汉滩病毒S基因5’端311bp编码蛋白的原核表达、纯化及其抗原性分析

Prokaryotic expression, purification and antigentic analysis of hantaan virus S gene 5’ terminal 311 bp fragment

目的 进一步了解汉滩病毒核蛋白氨基端的抗原特性 .方法 构建汉滩病毒 S基因 5 端 311bp片段的原核表达载体 ,并诱导表达和纯化了 Mr3.6× 10 4的融合蛋白 (汉滩病毒核蛋白氨基端 Mr 1.0× 10 4 与 Mr2 .6× 10 4 GST融合 ) ;用 m Ab,以 Western- blot和 EL ISA方法对该蛋白的抗原位点进行了鉴定 .结果 与完整核蛋白反应的 m Ab中仅有部分与 Mr 3.6× 10 4 融合蛋白反应 ,而与核蛋白氨基端 Mr 2 .6× 10 4片段反应的 5株 m Ab(1A8,A35 ,8E8,3A9,3G11)都能与 Mr3.6× 10 4 融合蛋白反应 .结论 汉滩病毒核蛋白氨基端 Mr1.0× 10 4 以内存在一个或几个线性表位 ,而羧基端可能主要起着维持完整核蛋白立体构象的作用 ,并影响识别立体表位的 m

AIM To further understand the antigen characteristic of the amino proximal of hantaan virus nucleoprotein. METHODS We constructed the prokaryotic vector of han taan S gene 5 terminal 311 bp, then inducingly expressed and purified the M r 3.6×10 4 fusion protein. Then, we further identified its antigentic epitopes by Western blot and ELISA methods using the nucleoprotein specific mAbs. RESULTS We detected that only parts of mAbs reacting with integrated nucleoprotein could react with M r 3.6×10 4 fusion protein, and all of those 5 mAbs (1A8, 3A9, A35, 3G11, 8E8) reac ting with nucleoprotein amino proximal M r 2.6×10 4 region could react with this fusion protein. CONCLUSION There exist one or more linear epitopes in the amino proximal M r 1.0 ×10 4 region of hantaan virus nucleoprotein, and the carboxy proximal region perhaps plays a significant role in maintaining the three dimensional constructions of nucleoprotein and influencing the combination of integrated nucleoprotein with mAbs which recognize the three dimensional epitopes.