摘要
表达犬细小病毒VP2蛋白(CPV-VP2),用于重组蛋白免疫小鼠制备单克隆抗体,并为犬细小病毒病的诊断奠定基础。采用PCR方法对CPV-VP2基因进行扩增,将CPV-VP2基因克隆到毕赤酵母分泌表达载体pPICZAA中,构建真核重组表达载体pPICZAA-VP2,将该重组质粒线性化后,转化巴斯德毕赤酵母(Pichia pastoris)X-33中,甲醇诱导表达CPV-VP2,SDS-PAGE和Western-blotting鉴定表达蛋白。结果,成功扩增了CPV-VP2基因,构建了真核重组表达载体pPICZAA-VP2,在毕赤酵母菌中表达出约68 000蛋白。Western-blotting鉴定表明,表达蛋白为目的蛋白VP2。表达菌株扩大表达体系于培养基中培养,上清液用70%过硫酸铵4℃沉淀浓缩,采用His选择镍-亲和层析柱分离纯化获得重组的酵母表达的带组氨酸标签的VP2蛋白,表达量约3mg/L。结果表明,在毕赤酵母中成功地表达了CPV-VP2蛋白,且能被犬细小病毒VP2单克隆抗体特异识别。
CPV-VP2 gene was amplified by PCR, cloned into Pichia pastoris expression vector pPICZAA,and an eukaryotic expression vector was constructed , named pPICZAA-VP21. The vector was lineazed and transformed into Pichia pastoris X-33 by electroporation method. The protein expression were induced in medium with methanol, SDS- PAGE and Western blotting a- nalysis of the culture supernatants confirmed that the VP2 protein of 68kD was expressed in Pichia pastoris. The secreted VP2 was concentrated from BMMY medium by 70% ammonium sul- fate precipitation. His-tagged VP2 protein was then isolated at 4℃ on a 20 mL HisSelect nickel- affinity column （Sigma） . HisSelect affinity chromatography yielded approximately 3 mg/L of protein. CPV-VP2 was successfully expressed in yeast Pichia astoris. The expressed CPV-VP2 protein shares reaction to monoclonal antibody against VP2.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2013年第11期1642-1646,共5页
Chinese Journal of Veterinary Science
基金
石家庄市科技支撑计划项目(09150293A)
河北科技师范学院科研创新团队项目(CXTD2012-01)
河北省自然科学基金资助项目(C2013407106)
