[Objective]To construct the mammalian expression vector of human cytoglobin(CYGB)gene with His tag,and transfected into CHO cells and HEK293 cells to express CYGB.[Method]The CYGB gene was synthesized using whole gene synthesis and inserted into the expression vector pcDNA3.4 through double enzyme digestion.After double enzyme digestion and sequencing verification,the gene was transfected into DH5α clone the strain,extract transfection grade plasmids,and trans-fected CHO and HEK293 cells for transient expression.Nickel column affinity chromatography to purify the CYGB,and the pu-rified CYGB was detected by Western Blotting and ELISA methods.[Result]Double enzyme digestion and agarose gel analysis showed that the pcDNA3.4-His-CYGB eukaryotic expression plasmid was successfully constructed.CHO cells were transi-ently transfected with pcDNA3.4-His-CYGB eukaryotic expression plasmid,and the expression level of CYGB was low,while that of HEK293 cells was high.Finally,HEK293 cells were used to express CYGB.The SDS-PAGE detection results showed that the molecular weight of CYGB protein was approximately 22 kDa,which was in line with expectations.The Western Blotting and ELISA detection showed that CYGB expressed in HEK293 cells had biological binding activity with anti CYGB monoclonal antibodies.[Conclusion]HEK293 cells are more suitable for expressing CYGB in mammalian cells than CHO cells,and the ex-pressed CYGB has Western Blotting and ELISA biological activities.
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