| Porcine rotavirus is a causative agent of piglets diarrhea caused by rotavirus, a member ofgenus Rotavirus, the family Reoviridae. The young animals is the most susceptible, many animalsare the natural host of porcine rotavirus, such as human beings, porcine, turkey, sheep and so on.The diarrhea caused by PRV is worldwide, and has badly harmed human life and animalhusbandry.Nowadays, some vaccines are used for precaution of PRV, but it is also out of control and itis necessary to develop the new vaccine and explore the new immunological mechanism for theprecaution and the control of PRV. Virus-like particles are the same or similar as the virus in themorphous, and it has good immunogenicity and reactionogenicity which has broad perspective inthe research of vaccine. It was reported that the PRV VLPs had been successfully constructed inabroad, and there were heartening achievements in the research of vaccine and the animalimmunity test. It provides the feasible theory for this study.According to the genome of VP4/6/7 PRV OSU strain G5 and the VP2 gene sequences offive human PRV stains published in GenBank, 4 pairs of primers were designed to amplifyVP4/6/7 genes of the PRV strain JS. In order to analyse biological properties of the VP2 gene,VP2 gene of strain OSU was also amplified. The gene was cloned into pMD18-T vector andsequenced. The result of Sequence analysis showed that the Vp2 genes of strain JS and strain OSUwere both consisted of 2717 bp, encoding 890 amino acids, and the similarity of nucleotides andamino acids were 99.9% and 99.91% respectively. Phylogenetic tree indicated that homology ofstrain JS and strain OSU was closer with the strain HP140 from porcine, although homology ofthat the strain wa from human was the highest. The entire Vp4 gene was consisted of 2342 bp,encoding 776 amino acids. Homology of 15 strains of the group A rotavirus was from 69.2% to99.5% in level of nucleotides, and homology of these strains was from 70.2% to 99.1% in level ofamino acids. The region of amino acids 45-250 was high various with mutations, insertions anddeletions, but the region of amino acids 241-247 was super-conserved. Phylogenetic treedemonstrated that the strain JS was the closest with the strain OSU and the strain JL94. Vp6 genewas consisted of 1320 bp, encoding 389 amino acids. Homology of 9 strains of rotavirus was from77.1% to 91.1% in level of nucleotides, and homology of these strains was from 89.7% to 99.5%in level of amino acids. Phylogenetic tree demonstrated that 10 strains of rotavirus could bereclassified to 4 groups, which was strain JS, strain B131, strain OSU and strain CN86. The Vp7gene was consisted of 1062 bp, encoding 326 amino acids. Homology with other 15 strains wasfrom 74.5% to 78.5% in level of nucleotides, and homology of these strains was from 75.2% to83.1% in level of amino acids. Phylogenetic tree demonstrated that genetic relationship of strainJS with the strains of ICB2185 and O-1 was closer, belonging to one group. These data indicatedthat PRV strain JS belong to G9 type. According to above sequencing informations, four pairs ofprimers with BamH I and Sal I sites at 5' terminal and 3' terminal respectively were designed toamplify the VP4/6/7 genes of the PRV strain JS. These genes were digested with BamH I and Sal Ienzyme respectively and then subcloned into pFastBac vector. Recombinant plasmids were respectively transformed into E. coli DH10Bac cells. After transposition, Recombinant Bacmids ofthe VP4/6/7 genes were obtained, and then these recombinant Bacminds were co-transfected intosf9 cells with lipofectin. After 72 hours, Recombinant virus was collected. Thses recombinantviruses were passaged three times blindly, and these recombinant viruses were identified withPCR, SDS-PAGE, and Western blot. These results demonstrated that the proteins VP2 and VP4 ofPRV JS strain were successfully expressed, and all recombinant proteins were reacted with theantisera of PRV strain JS.In order to study morphology character of PRV VLPs, the recombinant virus expressed Vp2protein was purified by sucrose gradient centrifugation. The result of electron microscopeindicated that the expressed Vp2 protein in sf9 cells could form 40 nm of VLPs in diameter, and itwas similar to native virus particles. |
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