| DNA vaccine is regarded as the third generation vaccine. A lot of progress has been made in the area of DNA vaccine in the past decade. DNA vaccine can elicit both cellular and humoral immunity and has several advantages, such as easy to produce, transport and store, easy to construct multivalent vaccines and easy to incorporate several adjuvant molecules. But two major factors restrict its development. One is the issue of safety. On the one hand, whether DNA vaccine will integrate into somatic cells' chromosome and cause genetic changes is not sure. On the other hand, the existence of antibiotic resistance gene within DNA vaccine is always a concern. The other factor is the immune efficacy is not so satisfactory. The underlying reason for this is the lack of effective and convenient immune methods. The common immune methods include muscle injection, intra-dermal and subcutaneous inoculation. But these methods have such shortcomings as the need of special equipment, the possibility of spreading blood-borne diseases and the lack of activation of both mucosal and systemic immunity. In this study both safety and efficacy issue of DNA vaccine were dealt with. First, a general DNA vaccine vector asd-pVAX1 containing no antibiotic resistance gene was constructed. Second, a Salmonella DNA vaccine transporting system based on asd-pVAX1 was developed by using the balanced-lethal system. Finally, a prokaryotic-eukaryotic double expression plasmid was successfully constructed for the development of novel recombinant Salmonella vaccines.1 Characterization of a novel DNA vaccine vector without antibiotic resistance gene and its transporting system based on SalmonellaThe antibiotic resistance genes in present DNA vaccine vectors are a great concern. By introducing asd gene from Salmonella typhimurium into DNA vaccine vector pVAX 1 and destroying kanamycin resistance gene at the same time, a new DNA vaccine vector asd-pVAXl which contained no antibiotic resistance gene was constructed. Then enhanced green fluorescence protein (EGFP) gene was inserted into its multiple cloning site (MCS). This new recombinant plasmid asd-pVAX 1-EGFP was harvested from Aasd E. coli X6212 in vitro and strong green fluorescence was observed in transfccted mouse mastocytoma P815 cells, asd-pVAX1-EGFP was used to immunize BALB/c mice intramuscularly and specific serum anti-EGFP antibody was elicited. The liter of anti-EGFP antibody was no lower than the antibody induced by pVAXl-EGFP.asd-p VAX1-EGFP was transformed into asd S. typhimurium X4550 and in vitro and in vivo tests showed asd-pVAX1-EGFP can be stably maintained within X4550. When X4550(asd-pVAX1 -EGFP) was used to infect COS-7 cells, EGFP positive COS-7 cells can be observed 24 hours post-infection.Two important conclusions can be made from these results. First, the worry of the antibiotic resistance gene was removed in this novel DNA vaccine vector asd-pVAXl and this vector can be amplified in large amount in vitro without the need of antibiotic pressure. Second, by using the balanced-lethal system, DNA vaccine based on asd-pVAXl can be stably maintained within X4550, thus solving the problem of "instability of DNA vaccine within host strain", which constituted a solid foundation for the further study of oral immunization.2 Characterization of a prokaryotic-eukaryotic double expression plasmidProkaryotic promoter Ptrc and multiple cloning site (MCS) was excised from plasmid pYA3334 and inserted into the downstream of eukaryotic expression promoter Pcmv in DNA vaccine vector pVAX1. Endonucleases digestion and nucleotide sequencing confirmed the Tightness of this ligation. This novel double expression plasmid vector was named as pVAXD and was 3,115 bp in length.To verify this novel vector EGFP gene was placed into the downstream of both promoters and consequently transformed into Salmonella typhimurium X4550 and transfected into COS-7 cell. The EGFP expression in X4550 (pVAXD-EGFP) was detected by flow cytometer, visible spectrum scanning and SDS-PAGE and its ex... |
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