Prokaryotic Expression Of Human Bone Sialoprotein And Establishment Of Colloidal Gold Detection Method

Human bone sialoprotein(BSP)can be used as a marker of cancer bone metastases,bone metabolic diseases.At present,BSP detection method mainly depends on ICH,RIA and ELISA,commercially available raw materials and kits are more expensive,no reports of BSP rapid detection methods.In this work,gene sequence of BSP was finding from NCBI,and the BSP prokaryotic expression bacteriaE.coli BL21-pET-15b-BSP has been constructed by PCR,digestion,ligation and transformation.The expression of BSP was induced by IPTG.The soluble protein was expressed at 20℃,150 rpm and 0.8 mM IPTG for 6 h.Protein was purified by Ni-NTA and the purity was 96.3%,and the concentration of BSP was 7.44 mg/mL.Molecular weight of recombinant BSP has been identifiedby SDS-PAGE is about 37 kDa.Using commercial BSP and antibodies,high titers with good specificity to different epitopes have been screened by ELISA,and the BSP prepared in this paper was proved to be of good immunogenicity.A colloidal gold qualitative detection method has been established.About this method,the sensitivity,repeatabilitywas 100 ng/mL and 95%,it could be stored at 4℃for 10 months.In this work,we obtained recombinant BSP,which is expected to be used as antigen material.A rapid semi-quantitative colloidal gold immunochromatography detection method for BSP has been established,which provided a theoretical basis and technical guarantee for clinical rapid detection of serum BSP.