ABSTRACT
Objective To construct the eukaryotic expression vector of human BRE1B labeled with pCMV-Tag-2B and detect its biological activity in melanoma cells preliminarily.Methods Ocular B16 melanoma cells were randomly divided into the experimental group,in which the cells were transfected with pCMV-Tag-2B-BRE1B and the control group,which was transfected with pCMV plasmid.The CDS coding region of human BRE1B gene was amplified by PCR using human mammary gland cDNA as a template for construction of the recombinant plasmid pCMV-Tag-2B-BRE1B.After transfected with pCMV-Tag-2B-BRE1B and pCMV plasmid in the experimental and control group,respectively,Western blot was applied to detect the expression of BRE1B protein,while cell counting kit-8 (CCK8) and colony assays were used to analyze the effects of recombinant plasmid pCMV-Tag-2B-BRE1B on the growth of B16 melanoma cells.Results The CDS coding sequence of human BRE1B gene was amplified by PCR successfully,which was equal to the expected size.Compared with the control group,the sequence from bacteria PCR was identified as positive,with the length of 4000 bp and 3050 bp by double enzyme digestion respectively.Moreover,the coding sequence of the human BRE1B gene was exactly the same as the inserted DNA sequence.Western blot results showed that the expression of recombinant plasmid pCMV-Tag-2B-BRE1B was successfully expressed in the experimental group,but there was no specific fragments in the control group.And cell counting kit-8 (CCK8) and colony assays showed that pCMV-Tag-2B-BRE1B recombinant plasmid could inhibit the growth of B16 melanoma cells.Conclusion The eukaryotic expression vector of pCMV-Tag-2B-BRE1B labeled with pCMV-Tag-2B is constructed successfully,and it has inhibitory effects on the growth of ocular B16 melanoma cells.