Development And Application Of Specific Antibodies Against Chicken High Mobility Group Box1

As a kind of nuclear protein, High mobility group boxl (HMGB1) can be released outside the cell membrane by necrotic cells or macrophages which are stimulated by inflammatory factors. After binding with RAGE or Toll-4, HMGB1would activate macrophage’s expression of inflammatory cytokines, chemokines, and adsorption factors. This progress can be suppressed to prevent the occurrence of lethal endotoxemia and sepsis. More and more research works had revealed that the secreted HMGB1was involved in development of many diseases with multiple functions. To date, the research reports focused on mammalian HMGB1, while poultry HMGB1was largely unknow. To explore the HMGB1in avian, this study made some expression constracts and developed monoclonal antibody against chHMGB1with purifide recombinant protein, and established sandwich-ELISA for detection HMGB1in avian.Part1:Gene clone and Prokaryotic expression of chHMGB1A pair of PCR primers was designed according to chHMGBl’s sequence from GenBank. The desired fragment was amplified form cDNA of CEF. After insertion into T-vector and sequencing, the fragment was digested and inserted into pGEX-6p-1and pET-32a respectively for prokaryotic expression in E.coli. SDS-PAGE and Western-Blot analysis showed that we got the soluble proteins and it would very helpful for the futher study.Part2:Development of specific antibody against chHMGB1According to the comparison with mammalian HMGB1’s amino acids sequence, a peptide of chHMGB1was selected to rabbit immunization for polyclonocal antibody development. Then purified polyclonal antibody named5527, which is against chHMGB1was acquired. Both recombinant protein and peptide was used to immunize Balb/c. After fusing the spleen cells with S/p20,10strains of hybridomas which secret monoclonal antibody against chHMGB1were acquired through screening of IFA and ELISA. Two of them have strong reactivity in indirect ELISA and IFA. The results of Western-Blot, IFA and immunohistochemistry indicated that those two monoclonal antibodies could specifically recognize both expressed and natural chHMGB1, whichprovided good reagents for the following research.Part3:Establishment of sandwich ELISA for the detection of chHMGB1The two monoclonal antibodies as previously study and polyclonocal antibody5527were purified and congigated with biotin. Grouping test confirmed that chHMGBl-2E5/Bio combined with chHMGB1-5527and chHMGB1-5527/Bio combined with chHMGB1-5527could effectively detect prokaryotic expressed and chHMGBl in cell also. The sandwich ELISA can detect as minimal as6.25ng of expressed chHMGB1. This assay provides a method for avian chHMGB1detection and we hope it will have futher use in the clinical diagnosis.