摘要
为获得纯化的山羊地方性鼻内肿瘤病毒(enzootic nasal tumor virus of goats,ENTV)表面蛋白(surface protein,SU protein)和抗SU蛋白的多克隆抗体,根据Gen Bank已登录的ENTV Shaanxi株SU基因序列,设计合成1对特异性引物,PCR扩增SU基因并将其连接于原核表达载体p ET-28a(+)中,构建重组质粒p ET-28a-SU,经鉴定正确后转化大肠杆菌Rosetta(DE3)株进行诱导表达,并进行SDS-PAGE分析。重组菌经IPTG诱导后成功表达出分子质量为43 ku的重组蛋白。将诱导表达的蛋白经镍柱亲和层析纯化、复性后免疫小鼠制备多克隆抗体。Western-blot分析结果表明,原核表达纯化的SU蛋白能够与制得的小鼠抗SU蛋白多克隆抗体特异性结合。上述结果表明,本试验成功获得了纯化的ENTV SU蛋白和小鼠抗ENTV SU蛋白的多克隆抗体,为进一步研究SU蛋白在ENTV感染过程中的作用和建立诊断方法提供了材料。
In order to obtain the purified SU protein and polyclonal antibody against the SU protein,according to the published sequence of SU gene of enzootic nasal tumor virus of goats Shaanxi strain,a pair of specific primers were designed.The SU gene was amplified from ENTV of Shaanxi strain.The SU gene sequence was cloned into p ET-28a（＋） vector.The prokaryotic expression plasmid p ET-28a-SU containing SU gene was successfully constructed.The expression of recombinant plasmid p ET-28a-SU in Rosetta（DE3） competent cell was induced and detected by SDS-PAGE.The expressed protein was purified by Ni-NTA affinity chromatography and refolded,then used to vaccinate the mouse.The polyclonal antibody against the SU protein was obtained.Western-blot analysis showed that the fusion protein could react with the mice anti-SU polyclonal antibody specifically.In conclusion,the purified SU protein and the polyclonal antibody against the SU protein were successfully obtained,which provide materials for research on infection mechanism and diagnosis.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2016年第12期1563-1567,共5页
Chinese Veterinary Science
基金
陕西省农业科技创新与攻关项目(2016NY-092)
陕西省重点产业创新链项目(2016KTZDNY02-06)
西北农林科技大学大学生创新创业训练计划项目(201610712019)
关键词
山羊地方性鼻内肿瘤病毒
SU蛋白
原核表达
纯化
多克隆抗体
enzootic nasal tumor virus of goat
SU protein
prokaryotic expression
purification
polyclonal antibody
