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当前位置: 首页 > 文章 > 8型蓝舌病病毒NS3蛋白的原核表达及鉴定 黑龙江畜牧兽医 2020 (6) 74-79
Position: Home > Articles > Prokaryotic expression and identification of serotype 8 Bluetongue virus NS3 protein Heilongjiang Animal Science and Veterinary Medicine 2020 (6) 74-79

8型蓝舌病病毒NS3蛋白的原核表达及鉴定

作  者：
史卫军;黄超华;曹琛福;杨俊兴;林彦星;王潇;花群义
单  位：
华南农业大学兽医学院;深圳海关动植物检验检疫技术中心
关键词：
蓝舌病病毒;NS3蛋白;载体构建;原核表达;鉴定
摘  要：
为了获得可初步鉴别诊断疫苗免疫和自然感染的蓝舌病病毒(BTV)抗原,试验将8型BTV S10基因中NS3蛋白对应的开放阅读框(ORF)克隆到原核低温诱导表达载体pColdⅠ中,构建重组表达质粒pCold-NS3,然后转化至大肠杆菌BL21(DE3)感受态细胞中,通过对IPTG浓度和诱导温度条件优化,确立重组蛋白NS3最佳表达条件,通过镍柱纯化后用Western-blot和间接ELISA鉴定其免疫活性。结果表明:成功构建了pCold-NS3重组质粒,重组菌株在温度为15℃、以1.0 mmol/L IPTG诱导24 h时重组NS3蛋白表达量最高,分子质量约为29 ku;纯化后的重组蛋白能与BTV自然感染阳性血清和His-tag单克隆抗体发生特异性结合。说明成功获得了具有良好反应原性的BTV重组NS3蛋白。
译  名：
Prokaryotic expression and identification of serotype 8 Bluetongue virus NS3 protein
作  者：
SHI Weijun;HUANG Chaohua;CAO Chenfu;YANG Junxing;LIN Yanxing;WANG Xiao;HUA Qunyi;Inspection and Quarantine Center for Animals and Plants of Shenzhen Customs;College of Veterinary Medicine, South China Agricultural University;
关键词：
Bluetongue virus(BTV);;NS3 protein;;vector construction;;prokaryotic expression;;identification
摘  要：
In order to obtain the preliminary differential diagnosis Bluetongue virus(BTV) antigen to distinguish between natural infection and vaccine immunity, S10 gene of BTV serotype 8 was inserted into the prokaryotic expression vector pColdⅠto obtain the recombinant plasmid pCold-NS3 and then transformed the plasmid into E.coli BL21(DE3). The optimal expression condition of recombinant protein NS3 was determined by optimizing IPTG concentration and induction temperature. The recombinant protein was purified by nickel column and identified by Western-blot and ELISA assay. The results showed that pCold-NS3 recombinant plasmid was successfully constructed. The recombinant strain showed the molecular weight was about 29 ku and the highest expression of the recombinant NS3 protein was obtained when the recombinant strain was induced with 1.0 mmol/L IPTG at 15 ℃ for 24 h. The purified recombinant protein could specifically bind to BTV naturally infected serum and His-tag monoclonal antibody. It indicated that BTV recombinant NS3 protein with good reactivity was successfully obtained.

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