Expression Of Avian Influenza Virus (H5N1) Hemagglutinin And Neuraminidase Gene In Plant

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 caused great losses and threats to the poultry industry and the public human health. Therefore, the development of efficient and safe systems for bird flu vaccine production with simple process and low price has important practical significance.With the rapid development of efficient plant expression vector systems and plant genetic transformation technology, various important proteins including vaccines and antibodies have been successfully produced in different plants and plant cells. Plant bioreactor has become an important, fast growing field of plant genetic engineering. Plant bioreactor has many advantages over other systems such as easy large-scale production, simple storage, easy use and low cost of production for foreign proteins. Plants can process post-translational modifications and proper protein folding, generating highly active molecules. Also, it is not involved in any ethical issues that were observed in transgenic animals. Thus, transgenic plants have become a new, efficient system as a bioreactor.Surface antigen hemagglutinin （HA） and neuraminidase （NA） genes of H5N1 subtype of an avian influenza virus from China were cloned into bacterial expression vector pGEX-6p-1, monocotyledon expression vector and dicotyledon expression vector, respectively. And the quantities of proteins expressed in bacteria by induction of IPTG as well as in plant leaves were purified and analyzed. Genetic transformation of calli derivde from wheat was also investigated.The results indicated that HA and NA could be expressed well in the bacteria, and affinity-purified by chromatography with GST matrix. The two genes were constructed to dicotyledon expression vector, and then were transformed into Agrobacterium tumefaciens. After the vacuum filtration of tobacco leaves for transient expression, total protein of tobacco leaves was extracted after three days. Purified proteins of both HA and NA were detected by Western blotting, and there are specific protein bands from the transiently expressed tabacco leaves, confirming proper expression of these two genes in tabocco leaves.By Agrobacterium-mediated floral dip, HA and NA genes constructed dicotyledon expression vector were transformed into Arabidopsis thaliana. The transgenic plants were screened by PCR after selection on antibiotic media, and the positive rates were 85.7% and 86.7%, respectively. Total leaf proteins of T₂ transgenic plants were isolated and used for ELISA screening, from which two transgenic plants with high expression levels were identified. Through RT-PCR and Western blot anlysis, both HA and NA proteins with expected molecular sizes were detected in the two plants. The results show that it is feasible to produce bird flu vaccine by Arabidopsis plants. The two genes constructed into monocotyledon expression vector were transformed into wheat calli by Agrobacterium-mediated method. The transformed plants were identified by PCR, suggesting the integeration of the foreign genes into wheat genome. The current studies could be used as a basis for further expression of avian influenza vaccines in plants.