| 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. It was first reported in Jiangsu China in 1984 and occurred later in other provinces successively. Today, almost every Chinese province has RHD report; RHD is widely distributed around the world and seriously threatens the farming of rabbits.In order to study the molecular biology character and virus epidemic feature of RHDV YL Strain, we had coloned the capsid protein VP60 gene of YL Strain by employing the method RT-PCR and then sequenced. YL Strain was the first RHDV strain being sequenced in the Northwest of China. The result showed that the nucleotide sequence of VP60 gene was 1 740 bp in length encoding a protein of 580 aa. The sequence had been submitted to GenBank (the accession number is DQ530363).We downloaded all other 39 published RHDV isolate sequences from GenBank and then constructed phylogenetic tree and drawn homology and divergence form by bioinformatics software. The result indicated that the VP60 gene sequences were very conservative, and the homology of the nucleotide among different isolates exceeded 90 %, but they can be divided into 5 gene groups. The gene group and region had negative connection. Exception WX84 Strain, all other Chinese RHDV isolates belonged to the V gene group. In this group, the hereditary distance between the strains isolated in recent years and NJ85 Strain was very close. The RHDV strains of China isolated in recent years probably come from the same strain NJ85. Meanwhile, we had predicted and analyzed the secondary structure and antigenic index of four strains from different areas of China. The result revealed there were some differences in secondary structure, but the antigen was the same.We also had studied the high expression of rabbit hemorrhagic disease virus capsid protein VP60 in E.coli. The prokaryote expression plasmid VP60-pET32a of VP60 gene was successfully constructed, and then inserted into the expression E.coli Strain BL21 (DE3). The target protein was highly expressed under the induction with 1 mmol/L IPTG.. SDS-PAGE analysis showed that the molecular weight of the recombinant VP60 protein was about 75 kD. The proportion of the recombinant VP60 protein reached to 39.8 % of the total bacterial proteins. Western blot analysis demonstrated that the recombinant VP60 had a specific reaction with antiserum of RHDV TP Strain.Through the research of different phytohormone combinations effect on the regeneration of Perfoliate Rosinweed, we had established its high-efficient regeneration system.The result showed that the medium of MS+6-BA(2.0mg/L)+NAA(0.1mg/L) could efficiently induce the differentiation of callus and buds and the medium of 1/2MS+IBA(0.1mg/L) could induce the regeneration of roots quickly. The vector VP60-pBI121 was constructed and then transformed to Perfoliate Rosinweed by agrobacterium-mediated method. The regenerated resistant plants could be obtained by pre-culturing for 3 d, co-culturing with Agrobacterium tumefaciens LBA4404 for 3~4 d, and then transfered to the callus inducement media containing 400 mg/L Carb and 40 mg/L Kan for 28 d. Under this condition, the rate of plant transformation was relatively higher and we had gotten two putative transgenic plants. This system was an elementary technology for production of oral plant vaccine for rabbit hemorrhagic disease with Perfoliate Rosinweed. |
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