| Avian influenza viruses could infect poultry, wild birds and even humans, resulted tremendous economic loss to poultry industry and brought threatens to human health, especially H5 and H9 avian influenza viruses. Vaccination as one of the most important and effective way to prevent and control the outbreaks and spread of avian influenza was wildly used by our country. DNA vaccine was a newly developed vaccine and caused the third revolution of vaccine community, it has a number of advantages that other vaccines did not have, and had the hope to be one of the powerful weapons to eliminate avian influenza in the future. Many researches indicated that low immunogenicity was one of the key factors that influenced the immune protective effect of DNA vaccine. To evaluate the effect of codon optimization of HA genes on immunogenicity and the immune protection of avian influenza DNA vaccine, we conducted this study.The HA gene was cloned from H5N1 avian influenza virus A/chicken/Guang Dong/E85/2011(H5N1), and then was optimized according to the codon bias of chicken and we named the optimized gene as opti HA5. The codon adaptation index of opti HA5 gene changed to 0.93 from 0.78, the frequency of optimal codons changed to 80% from 43%, the GC content changed to 54% from 40%. The non-optimized HA gene and optimized gene opti HA5 were linked to mammalian expression vector p CAGGK to constructed recombinant plasmids p CAGGKHA5 and p CAGGKopti HA5. The expression of HA protein of the two recombinant plasmids was detected in 293 T cells, and the results showed that the expression efficiency of p CAGGKopti HA5 was much higher than that of p CAGGKHA5. The 3 weeks old SPF chickens were immunized with the two recombinant plasmids twice, respectively. At 2 weeks after the first vaccination and 2 weeks after the second vaccination, the mean antibody titers of chickens in p CAGGKopti HA5 group were 3.3log2 and 5.0log2, which were higher than that of pCAGGKHA5 group. The concentrations of IFN-? and TNF-? of chickens in p CAGGKopti HA5 group were higher than that of p CAGGKHA5 group, and the concentrations of IFN-? and TNF-? of chickens in two recombinant plasmids groups were significantly greater than that of inactivated vaccine group. All chickens were challenged with 100 u L 106EID50 of E85 at 2 weeks after the second vaccination. The results showed that the protection rate and virus shedding rate of p CAGGKopti HA5 group was 70% and 30%, and that of p CAGGKHA5 group was 60% and 40%, respectively. All these results showed that the DNA vaccine has significant advantage in induction of the cellular immunity, and the codon optimization of HA gene could improve the HI titers and the concentrations of IFN-? and TNF-? induced by avian influenza DNA vaccine, and the immune protective effect to chickens.The HA gene was cloned from H9N2 avian influenza virus A/Chicken/Guangdong/G14-10/2012(H9N2), and then was optimized according to the codon bias of chicken and we named the optimized gene as opti HA9. The codon adaptation index of opti HA9 gene changed to 0.94 from 0.79, the frequency of optimal codons changed to 83% from 48%, the GC content changed to 55% from 41%. The non-optimized HA gene and optimized gene opti HA9 were coloned to mammalian expression vector p CAGGS and we constructed recombinant plasmids p CAGGHA9 and p CAGGopti HA9. The expression of HA protein of the two recombinant plasmids could be detected in 293 T cells, and the results showed that the efficiency of p CAGGopti HA9 was much higher than that of p CAGGHA9. The 3 weeks old SPF chickens were immunized with the two recombinant plasmids twice, respectively. At 2 weeks after the first vaccination and 2 weeks after the second vaccination, the mean antibody titers of chickens in p CAGGopti HA9 group were 3.8log2 and 4.8log2, which were higher than that of p CAGGHA9 group. The concentrations of IFN-? and TNF-? of chickens in p CAGGopti HA9 group were higher than that of p CAGGHA9 group, and the concentrations of IFN-? and TNF-? of chickens in two recombinant plasmids groups were significantly greater than that of inactivated vaccine group. All chickens were challenged with 200 u L 108EID50 of G14 at 2 weeks after the second vaccination. The results showed that the protection rate and virus shedding rate of p CAGGopti HA9 group was 60% and 40%, and that of p CAGGHA9 group was 50% and 50%, respectively. All these results showed that the the codon optimization of HA gene could improve the HI titers and the concentrations of IFN-? and TNF-? induced by avian influenza DNA vaccine, and the immune protective effect to chickens.From the above, compared with the non-optimized HA gene from H5N1 and H9N2 avian influenza viruses, the codon optimized opti HA5 and opti HA9 gene improved the expression efficiency of HA protein of recombinant plasmids, enhanced the immunogenicity of DNA vaccines, significantly improved the protective antibody levels and the cellular immunity response induced by avian influenza DNA vaccine, and the immune protective effect to chickens. |
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