Construction of mammalian co-expression plasmid pIRES-CEA-IL-2 and its expression in vitro / 中国癌症杂志

Purpose:To construct the mammalian co expression plasmid pIRES CEA IM 2 and detect the expression of the plasmid in vitro. Methods:Using cloning technique,the cDNA fragments of CEA gene and mouse molecule IL 2 gene were constructed into the mammalian co expression plasmid vector pIRES.The inserted target genes in the mammalian co expression plasmid were verified by nucleotide sequencing.C 26 cell line was transfected with this mammalian co expression plasmid using lipofecarnin reagent. The expression of CEA and IL 2 molecules were detected by ELISA technique.Results:The mammalian co expression plasmid pIRES CEA IL 2 was obtained by cloning technique. The nucleotide sequences of CEA gene and molecule IL 2 gene in this mammalian co expression plasmid had high homology with CEA standard strain (99.8%) and mouse IL 2 (99.9%) respectively. After transfection with this mammalian co expression plasmid, the CEA and IL 2 molecules were expressed in C 26 cells. Conclusions:The constructed mammalian co expression plasmid pIRES CEA IL 2 can express CEA and IL 2 molecules in vitro at the same time.

Control of coexpression plasmid of carcinoembryonic antigen and IL-2 gene by internal ribosome entrying site / 中国普通外科杂志

ObjectiveTo construct the eukaryotic coexpression plasmid CEA/IL-2, and to lay the foundation for further studying CEA nucleiotide vaccine , adjuvant and their effects of special antitumor immunity.MethodsThe eukaryotic expression plasmid (pcDNA3-CEA)containing the gene coding for CEA was obtained by RT-PCR and gene recombination techniques.Using enzymolysis,ligation and other techniques,an eukaryotic coexpression plasmid (pIRES-CEA/IL-2)containing two expression unites of CEA and IL-2 gene connected with internal ribosome site was constructed.ResultsThe coexpression plasmids were transformed into COS7 cells and expression of two proteins were demonstrated by ELISA, and flow cytometer and elecsy.CEA and IL-2 were (23.73?0.26)ng/ml,and(20.17?0.13)ng/ml respectively.ConclusionsThe eukaryotic expression plasmids pIRES-CEA/IL-2 could be successfully constructed and transformed into COS7 cells.Expression of two proteins were demonstrated with no difference on expression.

CEA DNA vaccine induces special cellular and humoral immune response to a carcinoembryonic antigen in mice / 中国癌症杂志

Purpose:To identify the expression of the DNA v accine in mice muscles and detect its ability to induce special cellular and hum oral immune response to carcinoembryonic antigens. Methods:Using cloning technique,the cDNA fragments of CEA gene and mouse co-stimulatory molecule IL-2 gene were constructed into the mammali an co-expression plasmid vector pIRES.Tongue muscle of mice were injected with our plasmid. The expression of CEA molecules in muscle was identified by immunoh istochemitry technique .The humoral responses were detected by ELISA technique, the special celluar immuse responses were detected by CTL and NK cytotoxity assa y. Results:We identified that our plasmids can express the CEA and IL-2 molecules in muscles of mice.CEA-antibody and IL-4 molecules appear in blood serum of mice,CTL activities and NK cytotoxity were much stronger in immun ized mice. Conclusions:The constructed mammalian co-expression plasmid pI RES-CEA , pIRES-IL-2,pIRES-CEA- IL-2 can express CEA and IL-2 molecules i n muscles and can generate cellular and humoral immunological responses to CEA p ositive carcinoma.