人钙周期素结合蛋白基因亚细胞定位载体的构建和鉴定

Construction and identification of hCacyBP gene subcellular localization vector

目的构建编码人钙周期素结合蛋白(hCacyBP)基因的亚细胞定位载体并进行序列测定。方法 hCacyBP的模板来自人结肠癌细胞株HCT-116的cDNA序列,根据hCacyBP基因序列设计合成引物,采用PCR方法扩增编码hCacyBP的基因片段;将hCacyBP基因定向克隆到亚细胞定位载体pCMV/myc系列:pCMV/myc/nuc(胞核定位载体)、pCMV/myc/cyto(胞质定位载体)。转化E.coli DH5α感受态细胞,在氨苄青霉素阳性LB培养基平板上筛选出阳性克隆。重组质粒经PCR及双酶切鉴定并进行序列测定。结果从HCT-116的cDNA模板扩增684 bp的hCacyBP基因片段,分别构建重组质粒pCMV/myc/nuc-hCacyBP、pCMV/myc/cyto-hCacyBP,PCR及双酶切鉴定产物大小与预期值相符合,测序分析进一步表明所克隆的基因为编码hCacyBP的基因片段。结论成功构建hCacyBP基因亚细胞定位载体,这将为研究结肠癌胞核、胞质内的hCacyBP功能和研究该基因异常高表达与结肠癌发生发展的关系奠定实验基础。

Objective To construct and sequence the hCacyBP - coding gene subcellular localization vectors. Methods The eDNA template from human colon cancer cell line HCT -116 was prepared by our laboratory. According to hCaeyBP gene sequenceing, primers were designed and synthesized, hCacyBP - coding gene was amplified by PCR and cloned into the subcellular localization vectors pCMV/myc series： pCMV/mye/nuc （nuclear localization vector） and pCMV/myc/cyto （cytoplasmic localization vector）. Both the two plasmids were transfected into E. coli DH5ct competent cells in LB medium plate and screened with ampicillin. Recombinant plasmids were identified by PCR/dual - endonuclease and sequencing. Results The size of hCaeyBP- coding gene amplified from the eDNA template of HCT- 116 by PCR was 684 bp, the constructed recombinant plasmids （pCMV/myc/nuc -hCacyBP, pCMV/myc/cyto -hCacyBP） were identified by PCR/dual - endonuclease, the size of product was consistent with the expected value, and sequence analysis showed that the cloned gene was the gene fragment encoding hCaeyBP. Conclusion The subcellular localization vectors are successfully constructed. It is the basis for the investigation of colon cancer cell nucleus/cytoplasm hCacyBP function and further study on the correlation between the abnormal up - regulation of gene expression and the oneogenesis of colon cancer.