| Since 1990's, avian influenza pandemic caused by H9N2 subtype of avian influenza virus has spread around our country and many other countries and has caused such a huge economic loss to powltry industry. A potential serious risk for animal and public health raises wide concern about sanitation issues. Coupled upgraded biosecurity strategy to vaccination in the territory is the major measure to control the outbreak of avian influenza. Since DNA vaccine has shown the ability to induce strong humoral, cellular and local mucosal immune response and has well security and stability, it can overcome many shortages of traditional vaccine. More and more researches have been done to test this technology in veterinary medicine. DNA vaccine delivered by attenuated intracellular bacteria can be directly delivered into special antigen presenting cells (APCs), so it improves the efficiency of DNA vaccine to a great extent. Some excellent attenuated salmonellae bacteria have been achieved to use as vaccine to guard against the infection of avian Salmonella or as prokaryotic expression vector for avian vaccine. The research that uses the attenuated salmonella bacteria as eukaryotic expression vector is being attempted in our laboratory. In this experiment two types of H9N2 subtype of avian influenza virus (AIV) DNA vaccines delivered by attenuated Salmonella typhimurium have been constructed, and their immunity efficacy have been evaluated in chicks.A pair of primers were designed and synthesized according to the previously published sequence in GenBank and used to amplify HA gene by reverse transcription-polymerase chain reaction (RT-PCR) from the genomic RNA of a H9N2 subtype AIV strain A/Chicken/Shanghai/3/2002 (H9N2) isolated from chicken. Adesired PCR product of 1.7kb was obtained, and then the PCR product was cloned into pGEM(?)-T easy vector system for sequencing. It was shown that its sequence has 94.7% and 94.6% of identity to that of HA gene of A/Chicken/Shanghai/4-4/01 and Chinese reference strain in lineage Ⅲ A/Chicken/Beijing/1/94 respectively, and 83.8% of identity to that of asian reference strain in lineage II A/Chicken/Korea/38349-p96323/96. Subsequently, this HA gene was subcloned into eukaryotic expression vector pVAX1 and asd-pVAX1 through KpnI and XhoI double digestion. The recombinant vectors pVAXl-HA and asd-pVAXl-HA were transfected COS-7 cells, and identified for the transient expression of AIV HA gene in indirect immunofluorescent assay. Finally, the recombinant vectors were transformed into final hosts of attenuated Salmonella typhimurium SL7207 and X4550, the recombinants were screened and designated as SL7207(pVAXl-HA) and X4550 (asd-pVAX1-HA) , respectively.In previous study, the safety and the dynamic changes in vivo of Salmonella typhimurium SL7207 and X4550 carrying recombinant plasmids had been evaluated.. In this study, the recombinant SL7207(pVAXl-HA) and X4550(asd-pVAXl-HA) were further evaluated their immunogenic and protective immune efficacy in chicks,and the recombinant eukaryotic plasmid pVAX1-IFN-γ was used as adjuvant. One day old commercial ISA brown chicks were administered orally with the vaccine every two weeks. The blood samples of each chick were collected from vein before the first vaccination, two weeks after the first vaccination and two weeks after the second vaccination. Five small intestine mucosal samples were collected from every group of chicks two weeks after the second vaccination. It was shown that high titers of antibodies with hemagglutination inhibition (HI) activity to AIV mucosal samples in small intestinal from immunized chicks, but low antibody titers in their sera. In contrast, there were high level of antibody titers in sera, but lower in mucosal samples in chicks immunized with killed oil-emulsified vaccine of AIV. The results suggested that DNA vaccines orally administered through attenuated Salmonella as delivering vehicle could elicit both mucosal immune response and systemic humoral immune response, but oil-emulsified vaccine injected intramuscularly mainly s...
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