Gene Cloning And Molecular Analytical Of Giant Panda Rotavirus Strain CH-1

Rotavirus (RV) belonging to the Reoviridae family, and is one of the leading cause of severe gastroenteritis in human and animals. Once giant panda infected rotavirus, often leading apastia, intense disgorging, then appear key feature of disease such as water-like diarrhoea, abdominal gaseous distention, diarrhoea protraction and so on, eventually die of multi-organ nonfunction, bring huge damage to giant panda's breeding and health. At present both at home and abroad the study of giant panda rotavirus is still tiny, has not carried out systematically research, thus brought great challenges to prevent and control of giant panda rotavirus. Based on the above considerations, this topic research aims to obtain the viral genome, bioinformatics analysis of its important protein gene, for giant panda rotavirus epidemiological survey, the virus protein antigenicity research and develop new type giant panda rotavirus vaccines. The content includes the following aspects:1 Genome coding sequence cloning of giant panda rotavirus strain CH-1According to the published sequence of rotavirus in GenBank,11 primers was designed. Then the RT-PCR method was carried out on giant panda rotavirus which propagated in MA-104. The 11 PCR fragments were purified, cloned into the pMD19-T simple vector and sequenced. The sequenced results were submitted to GenBank, and the accession number were VP1(HQ641297),VP2(HQ641294,VP3(HQ641295),VP4(HQ641296),VP6(GU188283),VP7(GU188284) NSP1(GU2O5762),NSP2(GU188281),NSP3(GU329525),NSP4(GU188282)和NSP5(GU329526)。2 bioinformatics analysis of giant panda rotavirus strain CH-1 VP4,VP6,VP7,NSP4In this research, VP4,VP6,VP7,NSP4 were used as Analysis object, the structure and function of them were carried out by means of bioinformatics analysis and the phylogenetic trees were obtained by CLUSTAL V software. The results indicated that VP4 contains a complete coding sequence (CDS) of 2331 bp, encoding 776 aa, Bioinformatics analysis showed that the VP4 gene codes for protein with molecular weight 86 768.8 u, grand average of hydropathicity was-0.265, while the maximum and the minimum hydrophobicity were 2.244 and-2.911, has no signal peptide and transmembrane regions. The antigenic determinants of VP4 locate in the N-terminal and C-terminal. Motif searching showed that VP4 protein may have 8 N-glycosylation sites, the phylogenetic tree showed that the evolution distance of giant panda rotavirus VP4 gene is the most homogeneous to porcine rotavirus; VP6 contains a complete coding sequence (CDS) of 1194 bp, encoding 397 aa, Bioinformatics analysis showed that the VP6 gene codes for protein with molecular weight 44 725.1 u, grand average of hydropathicity was -0.225, while the maximum and the minimum hydrophobicity were 1.822and -2.733, has no signal peptide and transmembrane regions, the antigenic determinants of VP6 locate in the N-terminal. Motif searching showed that VP6 protein may have 5 N-glycosylation sites, the phylogenetic tree showed that the evolution distance of giant panda rotavirus VP4 gene is the most homogeneous to porcine rotavirus; VP7 contains a complete coding sequence (CDS) of 981 bp, encoding 326 aa, Bioinformatics analysis howed that the VP7 gene codes for protein with molecular weight 37354.8 u, grand average of hydropathicity was -0.015, while the maximum and the minimum hydrophobicity were between -2.344 and 3.567. The protein has one signal peptide between 1 and 24 site, two transmembrane regions 4-23 site and 33～53 site and the antigenic determinants located on all of the protein. Motif searching showed that vp7 protein may have 2 N-glycosylation sites, The phylogenetic tree showed that the evolution distance of giant panda rotavirus VP7 gene is the most homogeneous to human rotavirus. NSP4 contains a complete coding sequence of 528 bp, encoding 175 aa. Bioinformatics analysis showed that the molecular weight was 20 223.7 u,6.84, grand average of hydropathicity of NSP4 was-0.240, while the maximum and the minimum hydrophobicity were between-2.400 and 2.622. The protein has no signal peptide, but has one transmembrane region, the antigenic determinants of NSP4 locate in the C-terminal. Motif searching showed that NSP4 may have 2 N-glycosylation sites. The phylogenetic tree showed that the evolution distance of giant panda RV NSP4 gene is the most homogeneous to porcine rotavirus.