Stability of the B. abortus S19 vaccine strain with a eukaryotic expression plasmid encoding the G glycoprotein from the rabies virus

Brucella abortus S19 is an intracellular vaccine strain against bovine brucellosis. Rabies is a lethal disease in cattle. Plasmids encoding the G glycoprotein from the rabies virus induce a protective immune response in different animal species. A vector called pBBR4-CMV-Ggp-SV40+, which encodes the G gene, regulated by the cytomegalovirus eukaryotic expression promoter, and which can be used to transform the B. abortus S19 vaccine strain, was constructed. The stability of the transformant strain was tested both in vitro and in vivo. In the in vitro assays, B. abortus S19 pBBR4-CMV-Ggp-SV40+ was grown for 5 sequential passages, and for the in vivo assays, female BALB/c mice were infected. Colony-forming unit counting and plasmid identification were performed in each passage and in the spleens at 7 days post-infection. To test the plasmid stability in the strain, all parameters were determined with and without antibiotic. The bacterial concentration was lower with antibiotic than without it, but the bacterial growth was more homogeneous. The plasmid was identified in antibiotic- and non-antibiotic-treated isolated colonies under both in vitro and in vivo conditions. The plasmid construct was also transfected into BHK-21 cells, which express the G glycoprotein. The B. abortus S19 pBBR4-CMV-Ggp-SV40+ strain showed stability, representing a suitable candidate vector for developing a bivalent vaccine against brucellosis and rabies. This is the first time that a Brucella species has been transformed with a eukaryotic expression plasmid.

Construction of a Recombinant Eukaryotic Expression Plasmid Containing Human Calcitonin Gene and Its Expression in NIH3T3 Cells

Aim. To construct a recombinant eukaryotic expression plasmid containing human calcitonin (hCT) gene and express the gene in murine fibroblast NIH3T3 cells.Materials and Methods. A murine Igκ-chain leader sequence and hCT gene were synthesized and cloned into pCDNA3.0 to form the pCDNA3.0-Igκ-hCT eukaryotic expression vector, which was transfected into NIH3T3 cells. The mRNA and protein expressions and secretion of hCT were detected. Primarily cultured osteoclasts were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells, and their numbers were counted and morphology observed.Results. The expression and secretion of hCT were successfully detected in pCDNA3.0-Igk-hCT-transfected NIH3T3 cells. The number of osteoclasts was decreased and the cells became crumpled when they were incubated with the supernatant of pCDNA3.0-Igk-hCT-transfected NIH3T3 cells.Conclusion. A recombinant eukaryotic expression vector containing hCT gene was successfully constructed and expressed in NIH3T3 cells. The secreted recombinant hCT inhibited the growth and morphology of osteoclasts.