| Avian influenza (AI) is a highly fatal infectious disease caused by Avian influenza virus (AIV) serotype A. It is also a severe infectious diseases mainly induced respiratory disease, digestive diseases, and reproductive system. Recently, variant strains of avian influenza virus can infect human beings and cause disease even death, which become a threat for humans and raise great public health concern.At present inactivated virus were effectively used to control the outbreak of AI and its epidemic in China, but there was a new challenge of how to determine the level of immunity and evaluate the immune situation. Hence, it was essentially to establish a method to rapid detect the antibody titer of immunized chicken. It was well known that the hemagglutinin (HA) protein was the most important antigen of AIV and can be used to induce neutralization antibody. HA1 was the main the compose of HA head structure contains epitopes, HA1 can be used to detect the antibody titer of AIV.This research focused on developing and evaluating a method for rapid detection of antibody titer of H5 subtype of AIV, which was based on an indirect method of colloidal gold immunochromatographic technique. The results were summarized as follows:1. The expression of AIV hemagglutinin gene fragment and its purificationRrecombinant plasmid pKG-HAl and pKG-HA1-dsp were transformed into E. Coli BL21(DE3), respectively. the purity of recombinant proteins were analyzed by SDS-PAGE after the fusion proteins were expressed and purified from inclusion bodies. The result shows that the recombinant proteins were about 62 KDa. The result of Western blotting demonstrates that the recombinant proteins have immunologic reactivity.2. Development and Preliminary application of immunochromatographic strips for rapid detection of antibody against H5 subtype AIVUsing trisodium citrate reduction method to prepare 20nm colloidal gold particles and then using mouse against chicken IgG Fc monoclonal antibody (McAb) tag it, the optimal label condition was established in which the pH for labeling McAb is 8.0, and the mount of McAb is 7μ/mL. Colloidal gold conjugate were used as the detector reagent, the recombinant proteins of HA1 or signal peptide removed HA1, which were purified with affinity purification and rabbit IgG against mouse were immobilized on the nitrocellulose membrane as test line and control line, respectively, to develop two kinds of immunochromatographic assay for rapid detection of AIV antibody. The result of the specificity shows that the strip were positive with standard positive serum of AIV H5 and negative to positive serums of AIV H9/NDV//IBDV/IBV/MG, serum from SPF chicken. In conclusion, a specificity, sensitivity and repeatability immunochromatographic strip was established successfully.3. Development and Preliminary application of immunochromatographic strips for rapid detection of antibody against H5 subtype AIV260 serum samples collected from Jingshan and xinzhou Hebei, which were detected with HI method the experiment with the development of subtype H5 avian influenza virus antibodies test strip. The experiment results indicated that the HI method detected positive 232 serum, the detection rate was 89.2% (232/260); GST-HA1 protein in the development of rapid detection test strip were detected 225 positive samples, the detection rate was 86.5% (225/260), both compliance rate was 96.98% (225/232). GST-HA1-dsp protein in the rapid test strip was prepared by a total of 222 positive samples detected, the detection rate was 85.4% (222/260), consistent with the HI method was 95.68% (222/232). This indicates that the two proteins developed little difference between the strip test results; avian influenza H5 antibodies developed rapid detection of colloidal gold test strip method consistent with the high rate of HI.
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