摘要
利用SUMO标签制备痘苗病毒(vaccinia virus,VACV)蛋白D8的纳米抗体,并分析其与抗原是否具有结合活性.设计并合成序列,构建原核表达质粒pKMD-SUMO-D8,将其转化大肠杆菌(Escherichia coli,E.coli)BL21(DE3)感受态细胞并诱导表达;纯化带有His标签和SUMO标签的抗体Anti-SUMO-D8,之后利用SUMO蛋白酶切除His标签和SUMO标签,进一步获得Anti-D8;最后,应用间接酶联免疫吸附试验(ELISA)及间接免疫荧光试验(IFA)对其结合活性进行检测并分析.结果显示,成功构建了原核表达质粒pKMD-SUMO-D8和重组菌株,在30 ℃条件下,终浓度为0.1 mmol/L的IPTG诱导表达菌株4 h时,Anti-SUMO-D8表达量最佳;获得了高纯度的Anti-SUMO-D8和Anti-D8纳米抗体;间接ELISA结果显示,Anti-SUMO-D8和Anti-D8与抗原均具有结合活性;在相同条件下,Anti-D8与VACV及E8L的结合活性高于Anti-SUMO-D8.间接IFA结果显示,VACV侵染48 h后,试验组部分细胞核在荧光显微镜下可见绿色荧光.利用SUMO标签可高效制备VACV蛋白D8的纳米抗体,且制备的纳米抗体具有一定的结合活性.本研究结果为纳米抗体的制备提供了新思路,也为深入研究VACV蛋白D8纳米抗体的生物学功能奠定了基础.
Abstract
This study aims to obtain nanoantibody of vaccinia virus protein D8 by SUMO tag and analyze its binding activity with antigen.The sequence was designed and synthesized to construct the prokaryotic expression plasmid pKMD-SUMO-D8,which was transformed into Escherichia coli BL21(DE3)receptor cells and induced to express.Anti-SUMO-D8 with His and SUMO tags were purified,and then all tags were removed by SUMO protease to further obtain Anti-D8.Final-ly,the binding activity was detected and analyzed by indirect ELISA and IFA.The results showed that the prokaryotic expression plasmid pKMD-SUMO-D8 and recombinant strains were construc-ted.Under the condition of 30 ℃,the expression of Anti-SUMO-D8 was best when induced by IPTG with 0.1 mmol/L for 4 h.High purity Anti-SUMO-D8 and Anti-D8 nanoantibodies were ob-tained.Indirect ELISA results showed that both Anti-SUMO-D8 and Anti-D8 had binding activity with antigen.Under the same conditions,the binding activity of Anti-D8 to vaccinia virus and E8L was higher than that of Anti-SUMO-D8.Indirect IFA results showed that 48 h after infection with vaccinia virus,some nuclei of the experimental group showed green fluorescence under fluorescence microscope.Nanoantibodies of vaccinia virus protein D8 could be efficiently prepared by SUMO tag,and the prepared nanoantibodies had binding activity.This study provides new idea for the preparation of nanoantibody,and also lays a foundation for further study of the biological function of vaccinia virus protein D8 nano-antibody.
基金项目
全国重点实验室课题基金资助项目(SKLPBS2223)
维普
万方数据