Study On Antibody-detection ELISA And Genetic Vaccination Of Swine Chlamydophila Abortus

Chlamydophila abortus (CAB) is one of the important pathogens causing the abortion of sow, cow and ewe and pregnant women. The establishment of rapid and accurate diagnostic method and the development of safe and efficient vaccine are essential measures for the prevention and control of this disease. In this study, the genes encoding major outer membrane protein (MOMP) and four different fragments of polymorphic outer membrane protein-90 (POMP90) of CAB were obtained by PCR. Applicabilities of the recombinant proteins were explored and the indirect ELISA methods for detecting the CAB antibody in sera of pigs were developed. Then the vaccinal plasmids with CAB momp gene were reconstructed, and such swine cytokines as interleukin-2 (IL-2) and interferon gammar (IFN-γ) encoding genes were individually inserted into the vaccinal plasmid for the double expression. Differences of the immune responses induced by the vaccinal plasmid with pcDNA3.1 and PCI-neo as the expression vector were researched, and the immune adjuvant effects of two cytokines and the lycium bartarum polysaccharides (LBPS) were explored.1. In this study, the full-length momp gene and four different fragments of pomp90 gene were amplified with the specific primers and the total DNA template of CAB strain CP/12. The obtained genes identified by sequencing after linked to the pMD18-T vector were linked to the prokaryotic expression vector pGEX-KG for expression. The recombinant plasmids were high-level expressed in the E.coli BL21. Western-blot of recombinant proteins exhitited strong immunogenicities. The indirect ELISA methods established using the five purified recombinant proteins as coating antigens were clinically used to detect the serum CAB specific antibody. While the recombinant MOMP protein was not suitable for the establishment of the ELISA method, the results showed that the methods developed with the four recombinant POMPs were sensitive, specific, accurate, which can overcome the shortcomings of IHA, such as low sensitivity, subjective judgments.2. Then the momp gene was cloned into the eukaryotic expression vector pcDNA3.1 and PCI-neo to reconstruct the vaccinal plasmids pcDNA3.1-MOMP and PCI-MOMP. The immunities were evaluated in mice model and the efficiency of genetic vaccines with various expression vectors was discussed. The results indicated that PCI-MOMP could induce higher humoral immune and celluar immune than pcDNA-MOMP did, but effective immune protection was not observed.3. To increasing the immunity of momp genetic vaccination, the swine IL-2 and IFN-γencoding genes were individually inserted into the vaccinal plasmid PCI-MOMP. The adjuvant effects of the two cytokines on the genetic vaccination through co-administration and double expression were explored in mice model. The results suggested that both humoral and celluar immune response were not increased by the two cytokines, and effective immune protection failed to be induced.4. The adjuvant effect of LBPS on the momp genetic vaccination by co-administration was also explored in mice model. The results showed that LBPS significantly increased the humoral immune response, however the higher celluar immune response and immune protection were not observed.In this paper, indirect ELISA methods were established for detecting the serum swine CAB antibodies. The immune effects of CAB momp genetic vaccine were explored in mice model, incuding the difference between the plasmids constructed with different expression vector, and the adjuvant effects of two cytokines and LBPS on the genetic vaccination. This study was the basis for the further research on immune prevention and diagnosis of swine Chlamydophila abortus infection.