Preparation Of BVDV E0 Antibody And Construction Of Interferon Knockout MDBK Cell Line

Bovine viral diarrhea?BVD?,also known as bovine viral diarrhea-mucosal disease,is a serious infectious economic disease caused by bovine viral diarrhea virus?BVDV?.BVD can cause immunosuppression,reproductive dysfunction,and persistent infection in infected animals.Itl have a large impact on livestock and can bring huge economic losses to the cattle industry around the world.The Office international desépizooties defines it as a Class B infectious disease.BVDV is a single-stranded positive-sense RNA virus with a length of 12.3 kb encoding a single large open reading frame?ORF?.The genome of BVDV contains5'-untranslated regions?5'-UTR?,open reading The open reading frame?ORF?and the 3'-untranslated regions?3'-UTR?,the proteins translated from the ORF are post-translationally processed by cells and viral proteases to produce mature proteins,including four structures?C,Erns,E1 and E2?and eight non-structural proteins?Npro,p7,NS2,NS3,NS4a,NS4b,NS5a and NS5b?.The envelope protein Erns?E0?,also known as gP48,is located at nucleotides811-1491 of the ORF and consists of about 227 amino acid residues.The Erns non-structural protein is mainly involved in the formation of the viral envelope structure.E0 is A highly conserved protein among the 12 BVDV proteins,which has a neutralizing epitope,and the neutralizing antibody produced by the animal immunization experiment as an antigen can have the ability to neutralize BVDV and CSFV.Analysis by amino acid sequence analysis software showed that there is a highly conserved domain in the E0 protein,and the highly conserved domain is an essential class for the research and development of subunit vaccines.Therefore,the E0 protein can be used as diagnostic antigens or prepare polyclonal antibodies.The CRISPR/Cas system is mainly composed of a CRISPR array element and a Cas gene family.The system consists of a set of Cas genes,non-coding RNAs and a unique array of repeating elements?direct repeats?.The CRISPR/Cas system relies on uptake of foreign DNA fragments into the CRISPR locus,followed by transcription and processing of these CRISPR repeat spacer arrays into short CRISPR RNA?crRNA?,followed by annealing with trans-activated crRNA?tracrRNA?and Direct sequence-specific silencing of Cas protein exogenous nucleic acids,in vitro studies have shown that a single synthetic guide RNA?gRNA?consisting of a fusion of crRNA and tracrRNA can direct Cas9-mediated target DNA cleavage and attenuate gene expression by causing gene frameshifts Or not expressed.E0 is one of the envelope proteins of BVDV,which is highly immunogenic and can induce neutralizing antibodies in infected animals.It can be used as an antigen to diagnose BVD.In this study,the prokaryotic expression vector was used to successf?Lly express and purify the E0 protein with higher purity.The soluble protein concentration was lower and the inclusion body protein concentration was higher.Therefore,the inclusion body protein was used as the antigen to immunize the mice and after the initial immunization.The immunization was boosted every two weeks,and finally a large amount of blood was collected.After ELISA and Western Blot verification,a polyclonal antibody against the E0 protein with high titer was successf?Lly obtained,which can be used for the detection of BVDV produced by the cell lines and animals.At present,the cost of BVDV vaccine is higher mainly because interferon can inhibit the replication of BVDV in cells.Therefore,this study uses CRISPR/Cas9technology to knock out BoIFN gene,so that interferon is knocked out or its expression is reduced.A series of gRNAs have been designed and the lentiviral plasmid LentiCRISPR V₂-gRNA has been successf?Lly constructed,The plasmid was integrated into the MDBK genome by lentiviral packaging and is currently being tested for knockout effects.