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目的:构建含人重组牙骨质蛋白1(recombination human cementum protein1,rhCEMP1)基因的真核表达载体,观察其在酿酒酵母细胞中的表达。方法:采用PCR方法扩增rhCEMP1基因,利用定向克隆技术将rhCEMP1基因插入到中间载体pTeasy中,再进一步转插入载体pWX530。重组的pWX530-rhCEMP1在大肠杆菌DH5α中扩增后,通过酶切电泳鉴定和DNA序列测定所构建的质粒。经鉴定正确的表达载体pWX530-rhCEMP1转入酵母感受态细胞中,酵母经氨基酸营养缺陷型筛选后培养表达。利用聚丙烯酰胺凝胶(SDS-PAGE)电泳和酶联免疫吸附测定(ELISA)分析蛋白表达情况,离子交换层析提纯蛋白。结果:构建的重组质粒成功转入酵母细胞,通过SDS-PAGE和ELISA检测rhCEMP1表达成功。结论:成功构建的含rh-CEMP1基因的真核表达载体pWX530-rhCEMP1,并能转入酵母细胞中成功表达。
Objective: To construct an eukaryotic expression vector containing recombination human cementum protein 1 (rhCEMP1) gene and observe its expression in Saccharomyces cerevisiae cells. Methods: The rhCEMP1 gene was amplified by PCR. The rhCEMP1 gene was inserted into the intermediate vector pTeasy by directional cloning technique and further inserted into the vector pWX530. The recombinant pWX530-rhCEMP1 was amplified in E. coli DH5α and identified by restriction enzyme digestion and DNA sequencing. The correct expression vector pWX530-rhCEMP1 was transformed into yeast competent cells. The yeast was screened by amino acid auxotrophy and cultured. Protein expression was analyzed by polyacrylamide gel (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA), and protein was purified by ion exchange chromatography. Results: The constructed recombinant plasmid was successfully transformed into yeast cells and the expression of rhCEMP1 was confirmed by SDS-PAGE and ELISA. CONCLUSION: The eukaryotic expression vector pWX530-rhCEMP1 containing rh-CEMP1 gene was successfully constructed and transformed into yeast cells for successful expression.