| Bovine viral diarrhea virus is a global viral diseases, it can affect cattle、sheep、rabbit、deer and so on many kinds of animals, the most serious harm to the cattleindustry. BVDV was found from yak by Wang qingjiang for the first time in1983, butBVDV infection has expand in yak because of neither adoption of measures to controlof BVDV nor BVDV vaccine applied. A long period of time, the research wasprimarily stayed in the field of etiological、epidemiological and serologic, but aboutmolecular biology’s study were few. so it is very necessary to study BVDV from themolecular biology. A series of studies have been carried out mainly on the BVDVYAK by molecular biology methods: First, we successfully cloned the full genomesequence of the BVDV YAK for the first time and analysis their biologicalinformation; last, E2protein of BVDV YAK has expressed in eukaryotic expressionsystem, and got one analysis about the express product. Results as following:1. Finishing the determination of whole genome sequence of BVDV YAKYak strains of BVDV genome is divided into19fragments were cloned, we getthe full genome sequence of the full length12305bp. Submitted to GenBank databaseaccession number for the JQ799141. Began to study from the total conservative baseof BVDV3’-UTR, successfully amplified genome3’-UTR of BVDV YAK, becausedesigned a special downstream primer.Compared with the strains of NADL, we foundinsertion at nucleotide of932-937bases at more than6, and determine its wholegenome structure as5’UTR-P20(Npro)-P14(C)-gp48(E0)-gp25(E1)-gp53(E2)-P7-P125(NS2-3)-P10(NS4A)-P30(NS4B)-P58(NS5A)-P75(NS5B)-3’UTR. Eight ATGcombinations in the5’-UTR structure and eight nucleotides of the3’-UTR structure ofrepetitive sequences, We got the whole genome sequence of BVDV YAK for the firsttime through the research, and laid a solid foundation for future in-depth and detailedto carry out yak BVDV research.2. Bioinformatic analysis of the strains of BVDV YAK genome sequence analyze and prognose structure of BVDV YAK, and comparatively analyze this wholegenome sequence with other8strains of BVDV genome sequences which record inGenbank so as to understand the identity and commonness among BVDV YAK strainand other BVDV whole genome and gene product. Homologous analysis show thatthe whole genome sequence homology was low among BVDV YAK whole genomesequence and other8strains of BVDV whole genome sequences, the homology ofnucleotide sequence was68.9%-74.4%; Evolutionary tree results show that thegenetic evolution relationship is farther between BVDV YAK strain and other8strains of BVDV. Comparatively analyzing change on the amino acid sequence ofstructural protein and noncoding region nucleotide sequence among BVDV YAKstrain and other8strains of BVDV. The results show the similarity was high onantigenic index and surface probability plot. It is indirect evidence that the structuralprotein is conservative among9strains BVDV. Analysis found that yak strains ofBVDV structural proteins hydrophilic and antigenic peak areas of high hydrophilicnature of the structural protein antigen into parallel,andthe structure of the protein hasfour sections of a highly hydrophobic region, to ensure that the structural proteinanchoring BVDV capsule membrane.3. E2protein was successfully expressed in P.pastoris GS115E2protein was successfully expressed in Pichia pastoris eukaryotic expressionsystems.E2gene of BVDV YAK strain was cloned into pPIC9K, and succeeded inestablishing the eukaryotic expression plasmid pPIC9K-sE2. Then transformed intoP.pastoris GS115，after inducing by methanol. Expression product was detectedthrough SDS-PAGE and Western-blot. A45KDa secreted proteins was expressedthrough SDS-PAGE; The result of Western-blot show that the E2protein ofsuccessfully expressed had good antigenicity.E2protein was successfully expressed inPichia pastoris eukaryotic expression systems, further enrich the BVDV yak strainspathogen molecular biology content and foundation for the future development ofsubunit vaccines and diagnostic antigens. |
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