SADS-CoV S1蛋白的原核表达及多克隆抗体制备  

Prokaryotic expression of SADS-CoV S1 protein and preparation of polyclonal antibody

作　　者：张睿玉 李龙驹 李程 周玲[1] 马静云[1] ZHANG Ruiyu;LI Longju;LI Cheng;ZHOU Ling;MA Jingyun(Laboratory of HealthyLivestock and Disease Control,College of Animal Science,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院畜禽健康养殖与疾病防控研究室,广东广州510642

出　　处：《中国兽医学报》2023年第2期229-233,共5页Chinese Journal of Veterinary Science

摘　　要：猪急性腹泻综合征(SADS)是由猪急性腹泻综合征冠状病毒(SADS-CoV)引起的仔猪急性腹泻、呕吐、脱水、体质量骤降,最终导致死亡的一类急性传染病。为制备SADS-CoV刺突蛋白(S1)的多克隆抗体,通过PCR扩增得到SADS-CoV S1基因片段,并将其克隆至原核表达载体pGEX-6p-1,构建出重组质粒pGEX-6p-1-S1。测序结果正确后将其转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,经GST标签凝胶纯化。纯化的S1蛋白与弗氏佐剂混合后经3次皮下注射免疫BALB/c小鼠后收集血清,通过免疫印迹试验(Western blot)和间接免疫荧光试验(IFA)进行检测。结果表明,重组的S1蛋白具有良好的反应原性和免疫原性,制备的鼠抗S1蛋白多克隆抗体也具备良好的反应活性和特异性,为后续SADS-CoV血清学诊断方法的建立及S1蛋白生物学功能研究奠定基础。Swine acute diarrhea syndrome(SADS)is an acute infectious disease caused by swine acute diarrhea syndrome coronavirus(SADS-CoV),which causes acute diarrhea,vomiting,severe dehydration,sudden weight loss and eventually death of piglets.In order to acquire polyclonal antibody against SADS-CoV spike protein(S1),the SADS-CoV S1 gene fragment was amplified by PCR and cloned into prokaryotic expression vector pGEX-6p-1 to construct recombinant plasmid pGEX-6p-1-S1.After the sequencing results were confirmed,the plasmid of pGEX-6p-1-S1 was transformed into Escherichia coli BL21(DE3)competent cells for induced expression.The fusion protein was purified by affinity chromatography on glutathione sepharose 4B.BALB/c mice were immunized by subcutaneous injection of the purified S1 protein mixed with Freund adjuvant for three times.Serum was collected and detected by Western blot and indirect immunofluorescence assay(IFA).The results showed that the recombinant S1 protein had good reactivity and immunogenicity,and the prepared mouse polyclonal antibody against S1 protein also had good reactivity and specificity,which laid a foundation for the establishment of follow-up SADS-CoV serological diagnosis method and the study of biological function of S1 protein.

关 键 词：猪急性腹泻综合征冠状病毒 S1蛋白 原核表达 多克隆抗体 

分 类 号：S852.65[农业科学—基础兽医学]