Research Of Transgenic Avian Influenza Virus (H5N1) Surface Antigen HA And NA Genes In Plants

Bird flu （Avian influenza, AI） is an infectious disease that occurred in wild birds and poultry. In recent years, the frequent occurrence of bird flu cases has caused great losses to the poultry industry and serious threats to the public human health. Therefore, the development of efficient and inexpensive systems for AI vaccines has great significance.With the improment of immunology and modern genetic engineering technology, various important vaccines have been successfully produced in different plants and plant suspension cultures. Plant bioreactor has many advantages over other systems such as complete eukaryotic cell expression system, Security, no exogenous pathogen pollution and easy large-scale production. Therefore, transgenic plants are considered an ideal protein production system for the foreign proteins. In this subject, surface antigen HA and NA gene of H5N1 subtype of avian influenza virus were cloned into monocotyledon expression vector with bar gene. Through the transformation of Yangmai158 by particle bombardment, we identified the pattern of gene expression. Also, the feasibility was evaluated using BY-2 for production system. Main results are the followings:1) The frequency of callus induction is 96.8%, and average frequency of regeneration is 37.4%. Yangmai158 has excellent characteristics for tissue culture.2) Through the biolistic method, pTRA-Bar-HA and pTRA-Bar-NA were transformed into treated callus. Compared the 20 d and 30 d callus on induction media, we found that the former in differentiation rate and positive rate were higher than the latter. The transgenic plants were screened by PCR after selection on PPT media, and the positive rates were 1.04% and 2.55%, respectively. We used the DNA from T₁ generation for Southern blot. The results indicated that transcription of foreign genes could be expressed well in plant.3) Agrobacterium strain GV3101 containing vectors carrying the target genes and a gene encoding the kanamycin selection marker was introduced into tobacco suspension cells. Five cell lines were selected based on kanamycin resistence and growth characteristic. Genomic PCR demonstrated that three cell lines were positive.The current studies could be used as a basis for further study of avian influenza vaccine in plants.