| Objective:To establish a suitable fermentation expression process,purification route and purity detection method for batch production of hepatitis B core virus like particles（HBc VLPs）,and to prepare HBc VLPs with good purity,which were loaded with CpG oligodeoxynucleotide（CpG-ODN）as drug carrier CpG@HBc VLPs to explore the clinical application of HBc VLPs nanocomposites in the treatment of breast cancer and other solid tumors.Method1.HBc VLPs were produced by E.coli expression system,and the effects of different initial medium p H,induction time,inducer concentration,induction temperature and induction time on cell growth and target protein expression were verified to determine the optimal fermentation conditions.2.Gel filtration chromatography,ion exchange chromatography,sucrose density gradient centrifugation and other protein purification techniques were used to purify the expressed target protein and enhance the purity of HBc VLPs.3.High performance liquid chromatography（HPLC）was used to detect the protein purity of each stage in the purification process of HBc VLPs,and the specificity,linearity and repeatability of the method were verified.4.HBc VLPs with good purity were prepared by depolymerization encapsulation method CpG@HBc VLPs nanocomposites.Using the prepared CpG@HBc VLPs were injected in situ into a mouse model of breast cancer CpG@HBc VLPs.The safety and efficacy of HBc VLPs nanocomposites were evaluated.Result1.A new process of HBc VLPs production by E.coli expression system was established.The optimal fermentation conditions were determined as follows:initial medium p H 7.2,induction time 1.8,IPTG inducer concentration 0.8 mmol/L,induction temperature 26℃,induction time 16 h.The OD600 value of the cell could reach 3.4,and the target protein expression could reach 0.36 g/L.2.A separation and purification route was established by gel filtration chromatography,ion exchange chromatography and sucrose density gradient centrifugation.The purity of HBc VLPs could reach more than 95%by HPLC.3.To verify the method of high performance liquid chromatography（HPLC）for detecting the purity of HBc VLPs,HBc VLPs showed a special absorption peak within the set integral range;when the sample concentration was 0.02-1 mg/ml,the solution concentration was linearly correlated with the absorption peak area,and the correlation coefficient was R2=0.999;the relative standard deviation（RSD）of peak area for six repeated injections was 0.47%.The specificity,linearity and repeatability of the method meet the requirements.4.CpG@HBc VLPs compared with the control group,HBc VLPs nanocomposites significantly inhibited the tumor growth of 4T1 tumor bearing mice,with a tumor reduction of 60.29%.Conclusion1.A complete set of hepatitis B core protein like particles expression,purification and quality inspection technology was established in this study,which can be used to prepare high purity HBc VLPs in batches.2.CpG@HBc VLPs nanocomposites have significant inhibitory effect on breast cancer. |
