APC11基因真核表达质粒构建及鉴定

目的克隆扩增APC11开放阅读框基因片段,构建其真核表达重组质粒,为后续研究其功能做准备。方法按照GenBank中人APC11序列设计引物,以HeLa细胞cDNA为模板,扩增出基因片段。该片段与真核表达载体pEGFP-C1连接,得到的重组质粒经双酶切和测序鉴定后用Western印迹检验表达。结果扩增片段长度为285bp,重组真核表达质粒经双酶切鉴定后测序鉴定正确。Western印迹证实pEGFP—C1-APC11在真核细胞内表达。结论成功克隆了APC11基因并构建了pEGFP—C1-APC11真核表达重组质粒,Western印迹证实其表达良好,对该基因功能的深入研究打下了坚实基础。

Construction and Identification of the APC11 Recombinant Eukaryot- ic Expression Plasmid

Objective To clone the ORF （123bp-377bp） of APC11 gene from the genomics, constructing the pEGFP-C1-APCll recombinant eukaryotic expression plasmid. Methods APC11 DNA fragment was amplified by PCR using specific primers, and then cloned into pEGFP-C1 vector respectively to yield the recombinant plasmid. The expressed products were identified by Western blot. Results A 285bp fragment of the APC11 gene was successfully amplified and cloned. The recombinant expression vector pEGFP-C1-APC11 was identified successfully by restriction enzyme digestion. The cloned gene fragment showed a 100 % homology with the human gene. （Genbank/ NCBI： NM_ 001002245）. Western blot showed that pEGFP-C1-APCll fusion protein was expressed. Conclusion The plasmids （pEGFP-C1-APCll） were successfully constructed and the fusion proteins were expressed. The studies has established the cloned genes and the expression system, which laid the fundation for futher functional studies of the APC11.