Prokaryotic Expression And T Cell Epitope Screening Of NP Gene Of H5N1 Subtype Avian Influenza Virus Isolates In Henan

Avian Influenza(AI) is an infectious poultry disease with change of general or respiratory organs caused by Avian Influenza virus(AIV), which belongs to Orthomyxoviridae Influenza virus genus, AI prevail all over the world and is one of the most dangerous disease that affect poultry farming, it can not only result in great damage to poultry farming, but also threaten human health by infecting common people.At present, the occurrence of AI is in rigorous situation, but there is no effective pharmaceutical medicine to treat the disease, so broad-spectrum vaccine that can be used to prevent high-risk AI is badly needed in order to control the disease and accelerate the development of animal husbandry and guarantee human health.Among main proteins composed of AIV, Nucleoprotein (NP) genes of influenza virus keep highly homology and strict conservatism, therefore polypeptide vaccine based on NP proteins is significant to prevent AIV infection.According to gene sequences of H5 subtype AIV published on GeneBank, NP gene was amplified from plasmid T-NP by polymerase chain reaction (PCR) method and then be used to form recombinant plasmid named pET32a-NP, the plasmid was transformed into Escherichia coli (E.coli) named Rosetta, the positive clones were identified by enzymes and PCR amplification. Sequencing results showed that target genes were inserted in the expression vector correctly, the recombinant was induced by isopropylthio-β-D-galac-toside (IPTG), The bacteria was gained at different time and was subsequently examined by SDS-PAGE,the result indicated that AIV-NP genes highly expressed in E.coli and was approximately 70kD in size, it could also react specifically with positive serum of H5N1 subtype AIV, which had good antigenicity. The chicken aged two months were immunized by expression proteins that regarded as antigen after purification through nickel column, the titer of antibody over 104 after immunizing three times.At the same time,T-cell epitope was predicted by computer and was produced by SPPS method.The concentration of PBMC was 2×106 on 96-hole cell plate after segregation, then adding the synthesized polypeptide dissolved by PBS after culturing overnight, and the amount reached to 20μg each hole,the ConA of 15μg/ml concentration could be regarded as positive control,the result showed that the reactions of P1-2 (NP36),P1-3(NP380),P3-9(NP155)were effective,which could possibly be T-cell epitope of AIV nucleoproteins.