| Bovine ephemeral fever(BEF)is a kind of popular by Bovine ephemeral fever virus(BEFV),through the spread of the arthropod acute,thermal sexually transmitted diseases,clinical symptom is given priority to with respiratory symptoms.Due to the rapid onset of the disease,which can be pandemic in a short period of time,it seriously threatens the healthy development of China’s cattle industry.Therefore,understanding the situation of BEF infection in cattle in intensive farms in Ningxia can provide a scientific basis for developing targeted prevention and control of BEF in the region.Infectious bovine rhinotracheitis(IBR)is an infectious disease caused by Infectious bovine rhinotracheitis virus(IBRV),which is one of the main diseases of bovine respiratory disease syndrome.The disease is widespread,after infection common secretion of nasal fluid,dyspnea,anorexia and other clinical symptoms.The clinical manifestations of cattle infected with BEF and IBR are very similar,and the clinical diagnosis is difficult to confirm.Therefore,it is necessary to establish a specific,rapid and effective double RT-PCR differential diagnosis method.G protein is the target of BEFV neutralizing antibody,and its G1 epitope only binds to BEFV serum.In order to achieve BEFV-G1 expression in vitro,this study constructed pET-G1 recombinant expression vector using prokaryotic expression vector,and induced expression in vitro.The results of the study were as follows:1.Serological investigation of bovine epidemic fever in some areas of NingxiaIndirect ELISA and PCR were used to detect 630 bovine serum samples collected from different regions of Ningxia.The results showed that the total positive rate of BEFV serum antibody was 16.98%(107/630)and the positive rate of BEFV nucleic acid was 12.38%(78/630).2.Establishment of a duplex RT-PCR assay for BEFV and IBRVIn this study,a pair of specific primers were designed based on the conserved regions of BEFV G gene and IBRV gB gene respectively,and the target gene fragments were cloned into pMD18-T vector to prepare pMD-BEFV and pMD-IBRV plasmid standards.The standard substance was used as the template to optimize the reaction conditions and procedures,and a duplex RT-PCR was established to detect BEFV and IBRV simultaneously,and the sensitivity,specificity,repeatability and clinical application of this method were carried out.The results showed that the minimum detection limits of pMD-BEFV and pMD-IBRV copies were 8.066 × 102 copies/μL and 5.99 × 102 copies/μL,respectively.There was no specific response to other common bovine viruses.The method was used to detect 92 samples with suspected clinical symptoms.Compared with the national standard PCR of BEFV and IBRV,the coincidence rates of BEFV and IBRV were 100%and 98.68%,respectively,indicating that the duplex RT-PCR of BEFV and IBRV has high sensitivity,strong specificity and good repeatability.It can be used for rapid clinical screening and epidemiological investigation of BEFV and IBRV.3.Construction and induction of BEF V-G1 gene expression vectorIn this study,the recombinant prokaryotic expression vector pET-G1 was constructed by PCR amplification of BEFV-G1 gene and cloning into pET-30a prokaryotic expression vector.Bioinformatics software was used to analyze pET-G1,and it was found that pET-G1 is a hydrophilic protein with 4 B cell antigen sites.The recombinant protein pET-G1 was induced to express,and soluble analysis showed that the protein was expressed in the form of inclusion body.pET-G1 expression reached its maximum when the final concentration of IPTG was 0.25mmol/L and 37℃for 7h.A single band at 17kDa was detected by SDS-PAGE and purified by Ni-NAT affinity chromatography.WesternBlot confirmed the reactivity of pET-G1 recombinant protein. |
