Construction Of DNA Vaccine With LipL21 From Leptospira And Study On Immunoprotectivity To The Guinea-pigs

Objectives:(1) To construct Eukaryotic Expression vector pcDNA3.1 (+)-lipL21 containing the outmembrane lipoprotein lipL21 gene of Leptospira and to express target protein in HeLa cells.(2) To observe immune response induced by DNA vaccine on the out membrane lipoproteinLipL21 of Leptospira in guinea-pigs,(3) To observe the protective effects of DNA vaccine on the out membrane lipoprotein LipL21of Leptospira in guinea-pigs from the infection by pathogenic Leptospira.Methods:Primers were designed by primer primierS.O software according to the lipL21 gene sequence of Leptospira interrogans serovar Lai. Polymerase chain reaction (PCR) was used to amplify the lipL27-encoding gene and about 561 bp lipL21 gene fiagment was isolated. The fragment of lipL21 was directly ligated into pUCm-T .After identified by PCR and restriction enzyme analysis, the fragment of lipL21 gene was subcloned into eukaryotic expression vector pcDNA3.1 (+) with linking reactions to construct pcDNA3.1 (+)-lipL21 The recombinant plasmid was transfected into HeLa cells using liposome .After expression of the lipL21 gene in HeLa cells was identified by immunocytochemistry and SDS-PAGE ,2-weeks old guinea-pip were immunrzed with pcDNA3.1(+)-lipL21 or pcDNA3.1(+) or PBS intramuscularly at 2 week interval for three times. MAT was used for the quantitative detection of the specific antibody in the sera of immune guinea-pigs. The proliferation response of spleen cells was detected by MTT and lymphocyte transformation experiment The existence of lipL21 gene in muscle of guinea-pigs was detected by PCR.Results: The lipL21 gene fragment (561bp) was successfully amplified. The eukaryotic...