利用猪源SUMO3标签蛋白可溶性表达猪腺病毒3型的Hexon基因高变区蛋白  

Soluble expression of Hexon protein of porcine adenovirus serotype 3 using the pigSUMO3 fusion tag

作　　者：李志要 孙阳阳 鲍恩东[1,2] 吕英军 LI Zhiyao;SUN Yangyang;BAO Endong;LYU Yingjun(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Tianjin Ringpu Bio-technology Co.Ltd,Tianjin 300308,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]天津瑞普生物股份有限公司,天津300308

出　　处：《畜牧与兽医》2020年第3期107-111,共5页Animal Husbandry & Veterinary Medicine

基　　金：江苏高校优势学科建设工程资助项目(PAPD)。

摘　　要：利用猪源小泛素化相关修饰蛋白可溶性表达猪腺病毒3型(PADV-3)的主要衣壳蛋白六邻体蛋白(Hexon)基因的高变区蛋白,为检测试剂盒和亚单位疫苗的开发奠定基础。本试验扩增出猪腺病毒3型的Hexon基因中的超变区(HVR1~HVR6)片段,并在其N端添加猪源的SUMO3的蛋白基因,连接到pET28a质粒,构建出pET28a-Hexon-HVR1-6、pET28a-pigSUMO3-Hexon-HVR1-6重组质粒,经过PCR以及双酶切验证并测序确认无碱基突变后,转入大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导进行表达,经超声波破碎后进行SDS-PAGE验证标签蛋白的促溶情况,将表达量最高的融合蛋白进行大量表达并纯化,Western blot鉴定分析。结果显示:在1100 bp左右可见扩增出来目的基因片段;SDSPAGE表明在32 kDa、37 kDa处可以看到目的蛋白,细菌破碎后发现SUMO3标签蛋白显著提高Hexon-HVR1-6蛋白可溶性;免疫印迹结果显示融合蛋白可以被His6标签的特异性抗体和PADV-3的阳性血清特异性识别。以上结果表明成功表达出了PADV-3的主要结构蛋白Hexon-HVR1-6,加入猪源的SUMO3标签后能显著提高重组蛋白的可溶性,且反应性和特异性良好。The hypervariable region protein that is the main capsid protein(Hexon)of porcine adenovirus 3(PADV-3)was solubly expressed using the small ubiquitin-related modifier protein,which provided foundation for the development of a detection method or a subunit vaccine of PADV-3.The hypervariable regions(HVR1~HVR6)of Hexon were amplified by PCR and by adding the N-terminal SUMO gene of pigSUMO3.Then they were ligated into pET28a using the T4 DNA ligase;and plasmids(pET28a-Hexon-HVR1-6)were constructed.The recombinant plasmids were verified by PCR through a pair of special primers of pET28a(T7/T7 terminal),transformed into E.coli BL21(DE3)after double enzyme digestion,and induced in expression by IPTG.The proteins were identified by SDS-PAGE and western blot.The results showed that the gene fragments could be seen of a size of about 1100bp.The SDS-PAGE assay showed the target proteins were expressed with a molecular weight of 32KDa or 37KDa.Hexon-HVR1-6 proteins with the pigSUMO3 fusion tag were abundantly solubly expressed in the whole cell lysates.The western blot analysis indicated that the fusion protein could specifically react with the anti-PADV-3 serum and His6 tag antibody.These results suggested that Hexon-HVR1-6 protein,the structural protein of PADV-3,was successfully expressed and the pigSUMO3 tag significantly improved the solubility of Hexon-HVR1-6.The fusion protein showed high activity and specificity.

关 键 词：猪腺病毒3型 六邻体蛋白高变区 SUMO3 可溶性表达 

分 类 号：S852.3[农业科学—基础兽医学]