DNA Vaccine Of Avian Reovirus σC Gene Delivered By Attenuated Salmonella Typhimurium And Its Immunogenicity

Avain viral arthritis which is also called infectious tenosynovitis, is cauded by Avian reoviruses in chickens and turkeys. Chickens affected with ARV can show various clinical signs including arthritis, gastroenteritis, tenosynovitis, hepatitis, myocarditis, malnutrition syndrome, chronic respiratory diseases and the increase of death rate,the decrease of egg laying, in addtion, this disease can also transmits by vertical transmission thus provoking considerable economic losses in poultry industry.σC protein is the major component of the outer capsid of ARV and can induce the host to produce specific neutralizing antibody.σB protein is the minor component of the outer capsid of ARV and can induce the host to produce group specific neutralizing antibody. Attenuated Salmonella typhimurium is now widely used as carrier of gene vaccine. So according to the characteristic of aB andσC proteins can induce antibody production and attenuated Salmonella typhimurium can be used as carrier of gene vaccine, this study constructed ARVσB andσC gene vaccines orally delivered by attenuated Salmonella typhimurium and investigated their immunogenicity in chickens.1 Construction of eukaryotic expression plasmids ofσB andσC gene of ARVA pair of primer was designed accordingσB gene sequence published in GenBank to amplifyσB gene. The amplifiedσB gene was then cloned into T vector, sequence analysis showed the amplified aB gene was 1130bp. ThenσB gene was ligated with prokaryotic expression plasmid pET32a(+) to construct prokaryotic expression plasmid of aB protein. The prokaryotic expression plasmid was then transferred into E coli BL21 to express aB protein. The optial expression condition was 0.6mM of IPTG and 4 hour of expression. Westting-blot anslysis showed the espressed ARVσB protein possesseed good reactionogenicity.ARVσB gene was ligated with eukaryotic expression plasmid pVAX to construct pVAX-σB. PCR amplification and enzyme digestion showed a 1100bp fragment appeared, showing eukaryotic expression plasmid pVAX-σB was successfully constructed. aC gene was amplified from existing eukaryotic expression plasmid pVAX-aC. Sequence analysis showd the amplifiedσC was 1138bp.σC gene was ligated with dual-cistron eukaryotic expression plasmid pVAXAB to construct bridge expression plasmid pVAXAB-σC, after identified by PCR amplification and enzyme digestion, a 1138bp fragment was acqired showing bridge expression plasmid pVAXAB-σC was successfully constructed. pVAXAB-aC was then ligated with aB gene to construct dual-cistron eukaryotic expression plasmid pVAXAB-σB-aC. After identified by PCR amplification and enzyme digestion a 1100bp appeared, showing the dual-cistron eukaryotic expression plasmid pVAXAB-σB-σC.The eukaryotic expression plasmids pVAX-σB, pVAX-aC and pVAXAB-σB-σC were then used to transfect COS-7 cells,the result of indirect immunofluorescence showed the newly constructed and existing plasmid can expression expected proteins and the proteins possesseed good reactionogenicity.2 Immunogenicity research of ARVσB andσC gene vaccine mediated by Attenuated Salmonella typhimuriumThe constructed eukaryotic expression plasmids were electroporated into S. typhimurium SL7207. Chickens were orally immunized with 1.0×109CFU of SL7207 (pVAX-σB). The resut showed the vaccine constructed was safe to chickens and the vaccine strain can persist at least 10 days. Chickens were orally immunized with 0.2 mL of Salmonella suspension (1×109 CFU) SL7207 (pVAX-σB),SL7207 (pVAX-aC) SL7207 (pVAXAB-σB-σC) three times at 2-week interval. The results of antibody detection showed SL7207 (pVAX-σC) generated higher IgG antibody titer compared with control groups PBS and SL7207(pVAX1)(P<0.05),whereas SL7207(pVAXAB-σB-σC) produed higher IgA antibody titer compared with control groups PBS and SL7207 (pVAX1) (P<0.01).Each vaccine group generated relatively high serum antibody IgG and mucous antibody IgA two weeks after second immunization, whereas SL7207 (pVAX-σC) generated higher antybody titer compareed with other groups (P<0.01) and antibody titers further improved two weeks after third immunization, while, SL7207 (pVAX-σC) and SL7207 (pVAXAB-σB-σC) vaccine group generated higher antibody serum antibody and mucous antibody titers compared with other groups. (P<0.01)...