Establishment Of A Visual Protein Chip Method To Differentiate Between Avian Influenza Nature Infection And Vaccine Immune Antibody

Avian influenza is a type of forma A avian influenza virus (AIV) caused by infectious diseases in poultry. Severity of the disease depends on the strain of the subtype, virulence of the virus, infected poultry species, age, gender, complications and other factors. In which highly pathogenic avian influenza (HPAI) is characterized by acute onset, and spread rapidly and high mortality, often appeared military destruction of infected flocks, brought about a tremendous economic losses to many countries and regions of the poultry industry. What the world most worried about is the Avian Influenza virus can cross species barriers to infect humans, so far, a number of countries around the world have reported human infected by Avian Influenza virus, Avian influenza poses a huge potential threat to human public health. At present, the main useful method of prevention and control avian Influenza is vaccines for immunization inoculation, immunized birds produced HI antibody in vivo, depending on HI experiment monitors the level of antibody. However, the body will also have HI antibody nature infected poultry, so we can't determine the increase of HI antibody was due to immunization or infection, which brings about difficult to avian influenza epidemiological surveillance and diagnosis. Therefore, It's very important for wild toxic method of identifying nature infection serum and vaccine immunization serum.Protein chip technology is a high throughput analysis of protein expression, protein interactions with protein and the analysis of enzyme activity.which is the powerful tool. Protein chip contains multiple capture probes can simultaneously detect different target protein, making it an effective method, which can diagnose many diseases and many diseases associated Proteins. However, the downside is that tradition protein chip test requires expensive scanning equipment. Immune colloidal gold technique is study of the markers based on namometer gold for the marker on the antigen or antibody material positioning, qualitative and quantitative. On this basis, Danscher improves and develops the immune gold silver staining, which greatly improved the detection sensitivity. The colloidal gold as a protein marker used protein chip research can throw off the classic chip results imperative and expensive laser scanning system, to achieve visual interpretation of the results. This study binding protein chips and immune colloidal gold technique, an algorithm can be raised distinguishing avian Influenza vaccine and nature infected with avian visual toxic protein chip diagnostic technology platform.Corrected by PCR and sequencing, the recombinant plasmid is transformed into BL21 (DE3) Escherichia coli, by IPTG induction, recombinant protein as inclusion body got into a lot of expression, molecular mass is about 34ku in line with expectations.Identified by Western-blotting, the recombinant protein has a good immune responsiveness of the protein expression. The visual diagnostic protein chip platform is established by using recombinant NS1 protein as antigen.In the experiment, the expression of NS1 protein was diluted 10 grads from 250μg/ml to 5000μg/ml, and other conditions was fixed for capturing optimal antigen concentration, and determining the 3000μg/ml was the optimal concentration. Known AI infections serum and immune sera each 25, SPF chickens serum, ND positive serum and IBD positive serum each 10 were detected by using protein chips, the test results were 100% in accordance by infected serum and immune serum, in dicating that the method has a good accuracy. SPF chicken serum, ND positive serum, IBD positive serum test hasn't nonspecific reaction, indicating a good specificity of protein chip. Infected serum collected at different time points were detected by using protein chip, would detected on day 8 of infection ,but detected by agar gel precipitin test (AGP) in the first 14 days of infection. Obviously, protein-chip detection is more sensitive than AGP method. Established diagnostic test platform for protein chip can quickly and accurately divide the nature infection and vaccination immune poultry, the test results can be determined by the naked eye alone, be helpful of using at any moments, which is the new breakthroughs for controlling and purification technology of avian influenza.