重组人Dll4真核载体的构建及其在CHO细胞中的表达

Construction and Expression of Recombinant Human Dll4 in CHO Cell

PCR法扩增rh Dll4基因片段,用EcoR I和Not I双酶切后克隆到真核载体p CA中,并对其进行双酶切和测序鉴定;用电转法将真核载体导入CHO细胞中,并利用Western blot法检测rh Dll4在CHO细胞中的表达水平。经双酶切和测序验证,成功构建p CA-rh Dll4真核表达载体。Western bolt验证表明,rh Dll4基因在CHO细胞中正常表达。经镍柱亲和层析纯化后,rh Dll4蛋白纯度达95%以上。MTT法检测,rh Dll4可以有效地抑制HUVEC增殖;同时可以促进Notch1受体的切割,表明所表达的rh Dll4蛋白具有生物学功能,为本实验下一步抗rh Dll4单克隆抗体的筛选奠定了基础。

The PCR was applied to amplify gene fragment of rh Dll4 that was inserted to eukaryotic vector p CA after digested with EcoR I and Not I. The recombinant plasmid p CA-rh Dll4 was identified by restriction endonuclease digestion and DNA sequencing.And the plasmid p CA containing rh Dll4 was electroporate to CHO. The expression of rh Dll4 was detected by Western blot. The rh Dll4 in the supernatant of cell culture was purificated by Ni-colum and was identified by SDS-PAGE. MTT method was used to measure rh Dll4 in inhibiting proliferation of HUVEC and Western blot was applied to determine rh Dll4 in promoting Notch1 cleavage. The results indicated that the recombinant eukaryotic expression plasmid p CA-rh Dll4 was constructed successfully and rh Dll4 expression was confirmed. And the purity of rh Dll4 achieved to 95%. Furthermore,the rh Dll4 could inhibit proliferation of HUVEC and promote Notch1 cleavage. Therefore,the rh Dll4 expressed in this study can be used to antigen for anti-rh Dll4 monoclonal antibody screening.