重组寻常型天疱疮抗原抗血清的制备与纯化

Preparation and Purification of Antibody against Pemphigus Vulgaris Antigen

寻常型天疱疮抗原 (pemphigusvulgarisantigen ,PVA )是桥粒中相对分子质量为 130 0 0 0的糖蛋白 ,其分子由细胞外区(extracellulardomain ,EC )、跨膜区和细胞内区三部分组成 ,主要发挥作用的区域在细胞外区。为了了解PVA细胞外区的致病性位点及其相应抗体的致病作用 ,制备抗血清是必需环节。本文在已重组表达的PVAEC1 2融合蛋白的基础上进行蛋白纯化 ,并免疫家兔 ,使之产生针对融合蛋白的特异性抗血清 ,经双向免疫扩散实验 ,其效价为 1:16～ 1:32。采用辛酸沉淀法和DEAE 5 2 纤维素阴离子交换层析进行抗血清IgG抗体成分的提纯 ,总量为 12 5～ 2 2 0mg ,经间接免疫荧光检测 ,其滴度为 1:40。从而获得高滴度、高特异性的兔抗PVAEC1 2融合蛋白抗血清IgG抗体成分。

Pemphigus vulgaris antigen (PVA) is a 130 kD membrane glycoprotein consisting of three components,extracellular domain(EC),transmembrane domain(TM) and intracellular domain(IC). Among these three portions,the most important one to express its function is EC. To understand the pathogenic epitopes in EC and the pathogenic effect of their antibodies,it is necessary to prepare antibody against PVA.On the basis of the expression of PVA EC 1-2 fusion protein(FP),this protein was purified by glutatbione affinity chromatography and was used to immunize the New Zealand white rabbits. The effective titres of antibody thus prepared were 1∶16～1∶32 by double immunodiffusion testing,and the IgG fraction was purified by precipitation with caprilic acid and DEAE-52 non-exchange chromatography. The total amount of IgG was 125～220 mg with titre of 1∶40 by indirect immunofluorescence testing. A high titre,specific rabbit anti-PVA EC1-2 FP IgG antibody was thus prepared efficiently.