抗ＨＢＶ　Ｇ１４５Ｒ　ＨＢｓＡｇ多克隆抗体的制备与鉴定.docVIP

抗ＨＢＶ　Ｇ１４５Ｒ　ＨＢｓＡｇ多克隆抗体的制备与鉴定 作者：李佩珊　龚劲松　李时君　张波　陈妍　项美娟　李方和 　　【摘要】 目的 实验性rG145R变异抗HBs多克隆抗体。方法 采用纯化全基因重组G145R变异HBsAg常规皮下免疫制备小鼠多克隆抗体，以间接ELISA、SDS-PAG及Western blot等多种实验对制备物进行鉴定，并初步应用于转染CHO细胞的化学染色。结果 制备物在Western blot、ELISA中和试验与抗原替代ELISA中与重组G145R变异HBsAg及重组野生HBsAg等均有很好的特异性与反应性；对此两种抗原的ELISA效价前者显著高于后者（分别为51200与6400）；将其用于2A8细胞(转染并分泌G145R HBsAg)爬片的PAP染色，亦取得较理想的实验结果。结论 采用重组G145R变异HBsAg免疫成功制备出较高效价的抗体。鉴于该抗体同时与野生重组wHBsAg存在较强交叉反应，其与野生抗HBs的混合应用能增ELISA免疫逃逸变异的检测能力。 　　【关键词】乙型肝炎病毒；基因变异；合成肽；多克隆抗体 　　 　　【Abstract】 Objective To prepate the mouse polyclonal Anti G145R rHBsAb and analyze its immunological characteristics． Methods The recombinant G145R mutant HBsAg were respectively used to develop poly-clonal antibodys through immunize BALB/c rats, and the polyclonal antibody were confirmed by empirical methods, include western blot, ELISA and SDS PAGE, and preliminary used in chemical staining for the CHO.Results The recombinant G145R mutant HbsAb poly were indentified by western blot and ELISA, it showed a good specific reaction performence to both of G145R rHBsAg and r-wHBsAg． And the potency of the reaction with G145R rHBsAg was obviously higher than r-wHBsAg(they were 51200 and 6400)．And it had a good result of the PAP staining of cell 2A8 also． ConclusionThe satisfactory antibody could be created by immunizing with G145R rHBsAg． Because of the cross reaction with r-wHBsAg, the recombinant G145R mutant HbsAb poly which is mixed with r-wHBsAg can enhance the detectivity of ELISA. 　　【Key words】 Hepatitis B virus; Gene variation ;Synthetic peptide; Polyclonal antibody 　　 　　HBV具有较高的变异趋势，发生在S蛋白“a”决定簇基因区段的突变可能导致其HBsAg抗原性改变而形成免疫逃逸株（immune escape mutant strain, IEMS） [1] 。由S基因第nt 587位G-A突变所产生G145R变异株（G145R IEMS）是其中发生频率最高，抗原性飘逸最彻底的IEMS之一 [1～4] 。受实验材料限制，对该变异的研究仅限于其变异机制及其分子流行病学调查。为进一步拓展对其基础与临床研究范围，笔者进行抗重组G145R变异S蛋白多克隆抗体（抗rG145R HBs poly）的制备研究，现报道如下。 　　1 材料与方法 　　1．1 实验材料 重组G145R变异S蛋白（r-G145R-HBsAg）由同济 医院 医学实验中心采用亲和层析法制备 [3] ；HBsAg “a”决定簇合成多肽（G145R-SP24，包含123～147位氨基酸）、腺病毒抗原初提物（ADV-Ag）、2A8