摘要
为获得稳定表达猪德尔塔冠状病毒(PDCoV)核衣壳(N)蛋白的非洲绿猴肾(Vero)细胞系,本研究将PDCoV-N基因克隆入慢病毒载体中,获得重组质粒pLVX-PDCoV-N,利用慢病毒包装系统转染293T细胞,包装成表达N蛋白的慢病毒颗粒,慢病毒感染Vero细胞,嘌呤霉素加压筛选目的细胞。RT-PCR扩增N基因和测序表明细胞系基因组中存在N蛋白编码序列,Western blot和IFA试验表明N蛋白可在细胞系中稳定表达。应用制备的细胞系对临床PDCoV阳性血清样品进行检测,与ELISA检测结果符合率达到100%。本研究成功建立了稳定表达PDCoV N蛋白的Vero细胞系,为PDCoV N蛋白生物学特性研究和PDCoV的临床检测、流行病学调查奠定了基础。
To obtain a African green monkey kidney cell(Vero)line stably expressing nucleocapsid(N)protein of Porcine deltacoronavirus(PDCoV),the PDCoV N gene was cloned into a lentiviral vector to obtain the recombinant plasmid PLVX-PDCoV-N.Then,the recombinant plasmid PLVX-PDCoV-N was transfected into 293T cells by lentivirus packaging system and packaged into lentivirus particles for N protein expression.Vero cells were infected then with the lentivirus and the target cells were screened by puromycin.The results of RT-PCR and sequencing of PCR product showed the genome of the resulting Vero cell line containing N gene.The N protein that was stably expressed in the Vero cell line was confirmed by Western blot and IFA.The Vero cell line was used to detect clinical PDCoV positive serum samples and the coincidence with the results of ELISA was 100%.The study successfully established a Vero cell line stably expressing PDCoV N protein,which laid a foundation for further research of biological characteristics of N protein and clinical detection and epidemiological investigation of PDCoV.
作者
钱炳旭
薄宗义
白雪雁
张成成
郭梦娇
李梦娇
廖凯
薛峰
吴艳涛
张小荣
QIAN Bingxu;BO Zongyi;BAI Xueyan;ZHANG Chengcheng;GUO Mengjiao;LI Mengjiao;LIAO Kai;XUE Feng;WU Yantao;ZHANG Xiaorong(College of Veterinary Medicine of Yangzhou University,Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou 225000,China;International Cooperative Laboratory for Animal Health and Food Safety,Nanjing Agricultural University,Nanjing 210095,China;Joint International Research Laboratory of Agriculture and Agri-Product Safety,Ministry of Education of China,Yangzhou University,Yangzhou 225000,China)
出处
《中国动物传染病学报》
CAS
北大核心
2024年第1期56-61,共6页
Chinese Journal of Animal Infectious Diseases
基金
扬州大学高端人才支持计划
江苏高校优势学科建设工程资助项目。
作者简介
钱炳旭,男,博士研究生,主要从事动物传染病防控研究;薄宗义,男,助理研究员,主要从事动物传染病防控研究;通信作者:张小荣,E-mail:zxr@yzu.edu.cn。
