重组G145R HBsAg亲和纯化方法的建立及其应用

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

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。

Objective To develop the afiqnity chromatography for purification of G145R mutant of recombinant hepatitis B sur- face antigen（HBsAg）. Methods Collect G145R mutant of HBsAg from culture supematant of 2A8 cells by salting out with ammonium sulfate and purify by afiqnity 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 afiqnity 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 afiqnity 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.