Expression And Immunological Characteristics Of Avian Influenza Antigen Genes In Chlamydomonas Reinhardtii

Avian influenza known as fowl plague is a heavy contagious, acute, infectious disease caused by avian influenza virus. In recent years, bird flu virus has been threatening the health of the birds, but also caused great harm to human’s life and safety. As for this, preliminary diagnosis has been established for the propagation and pathogenic mechanism of avian influenza virus in many countries and regions. Although the application of antiviral drugs and vaccines are available and quite efficient now, limitations such as high cost and limited supply are still exsiting. Therefore, the production of edible protein of avian influenza antigen by genetic engineering is of important value in the theory and application. In this study, The immuno-adjuvant gene (Staphylococcus aureus enterotoxin C2gene) and avian influenza antigen gene(HAV) were integrated into the Chlamydomonas reinhardtii cc849with cell wall deficient. Then, the transformant with antigen specificities was fed for animals. The details are as follows:(1) Specific primers were designed according to the S. aureus enterotoxin C2(SEC2) gene sequence (GenBank accession number:AY450554) to clone SEC2gene. At the same time, attenuation was carried out by site-directed mutagenesis techniques with a substitution of histidine for casein acid at codon118. Then recombinant expression plasmid Pet28Asec2was transformed into competent cell BL219(DE3). The26kD target protein was purified by affinity chromatography after induced by IPTG and confirmed by SDS-PAGE analysis, which laid the foundation for eukaryotic expression in further experiment.(2) The avian influenza antigen (HAV) gene was obtained by artificial chemical synthesis and codon optimization. Both HAV gene and SEC2gene was inserted into the C. reinhardtii genetic expression vector PH124to construct expression vector PH124SEC and PH12AHAV respectively. And then the vector was transformed into cell wall free C. reinhardtii cc849with bead mill method. After that, the genes was Integrated by homologous recombination. Tran-SEC2and Tran-HAV monoclonal alga were got through antibiotic selection. The result of Southern blot analysis showed that the SEC genes and HAV genes have been successfully integrated into the cell wall free C. reinhardtii cc849. Moreover, RT-PCR indicated that foreign genes in C. reinhardtii had transcription activity. Western blot analysis proved that SEC2genes and HAV gene in C. reinhardtii could express the target proteins with the molecular weight of26kD and60KD respectively.(3) BALB/C mice was fed with mixture cultures (1:1) of trans-SEC2and trans-HAV, trans-SEC2algae, trans-HAV algae and0.9%sterile saline respectively. After that, T lymphocytes in spleen of the mice was detected by flow cytometry. The results showed that…SEC2attenuated superantigen expressed in Tran-SEC2algae stimulated the production of T lymphocytes in murine. The CD4/CD8ratio was significantly reduce than the control, while the number of CD3+and CD8+cells was significantly increased. Then the Western blot was applied to analyze the antibody tilter of mice serum based on the degree of light and shade of hybridization bands. The results showed that the hybridization band of group fed with mixture transgenic algae was brighter than that of group fed with HAV transformant only. That is to show the expressing protein of super-antigen SEC2gene does enhance the immunogenicity of the avian flu antigen gene HAV, stimulate non-specific T cell proliferation in mice and raise the immune of mice.These resultsindicated that the mixture of SEC2transgenic algae and the HAV transgenic algae could stimulate the animals to produce the antibody of avian influenza virus, and enhance the immune responses. Transgenic algae featured as a plant vaccine for avian influenza, laid an important foundation for the further development of the algae feeds with the function of preventing avian flu.