摘要
[目的]在大肠杆菌中表达猪丹毒丝菌spaA基因并纯化重组蛋白。[方法]利用PCR扩增猪丹毒丝菌临床分离株spaA基因,构建重组质粒p GEX-4T-1-spaA,转化大肠杆菌BL21(DE3)进行诱导表达融合蛋白GST-SpaA,并优化表达条件。最后采用GST琼脂糖纯化树脂纯化,SDS-PAGE和Western Blotting检测。[结果]成功扩增spaA基因,获得重组表达菌株BL21(DE3)/p GEX-4T-1-spaA;在菌体OD600为0.9时,加入IPTG至终浓度0.1 mmol/L,34℃诱导6 h的条件下表达效果最好。经SDS-PAGE检测和纯化后得到大小为97 kDa的GST-SpaA;Western Blotting检测结果表明,GST-SpaA具有良好的免疫原性。[结论]成功在大肠杆菌中表达了SpaA蛋白,经纯化得到具有免疫原性的重组蛋白,为后续研制猪丹毒丝菌SpaA蛋白亚单位疫苗奠定基础。
[Objective]To express the spaA gene of Erysipeiothrix rhusiopahiae in E.coli and purify the recombinant protein.[Method]SpaA gene was amplified form E.rhusiopahiae by PCR and cloned into the expression vector to get the recombinant plasmid p GEX-4 T-1-spaA,then transformed into E.coli BL21(DE3)strain and induced to express fusion protein GST-SpaA.The expression conditions were also optimized.Finally the GST-SpaA was purified by GST resin column and detected by SDS-PAGE and Western Blotting.[Result]The spaA gene was successfully amplified by PCR and the recombinant expression strain BL21(DE3)/p GEX-4 T-1-spaA was constructed correctly.The gene was highly expressed under the condition of inducing at 34℃for 6 hours with 0.1 mmol/L IPTG when the cell concentrate OD600 reached to 0.9.A 97 k Da target protein could be detected by SDS-PAGE and purified by GST resin column.The Western Blotting analysis showed that the GST-SpaA has good immunoreactivity.[Conclusion]The spaA gene was successfully expressed in E.coli.The recombinant protein with immunoreactivity was obtained after purification.It was a potential vaccine candidate for engineering subunit vaccine for E.rhusiopahiae.
作者
刘博婷
李肇高
严舒焕
李荣君
蔡巩林
林锦铨
彭凌
LIU Bo-ting;LI Zhao-gao;YAN Shu-huan;LI Rong-jun;CAI Gong-lin;LIN Jin-quan;PENG Ling(Yingdong College of Biology and Agriculture,Shaoguan University,Shaoguan 512005,China;Joint Laboratory of Animal Infectious Diseases Diagnostic Center,Harbin Veterinary Research Institute,Caas-Shaoguan University,Shaoguan University,Shaoguan 512005,China)
出处
《生物技术》
CAS
2020年第1期12-16,共5页
Biotechnology
基金
广东省自然科学基金项目(2017A030307041)
粤北生猪生产及疫病防控协同创新发展中心资助项目
2018年度广东省大学生创新创业训练计划资助项目(201810576039)
韶关学院生态学重点扶持学科(230079030101)
2020年广东省科技创新战略专项资金-“攀登计划”专项资金(pdjh2020b0537)。
关键词
猪丹毒丝菌
表达
spaA基因
纯化
Erysipeiothrix rhusiopahiae
expression
spaA gene
purification
