Construction And Immunological Evaluation Of Recombinant Adenovirus Expressing Hemagglutinin Gene Of H5n1Subtype Avian Influenza Virus

Avain influenza is one of the most important infectious diseases of poultry, which is caused by AIV.Since the first report in Italy in1878, the world animal husbandry had been caused great loss by AIV.And the highly pathogenic avian influenza （HPAI） can infect pigs, dogs and even human in additionto fowls. In1997, the man was found be infected with AIV in Hong Kong.Then the World HealthOrganization（WHO） began to pay more attention toAIV. After that, the disease occurred inAsia areaoccasionally. But from December2003, AIV outbreaked all over the world, and even caused manyhuman deaths. So seeking ease of production, low production costs and efficient AIV vaccine is ofimportant significance for human to prevent and controlAIV.To construct recombinant adenovirus shuttle plasmids pShuttle-CMV-HA and pShuttle-CAG-HA,HAgene ofA/Chicken/ZF/1/2011（H5N1） was amplified by RT-PCR.On the one hand, HA gene wasdirectly inserted into the adenovirus shuttle plasmid pShuttle-CMV to form recombinant shuttle plasmidpShuttle-CMV-HA. On the other hand, HA gene was subcloned into the plasmid vector pCAGGS.ThenThe gene fragment containing HA gene and CAG promoter were inserted into adenovirus shuttleplasmid pShuttle to form recombinant shuttle plasmid pShuttle-CAG-HA.The recombinant shuttleplasmids were linearlized with Pme I and transformed into BJ5183,which contained adenovirusbackbone vector Adeasy-1to achieve the homologous recombination and obtain recombinantadenovirus genome plasmids named Ad-CMV-HA and Ad-CAG-HA. Then the Ad-CMV-HA andAd-CAG-HA were subsequently linearlized with PacⅠand transfected into AD293cells to getrecombinant adenoviruses rAd-CMV-HA and rAd-CAG-HA, expressing HA controlled by CMV andCAG promoters. RT-PCR, IFA and Western-blot were used to indicate that the HA gene was expressedwell in AD293cells and the expression of HA gene had correct biological activities. The TCID50of therAd-CMV-HAand rAd-CAG-HAwere evaluated as2.38×108/ml and2.56×108/ml.BALB/c mice and SPF chickens were vaccinated intramuscularly with the recombinantsrAd-CMV-HA and rAd-CAG-HA. Three weeks after the primary vaccination, BALB/c mice andchickens were vaccinated intramuscularly again, and simultaneously two groups vaccinated withadenovial vector rAd-y and PBS were included in the animal trials as control groups. The serumantibody levels were tested by hemagglutination inhibition test （HI）. The mice were challenged withA/Chicken/Anhui/2009（H5N1） virus and the chickens were challenged with A/Goose/QFY/2004（H5N1） after vaccination. The results of Immunization showed that the immune effect achieved thebest state following the boost vaccination in BALB/c mice, and rAd-CMV-HA and rAd-CAG-HAinduced high antibody levels which both could reach1:254. It was demonstrated that the rAd-CMV-HAand rAd-CAG-HA induced the antibody levels in SPF chickens which achieved1:60and1:74,respectively. The statistical analysis showed that the difference between the immune groups and controlgroups was obvious （P<0.01）. Weight changes of the immuned BALB/c mice were insignificant afterinfected with AIV, but the control groups were reduced with24.3%and22.5%on the average.It demonstrated that the rAd-CMV-HA and rAd-CAG-HA could protected mice from AIV infection. Afterchallenge with the H5N1subtype AIV, rAd-CMV-HA and rAd-CAG-HA produced protective efficacyin SPF chickens, and the protective rate was70%and80%, respectively. However, all of the controlchickers were dead in two days. These results showed that rAd-CMV-HA and rAd-CAG-HA canefficiently protect chickens against AIV infection.