摘要
【目的】获取高纯度猴痘病毒(Monkeypox virus, MPXV)A5L蛋白,制备小鼠抗A5L高效价抗血清,为MPXV A5L蛋白功能研究及相关诊断试剂的研发提供材料。【方法】构建重组质粒pET-28a-A5L,转化大肠杆菌BL21(DE3)感受态细胞进行诱导表达,通过分析不同IPTG诱导浓度、诱导温度、诱导时间条件下重组蛋白A5L表达情况,筛选最佳表达条件。通过Western blotting鉴定重组蛋白A5L表达,利用SDS-PAGE分析重组蛋白可溶性。A5L蛋白经His-Bind镍柱亲和层析纯化后免疫小鼠,通过ELISA法检测抗体效价。【结果】双酶切和测序结果显示,原核表达载体pET-28a-A5L构建成功。Western blotting结果显示重组蛋白A5L在大肠杆菌BL21(DE3)感受态细胞中成功表达;A5L蛋白在诱导温度42℃、诱导剂(IPTG)浓度为1.6 mmol/L、诱导16 h后表达量最高,大小为40 ku。SDS-PAGE分析结果显示,A5L蛋白主要以包涵体形式存在,最佳咪唑洗脱浓度为50 mmol/L。ELISA结果显示,制备的鼠抗A5L蛋白抗体最高效价达1∶205 440。【结论】本研究成功构建了重组质粒pET-28a-A5L,并在原核表达系统中成功表达重组蛋白A5L,制备的A5L多克隆抗体具有较好的免疫原性,研究结果为进一步探究MPXV A5L蛋白的生物学功能奠定基础。
【Objective】The purpose of this experiment was to obtain high-purity Monkeypox virus(MPXV)A5L protein and prepare mouse anti-A5L highly valent antiserum,which provided materials for the function study of MPXV A5L protein and the research and development of related diagnostic reagents.【Method】The recombinant plasmid pET-28a-A5L was constructed,and the correctly sequenced recombinant plasmid was transformed into Escherichia coli BL21(DE3)competent cells for induction expression.The expression of recombinant protein A5L was analyzed under different IPTG induction concentration,induction temperature and induction time conditions,and the optimal expression conditions were screened.The expression of recombinant protein A5L was identified by Western blotting,and the solubility of recombinant protein was analyzed by SDS-PAGE.A5L protein was purified by His-Bind nickel column affinity chromatography and immunized mice.Antibody titers were detected by ELISA.【Result】The results of double digestion and sequencing showed that the prokaryotic expression vector pET-28a-A5L was successfully constructed.Western blotting results showed that recombinant protein A5L was successfully expressed in Escherichia coli BL21(DE3)competent cells.A5L protein expression was the highest at 42℃,IPTG concentration of 1.6 mmol/L and 16 h after induction,with a size of 40 ku.SDS-PAGE analysis showed that A5L protein mainly existed in the form of inclusion bodies,and the optimal imidazole elution concentration was 50 mmol/L.ELISA results showed that the most effective value of the prepared murine anti-A5L protein antibody was 1∶205440.【Conclusion】The recombinant plasmid pET-28a-A5L was successfully constructed and the recombinant protein A5L was successfully expressed in the prokaryotic expression system.The prepared A5L polyclonal antibody had good immunogenicity,and the results laid a foundation for further investigation of the biological function of MPXV A5L protein.
作者
熊嘉琦
贾梦乐
杨领弟
王毅豪
孙静婷
王婷
黎美凤
孔令保
彭棋
XIONG Jiaqi;JIA Mengle;YANG Lingdi;WANG Yihao;SUN Jingting;WANG Ting;LI Meifeng;KONG Lingbao;PENG Qi(College of Bioscience and Engineering,Jiangxi Agricultural University,Nanchang 330045,China;Nanchang Key Laboratory of Animal Virus and Genetic Engineering,Nanchang 330045,China)
出处
《中国畜牧兽医》
CSCD
北大核心
2024年第1期365-372,共8页
China Animal Husbandry & Veterinary Medicine
基金
国家自然科学基金项目(32202823、31860038)
江西省自然科学基金项目(20232BAB215001)。
关键词
猴痘病毒
原核表达
A5L蛋白
免疫原性
Monkeypox virus
prokaryotic expression
A5L protein
immunogenicity
