Cytokines Expression In Hepatitis B Virus X Gene Hydrodynamic Injection Mouse Model And The Construction Of GST-MIG, GST-IP10 Recombined Protokaryon Vector

【OBJECTIVE】To establish a gene expression analyse of chemokines and cytokines in HBx hydrodynamic injection mouse model through microarray;To construct the plamid of pET42a-MIG�'�pET42a-IP-10 which can express fusion protein for antibody production ,thus inverstigate the potential effect of Mig and IP-10 on live disease .【METHODS】1. Establish a HBx hydrodynamic tail vein injection mouse model successfully,meanwhile,establish HBx-lacked mouse model as internal control and take the untreated mice as blank control.2. Liver tissue obtained from untreated and HBx hydrodynamic injection mouse were initially analysed by DNA microarray techlology and subsequently confirmed the interested up-regulated gene by quantitative realtime PCR and then verified the chemkines and cytokines by semi quantitative RT-PCR.3. The expression of Mig and IP-10 in liver was determined by Immunohistochemistry.Furthermore,IFN-γ,the inducer of Mig and IP-10 in liver was detected via ELISA.4. Construct the recombinant plasmid pET42a-MIG and pET42a-IP-10 expressing fusion protein in E.coli BL21 for antibody production.5. Transwell:take the PBMC from the stimulated mouse as target cell,detect the chemotaxis of fusion protein.【RESULTS】1. We establish a mouse model of acute Hepatitis B virus X infection by hydrodynamic tail vein injection of HBx genome plasmid verified by HBx RT-PCR of the liver tissue.2. The consequent results indicated that abundant genes(611,including the up-regulate and down-regulate genes) changed in HBx mouse model and most selected chemokines were observable up-regulated via Realtime PCR verification;then we selected 4 chemokine genes including Ccl2,Ccl9,MIG and IP-10 via the further verification of RT-PCR.3. The immunohistochemistry detection results of of MIG and IP-10 were coinside with their gene detection results.4. IFN-γexpression was up-regulated at protein level in liver tissue,this indicate that the expression of these two chemokines and IFN-γwere induced after HBx infection.5. The recombinant plasmid pET42a-MIG and pET42a-IP-10 were successfully constructed and fusion proteins were obtained.6. Transwell expriment indicated that the two GST fusion proteins were chemotasis,thus had biological effect.【CONCLUSION】These results indicate that chemokines especially MIG and IP-10 play a crucial role in the liver injury mediated by the immune response of antiviral effect . The recombinant plasmid pET42a-MIG and pET42a-IP-10 can be used to product antibody for further study of MIG and IP-10 in the immune response afer HBV affection.