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]

旨在明确猪场腹泻原因并分离猪轮状病毒(PoRV)。采集腹泻猪粪便,经RT-PCR检测为阳性的样品接种于单层细胞,通过细胞病变(CPE)和间接免疫荧光(IFA)来检测病毒增殖;RT-PCR扩增VP4和VP7基因并测序以确定G/P基因型,生物信息学软件分析VP4和VP7基因的遗传进化特征;传代病毒灭活后制苗接种家兔制备多克隆抗体,测定中和抗体,并经Western blot和IFA鉴定其特异性。结果:猪流行性腹泻病毒(PEDV)、传染性胃肠炎(TGEV)、猪德尔塔冠状病毒(PDCoV)、伪狂犬病毒(PRV)和牛病毒性腹泻病毒(BVDV)均为阴性,只有PoRV为阳性;过滤粪便样接种单层MA104细胞,连续传5代均可观察到以细胞圆缩、最后瓦解脱落为特征的CPE,IFA检测到PoRV的特异性荧光,表明病毒分离成功,命名为JSNJ2019;分离株连续传代,病毒滴度逐步升高,介于105~106.5 TCID50/mL;分离株JSNJ2019为G1P[7]基因型;3次免疫后家兔血清的中和抗体滴度最高可达211,Western blot出现特异性条带且IFA荧光亮而特异。本研究成功分离PoRV JSNJ2019(G1P[7])且免疫原性良好,制备的多克隆抗体特异性强,为研究PoRV致病机制奠定基础。

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 RTPCR 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.