Differentiate N1and N2Subtypes Antibody Of Avain Influenza Virus By Protein Microarray

Avian influenza (AI) is an infectious viral disease of birds caused by type A strain of the influenzavirus. The highly pathogenic avian influenza virus is already panzootic in poultry with attendanteconomic consequences. It continues to cross species barriers to infect humans and other mammals.Therefore, Avian influenza virus has rightly received attention as a potential pandemic threat. As a RNAvirus, mutations in influenza occur frequently. Recombination and mutations in the surface proteinsallow the virus to elude some host immunity. Therefore, it is critical to detect the virus and identify thesubtype, the classical and gold rule of subtype identification were hemagglutinin inhibitation (HA-HI)and neuraminidase inhibitation (NA-NI) tests, but the operation is complicated and poor feasibility. Sothis study developed a new type of protein microarray, which was used to differentiate between N1andN2Avian influenza virus neuraminidase antibody.In order to obtain the neuraminidase protein N1and N2, recombinant baculovirus expressionsystem was developed. The DNA fragments of N1from H5N1AIV and N2from H9N2AIV wereamplified by RT-PCR. The genes of interest then were cloned into transfer vector, and the recombinantvectors were introduced into the bacmid in DH10B cells. Sf9cells were transfected with therecombinant bacmid DNA and the recombinant protein N1and N2were harvested. The protein N1andN2were analyzed by western bolt and neuraminidase inhibition test, the results showed the recombinantprotein N1and N2respectively had good reactivity with antiserum of H5N1and H9N2AIV. Here aprotein microarray method which is used to differentiate between N1and N2AIV neuraminidaseantibody was developed. Then the microarray was used to test positive serum samples of1to15AIVsubtypes and20chicken clinic samples, and the results showed100%compliance with NI test, while noreaction results were observed with blank SPF chicken serum and anti-IBDV serum, anti-NDV serum.The results showed that this method is specific and accurately.This method is very effective to identify the subtypes of neuraminidase, which reduces theexperiment time from2days to a few hours, and the operation avoids some toxic reagents are used. Theresults of this method from clinic serum samples were in accordance with those of the NI test, whichindicates that it is practicable to apply the protein microarray in the detecting of clinic serum samples.High-throughput differential detection of N1-N9subtypes can also be achieved by adding protein ofother subtypes into it.