Construction and identification of pneumocytis carinii eukaryotic expression plasmids of pVAX-p55-v3 and pVAX-p55-v0 antigenic gene / 中国免疫学杂志

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.