Study On The Immunogenicity Of Different Molecular Adjuvants For ALV-J ENV Gene Nucleic Acid Vaccine

Avian Leukosis virus subgroup J(abbreviate as ALV-J), a new subset of avian leucosisvirus which was first extracted from Great Britain white feather broilers in1988, is the maincause of marrow cell hyperplasia and renal tumor of white feather broilers. Same as A and Bsubsets, ALV-J is also an exogenous avian leucosis virus and able to transmit vertically andhorizontally. And with horizontal transmission speed far beyond that of A and B subsets, thisdisease has rapidly spread in many countries, making poultry industry faced with a seriousproblem.At the moment when there is still no anti-ALV-J vaccine for sale, people mainly dependpurification of breeding hens to control and eradicate ALV-J, yet large expenses are cost andcomplete purification is difficult or even impossible. A large number of scientists are devotingactively to the research of ALV-J vaccine. However the researches showed that eradication ofALV-J alive is destroying its ability of inducing antibody, therefore its own complicatedantigenic structure of ALV-J failed all the attempts to get the expected attenuated vaccine.Thus, it is urgent to develop a new efficient ALV-J vaccine.As one of the new vaccines developed after1990s, nucleic acid vaccine has been foundmany advantages such as convenient preparation, durable immunity and less side effects, andshortcomings like slow production and low level of antibody which needs efficient adjuvantto overcome. In recent years, with more and more bioactive small molecules having beenproved able to enhance the immune effect of vaccine and more and more researches thatshowed selection of adjuvant can enhance the immune effect of nucleic acid vaccine, all kindsof new molecular adjuvants have been developed constantly. From these new adjuvants, IgGFc fragment, C3d fragment, Bursin and TP-5were chosen for the research. Among them, IgGfusion protein can enhance the immunogenicity of antigenic protein; researches of C3dfragment as new molecular adjuvant are in large number; and Bursin and TP-5were used asbioactive small molecules to enhance immunity of organism and widely applied in the immune therapy of severe infection, hepatitis and tumor.pcDNA3.1recombinant plasmid of ALV-J(DNA vaccines) which contains differentmolecular adjuvants was constructed using PCR, RT-PCR and overlapping PCR, and it wasidentified by double digests. In addition, indirect immunofluorescence assay (IFA) could befound within the cells after293T cells transfected by the recombinant plasmids. Furthermore,mice were injected with these recombinant plasmids. ENV-specific ELISA antibody, IFN-γlevel and IL-4level in the sera were detected.Pig C3d and chicken IgG Fc fragment gene were successfully cloned and differentmolecular adjuvant (C3d, FC, BS, TP) were connected to ALV-J ENV, which cloned intoeukaryotic expression vector of pcDNA3.1. It’s proved that ALV-J ENV recombinant ofplasmids containing different molecular adjuvant (C3d, FC, BS, TP) was successfullyconstructed by enzyme digestion test.A large number of plasmids were extracted and transfected into HEK-293T cells, andusing indirect immunofluorescence to prove the recombinant plasmid can be expressed incells after48hours. Using quantified and purified plasmids to immunized mice, using ELISAkit to determine specific antibodies of ALV-J ENV gene, IL-4and IFN-γin mice in vivo.The results showed the amount of valence of antibody reach its climax after the third immunetreatment, and pcDNA3.1-Fc-ENV was the highest,which was significantly higher thancontrol group and pcDNA3.1-ENV group. The amount of valence of antibody, IL-4and IFN-γincreased in positive group with the immune time, and it reach its climax after the thirdimmune. Immune effect was better in molecular adjuvant positive group(pcDNA3.1-Fc-ENVand pcDNA3.1-C3d-ENV) than control group without molecular adjuvant(pcDNA3.1-ENV),and immune effect treated group was better than control group. Immune efficiency ofpcDNA3.1-Fc-ENVwas the best, and antibody content of IL-4, IFN-γ group increased to33.6pg/mL,32.8pg/mL respectively.ALV-J ENV gene vaccines with different molecular adjuvant were successfullyconstructed and immune effects of different plasmid were compared, among all, immuneeffect of ALV-J ENV gene vaccine with IgG-Fc adjuvant was best.