| Avian influenza （AI） was an acute infectious disease caused by type A influenza viruses.According to different circumstances divided into non-pathogenic pathogenic avian influenza andlow pathogenic avian influenza and highly pathogenic avian influenza. Low pathogenic avianinfluenza virus H9N2first appeared in the1960s to the early1990s, but around the world since thelate1990s chicken H9N2avian influenza virus in a large area. Serological survey of avian flufound that the H9N2positive flocks in avian influenza-positive flocks proportion more than90%,to prove that this subtype was the impact of China’s poultry industry AIV subtypes. In this study,by the preparation of monoclonal antibodies against H9N2subtype of avian influenza virusdesigned to avian influenza surveillance and diagnostic methods provide a technical basis toestablish.H9N2avian influenza virus to melt diluted allantoic cavity inoculation9to11-day-old SPFchicken embryos,37℃incubated. Mounted separate collection of H9N2avian influenza virus inthe allantoic fluid to the supernatant after centrifugation, and the precipitate was dissolved in anappropriate amount of PBS stored at-70℃standby. After the purified virus with an equal amountof Freund’s complete adjuvant emulsified for the first immunization. Each mouse100μg.14d aftersecondary immunization using Freund’s Incomplete Adjuvant emulsified antigen, the dose of100μg/PCS. Again interval14d the third immunization,the second immunization with the samedose and way to strengthen the immune cell fusion. Three immune to select the highest antibodytiter of mouse spleen cells with SP2/0cells after fusion. The first seven days after fusion with HATculture medium half the medium was changed and then began to detect in the tenth day. Afterpurification of the H9N2avian influenza virus antigen, negative sera of mice were dilution.Select the OD450value around1.0, and P/N value largest antigen as its optimal workingconcentration.8to11weeks old BALB/c mice, each intraperitoneal injection of0.5ml of sterileliquid paraffin,7-10d after intraperitoneal injection of5×106hybridoma cells. Collected theascites and centrifuged to remove grease precipitation. Collection good the supernatant withestablished ELISA method to detect ascites titer, and stored at-70℃spare. The MDCK cells wereplated to6well plates,do indirect immunofluorescence test at the microscope, where a bright greenfluorescent judged as positive, non-fluorescent judged as negative. To further identification of two monoclonal antibodies whether the anti-H9N2avian influenza virus NP monoclonal antibody,recombinant eukaryotic expression plasmid pCDNA-NP in MDCK cells transfected MDCK cellstransfected with empty pCDNA plasmid as a negative control.24h after transfection, the cells werefixed and the IFA, and finally observed under a fluorescence microscope. Where have bright greenfluorescent ruled the positive, no fluorescence judged as negative.After immunization with fusion, using the limited dilution method for3consecutivesubcloned, a total of two stable hybridoma cell lines secreting anti H9N2AIV antibody, named1E7and3D6. The hybridoma cell culture supernatants and ascites were2-fold dilution series, withthe indirect ELISA method to the determination of OD450value. The1E7cell supernatant titer1:512ascites titer of1:25600,3D6cell supernatant titer1:256, ascites titer of1:12800. Made bythe H9N2avian influenza virus infection in MDCK cells by indirect immunofluorescence, theresults showed that the two monoclonal antibodies could produce a bright green fluorescence,indicating that these two monoclonal antibodies with the occurrence of the H9N2AIV someprotein combination. IFA test,1E7and3D6appeared bright green fluorescent pCDNA-NPeukaryotic plasmid transfected MDCK cells do. Control MDCK cells transfected with emptypCDNA plasmid no fluorescence. Western-Blot experiments of two monoclonal antibodies werenot specific bands. |
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