畜牧与兽医2024，Vol.56Issue(4)：101-107,7.
猪轮状病毒G1P[7]株分离鉴定及多克隆抗体制备
Isolation,identification and preparation of polyclonal antibody against porcine rotavirus G1P[7]
摘要
Abstract
This study was aimed to identify the causes of diarrhea in pig farms and to isolate porcine rotavirus(PoRV)in order to accumu-late biological materials for the prevention and control of the disease.Feces samples were collected from pigs inflicted with diarrhea for RT-PCR detection.The positive samples were inoculated into monolayer cells and the virus proliferation was detected by the cytopathic effect(CPE)and indirect immunofluorescence(IFA).The VP4 and VP7 genes were amplified by RT-PCR and were sequenced to determine their G/P genotypes.The genetic evolution characteristics of VP4 and VP7 genes were analyzed by bioinformatics software.The rabbit polyclonal antibody prepared by immunizing with inactivated JSNJ2019 vaccine was evaluated by Western blot and IFA.The results showed that porcine epidemic diarrhea virus(PEDV),transmissible gastroenteritis virus(TGEV),porcine coronavirus(PDCoV),porcine pseudorabies virus(PRV)and bovine viral diarrhea virus(BVDV)were all negative,and only PoRV was positive.A single layer of MA104 cells was inocula-ted with filtered fecal samples,the typical CPE was characterized by round contraction,and finally its collapse and shedding was observed af-ter 5 successive generations.The specific fluorescence of PoRV was detected by IFA,indicating successful isolation of the virus,which was named JSNJ2019.The virus titers of the isolates increased gradually after successive passage,ranging from 105-106.5 TCID50/mL.The strain JSNJ2019 was of the G1P[7]genotype.After three times of immunization,the titer of the neutralizing antibody in the rabbit serum(SNT)was up to 211.Western blot showed specific bands,and IFA fluorescence was bright and specific.In this study,PoRV JSNJ2019(G1P[7])was successfully isolated with good immunogenicity,which enriched the epidemiological data of PoRV and laid the foundation for the development of vaccines.Moreover,the prepared polyclonal antibody had strong specificity,and could serve as a reliable test tool for studying the pathogenesis of PoRV.关键词
猪轮状病毒/分离鉴定/基因型/免疫原性,多克隆抗体Key words
porcine rotavirus/isolation and identification/genotype/immunogenicity/polyclonal antibody分类
农业科技引用本文复制引用
李素芬,李彬,汤学超,周金柱,王丹丹,朱雪蛟,陶然,李运川,郭容利,张雪寒..猪轮状病毒G1P[7]株分离鉴定及多克隆抗体制备[J].畜牧与兽医,2024,56(4):101-107,7.基金项目
国家重点研发计划(2022YFD1800601) （2022YFD1800601）
