细粒棘球蚴(CE)重组抗原B(rAgB)的制备及其免疫印迹检测的初步探讨

Recombinant antigen B preparation and conformation

目的 :包虫病在我区发病率相当高 ,是牧区常见的一种人畜共患寄生虫病。目前国内 ,用血清学方法诊断细粒棘球蚴病所用抗原 B均为自然抗原 ,自然抗原必需从患病动物脏器的囊液内提取 ,其不仅获得途径困难、数量有限 ,而且容易污染环境并且所获抗原成分不纯。因此 ,人工合成 r Ag B并应用于临床检测具有深远意义。方法 :( 1)构建重组体 :用基因工程的方法构建融合质粒 p Mal Ag B( r Ag B.MBP) ,p MAL - c2 X作为表达目的基因的载体 ,r Ag B.MBP为重组抗原 B及麦芽糖连接蛋白的基因 ,r Ag B基因序列为有意义片段 ,MBP基因是淀粉柱亲和纯化所必需的 ;再转染宿主菌 JM10 9,得到重组体 JM10 9- p Mal Ag B( r Ag B.MBP) ;( 2 )目的基因的诱导表达 :JM10 9-p Mal Ag B( r Ag B.MBP)接种于含氨苄青霉素和葡萄糖的 L B培养基中 37℃气浴振荡器内过夜培养 ,次日以 4%接种量转入含氨苄青霉素和葡萄糖的 L B培养基中 37℃气浴振荡器内培养至 OD60 0 为 0 .5～ 0 .7时 ,加 IPTG诱导 (终浓度为 0 .3m M) 3h,得到融合蛋白 ;( 3)采用亲合层析法纯化抗原 ,直链淀粉树脂作为亲和柱子填充料 ;( 4)免疫印迹 Western- Blot法检测血清。结果 :( 1)经过诱导表达、纯化 ,获得了人工合成的具有生物活性的蛋白质 - r Ag B;( 2 )

Objective: Nature antigen B was extracted from Echinococcus granulosus hydatid cyst fluid. The production was effected by objective factor. Antigenic components of it were unpurification. Methods: Construction the recombinantion JM109 pMalAgB(rAgB.MBP)by Gene Project Path; Fusoin protein purification using amylose resin affinity; rAgB. MBP conformation by Western Blot method. Results: Recombinant AgB not only has bio antigenicity, but also was purification. Recombinant AgB showed approximately 91.6%(44/48)sensitivity and 93.8%(30/32)specificity by Western Blot method from conformed CE patients; showed no cross reactivity with sera from AE and tumor patients. Conclusions: Recombinant AgB was successfully expressed and purified from JM109 pMalAgB(rAgB.MBP)in China. The serologic sensiticity of rAgB was higher and its specificity was better in Western Blot. Recombinant AgB is a potential replacement for native antigen B.