| Bluetongue(BT), is a ruminant infectious disease that is characterized with hyperpyrexia, leucocyte decrease and ulcerative changes. BT is non-contact infectious spread by insects, like culicoides. Sheep and cattle are susceptible animals, especially purebred sheep. BTV has many serotypes and now 26 distinct serotypes have been found. There is not any protection among different. In our country, BTV1 and 16 are the mostly epidemic serotypes. BTV has spread and widely prevalent become many in countries. Before 2006, BTV4 spread widely in Greece, Spain, France, other countries. After 2006, it caused a great loss to the economy that BTV8 broke out in European countries. The OIE has listed BTV as a mark disease, and it is one the first of class of animal infectious disease.The geonem of TBV composed by 10 parts ds RAN, it encode seven prteions strutucral and four prointes nonructstural(NS1, NS2, NS3/NS3 a and NS4). VP7, prteoin group-sepicfic of BTV, encoded by S7 gene. It composed by 349 amino acids, located of the surface the on virus. Above 94% aimno acdis of difierent serotypes BTV VP7 are hihlgy cosernved. VP2 is the serotype-specific protein of BTV, encoded by L2 gene. It composed by 950 to 960 amino acids, on the otuer of the viurs casipd. Amino acids of difierent serotype VP2 changes in the range of 22.7% to 72.9%. In the study, BTV8 VP7, BTV4 VP2, BTV8 VP2 were expsresed in prorykaotic system expression, and the monoclonal against antibodies VP7 and VP2 prepared on were basis of recombinant the proteins. This study provides material foundation for the establishment of serological diagnosis method, and provides technical reserve for preventing new serotype inflow in our country in quarantine at ports.BTV8 S7 gene, BTV4 L2 gene and BTV8 L2 gene were composed according to the gene sequences published in Gen Bank. Primers were designed to amplified 4A, 4B, 4C and 8A, 8B, 8C gene sequences of L2 form BTV4 and BTV8, and they were exrespsed in prokyoartic sytesm exprsesion. S7 gene and segmented L2 gene were into cloned expression prokaryotic vector p ET-28a(His-tag) and p MAL-c5x(MBP-tag) to construct recombinant plamids. The potisive recobimnant plaimds transorfmed were itno E.coli BL21 competent cells and induced with 1.0mmol/L IPTG. Recombinant protein His-VP7, His-4A, His-4B, His-4C, His-8A, His-8B, His-8C and MBP-VP7, MBP-4A, MBP-4B, MBP-4C, MBP-8A, MBP-8B, MBP-8C were exesprsed whit succssfeully SSD-PAGE and Wteesrn bolt. The proteins recombined with MBP-tag were by amylase purified resin, and the proteins recombined with His-tag were cutting by purified gel slices.Six weeks old BALB/c mice were immunized with the purified recombinant proteins VP7 and VP2 with His-tag. Splenocytes from the immunized mice and SP2/0 myeloma cells wre fused by cell fusion technique. Purified recombinant proteins VP7 and VP2 with MBP-tag were treated as detective antigens, so that indirect ELISA detection methods could be established to screen hybridoma cells which monoclonal secreting against antibodies(Mc Ab) VP7 and VP2. Five hybridoma cells secreting which Mc Abs against BTV8 VP7 were stably designated 3D7, 3F4, 5C9, 6B5 and 6D11. Three hybridoma cells which secreting Mc Abs against BTV4 VP2 stably were designated 1G7, 8G3 and 5E6. Two hyidobrma cells sreecting which Mc Abs aginast BTV8 VP2 stably were designated 2B8 and 3G11. Results of shedow Wetesrn blot that of all the obnetaid Mc Abs could specicalifly react with the corrndespoing proteins recobimnant. Results of IFA and indirect ELISA detection showed that Mc Ab 3F4 reacted specifically with BTV4, and had no reaction with cross Ibaraki virus, Akabane virus, Porcine Rotavirus. Isotype of Mc Abs was identified by Mouse Monoclonal Antibody Isotyping Reagents. Among the Mc Abs against BTV8 VP7, the isotypes of 3D7, 3F4, 5C9 and 6D11 are Ig G1, and 6B5 is Ig M. Among the Mc Abs against BTV4 VP2, the isotypes of 1G7 and 8G3 are Ig G1, and 5E6 is Ig G2 b. Among the Mc Abs against BTV8 VP2, the isotype of 2B8 and 3G11 are Ig G2 b. |
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