重组G145R HBsAg亲和纯化方法的建立及其应用

目的建立重组乙型肝炎病毒G145R变异HBsAg抗体亲和纯化方法。方法用抗-HBs单克隆抗体(D12- McAb)制备亲和层析胶,对2A8细胞(分泌G145R变异HBsAg)培养上清盐析物进行亲和纯化。采用SDS-PAGE、Western blot及ELISA,对纯化产物的纯度、特异性、含量和回收率进行鉴定,并与同法纯化的HBsAg阳性血清及r-wHBsAg提取物进行比较。结果D12-McAb对重组真核表达G145R变异HBsAg、HBsAg阳性血清及r-wHBsAS三者具有相似的亲和性,产物纯度分别为90.3%、95.2%和93.1%,回收率分别为43.3%、72.0%和66.4%。结论已成功地建立了重组G145R变异HBsAg抗体亲和纯化方法,为G145R变异以及其他HBV免疫逃逸变异感染的深入研究奠定了重要的技术基础。

Development and Application of Affinity Chromatography for Purification of Recombinant G145R HBsAg

Objective To develop the affinity chromatography for purification of G145R mutant of recombinant hepatitis B sur- face antigen(HBsAg).Methods Collect G145R mutant of HBsAg from culture supernatant of 2A8 cells by salting out with ammonium sulfate and purify by affinity chromatography using the gel coupled with anti-HBs monoclonal antibody D12-McAb.Determine the purity by SDS-PAGE,the specificity by Western blot,and the recovery by ELISA,and compare the results with those of HBsAg positive sera and r-wHBsAg purified by the same method.Results D12-McAb showed similar affinity to G145R mutant of HBsAg,HBsAg positive sera and r-wHBsAg.The purities of G145R mutant,HBsAg positive sera and r-wHBsAg were 90.3% ,95.2% and 93.1% ,and their re- coveries were 43.3%,72.0% and 66.4% ,respectively.Conclusion The affinity chromatography for purification of G145R mutant of HBsAg was successfully developed,which laid an important technical foundation of further study on infection with G145R and other im- munologic escape mutant of HBsAg.