| Rabbit hemorrhagic disease (RHD) caused by Rabbit hemorrhagic disease virus (RHDV) is characterized by an acute course, hemorrhagic septicema and a high mortality in adult rabbits whereas infected rabbits below the age of about 2 months usually survive. It was first reported in China in 1984 and occurred later in other countries successively. Today, RHD is widely distributed around the world and seriously threaten the farming of rabbits.In our study the capsid protein (VP60) gene of the Chinese early isolate NJ85 of RHDV was amplified by reverse transcribed polymerase chain reaction (RT-PCR), cloned and sequenced. VP60 gene of NJ85 was in size of 1740nt and encoding 579aa. Comparison with other Chinese field isolates WX84 and TP showed that the homology were 92.7% and 97.2% for nucleotide sequence; 96.1% and 98.6% for amino acid sequence respectively. Alignment with other 16 sequences of RHDV isolated from other countries registered in GenBank exhibited that the homology was 83.7%~97.0% for nucleotide and 90.5%~ 99.0% for amino acid sequence. The results indicated that the sequence of VP60 in different RHDV isolates was highly homologous. In the all six regions of VP60, low degree of variation of VP60 was found in the region A, B, D, F and relative high degree of variation in the region C and E. Based on VP60 sequence, phylogenetic analysis showed that the historic RHDV isolates were divided into 3 branches of the lineage of RHDV according to the amino acid sequences and 4 branches according to the nucleotide sequences. There was no obvious correlation between geographical region, historic period and homological degree. Three Chinese field isolates lay in 2 different branches of the RHDV lineage, in the meanwhile EBHSV formed another lineage. The molecular weight, isoelectric point, hydrophobicity, 2-dimensional and 3-dimensional structure of NJ85 VP60 were determined and predicted on the theory of bioinformatics. The major secondary structure, B-sheet contributed to the stability of VP60. Based on the structural model, the architecture of NJ85 capsid was 32 cup-shaped depressions. The capsid was composed of 90 A/B5 and C/C2 dimers formed by the single unit of VP60. The conformation of thesingle unit in dimer existed three forms of A, B and C. The single unit of VP60 had a protruding (P) domain connected by a flexible hinge to a shell (S) domain. P domain was subdivided into two subdomains, P1 and P2. P2 subdomain that located the surface of the capsid contained the determinants of strain specificity and erythrocyte binding site.Prokaryotic expression vectors are usually advantageous to yield the product of foreign purpose gene in large quantities, timesaving and low costs. For this purpose, the full-length of VP60 gene cloned from isolate NJ85 was subcloned into the high-level expression vector pET28. The expression plasmid pET-VP60 was constructed and highly expressed under the induction with IPTG. The proportion of the recombinant VP60 protein reached to 40 percent of the total bacterial proteins under the optimized inductive condition. SDS-PAGE analysis showed that the molecular weight of the recombinant VP60 protein was approximate to 62 kD, and it formed inclusion body in E.coli. The specific reaction of the recombinant VP60 protein with antiserum of RHDV was confirmed by immunoblotting assay. However, The earlier studies on the expression of VP60 the yield of recombinant VP60 was below 21%.Until now the hemagglutination inhibition (HI) test was the popular method for the detection of antibody against RHDV. But it had many inconveniences and disadvantages in practice. In order to overcome the questions, an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibody against RHDV was developed using the recombinant VP60 expressed highly in E.coli as antigen. The optimized conditions for the assay were that the wells of ELISA plate were coated with recombinant VP60 (1.0ug/mL), 10% normal bovine serum was added as blocking agent, and the serum samples were preincubated wi...
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