核心蛋白聚糖原核表达体系的构建

Construction of the recombinant prokaryotic expression plasmid pET-21a(+)-DCN

目的通过克隆核心蛋白聚糖(decorin,DCN)基因,构建原核表达体系并表达DCN蛋白。方法利用特异引物,通过反转录-聚合酶链反应(RT-PCR)方法扩增编码DCN分子的外显子序列,与表达载体pET-21a(+)重组,构建原核表达体系pET-21a(+)-DCN的重组质粒。经双酶切、测序鉴定基因序列,将重组质粒转入E.coliBL21(DE3)中,用IPTG37℃诱导获得DCN融合蛋白。结果克隆了hDCN基因,成功构建了pET-21a(+)-DCN重组质粒,获得了稳定的pET-21a(+)-DCN原核表达体系,并得到了纯化的融合蛋白。结论建立了pET-21a(+)-DCN稳定的原核表达体系,表达并纯化了DCN。

Objective To clone and express the human decorin extron gene.Methods Using decorin specific primer and by reverse transcriptase ploymerase chain reaction （RT-PCR） ,the full length of cDNA of decorin extron was obtained,then recombined plasmid -decorin was constructed and sequenced to identify the gene sequence. Furthermore,pET-21a （＋）-decorin was transformed into E.coli BL21 （DE3）and after higher temperature and IPTG induction,the fusion protein was expressed. Results The recombinant vector was constructed and identified using genetic cloing technology,and the target peptide was produced in E.coli BL21 （DE3）in this experiment. The fusion protein was high expressed. Conclusion The prekaryotic expression vector with pET-21a（＋）-decorin can effectively express decorin fusion protein.