采用酵母表达载体pWX530构建表达人重组牙骨质蛋白1  

The expression of rhCEMP1 with the eukaryotic expression vector pWX530 in yeast

作　　者：欧伟[1,2] 刘玉[1] 孙卫斌[1] 杨建良 Xuebin Yang David Wood 

机构地区：[1]南京大学口腔医学院,江苏南京210008 [2]德阳市人民医院口腔科,四川德阳618000 [3]无锡和邦生物科技有限公司,江苏无锡214064 [4]利兹大学口腔医学院

出　　处：《口腔生物医学》2013年第1期8-10,共3页Oral Biomedicine

基　　金：国家自然科学基金(51142013;81271155);江苏省自然科学基金(BK2010118);南京市医学科技发展一般性课题(YKK10126)

摘　　要：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 the recombinant eukaryotic expression vector containing the recombination human cementum protein 1 （rhCEMP1） and to observe the expression of rhCEMP1 in yeast, Methods ：RhCEMP1 cDNA was amplified by PCR, and correctly inserted into corresponding sites of intermediate vector pTeasy after restriction endonuclease digestion, and then inserted into corresponding sites of eukaryotic expression vertor pWX530 after another restriction endonuclease digestion. The recombinant vector pWX530-rhCEMP1 was confirmed to contain rhCEMP1 DNA sequence by agarose gel electrophoresis and DNA sequence analysis. The correct vector pWX530-rhCEMP1 was transfectd into yeast competent cells,and then the yeast was cultured after amino acid auxotroph screening. The expression of rhCEMP1 was determined by SDS-PAGE and enzyme-linked immunosorbent assay（ELISA） ,and the rhCEMP1 was purified by ion ex- change chromatography. Results ： The recombinant plasmid was successfully transferred to the yeast cells. The rhCEMP1 was successfully expressed by SDS-PAGE and ELISA analysis. Conclusions：The recombinant vector pWX530-rhCEMP1 was constructed successfully. The recombinant plasmid pWX530-rhCEMP1 could be transfected into yeast and expressed.

关 键 词：牙骨质蛋白1 真核表达载体 酵母 

分 类 号：Q78[生物学—分子生物学]