赤羽病病毒囊膜糖蛋白G1基因的截短表达、纯化及间接ELISA诊断方法的建立  

Expression and Purification of Truncated Glycoprotein G1 Gene of Akabane virus and Development of an Indirect ELISA

作　　者：徐树兰[1] 童铁钢[1] 白宇[1] 辛九庆[1] 王群[1] 张维军[1] 孙庆歌[1] 吴东来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所国家兽医生物技术重点实验室,哈尔滨150001

出　　处：《农业生物技术学报》2008年第4期562-567,共6页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2006AA10A203);黑龙江省自然科学基金重点项目(No.ZJN-0602-01)资助

摘　　要：为获得赤羽病病毒(Akabane virus,AKAV)囊膜糖蛋白重组抗原作诊断应用研究,通过软件分析AKAV OBE-1株的囊膜糖蛋白G1的氨基酸序列,筛选出抗原性较好的基因片段G1-2作为目的片段,经RT-PCR扩增后,插入pMD18-T载体。经测序鉴定正确后,将该片段定向亚克隆于pET-28a(+)表达载体中,转化至BL21(DE3)感受态细胞中进行诱导表达。经SDS-PAGE和Western blot分析,诱导表达产物以包涵体的形式存在,大小约为44 kD,且具有免疫学活性。亲和纯化后的融合蛋白浓度为2 mg/mL,纯度为92.6%。以该融合蛋白作为诊断抗原,建立了间接ELISA诊断方法。确定的抗原包被浓度为20μg/mL,血清最佳稀释度为1∶200。交叉试验表明,该方法对牛常见的6种疾病阳性血清无交叉反应。应用该方法对病毒微量中和试验检测过的云南(77份)和内蒙(70份)牛血清样品进行了检测。以中和试验为参照,通过统计学处理,得出两地临界值分别为0.493和0.488,特异性为73%和86.9%,二者的符合率分别为80%和85.9%。To obtain a recombinant glycoprotein G1 of Akabane virus （AKAV） for diagnostic purpose, the gene fragment coding high antigenic domain ofAKAV Gl protein was amplified from the AKAV genome by RT-PCR after analyzing glycoprotein G1 with bio-soitware. The amplified product was cloned into pMD18-T vector and sequenced. Then the gene fragment was subcloned into pET-28a （＋） vector directionally. The recombinant plasmid was transformed into Escherichia coli BL21 （DE3）. SDS-PAGE and Western blotting analysis indicated that the fusion protein was insoluble and approximately 44 kD in molecular weight and had immtmologieal activity. After purified by affinity chromatography, the concentration of purified protein was 2 mg/mL and the purity was 92.6%. An indirect enzyme linked immunosorbent assay （ELISA） was developed using the purified recombinant protein. The optimal reaction conditions of ELISA were determined： the coating concentration of the purified recombinant protein was 20μg/mL; the dilution fold of the serum samples was 1：200. There was no cross reaction with positive sera of other six infectious diseases. Bovine serum samples from Yunnan （77） and Inner Mongolia （70） had been detected by serum neutralizing （SN） test before detected by the ELISA. In accordance with SN test, the positive threshold of the ELISA was determined as 0.493 and 0.488 in two districts respectively; the speciality of the ELISA was 73% and 86.9% respectively. And the agreement ratio between the two methods was 80%（52/65） and 85.9%（55/64） respectively.

关 键 词：赤羽病病毒 囊膜糖蛋白 表达纯化ELISA 

分 类 号：S188[农业科学—农业基础科学]