新型冠状病毒核衣壳蛋白在卡介苗中的表达及鉴定

Expression and identification of SARS-CoV-2 nucleocapsid protein in BCG

目的构建并鉴定表达新型冠状病毒核衣壳(nucleocapsid,N)蛋白的基因重组BCG。方法利用PCR技术从质粒pUC57-N(Kan^(+)抗性)中获得新型冠状病毒N蛋白全长基因。将N基因连接到大肠埃希菌-BCG穿梭表达质粒pMV261,进行酶切、PCR及测序鉴定。重组质粒经电转化导入感受态BCG,通过蛋白印迹法鉴定目的基因在重组BCG中的表达。结果经过PCR扩增获得1260 bp的N基因,构建的重组表达质粒pMV261-N序列完整,插入的基因片段与美国国家分子生物学信息资源中心报道的新型冠状病毒N蛋白全长基因的大小及序列一致。重组质粒能够在BCG中表达相对分子质量约为46000的目的蛋白,蛋白印迹法显示该蛋白能被抗新型冠状病毒N蛋白单克隆抗体识别。结论构建的重组BCG能稳定表达新型冠状病毒N蛋白,为开发新型冠状病毒N基因重组BCG疫苗奠定了基础。

Objective To construct and identify the recombinant BCG vaccine with nucleocapsid(N)protein gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).Methods SARS-CoV-2 N protein full-length gene was obtained from plasmid pUC57-N(Kan^(+))by PCR.The N gene was connected to the shuttle expression plasmid pMV261 of Escherichia coli and BCG,further identified by enzyme digestion,PCR and sequencing.The recombinant plasmid was transformed into the competent state of BCG,and the expression of the target antigen in the recombinant BCG was identified by Western blot.Results N gene with 1260 bp was obtained by PCR amplification,and the recombinant plasmid pMV261-N had complete sequence,and the fragment inserted was consistent with N gene full size and sequence of America National Center for Biotechnology Information.The recombinant plasmid could express the target protein in BCG,and its relative molecular weight was about 46000.Western blot showed that the protein could be recognized by anti-N protein monoclonal antibody.Conclusion The recombinant BCG can express the fusion protein stably,which lays the foundation for the development of recombinant SARS-CoV-2 N protein BCG vaccine.