摘要
根据猪瘟病毒衣壳蛋白(Core)基因序列设计一对特异性引物,RT-PCR从猪瘟兔化弱毒中扩增出完整的Core基因,经酶切和序列测定后,克隆到携带His标签的原核表达载体p ColdⅠ中,成功构建了重组质粒p Cold-Core,并转化到大肠杆菌Rosseta 2(R2)中;利用IPTG诱导表达目的蛋白并鉴定表达效果。结果表明:经终浓度为0.1 mmol/L IPTG诱导24 h后,Core基因在R2中获得分泌性可溶表达,表达量占菌体蛋白的32.6%,SDS-PAGE显示目的蛋白相对分子质量为20 ku。Western blot结果显示Core蛋白可以分别与His单抗和猪瘟病毒阳性抗血清发生特异性反应,均出现单一反应带。将目的蛋白切胶后免疫Balb/c小鼠,制备特异性抗血清,Western blot结果显示该抗血清与衣壳蛋白反应后有明显的特异性条带,经激光共聚焦鉴定抗血清能够与胞浆中的猪瘟病毒粒子发生反应。结果表明,Core基因表达成功,制备的抗血清具有生物学功能。本研究为深入探讨Core蛋白在猪瘟病毒致病机制上的作用奠定了基础。
Based on a pair of specific primers, the Core gene （307 bp） was amplified from the plasmid pMD-Core containing Core gene of classical swine fever virus （CSFV） , cloned into the prokaryotie expression vector of pCold I carrying the His label and verified by enzyme digestion and DNA sequencing. Then this recombinant plasmid pCold-Core was transformed into Escherichia coli Rosetta 2, and CSFV Core fusion protein was induced to express with 0. lmmol/L IPTG under 15℃ for 24h. The results showed that the amount of expressed protein was up to 32. 6% of total protein. The product had a molecular mass about 20kD as expected. The target protein was separated in gel slices and used to immunize BALB/c mice. The polyclonal antibody was prepared, and its specificity was demonstrated by Western blot and confocal microscope. The successful preparation of the polyclonal antibody lays a foundation for further understanding of the role of Core protein in CSFV pathogenesis.
出处
《畜牧与兽医》
北大核心
2015年第6期7-11,共5页
Animal Husbandry & Veterinary Medicine
基金
江苏省自然科学基金(BK20131317)
国家自然科学基金青年基金(31001062)
中央高校基本科研业务费自主创新重点项目(KYZ201416)
关键词
猪瘟病毒
Core蛋白
原核表达
抗血清
classical swine fever virus
Core protein
prokaryotic expression
polyclonal antibody
