抗新型冠状病毒S蛋白scFv重组基因的构建  

Construction of single-chain fragment variable recombinant gene against SARS-CoV-2 S protein

作　　者：犹桃元 杨光勇 何光志 YOU Taoyuan;YANG Guangyong;HE Guangzhi(Department of Preventive Medicine,Basic Medical School,Guizhou University of Traditional Chinese Medicine,Guiyang 550025,China)

机构地区：[1]贵州中医药大学基础医学院预防医学教研室,贵州贵阳550025

出　　处：《沈阳医学院学报》2025年第1期32-37,共6页Journal of Shenyang Medical College

基　　金：贵州省科技计划项目(No.黔科合支撑[2020]4Y187号)。

摘　　要：目的:构建抗新型冠状病毒S蛋白单链抗体(scFv)重组基因。方法:采用基因工程抗体技术,对BALB/c小鼠利用新型冠状病毒S蛋白抗原进行免疫接种,通过提取脾脏总RNA取得抗体可变区(V)重链(VH)和轻链(VL)基因序列,经过多种引物PCR扩增并筛选得到对应的VH和VL基因序列,再经过提纯回收后,通过Linker将带有限制性酶切位点的抗新型冠状病毒S蛋白VH和VL引物基因与VH、VL基因用重叠延伸PCR方法成功连接得到scFv重组基因,测序结果通过NCBI网站使用BLAST在基因库对比分析。结果:成功获得抗新型冠状病毒S蛋白VH和VL基因,并通过重叠延伸PCR技术(SOE-PCR)方法连接得到scFv,scFv经过BLSAT分析显示其基因同源性大于80%。间接ELISA检测结果显示,原核表达上清有明显的结合。结论:本研究为新型冠状病毒的诊治及研发新型相关抗体提供技术基础。Objective:To construct a recombinant gene of single-chain fragment variable(scFv)against the SARS-CoV-2 spike protein(S protein).Methods:Using genetic engineering antibody technology,BALB/c mice were immunized with SARS-CoV-2 S protein antigen.The total RNA of the spleen was extracted to obtain the heavy chain(VH)and light chain(VL)gene sequences of the antibody variable region(V).Through multiple-primer PCR amplification and screening,the corresponding VH and VL gene sequences were obtained.After purification and recovery,the VH and VL primer genes of anti-SARS-CoV-2 S protein with restriction enzyme cutting sites and the VH and VL genes were successfully connected by the overlap extension PCR method to obtain the scFv recombinant gene.The sequencing results were compared and analyzed in the gene bank by using BLAST on the NCBI website.Results:The VH and VL genes of anti-SARS-CoV-2 S protein were successfully obtained,and scFv was obtained by connecting through the overlap extension PCR technique(SOE-PCR).The BLSAT analysis of scFv showed that its gene homology was greater than 80%.The indirect ELISA detection results showed that there was obvious binding in the prokaryotic expression supernatant.Conclusion:This study provides a technical basis for the diagnosis and treatment of SARS-CoV-2 and the research and development of new related antibodies.

关 键 词：新型冠状病毒 刺突蛋白蛋白 单链抗体 

分 类 号：R392[医药卫生—免疫学]