K-ras原核表达载体的构建及生物学功能研究

Construction of Human K-ras Eukaryotic Expression Vector and its Biological Function Research

目的:构建带Myc标签的K-ras原核表达载体,并初步鉴定其生物学活性。方法:利用PCR技术从人乳腺文库中扩增人K-ras结构域的基因编码序列,插入载体p XJ-40得到重组质粒,经Bam HⅠ和KpnⅠ双酶切鉴定后,Western印迹检测其在人293T细胞中的表达;利用GST pull down实验检测与Raf1-RBD的结合情况,验证其生物性活性。结果:用PCR技术从人乳腺文库中扩增得到约570 bp的目的片段,插入载体p XJ-40后构建了Myc-K-ras重组质粒,并经酶切鉴定及测序证实无误;Myc-K-ras能在293T细胞中正确表达,并能与Raf1-RBD结合,具有生物学活性。结论:为进一步研究K-ras的生物学功能奠定了重要基础。

Objective： To construct the eukaryotic expression vector of human K-ras labeled with Myc tag and preliminary detect the biological function of the expression product Myc-K-ras.Methods： Human K-ras gene was obtained from human mammary gland cDNA library by PCR and cloned into pXJ-40 vector. After the eukaryotic expression vector mediated expression of Myc-K-ras was observed in the human kidney 293T cells lines. The interactions of the K-ras with Raf1-RBD were identified by GST-pull-down assay.Results： The DNA fragment of about 570 bp was amplified from human mammary gland cDNA library by PCR, K-ras eukaryotic expression vector labeled with Myc was constructed and the expression of human K-ras protein was detected by Western blot in the human kidney 293T cells lines. Myc-K-ras fusion protein of about 21 kD was induced and identified by SDS-PAGE analysis, its activity was consistent with the report.Conclusion： The results has laid foundation for the further study on the function of K-ras.