The Construction Of Expression Vectors Of Avian Influenza Virus Hemagglutinin Fusion Gene And Tts Expression In Tobacco And Lotus

Avian influenza virus hemagglutinin (AIV HA) is the most important protective antigen of AIV, which can induce the production of specific antibodies, as well as stimulate the cytotoxic T lymphocyte (CTL) Reaction. It will be a good immune protection to the same strain of different H subtypes from vaccine which was developed from HA. The study of expression in transgenic plant and enhance the expression of HA is of great significance for the research of transgenic plant vaccine against AIV. This study is based on the establishment of genetic transformation system of Legumes Lotus and the H5N1 subtype of avian influenza virus hemagglutinin (AIV HA) gene-modified, in this study we construct several AIVHA and GFP fusion gene expression vectors with taking advantage of KDEL, MAR, BBE targeting sequence and other expression regulation components, which transformed into tobacco and lotus via Agrobacterium-mediated transfer methods. The antigen expression in transgenic plants has been significantly improved, and the results are as follows:1. PCR amplification were performed by designing specific primers, which results in CaMV 35S promoter with two enhancers, polyA and Nos terminator sequence, green fluorescent protein (GFP), nuclear matrix binding region (MAR) RB7. The ? and BBE signal sequence were modified and synthesized manually according to codon usages preferred by plant.2. Four plant expression vectors were constructed by genetic recombination technology: pNG ( based on plant expression vector pCAMBIA2301, CaMV35S promoter with double enhancers, ? sequence, NAIVHA-GFP fusion gene, PolyA, Nos terminator); pNGK( based on pCAMBIA2301, CaMV 35S promoter, NAIVHA-GFP-KDEL fusion gene, Nos terminator); p2MNGK(based on pCAMBIA2301, CaMV35 promoter with double enhancers, ? sequence, NAIVHA-GFP-KDEL fusion gene, PolyA, Nos terminator, MAR RB7); p2MNGB (based on pCAMBIA2301, CaMV35 promoter with double enhancers, ? sequence, BBE-NAIVHA-GFP fusion gene, PolyA, Nos terminator,MAR RB7).3. pNG, pNGK, p2MNGK, p2MNGB were transformed into tobacco NC89 and lotus"Leo"via Agrobacterium-mediated transfer methods.13, 54, 20, 22 positive genetic modified tobacco seedlings were detected and acquired by Kan resistance selection, PCR and RT-PCR, respectively. At the same time, 12,15,19,18 genetic modified lotus were acquired too.4. The expression of fusion protein NAIVHA-GFP was observed by fluorescence confocal laser microscopy. Results showed that strong green fluorescence in the cell membrane and the cell space in transgenic tobacco and Lotus, rather than the endoplasmic reticulum and vacuole as we expected before, we first found the avian flu hemagglutinin gene play an important role in the subcellular localization. At the same time, it will simplify Antigen purification because of the expression of avian influenza hemagglutinin gene in the cell membrane and cell space.5. The expression detection of NAIVHA-GFP in transengic tobacco and lotus were performed by SDS-PAGE and Western Blot, results showed that the expression of transgenic obacco and lotus, which transformed with pNG,pNGK,p2MNGK and p2MNGB, were increased significantly compared to pC234ANAIVHA which constructed before. The expression of pNG in tobacco and pNGK in tobacco are similar, as well as p2MNGK in tobacco and p2MNGB in tobacco, and p2MNGK in lotus and p2MNGB in lotus. The expression of p2MNGK and p2MNGB in tobacco are 2 times that of pNG and pNGK in tobacco, which indicates that MAR plays a significant role in enhancing the expression of avian influenza hemagglutinin gene. The expression of p2MNGB in lotus are 3.5 times that of pNG and pNGK in tobacco, which indicates that lotus is the optimal Bioreactor material for its high expression of foreign protein.