重组汉坦病毒核蛋白的原核表达及纯化  

Prokaryotic expression and purification of recombinant hantavirus nucleoprotein

作　　者：常东英[1] 张健锋[1] 吴菲[1,2] 王玉霞[1,3] 王振萍[1] 迟祥[1] 贾媛[1] 曹玉锋[1] 陈子杨[1] 刘国瑞[1] 唐剑光[1] 时成波[1] 

机构地区：[1]长春生物制品研究所有限责任公司生物技术室,130000 [2]武汉生物制品研究所有限责任公司乙脑疫苗室,430207 [3]长春博德生物技术有限责任公司血型室,130000

出　　处：《国际生物制品学杂志》2016年第4期162-166,共5页International Journal of Biologicals

摘　　要：目的原核表达重组汉滩型病毒（Hantaanvirus，HTNV）核蛋白（recombinant HTNV nuclear protein，rHTNNP）和重组汉城型病毒（Seoulvirus，SEOV）核蛋白（recombinant SEOV nuclear protein，rSEONP），并进行纯化。方法从HTNVPS-6株和SEOVL-99株灭活病毒液中提取总RNA，依据病毒S片段多变区基因序列两端保守序列设计引物，用逆转录PCR法扩增此S基因片段，构建原核表达质粒pET-Trx-his—HTNNP和pET-Trx-his—SEONP，并在大肠埃希菌中诱导表达。表达产物经凝胶电泳初步鉴定和镍柱亲和层析纯化后，用双向免疫扩散试验检测其抗原特异性。结果重组表达质粒经菌落PCR分析和测序证明构建正确。表达的rHTNNP和rSEONP相对分子质量约为26000，表达量分别约占菌体总蛋白的30％和20％，主要以可溶性形式表达，且纯度可达约70％。双向免疫扩散试验证实，这2种重组蛋白具有较好的抗原性。结论成功构建了表达rHTNNP和rSEONP的原核表达质粒，并获得了较高纯度的rHTNNP和rSEONP，为汉坦病毒型别鉴定试剂盒的开发奠定了基础。Objective To express recombinant Hantaan virus （HTNV） nuclear protein （rHTNNP） and recombinant Seoul virus （SEOV） nuclear protein （rSEONP） in prokaryotic cells, and purify expressed proteins. Methods Total RNAs were extracted from inactivated HTNV PS-6 strain and SEOV L-99 strain. The S genome segments were then amplified by reverse transcriptase-PCR and cloned into expression vector pET-48b（＋）. The constructed recombinant plasmids pET-Trx-his-HTNNP and pET-Trx-his-SEONP were transformed and expressed in E. coll. Expression products were preliminarily analyzed by SDS-PAGE and purified by nickel ion affinity chromatography. Antigenic specificity was detected by double immunodiffusion test. Results Clony PCR analysis and DNA sequencing proved that both recombinant plasmids were constructed correctly. The relative molecular masses of rHTNNP and rSEONP were both about 26 000. rHTNNP and rSEONP were about 30% and 20%of total protein, respectively, mainly soluble. Purified products reached near 70% purity with good antigenicity. Conclusion The prokaryotic expression plasmids pET-Trx-his-HTNS and pET-Trx-his-SEOS are correctly constructed and high purity rHTNNP and rSEONP are obtained, laying the foundation for developing identification kit of hantavirus classification.

关 键 词：汉坦病毒属 核壳蛋白质类 基因表达 质粒 纯化 

分 类 号：R373.9[医药卫生—病原生物学]