| RV comprise of the genus Rotavirus within the family Reoviridae. Rotaviruses(RVs) are enteric pathogens causing diarrhea in various host species, including birds and mammals. Porcine rotaviruses(Po RV) is a major cause of serious diarrhea, vomiting, fever, dehydration in weaning and post-weaning enteritis in piglets, which cause high fatality rate, as one of the main infectious disease in the pig, resulting great economic losses.The genome consists of 11 segments of double-stranded RNA, encoding six structural proteins and six nonstructural proteins. The nonstructural protein NSP3 is a protein of glycosylation, the protein and virus particles infected cells, can shut down a host cell m RNA translation. NSP5 is phosphorylated and hyperphosphorylated to various degrees, rich in Ser and Thr residues, during the course of rotavirus replication, NSP5 is involved in many processes of infection, playing an indispensable role. Those results provided a basis for the further study on the structure and function of the NSP3 and NSP5.The RV strains NMTL was used as template for PCR. After digestion with Bam H I and Sal I, the resulting PCR products were cloned into prokaryotic expression vector p GEX-6p-1. Resulting in recombinant plasmids designated as p GEX-NSP3 and p GEX-NSP5, then transformed them into the DH5α cells. After induction with a final concentration of 1m M Isopropyl β-D-1-thiogalactopyranoside(IPTG).for 8-10 hours in LB-medium, empty vector transformed bacteria was used as control. The expressed r NSP3 and r NSP5 were analyzed with SDS-PAGE. After the purified, the SDS-PAGE analysis show that the recombinant proteins were expressed successfully in E. coli cells, appearing as a band near 60 k Da(NSP3) and 50 k Da(NSP5). The r NSP3 and r NSP5 could react with porcine anti-Po RV serum as shown by WB analysis, which implies that it had similar antigenicity to the native NSP3 and NSP5 proteins of Po RV.The gel slices were crushed and added to an appropriate volume of sterilized phosphate buffered saline(PBS) and used for immune antigen and immune to the mice, Female Six-week-old healthy BALB/c mice were primed subcutaneous injection with purified r NSP3 and r NSP5 proteins. Three booster immunizations were given at two weeks intervals with the same antigen.After three immunizations, the immunized mice sera were collected and determined using indirect ELISA. A final immunization was injected intraperitoneal. Three days after the final dose,with one additional intraperitoneal immunization, the mice were euthanized and their splenocytes were harvested and fused with SP2/0 myeloma cells. In addition used for coated antigen with indirect ELISA. By hybridoma technique, two MAbs were obtained, and identified were lg G1. Immunofluorescence assays and Western blot showed that the MAbs reacted well with native viral NSP3 and NSP5 protein.Six overlapping fragments covering the NSP3 and NSP5 proteins, were expressed as GST-fusion peptides, and subjected to western blot with MAb as the primary antibody. Those results revealed that the minimal linear epitope required for reactivity with the 5B8 was 290 QDYDRTFL 297 and 5E11 was 61 GPSDSA 66.To preliminary identify the antigenic eptiopes against those MAbs, a series of truncated NSP3 and NSP5 proteins were expressed. Then using western blot method, the antigenic epitope sequence of 290 QDYDRTFL 297 of 5B8 was identified, the antigenic epitope sequence of 61 GPSDSA 66 of 5E11 was identified.By hybridoma technique, the MAb of 5B8 and 5E11 were obtained. Those results provided a basis for the further study on the structure of the protein and function. The MAbs can be used for detection and functional analysis of Po RV. Precise mapping of the epitopes on the proteins were important for understanding the antibody-mediated protection of the host against the virus and for providing an effective epitope-based way to diagnostic rotavirus. |
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