Study On Vaccine Of Recombinant Adenovirus Containing MOMP Gene Of Chlamydia Psittaci Of Chicken Origin

A chlamydia psittaci were isolated from affected chickens and the PCR method for chicken chlamydiosis was established. Recombinant adenovirus containing major outer membrane protein (MOMP) gene of Chlamydia psittaci of chicken origin were successfully constructed with human adenovirus type 5 vector which lack El and E3 genes. The MOMP genes were expressed in HEK293 cells and the recombinant adenovirus were evaluated in vitro and in vivo. It is the first time to construct recombinant adenovirus containing MOMP gene of avian chlamydia psittaci on the world.(1) Avian Chlamydia psittaci were isolated from chicken which were suspected suffering from Chlamydia psittaci through iodine staining, chicken embryo vaccination, sulfadiazine sodium test and electron microscopic observation. The virulence of hnq strain was measured in chicken embryo, which can reach 1.8× 10^-7/0.2ELD₅₀, CpL strain can reach 5.6×10¹⁰/0.2 ELD₅₀. The CpL strain was chosen as research object. The PCR method for chicken chlamydiosis with good specificity and sensitivity was established.(2) The MOMP genes were cloned and sequenced. Comparisons of the MOMP amino acid sequence of CpL with other strains of avian chlamydia psittaci showed that there was 89.7%, 88.7%, 82.3%, and 94.4% of homology of MOMP of CpL with duck strain, turkey, pigeon, and parrot, respectively.(3) Using the way of gene ligation and recombination, avian chlamydia psittaci MOMP genes were inserted into the region between CMV promoter and SV40 Poly (A) of pShuttle-CMV vector to form a gene expression cassette. The positive pShuttle-CMV vector and skeleton vector of adenovirus were recombined in BJ5183 competent cells. Then positive recombinant adenovirus was picked out and named as Ad-MOMP. Sequence detection showed that the sequence of MOMP gene in recombinant adenovirus hadn't any mutation. Detecting the target genes and products expressed in HEK293 cells with an indirect immunofluorescent assay and PCR technique after transfection, it was proved that the recombinant adenovirus could express the target genes efficiently. Neutralization test showed that there was no cross response between EDS₇₆ positive serum and the recombinant adenovirus in vitro. The stability test showed that the recombinant adenovirus with MOMP gene could stably passage in HEK293 cells.(4) Anti-MOMP antibodys of vaccinated SPF chicks were detected on 21^th, the result showed that it can reach 1 : 32. SPF chicks vaccinated with 6.8×10⁹TCID₅₀ Ad-MOMP recombinant adenovirus could resist 5.6×10¹⁰ELD₅₀ CpL strain challenge on the day 21 post vaccination(dpv) and the protection rate was 9/10. But no chicks of both wild type adenovirus and HEK293 groups were protected from the challenge. No antibody against EDS₇₆ was detected, which suggested that inoculation of Ad-MOMP recombinant adenovirus to chicks would not inhibit vaccination againstEDS76- Intramuscular and subcutaneous administrations were chosen in the test. The results suggested that the efficacy of both administrations was same. In order to meet practicability, we selected Intramuscular administration of the vaccine in the test.(5) According to the test results of SPF chicks, 7 days old chicks were vaccinated with 6.8 X lO9TCID5o Ad-MOMP recombinant adenovirus and challenged with the vaccine strain after 21 days. The results showed that chicks of the immunized group could resist 1.1 X 10I0ELD50 of the vaccine strain attacking while the control group failed after 6 months. The protection rate was 8/10. Antibody duration test showed that anti-MOMP antibodys can reach 1 : 64 on 21th day post vaccination. Then began down slowly still keeping a protective level on the 180th day. Specific antibody and T lymphocyte proliferation were detected with the IHA for Chlamydia antibody and MTT. The results showed that both of them had responses to Ad-MOMP recombinant adenovirus. The safety tests confirmed that the Ad-MOMP recombinant adenovirus had no side-effects on the inoculated chicks' health and gain and no recombinant adenovirus was discharged outside. In conservation period test, when the Ad-MOMP recombinant adenovirus was kept at -20°Cfor a year, its violence titer fell a little down, but immunity efficacy of the vaccine was un-reduced.