The Detection Antigen Preparation Of Type A And Subtype H Of Avian Influenza Virus

Avian influenza virus (AIV) is the pathogen of avian influenza (AI). The highly pathogenic H5N1 subtype AIV, which infected human beings and made them dead, can cause the fowl dead largely and influence the fowl industry severely. The AIV genome which encodes 10 proteins related to the viral structure and function is comprised of eight negative-strand RNA. The nucleoprotein (NP) and the matrix protein (M) are the type-specific antigens, and the haemagglutinin (HA) was the main protein of surface spikes.In this study, HA and HA1 gene of H5 AIV were expressed in insect cell by pBlueBacHis2A baculovirus vector. The Western Blotting provided the antigenicity of products without reaction to H7 and H9 antiserum of chicken and duck. Dot-ELISA showed that the expressed HA1 had good and special character at the non-denatured condition. Through the amino acid identity analysis of HA1 and HA2 genes of 15 subtypes typical AIV (H1～H15), it was found that the amino acid identity of HA1 was 9.4% to 40.2% and the amino acid identity of HA2 was 29.8% to 92.7%; and the result also showed that HA1 was the main domain of all H-subtypes. Previous study demonstrated that all the epitopes of HA were in the HA1 domain. All the above showed that the HA1 was more superior as detection antigen than HA.M1 of AIV was expressed in E coli BL21(DE3) by pET-30a(+) prokaryotic expression system and purified by affinity chromatograph of Ni²⁺ with 6 tandem histidine residues. Western Blotting provided the antigenicity of purified product. The purification could be used as the detection antigen of H5, H7 and H9 subtypes AIV anti-serum of chicken and duck. NP of AIV was expressed in E coli BL21(DE3)pLysS and Rosetta(DE3)pLysS. The expression capacity in Rosetta(DE3) pLysS which contained codons rarely used in E coli was better than in BL21(DE3)pLysS. The purified NP by Ni²⁺ affinity chromatograph could be used as the detection antigen of H5, H7 and H9 subtypes AIV anti-serum of chicken and duck.The expressed HA and HA1 could be used as the detection antigen of H5 subtype AIV anti-serum, and the NP and M1 could be used as the detection antigen of type A avian influenza virus. The detection model of "M1-duck AIV antiserum-HRP tagged rabbit IgG to duck" to duck serum was not seen before, and the study will have significance for the serologic detection and monitoring of duck.