To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites ...To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.展开更多
Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded po...Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-展开更多
ZINC finger proteins play an important role in the regulation of gene expression by binding to DNA/RNA in a sequence-specific manner. Now nearly 200 human zinc finger genes have been cloned. According to the differenc...ZINC finger proteins play an important role in the regulation of gene expression by binding to DNA/RNA in a sequence-specific manner. Now nearly 200 human zinc finger genes have been cloned. According to the difference of the conservative domain, the zinc finger proteins can be classified into several types.Most of zinc finger proteins belong to the C<sub>2</sub>H<sub>2</sub> type containing the consensus amino acid sequence YECX<sub>2</sub>CX<sub>3</sub>FX<sub>5</sub>LX<sub>2</sub>HX<sub>3</sub>HTGEKP, which is rather conservative especially in the link region between two zinc finger motifs （TGEKP）展开更多
文摘To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.
基金supported by grants from the Science and Technology Commission of Shanghai Municipality (13ZR1462900)the Shanghai Institutes for Biological Science (SIBS),Chinese Academy of Science (CAS) (2013KIP317)+1 种基金the support of the SA-SIBS scholarship programYouth Innovation Promotion Association of CAS (2016249)
文摘Dear Editor,Coxsackievirus A16(CA16)is one of the major viral pathogens associated with hand,foot,and mouth disease.CA16 belongs to the Enterovirus genus of the Picornaviridae family and possesses a single-stranded positivesense RNA genome(Mao et al.,2014).Reverse genetics is an important tool for CA16 research.Previously,a reverse genetics T7 polymerase-based system was de-
文摘ZINC finger proteins play an important role in the regulation of gene expression by binding to DNA/RNA in a sequence-specific manner. Now nearly 200 human zinc finger genes have been cloned. According to the difference of the conservative domain, the zinc finger proteins can be classified into several types.Most of zinc finger proteins belong to the C<sub>2</sub>H<sub>2</sub> type containing the consensus amino acid sequence YECX<sub>2</sub>CX<sub>3</sub>FX<sub>5</sub>LX<sub>2</sub>HX<sub>3</sub>HTGEKP, which is rather conservative especially in the link region between two zinc finger motifs （TGEKP）
