Studies On Recombinant Duck Plague Virus Vector Expressing H7HA Or H9HA Gene From Avian Influenza Virus And Their DNA Vaccination

Avian Influenza (AI) is the poultry infection or disease syndrome caused by avianinfluenza virus A. Since it was first recognized as a serious disease of chickens in Italy in 1878,this disease has circulated in many countries and areas, causing enormously economic losses inpoultry industries. The highly pathogenic avian influenza (HPAI) is known for its sudden onsetof morbidity and high mortality, which can wipe out the whole chicken colony. Therefore,HPAI has been listed as one of the category A animal infectious diseases by the WorldOrganization for Animal Health (OIE).In this study, we firstly amplified two HA gene fragments (H7HA and H9HA subtypes) thatencode the protective epitopes of the AIV by RT-PCR. The two gene fragments were thensubcloned into pMD18-T and sequenced. The results showed that: (1) the coding region of theH7HA gene of strain is 1692 bp in length, encoding protein of 563 amino acids. Comparison ofthis gene and its product with the counterparts of other 17 known reference strains revealedhomologies of 76.3% to 84.3% at nucleotide level, and 83.3%to 93.2% at amino acid level. (2)The coding region of the H9HA gene of strain CK/MJ/0823/00(H9N2) is 1683 bp in length,encoding a polypeptide of 560 amino acids. Comparison of this gene and its product with thecounterparts of other 17 known reference strains revealed homologies of 67.1% to 95.1% atnucleotide level, and 91.9% to 99.7% at amino acid level.The H7HA and H9HA gene fragments from pMD18-T were subcloned into expressionvector p-3-3.1, which were named p-3-3.1-H7-HA and p-3-3.1-H9-HA, respectively. Therecombinant plasmids p-3-3.1-H7-HA and p-3-3.1-H9-HA were digested with XhoⅠand theH7HA and H9HA components were purified. The H7HA and H9HA components were thencloned into plasmid pUC-TK2.9-LacZ at the XhoⅠsite containing a duck plague virusreproduce negative necessary border. By this way two transposing constructs were successfullymade: one contained an eukaryotic expression component of H7HA gene, a LacZ reporter gene,and a duck plague virus reproduce negative necessary border (pUC-TK2.9-LacZ-H7-HA); theother one contained an eukaryotic expression component of H9HA gene, a LacZ reporter geneas well as a duck plague virus reproduce negative necessary border (pUC-TK2.9-LacZ-H9-HA).Finally, the H7HA和H9HA gene components were cloned into the eukaryotic expressionvector pcDNA3.1(+) under the control of the CMV promoter. The resulting constructs canserve as DNA vaccines and induce the protective immune response to the epitopes of H7HAand H9HA in the host cells.When the two DNA vaccines were used to transfect MDCK cells, the expressions of theH7HA/H9HA were detected at 48 hours after transfection. The expression was demonstrated by immunofluorescence. As negative controls, the empty pcDNA3.1(+) vector and theuntransfected MDCK cell showed no fluorescence. These experiments indicated that both DNAvaccines work well in vitro, serving as a prerequisite for further animal experiments.To further evaluate the efficacy of the two DNA vaccines in vivo, they were administratedintramuscularly to BALB/C mice (at age of 6-8 weeks) at doses of 100μg/mouse. EmptypcDNA3.1(+) vector and PBS were used as negative controls. Together, three doses wereadministrated at two weeks intervals. Bloods were taken from mice each time directly beforeinjection and once a week after the last injection. The kinetic changes of H7HA/H9HAantibodies and peripheral CD4~+, CD8~+ T cells was analyzed by HI and ELISA. The resultsshowed that: (1) the HI titer increased rapidly after administration of the vaccines, while onlylimited or no measurable titer was detected before the vaccine injection. (2) ELISA antibodyincreased in all groups of vaccination. (3) Both CD4~+ and CD8~+ T cells were up-regulated ineach vaccination group. In conclusion, our study demonstrated that the two DNA vaccinescould induce humoral and cell-mediate immune response in BALB/C mice.In summary, we have successfully cloned the H7HA and H9HA gene fragments thatencode protective epitopes of strains AIV CK/MJ/0823/00(H9N2). We then constructed twotransposing plasmids containing the H7HA/H9HA components and a recombinant duck plaguevirus. The DNA vaccines were successfully constructed via inserting the H7HA/H9HA geneinto the multiple cloning sites of the eukaryotic expression vector pcDNA3.1(+), whoseexpression was controlled by the CMV promoter. The two DNA vaccines contain the protectiveepitopes of AIV H7HA and H9HA. Their expression efficacy was examined by eukaryotic celltransfection and animal experiments. In conclusion, our study provides important experimentalmaterial and useful data for further development of vaccines and diagnostic reagents against AIdiseases.