摘要
目的 大肠杆菌细胞表达和镍柱纯化猴痘病毒(MPXV)A23R蛋白,制备小鼠抗MPXV A23R的高效价抗血清。方法构建重组质粒pET-28a-MPXV-A23R,并转化至大肠杆菌BL21中诱导表达,优化蛋白表达条件后获得大量蛋白。镍柱纯化重组蛋白A23R并进行Western blot法鉴定。纯化后的A23R蛋白免疫小鼠,制备A23R多克隆抗体,采用ELISA检测抗体免疫滴度。结果 在0.6 mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)、诱导温度37℃、诱导20 h时,获得的A23R重组蛋白表达量最大,纯化后的蛋白纯度约为96.07%,经Western blot法鉴定为目的蛋白。重组蛋白免疫小鼠,第6周时IgG抗体的效价达到1∶102 400。结论 成功获得高表达和高纯度的重组A23R蛋白,并制备了小鼠抗MPXV-A23R的高效价血清。
Objective To express the monkeypox virus(MPXV)A23R protein in Escherichia coli and purify by Ni-NTA affinity column,and to prepare mouse antiserum against MPXV A23R.Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein.After optimizing the conditions of expression,A23R protein was highly expressed.Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis.The purified protein was used to immunize mice for preparing the A23R polyclonal antibody,and the antibody titer was detected by ELiSA.Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-β-D-thiogalactoside(IPTG),37℃and 20 hours.The purity of the protein was about 96.07%and was identified by Western blot analysis.The mice were immunized with recombinant protein,and the titer of antibody reached 1:102400 at the 6th week after immunization.Conclusion MPXV A23Ris expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.
作者
王毅豪
李名志
贾梦乐
杨领弟
熊嘉琪
王婷
王宇
刘树荣
郭韫丽
孔令保
黎美凤
WANG Yihao;LI Mingzhi;JIA Mengle;YANG Lingdi;XIONG Jiaqi;WANG Ting;WANG Yu;LIU Shurong;GUO Wenli;KONG Lingbao;LI Meifeng(Institute of Pathogenic Microorganism,Nanchang City Key Laboratory of Animal Virus and Genetic Engineering,Department of biotechnology College of Bioscience and Engineering,Jiangxi Agricultural University,Nanchang 330045,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第7期642-648,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(31860038)
南昌市动物病毒与基因工程重点实验项目(2021-NCZDSYS-008)
江西省自然科学基金(20224BAB215036)
江西省教育厅科技基金(GJJ2200442)。
