摘要
猪水泡病(swine vesicular disease,SVD)与口蹄疫临床症状相似,临床中很难对该病进行快速、有效的诊断,诊断方法的建立有助于SVD的预防和控制。猪水泡病病毒(SVDV)VP1蛋白是SVD诊断试剂最常用的抗原之一,有效制备VP1抗原是诊断方法建立的关键。本研究将SVDV的VP1基因插入载体pET32a(+),获得重组质粒pET32a(+)-VP1,转化大肠杆菌BL21(DE3)细胞后经诱导表达条件的摸索,确定了VP1蛋白可溶性表达的最佳条件为:16℃下以0.5 mmol/L的IPTG诱导16 h。通过该条件诱导表达纯化获得了可溶性表达的SVDV VP1蛋白,且经2次柱纯化后获得的蛋白的纯度在90%以上;经Western-blot鉴定,目标蛋白具有很好的反应原性。总之,本研究成功获得了高水平可溶性表达的SVDV VP1蛋白,为SVD检测方法的建立和亚单位疫苗的研发奠定了基础。
Swine vesicular disease(SVD) has similar clinical symptoms to foot-and-mouth disease.It is difficult to diagnose this disease quickly and effectively in clinical practice.The establishment of diagnostic methods is helpful to the prevention and control of SVD.SVDV VP1 protein is one of the most commonly used antigens in diagnostic reagents for this disease.Effective preparation of VP1 antigen is the key to the establishment of diagnostic methods.In this study,the VP1 gene of SVDV was inserted into the vector p ET32 a(+) to obtain the recombinant plasmid p ET32 a(+)-VP1.After transforming into E.coli BL21(DE3) cells,the optimal conditions for the soluble expression of the VP1 protein were confirmed,inducing for16 h at 16 ℃ with 0.5 mmol/L IPTG.The soluble expression of SVDV VP1 protein was obtained by inducing expression and purification under this condition,and the purity of the protein was more than 90% after two times of nickel column purification.The target protein was identified by Western-blot as having good antigenicity.This study successfully obtained high-level soluble expression of SVDV VP1 protein,which laid the foundation for the establishment of detection methods and the development of subunit vaccines.
作者
景伟
白满元
郭慧琛
孙世琪
孙晓林
JING Wei;BAI Man-yuan;GUO Hui-chen;SUN Shi-qi;SUN Xiao-lin(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology/National Foot-and-mouth Disease Reference Laboraiory/Lanzhou Veterinary Research Institute,Chinese Academy of Agricullural Sciences,Lanzhou 730046,China;College of Animal Science,Yangtze University,Jingzhou 434025,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2021年第10期1310-1315,共6页
Chinese Veterinary Science
基金
国家自然科学基金项目(32072847,32072859,32002272)。
关键词
猪水泡病病毒
原核表达
可溶性表达
VP1蛋白
swine vesicular disease virus
prokaryotic expression
soluble expression
VP1 protein
