequencing After identification of positive clones, plasmids were extracted and transiently transfected into 293T cells using Lipofectamnie 2000TM reagent. After 24 h, Western Blot was used to detect the expression of the plasmids in eukaryotic cells. Results The recombinant plasmid pCMV-Tag-2B-SIK2 was successfully constructed and successfully expressed in eukaryotic cells. Conclusion The construction of SIK2 exogenous expression vector was successful, which laid the experimental foundation for further study of its function.