核心蛋白聚糖真核表达载体的构建及其在CHO细胞中的表达

Construction of decorin expression vector and its expression in CHO cells

目的:构建人核心蛋白聚糖(DCN)真核表达载体,并在CHO细胞中进行表达,为进一步研究其抗肿瘤作用奠定基础。方法:以含有人DCN cDNA的质粒pBluescript为模板,采用聚合酶链式反应(PCR)扩增目的基因片段。真核表达载体pcDNA3及PCR产物经XbaⅠ和EcoRⅠ双酶切后连接,转化大肠杆菌JM109,获得重组载体pCDNA-DEC,进行酶切鉴定和测序鉴定。脂质体lipofectamine介导重组载体转染CHO细胞,经G418(800mg·L-1)筛选建立稳定转染细胞株。采用细胞免疫组织化学方法检测稳定转染CHO细胞中DCN表达。结果:PCR获得与预期大小一致约1000bp的特异性DNA片段;重组载体pCDNA-DEC经双酶切鉴定及测序证实,人DCN基因cDNA片段正确插入真核表达载体中;在稳定转染的CHO细胞株中可见DCN蛋白表达。结论:成功构建DCN真核表达载体pCDNA-DEC,并建立稳定转染CHO细胞株。

Objective To construct a eukaryotic expression vector of decorin （DCN）, and observe its expression in CHO cells, in order to provide a basis for further study on the anti-tumor effect of DCN. Methods DCN cDNA was amplified by PCR. The human full-length DCN cDNA ligated into pBluescript was used as template. The fragment was ligated to the expression vector pCDNA3 previously digested with Xba Ⅰand EcoR Ⅰ The ligation mixture was transformed into competent E. coli JM109 cells. Transformants containing inserts were confirmed by restrictive digestion and DNA sequencing. The expression vector was transfected into CHO cells using lipofectamine, and transfected cells were cultivated in DMEM containing G418 （800 mg· L^-1） for about 2 months. Immunohistochemistry method was used to detect the expression of DCN protein in stably transfected cells. Results The PCR product was about 1 000 bp. The recombinant expression vector was identified by restrictive digestion and DNA sequencing. DCN protein was detectable in stablely transfected cells. Conclusion The recombinant eukaryotic expression vector pCDNA-DEC is constructed successfully and stablely transfected CHO cells are established.