猫传染性腹膜炎病毒N蛋白在昆虫细胞-杆状病毒系统中的表达与鉴定

本研究旨在利用昆虫细胞-杆状病毒表达系统表达猫传染性腹膜炎病毒(Feline infectious peritonitis virus,FIPV)N蛋白,并制备抗该蛋白的多克隆抗体,用于FIPV临床抗原/抗体诊断及N蛋白功能研究。参考GenBank中FIPV的N基因序列,选择FIPV毒株N基因(登录号:KC461235.1),并对该N基因进行密码子优化、基因合成,酶切后将N基因连接至pFastBac1载体,转化大肠杆菌DH5α感受态细胞,经氨苄西林抗性筛选阳性克隆,提取质粒经酶切及测序鉴定正确后,转化大肠杆菌DH10Bac感受态细胞,经蓝白斑筛选及PCR鉴定后,最终成功构建重组杆状病毒质粒Bacmid-N,转染Sf9细胞包装杆状病毒,于28℃温箱培养4 d后收集感染Sf9细胞上清,并通过镍离子亲和层析纯化获得重组N蛋白。经SDS-PAGE及Western blotting鉴定结果表明,本研究成功利用昆虫细胞-杆状病毒表达系统表达出大小约为51 ku的FIPV重组N蛋白。将该蛋白与弗氏佐剂按一定比例混合后,免疫6周龄BALB/c小鼠。用间接ELISA方法检测小鼠血清抗体效价可达1∶102400;利用Western blotting和间接免疫荧光试验对N蛋白多克隆抗体进行检测分析,结果表明,真核表达的重组蛋白免疫原性良好,多克隆抗体具有良好的反应原性,可特异识别FIPV感染细胞。本研究为FIPV抗原/抗体诊断试剂盒的研发奠定了基础。

Expression and Identification of Feline Infectious Peritonitis Virus N Protein in Insect Cell-Baculovirus Expression System

The aim of this study was to express Feline infectious peritonitis virus(FIPV)N protein by insect cell-baculovirus expression system,and prepare polyclonal antibody against FIPV for clinical antigen/antibody diagnosis and N protein function research.According to the N gene sequence of FIPV in GenBank,the N gene of FIPV strain(accession number:KC461235.1)was selected,and the N gene was optimized by codon optimization and gene synthesis.After enzyme digestion,the N gene was connected to pFastBac1 vector and transformed into E.coli DH5αcompetent cells.The positive clone was screened by ampicillin resistance.After being identified by restriction enzyme digestion and sequencing,the extracted plasmid was transformed into E.coli DH10Bac competent cells.The recombinant Bacmid-N was successfully constructed after blue and white spot screening and PCR identification.Sf9 cells were transfected with baculovirus and cultured at 28℃for 4 days.The supernatant of Sf9 cells was collected and purified by Ni ion affinity chromatography.The results of SDS-PAGE and Western blotting showed that the recombinant N protein(51 ku)of FIPV was successfully expressed in insect cell-baculovirus expression system.Six weeks old BALB/c mice were immunized with the protein and Freund’s adjuvant.The titer of serum antibody was 1∶102400 detected by indirect ELISA.Western blotting and indirect immunofluorescence assay were used to detect and analyze the polyclonal antibody against N protein.The results showed that the recombinant protein expressed in eukaryotic cells had good immunogenicity and the polyclonal antibody had good reactivity,which could specifically identify FIPV infected cells.This study laid a foundation for the development of FIPV antigen/antibody diagnostic kit.