摘要
目的表达和纯化猴痘病毒(monkeypox virus,MPXV)A29L_(17-49)蛋白,通过其制备MPXV多克隆抗体,检测抗体的结合与中和活性。方法合成MPXV的A29L_(17-49)基因片段,构建pGEX-6p-1-A29L_(17-49)原核表达载体,采用SDS-PAGE和Western blot鉴定目的蛋白,并用GST亲和层析法纯化重组蛋白;然后用纯化的A29L_(17-49)蛋白多次免疫日本大耳白兔制备多克隆抗体,测定其结合和中和抗体活性。结果测序结果表明,成功构建了原核表达载体;经SDS-PAGE和Western blot鉴定,成功表达A29L_(17-49)融合蛋白;可溶性分析结果显示,该蛋白表达产物主要存在于上清。制备的多克隆抗体结合抗体效价为1∶10~6,中和抗体最高滴度为200。结论在原核表达系统中成功表达MPXV A29L_(17-49)蛋白,制备了具有结合活性和中和活性的多克隆抗体,可用于MPXV血清学检测的进一步研究。
Objective To express and purify monkeypox virus(MPXV)A29L_(17-49)protein,and to prepare MPXV polyclonal antibody,and to detect the binding and neutralizing activity of the antibody.Methods The A29L_(17-49)gene fragment of MPXV was synthesized,the pGEX-6p-1-A29L_(17-49)prokaryotic expression vector was constructed,the target protein was identified by SDS-PAGE and Western blot,and the recombinant protein was purified by GST affinity chromatography.Then,polyclonal antibodies were prepared by immunizing Japan big-eared white rabbits with purified A29L_(17-49)protein,and their binding and neutralizing antibody activities were determined.Results The results of next-generation sequencing showed that the prokaryotic expression vector was successfully constructed.and the A29L_(17-49)fusion protein was successfully expressed by SDS-PAGE and Western blot.The results of soluble analysis showed that the protein expression product mainly existed in the supernatant.The polyclonal antibody binding antibody titer was 1∶10~6,and the highest titer of neutralizing antibody was 200.Conclusion MPXV A29L_(17-49)protein was successfully expressed in the prokaryotic expression system,and polyclonal antibodies with binding and neutralizing activities were prepared,which can be used for further research on the serological detection of MPXV.
作者
唐一
季雅琦
段盼盼
李玉薇
卢佳
王泽鋆
TANG Yi;JI Yaqi;DUAN Panpan;LI Yuwei;LU Jia;WANG Zejun(Biosafety(level 3)Laboratory,Wuhan Institute of Biological Products Co.,Ltd.,Wuhan 430000,Hubei Province,Chind)
出处
《微生物学免疫学进展》
CAS
2024年第5期19-25,共7页
Progress In Microbiology and Immunology
基金
湖北省重点研发项目(2023DJC159)。
