ABSTRACT
Objective:To construct eukaryotic expression plasmids of pneumocystis carinii p55-v3 and p55-v0 antigenic genes and to identify their expression in COS-7 cells at mRNA level.Methods:Pneumocystis carinii total RNA was used as the template to amplify p55-v3 and p55-v0 antigenic gene by RT-PCR.The products were connected to pTA2 vector and then cloned in pVAX1 eukaryotic expression vector to construct recombinant plasmids as pVAX-p55-v3 and pVAX-p55-v0.After propagated in E.coli DH5α,the recombinant plasmids were transfected into COS-7 cells.After 24 h incubation,the RT-PCR was performed to identify the mRNA expression of p55-v3 and p55-v0 antigenic gene.Results:The recombinant plasmids were qualified by restrictive endonuclease digestion and sequencing.And when compared with that in GenBank,the homology of p55-v3 antigenic gene was 99.9% in nucleotides and 100% in amino acid.The homology between p55-v0 antigenic gene and the one reported previously in nucleotide and amino acid seguence were 99.8% and 100%.The results of RT-PCR confirmed that p55-v3 and p55-v0 antigenic genes were transfected into COS-7 cells successfully and the genes were expressed in the cells.Conclusion:In this study,the recombinant plasmids of pVAX-p55-v3 and pVAX-p55-v0 are conducted successfully and expressed in the COS-7 cells,which provide a basis for clarification of immunologic function of p55-v3 and study of DNA vaccine.
ABSTRACT
Objective To construct a fusion minigene expression vector of seven HCV NS3 epitopes and express the fusion gene in the eukaryotic cell line, in order to form a foundation for the investigation of the DNA immunization for HCV prevention. Methods Three pairs of complementary oligonucleotides primers covering all seven HCV NS3 epitopes were synthesized. Overlapping extension PCR were performed to construct the fusion minigene and cloned in pEGFP-N3 and pBuDCE vector respectively. The fusion protein was detected by Western blotting and flow cytometric analysis. The transfection efficiency was assessed with by flow cytometric analysis. Results The HCV NS3 epitope fusion minigene was obtained and inserted into pEGFP-N3 and pBuDCE vector respectively. The recombinant plasmid pEGFP-DR4 and pBuDCE-DR4 were expressed in 293T and 046W cell lines respectively. The Western blot result indicated that the expected 36 kDa minigene/EGFP fusion product was expressed from pEGFP-DR4, while 13kDa product from pBuDCE-DR4 respectively. Conclusion The HCV NS3 epitope fusion minigene was expressed in the eukaryocyte expression system.