Clone And Expression Of Structural Protein Of Bovine Viral Diarrhea Virus And Establishment Of Nucleic Acid Detection Method

Bovine viral diarrhea virus infection causes bovine viral diarrhea,which is also one of the main pathogens causing bovine respiratory disease syndrome(BRDC).The clinical manifestations of bovine viral diarrhea are complex,including abortion of pregnant cattle,mummification of fetal cattle,congenital defects of calves,bidirectional fever,diarrhea dehydration,ataxia,immunosuppression and persistent infection of infected cattle,The disease is widespread in the world.At present,pathogens have been detected in cattle in more than 20provinces,municipalities and autonomous regions in China,Artiodactyls such as cattle,sheep,pigs and deer are also infected to varying degrees,which seriously affects the healthy development of animal husbandry.ORF in BVDV genome encodes structural protein and non-structural protein,Structural proteins include core protein C and glycosylated envelope proteins E^rns,E1 and E2,It plays an important role in adapting the virus to the environment and maintaining its own stability.In this experiment,MDBK cells were used to measure the proliferation and titer of BVDV strain,Full-sequence primers were designed to clone and identify the genes encoding structural proteins.On this basis,bioinformatics analysis tools are used to predict and analyze the genome sequence and protein structure,properties and functions.The result showed that after the MDBK cells were inoculated and cultured for 24h,cytopathic effects began to appear.The measured BVDV TCID₅₀=10^-5.112/0.1m L,the structural protein gene bands amplified by PCR were 306bp,681bp,585bp and 1122bp,respectively,sequencing results were compared with 17 BVDV-1,BVDV-2,CSFV and BDV strains in NCBI database.The homology with BVDV-1 NADL strain was 100%,99.71%,97.78%and 100%,respectively;the phylogenetic tree showed that it was closely related to the evolution of BVDV-1 NADL strain,The evolutionary relationship with CSFV and BDV is relatively distant.BVDV N^proand E^rnsproteins are conservative and have characteristic functions,It is a differential protein that can be distinguished from viruses of the same family and different genera,N^proand E^rnsRNase play an important role in blocking interferon induction and causing host persistent infection.In this experiment,the recombinant expression vectors of BVDV N^proand E^rnswere constructed.Expression of N^proand E^rnsproteins by mammalian eukaryotic expression system.Eukaryotic expression plasmids p CMV-Myc-N^proand p CMV-Myc-E^rnswere successfully constructed,the recombinant plasmid was transiently transfected into HEK 293 cells,after Western blot,the target bands appeared at 21k D and 27k D respectively.The common diagnostic methods of BVD include clinical diagnosis,etiological examination,immunological examination and molecular biological examination.In this experiment,three nucleic acid detection methods of BVDV RT-PCR,q RT-PCR and RT-LAMP were established,and the detection methods were optimized,verified and compared.Experiments have confirmed that the three established detection methods have good specificity;The detection of standard plasmid samples with different concentrations showed that q RT-PCR had the highest sensitivity(8.134×10¹copies/?L).They are 10 and 1000 times higher than those of RT-LAMP and RT-PCR,followed by RT-LAMP(8.134×10²copies/?L)and RT-PCR(8.134×10⁴copies/?L);Kappa and Mc Nemar calibration tests were carried out on the test results of 67 clinical samples,and the results showed that the three methods had high clinical detection rate and good consistency of test results.When diagnosing BVDV,it is suggested that RT-LAMP should be used under limited conditions,and q RT-PCR should be further used when laboratory conditions permit.