Study On Mucosal Immunization Of Recombinant Proteins Of Avian Influenza Virus And Development Of NP-ELISA Detecting Antibodies Against Avian Influenza Virus

Avian influenza (AI) is one of fatal diseases caused by avian influenza virus (AIV) in avian and breaks out around the world. Since AI infected human causing disease and death in Hongkong in 1997, there have been hundreds of people infected and killed by AIV, which suggests AI become a threat for humans, and raise great public health concern.At present, the normal methods for AIV-diagnosis, such as the viral isolation and identification, hemagglutinin inhibition (HI), agar gel precipitin (AGP) and reverse transcription polymerase chain reaction (RT-PCR), are limited in spot application because of their time-consuming, complicated manipulation or requiring expensive instruments. Then the development of rapid diagnosis for AIV has become one of the problems these need to be solved urgently. Gold-immunochromatographic assay (GICA) is convenient, rapid and direct-viewing. However, the preparation of monoclonal antibody (McAb) against the different epitopes is the prerequisite for GICA. Eight McAbs against the different epitopes of H9N2 AIV developed in this study are high titer and strongly specific which established the basis for the development of GICA. At the same time of studying the antigen-diagnosis against AIV, serological detection was still an important monitoring method. Therefore, the present study developed NP-ELISA serological detection method using the characteristic of type-specificity for nucleoprotein (NP). The development of NP-ELISA can be used to monitor AIV antibodies and provide reliable means for the epidemiological investigation of AI.Hemagglutinin (HA) is the most important immunogen to produce the neutralization antibodies, and the main determinative factor of the viral mutation and the specificity of virulence and host. The vaccine using HA gene or HA protein can confer 100% protection against the different isolate of the same subtype. However, the antibodies against the different HA subtypes can not confer the cross-reaction and protection, which raise the difficulty of the detection and prevention of AI. To develop the new vaccines conferring the cross-protection, the researchers found the surface membrane protein M2. M2 protein which has potential antigenicity is one of the conserved proteins of influenza virus. The antibodies against M2 can confer the protection against different subtype and inhibit obviously the viral replication in the respiratory tract. Then M2 protein is an ideal candidate antigen to investigate vaccine with cross-protection. The present study succeeded in the alone- or fused- expression of HA and M2 providing the basis for the development of cross-protection vaccines.The mucosal immunity appeared to be important because AIV infected animals through the respiratory tract and digestive tract. Cholera toxin B subunit (CTB) can induce mucosal responses and the production of secretory IgA (sIgA) which is the primary factor in mucosal immunity. SIgA can exist in respiratory and digestive system over a long period of time for their repellence against proteinase. Because sIgA can prevent AIV...