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Construction and Evaluation of Immune Efficacy of DNA Vaccine from Paratuberculosis

Author: WangZuo
Tutor: ZhangShouFa
School: Yanbian University
Course: Preventive Veterinary Medicine
Keywords: paratuberculosis fusion DNA vaccines immune effect
CLC: S855.12
Type: Master's thesis
Year: 2009
Downloads: 19
Quote: 1
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Abstract


Paratuberculosis is a chronic wasting disease primary caused by Mycobacterium avium subsp,paratuberculosis,that is prevalent throughout the world,particularly in developing countries.This disease causes tremendous economic hardship to the livestock industry.Hence,the development of strategies to control the disease is tremendously important to preventing this disease.Currently,no clinical vaccines against Paratuberculosis is available.In the present study,the DNA fragments of map0862-2154c and map2751-2762c were amplified by PCR from the recombinant plasmid of pET32a-0862-2154c and pET32a-2751-2762c.These two fragments were inserted into pCDNA3.1(+) vector to construct recombinant plasmids pCDNA-mapO862-2154c(pC0862-2154c) and pCDNA-map2751-2762c(pC2751-2762c).Then the two fusion protein r0862-2154c and r2751-2762c were expressed in prokaryotic system.Bovis were vaccinated with the fussion DNA vaccine,recombination subunit vaccines,inactivated vaccine,BCG and the control vector pCDNA3.1(+) respectively.The animals in each group were immunized tree times with two week intervals.The group of inactivated vaccine and BCG were only vaccinated once.The serum antibody titers were determined using indirect enzyme-linked immunosorbent assay(iELISA) coated with the two fusion protein r0862-2154c and r2751-2762c every week.Two weeks after each immunization,splenic lymphocytes proliferation(SLP) assay were detected by MTT method using the mixture of two fusion protein r0862-2154c and r2751-2762c as stimulator.Also IFN-γ,IL-2,IL-4 and IL-6 assay were detected.Three weeks after the third immunization,intracutaneous transformation reactions was tested using the PPD as stimulator.The data demonstrated that immunization with recombination subunit vaccines results in significantly higher serum antibody levels. Lymphocyte proliferation(SI value),IFN-γand IL-2 levels immunization with fussion DNA vaccine were higher than other groups.And the index of IL-4 and IL-6 were also higher than other groups,but the levels were very low.The data of intracutaneous transformation reactions demonstrated that thickness of bovis skins were same with the control,but the bovis vaccinated with inactivated vaccine and BCG showed positive transformation reactions.The data demonstrate that fusion DNA vaccines induced humoral and cellular immune responses,cytokine secretion were higher than other groups.And fusion DNA vaccines did not react with PPD,suggesting that fusion DNA vaccines represent a promising approach for the prevention of paratuberculosis.So our experiment may be useful for the development of paratuberculosis vaccine.

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CLC: > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Veterinary Infectious Diseases > Bacterial Diseases > Bacillary disease
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