Expression and identification of human EpCAM eukaryotic recombinant plasmid / 国际检验医学杂志

Objective To construct human EpCAM eukaryotic recombinant plasmid ,and to study the expression of EpCAM pro‐tein in HepG2 cells .Methods The recombinant plasmid ,named pcDNA3 .1(+ )‐EpCAMwas constructed by cloning EpCAM gene into eukaryotic vector pcDNA3 .1(+ ) .Then HepG2 cells were transfected with EpCAMrecombinant plasmid .The eukaryotic ex‐pression of EpCAM protein was verified by immunofluorescence ,Western Blotting and flow cytometry .Results The construction of EpCAM recombinant eukaryotic plasmid was identified by restriction enzyme digestion .The results of immunofluorescence , Western Blotting ,and flow cytometry consistently indicated that EpCAM protein were successfully expressed in HepG2 cells .Con‐clusion Human EpCAM eukaryotic vector is constructed and EpCAM protein could be expressed well in HepG2 cells ,which laid a foundation for further research on the function ofhepatocarcinoma cells highly expressing EpCAM .

Immunogenicity of a vaccine of S fragments from a new type reovirus generates humoral and cellular immune responses in mice / 中国实验动物学报

Objective To explore the humoral and cellular immune responses of a vaccine of S fragments from a new type reovirus R4 strain in mice.Methods Four recombinant plasmids were constructed by respectively cloning S 1, S2, S3,S4 genes into pcDNA3.1+, and mice were intramuscularly immunized with the recombinant plasmids in a dose of 100 μg/mouse.Control vector pcDNA3.1 +and phosphate buffered saline (PBS) were used as negative controls.The spe-cific antibody level and IgG subclass (IgG1, IgG2a, and IgG2b) were detected by ELISA, and cellular immune responses to R4 were assessed using an interferon ( IFN)-γELISpot assay .Results All recombinant plasmids induced significantly higher levels of anti-R4 IgG compared with that of the controls (pcDNA3.1 +and PBS), and the titers were highest in the mice immunized with S1 and S3.On the other hand, S1 gene induced highest IgG2a antibody and the cellular immune re-sponse was best .Conclusions After the mice immunized with S 1 gene recombinant plasmid , this plasmid can initiate both cellular and humoral immune responses in mice .S1 gene recombinant plasmid is a promising vaccine candidate .