摘要
本研究旨在通过构建pGEX-4T-1-Cap原核表达载体,制备猪圆环病毒3型(PCV3)重组核衣壳(Cap)蛋白抗原。将密码子优化的PCV3 Cap全基因插入pGEX-4T-1载体,构建重组质粒pGEX-4T-1-Cap,转化大肠杆菌BL21(DE3)感受态细胞中,筛选出最佳诱导条件,通过谷胱甘肽琼脂糖树脂纯化重组Cap蛋白。结果表明,成功构建重组质粒pGEX-4T-1-Cap;在IPTG终浓度为0.1 mmol/L、16℃诱导20 h的条件下,可获得大量可溶性重组Cap蛋白,SDS-PAGE结果表明分子质量在51.6 ku,与预期相符;Western blotting分析表明经纯化的Cap蛋白与鼠抗GST单克隆抗体、PCV3兔源阳性血清呈阳性反应,进一步证实了重组蛋白的表达。本研究表达并纯化了PCV3 Cap重组蛋白,为新型基因工程亚单位疫苗的研制和ELISA血清学诊断方法的建立奠定了基础。
The purpose of this study was to prepare porcine circovirus type 3(PCV3)capsid protein by construct a prokaryotic expression vector pGEX-4T-1-Cap.Recombinant plasmid pGEX-4T-1-Cap was constructed by inserting codon optimized PCV3 Cap gene into pGEX-4T-1 vector,and then transformed into Escherichia coli BL21(DE3)competent cell.The optimal induction conditions were screened out,and the recombinant Cap protein was purified by glutathione agarose resin.The results showed that the recombinant plasmid pGEX-4T-1-Cap was successfully constructed.A large amount of soluble recombinant capsid protein could be obtained under the condition that the final concentration of IPTG was 0.1 mmol/L and induced at 16℃for 20 h.SDS-PAGE results showed that the molecular mass of the recombinant capsid protein was 51.6 ku with the same size as expected.Western blotting analysis showed that the purified Cap protein reacted positively with mouse anti-GST monoclonal antibody and PCV3 rabbit-derived positive serum,further confirming the expression of the recombinant protein.Therefore,the recombinant protein PCV3 Cap expressed and purified in this study,it laid a foundation for the development of new gene engineering subunit vaccine and the establishment of ELISA method.
作者
谷思颖
屈勇刚
魏其
吴圆圆
于会举
李岩
GU Siying;QU Yonggang;WEI Qi;WU Yuanyuan;YU Huiju;LI Yan(College of Animal Science and Technology,Shihezi Univerisity,Shihezi 832003,China;The General Station of Animal Husbandry and Veterinary Medicine of Xinjiang Production and Construction Corps,Urumqi 830063,China)
出处
《中国畜牧兽医》
CAS
北大核心
2020年第8期2660-2665,共6页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金“新疆重要自然宿主和媒介昆虫携带人兽共患病毒的病原生态学与分子流行病学研究”(U1503283)。
