| Avian Influenza is an infection and/or disease syndrome in both wild birds and poultry, which caused by avian in fluenza virus type A of the Orthomyxoviridae family. Highly pathogenic avian influenza (HPAI) is classified to "List A" disease by the Office International des Epizooties (OIE). Several methods are employed to monitor for avian influenza, which mainly include antigen directed and antibody directed diagnosis, Conventional methods to detect virus specific antibody include Hemagglutinin Inhibition (HI) test, Enzyme-Linked Immunosorbent Assay (ELISA) and Agar Gel Diffusion Precipitation (AGP) test.Protein Chip, so called Protein Microarray, is a kind of time saving, quantative, high parallel, and high throughput new technology for the detection of proteins or peptides, which can be applied to the detection of hundreds of disease markers simultaneously. The establishment of protein chip include the selection of suitable substrate, the delivery of capture molecular, the hybridization of antibody and the removal of unspecific binding. Employing protein microarray to detect animal viral disease is one of the most important applications of microarray technology.In this study, it was aimed to setup a protein microarry method, which made use of vitro expressed recombinant protein as capture antigen, to detect the avian influenza virus specific antibody in the chicken blood sample. The main steps included expressing recombinant hemaggulitin antigen in vitro by cloning the virus surface protein HA gene into the expression vector pET30 and printing the purified recombinant protein antigen onto the chip substrate to capture the virus specific antibody. The processes included:(1) The preparation of protein antigenCloning three subtypes of hemaggultinin gene from three virus strains by RT-PCR, respectively diggested the PCR product and pET30 vector by restriction enzyme Hind III and Xho1, and then the purified products were linked by T4 ligase. The recombinant plasmids were checked by PCR, nucleotide restriction enzyme cut reaction and sequencing. The positive recombinant plasmids were transformed into BL21 (DE3) and induced by IPTG. Use sonication and other chemical lysis reagent to purify the inclusion body primarily. Later, the protein was further purified by Ni-NTA affinity chromatography method. Finally recombinant proteins were dialysised to refold. Western blotting showed that the recombinant proteins had good antigenicity.(2) The fabrication of protein microarray and optimization of conditionsSpotting the capture antigen onto the chip substrate which was chemically modified with amino- or epoxy-; Optimizing the chip parameters by the test of different pins, substrates, blocking buffers, printing buffers, and compare the feature morphology by printing under different relative humidity; Nextly, the concetration ratio of antigen to antibody was optimized; Finally, the chip's linear detection region and sensitivity were checked by the detcetion of blood-serum samples which were diluted according to concentration gradient.In conlusion, the three subtypes of avian influenza virus Hemagglutinin were well expressed in viro, and the condition of expression, purification and refloding were co-optimized. Primary results showed that our home-build recombinant protein microarry had good feature morphology and high reproducibility. Besides the detetion range of recombinant protein microarray were suprior to the indirect ELISA which was set up with the same capture antigen, and the sensitivity of microarray was 2.5 times more than indirect ELISA. The progress of this research laid solid foundation for the setup of higher density protein microarray. |
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