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作 者:张琳[1,2] 余斌[2] 刘跃生[3] 张存[2] 华炯钢[2] 崔言顺[1]
机构地区:[1]山东农业大学动物科技学院,山东泰安271018 [2]浙江省农业科学院畜牧兽医研究所,浙江杭州310021 [3]嘉兴职业技术学院,浙江嘉兴314036
出 处:《中国兽医杂志》2014年第4期6-9,共4页Chinese Journal of Veterinary Medicine
基 金:浙江省科技厅重大科技专项项目(2011C14011);浙江省"三农六方"项目;嘉兴市科技研究计划(2012AY1063)
摘 要:用RT-PCR扩增鸭源坦布苏病毒YY5株的E蛋白基因片段,分别插入到表达载体pMBP-c、pET-GST、pET-His、pET-DsbA中,构建了一系列原核表达载体,转化人大肠杆菌E.coli Trans10或DE3(plysS)宿主菌。工程菌株经诱导后进行SDS-PAGE,结果显示MBP-E融合蛋白得到了有效表达并且以可溶性蛋白的形式存在,且能与自然感染康复的鸭源多抗和鼠源单抗反应。以MBP-E免疫小鼠制备的高免血清,在体外可以中和DDTMUV,中和抗体效价达到1:46。重组蛋白MBP-E具有良好的免疫反应原性、免疫原性和体外免疫保护特性,这为进一步研制DTMUV重组亚单位疫苗奠定基础。The envelope protein gene of duck Tembusu virus(DTMUV) was amplified by RT-PCR from DDTMUV YYS-infected DF-1 ceils and cloned into several prokaryotic expression vectors pMBP-c, pET-GST, pET-His, pET-DsbA separately to construct recombinant expression plasmids pMBP-E, pET-GST-E, pET-His-E arid pET-DsbA-E. Then the constructs were transformed into bacteria E.coli Trans 10 or BL21 (DE3)plysS. The bacteria harboring each expression vector were induced by IIYFG and protein expres sion was detected by SDS-PAGE. The results show the MBP-E fusion protein was expressed in soluble form and eapable of reacting specifically with anti-DDTMUV antiserum from naturally DDTMUV-infected rehabilitation ducks and mouse anti-DDTMUV mono clonal antibody. The antiserum prepared by immunizing mouse with MBP-E could neutralize DDTMUV in vitro and its titer measured by neutralization test reached 1:46, indicating that the MBP-E fusion protein has good immunogenicity and reactionogenicity which lay a foundation for developing sub-unit vaccines of DDTM U V.
分 类 号:S858.32[农业科学—临床兽医学]
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