摘要
目的以纯化的内质网相关的干扰素诱导病毒抑制蛋白(viperin)免疫小鼠制备其多克隆抗体并鉴定其特异性。方法对BALB/c小鼠颅内注射鸭坦布苏病毒,刺激小鼠脑组织产生viperin,通过反转录PCR从小鼠大脑组织克隆viperin,并将其插入至pGEX-6P-1原核表达载体,转化至大肠杆菌Rosetta。用异丙基硫代半乳糖苷(IPTG)对重组蛋白诱导表达,对其进行可溶性分析,以氯化钾染色切胶法对大量表达蛋白进行纯化;将纯化的重组viperin蛋白经腹部皮下免疫小鼠制备多克隆抗体,利用间接ELISA测定多抗效价,Western blot法和间接免疫荧光法(IFA)检测BHK-21仓鼠肾成纤维细胞瞬时表达的viperin蛋白鉴定viperin抗体的特异性和敏感性。结果成功克隆并表达小鼠viperin基因,制备的抗体效价可达1∶25600,小鼠抗viperin多克隆抗体可特异性识别BHK-21细胞表达的viperin蛋白。结论成功制备特异性较好的小鼠抗viperin多克隆抗体。
Objective Mice were immunized with purified virus inhibitory protein endoplasmic reticulum associated interferon inducible(viperin)to prepare polyclonal antibody and identify specificity.Methods BALB/c mice were injected with duck tembusu virus to generate viperin in mouse brain by intracranial injection.Viperin gene,cloned from mouse brain tissue by reverse transcription PCR,was inserted into pGEX-6p-1 prokaryotic expression vector and transformed into E.coli Rosetta.The recombinant viperin protein was induced by isopropyl thiogalactoside(IPTG)and its solubility was analyzed.The protein was purified by potassium chloride(KCl)staining and gel cutting method.Polyclonal antibody was prepared by immunizing mice with purified recombinant viperin protein subcutaneously through abdomen,and the titer of polyclonal antibody was determined by indirect ELISA.Western blot analysis and indirect fluorescence assay(IFA)were used to detect the transient expression of viperin protein in BHK-21 cells to identify the specificity and sensitivity of the prepared polyclonal antibody against viperin protein.Results The mouse viperin gene was successfully cloned and the viperin protein was expressed.The titer of the prepared anti-viperin polyclonal antibody reached 1∶25600.The mouse anti-viperin polyclonal antibody could specifically recognize the transient expression of viperin protein in BHK-21 cells.Conclusion Mouse polyclonal antibody against viperin protein with high specificity and sensitivity was successfully prepared.
作者
刘丹
于胜祖
方天
王蓓
张淼
吕炫
祝萌
王晴
王桂军
LIU Dan;YU Shengzu;FANG Tian;WANG Bei;ZHANG Miao;LYU Xuan;ZHU Meng;WANG Qing;WANG Guijun(College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036;Key Laboratory of Veterinary Pathobiology and Disease Control of Anhui Province,Hefei 230036,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2022年第9期842-847,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(32072874)。
