| Brucellosis is a zoonosis caused by Brucella spp. Brucellosis has occured widly inthe world and done harm to stock raising and human health causing serious economiclosses. Brucellosis is prevalent in 170 countries in the world and in 28 province in ourcountry, the spreading of brucellosis is more and more serious in recent years.The genus Brucella consists currently of 7 nomenspecies and 21 biotypes includingmarine mammal genus, classified on the basis of microbiological features and hostpreference. Brucella is a Gram-negative, facultative intracellular bacterium that infectsmacrophages where the bacteria persist and effectively evade the host immune elimination.This pathogen infects ruminants, causing abortion and infertility, and in humans, leading tochronic stages of the disease known as undulant fever. Brucella has been postulates to be apotential agent of blowarfare and bioterrorism.The most important way to control of the disease in domesticated animals is the useof vaccines. At present, although attenuated live vaccines (B.bortus strain 19 andB.melitence Rev.1) against brucellosis are effective for controlling the disease, there aremany potential problems such as vaccine safety for pregnant female animals and youngeranimals, and so on. So it is a very important topic to study and develop new generationbrucella vaccine for controlling and finally eradicating brucellosis.At present, the study of new brucella vaccines focuses on the attenuated live vaccinesand gene engineering vaccines. The protection of the former is excellent, but the security isa question. The security of the latter is reliable, but the protection is incomplete. So wepresume that it is a feasible method to study multivalence gene engeering vaccines andeffective antigen presentation system which will show a very important significant forcontrolling brucellosis.In this study, L7/L12,BLS and BLS-L7/L12 genes were cloned from B.meliteniseM16 and inserted into prokaryotic expression vector pET32a to be expressed in E.coliBL21, and 3 recombinant proteins were expressed succesfully. At the same time, these 3genes were inserted into the eukaryotic expression vector pVAX1, transfected therecombinant plasmids to COS-7 cell. The proteins expressed by the identification ofWestern blot analyses and immunchemistry.BLS-L7/L12 gene was cloned into one plasmid and transformed into attenuated S.typhimurium vaccine which could induce mucosal and cellular immunoresponse. Theplasmid was steadily maintained by a host-plasmid balanced-lethal system based asd gene.BLS-L7/L12 was simultaneously expressed in these vaccine strains.BALB/c mice was immunized with these candidate vaccines and both humoral andcellular immune responses were elicited. Experimental animals exhibited specific antibodyafter the first immunization and increased steadily. The DNA vaccines and AttenuatedSalmonella live vector vaccine elicited a T-cell proliferative responses and also induced agamma interferon production upon stimulation with the specific antigens using ELISPOTassay. The ratio of CD4~+/CD8~+ were lower than that of the control mice. Furthermoreimmunoglobulin G isotype analysis indicated that the ratio of IgG1 and IgG2a in proteinvaccinated group was higher than 1 and that of DNA vaccine groups and the attenuatedSalmonella live vector vaccine groups was lower than 1.Whish suggested that theinduction of a typical T-helper 1-dominated immune response in DNA vaccinated groups.The immune protection assay displayed that all the candidate vaccines could induceeffective immune protection against the attack of 544A in the immunized mice. The DNAcandidate vaccines were more excellent than the protein vaccines, and bivalent vaccineswere better than monovalent vaccines. And the attenuated Salmonella live vector bivalentgene vaccine was the best, but the protection was incomplete. This study laid thefoundation for further research on immunogenicity of L7/L12,BLS and developingeffective prevention methods to the infection of brucellosis based on vaccine strategy.
|
PDF Full Text Request