Identification Of The Group-specific Conformational Epitope Of Bluetongue Virus VP7

Bluetongue (BT) is a kind of ruminant animals fulminating infectious disease caused bybluetongue virus which belongs to Orbivirus genus, the Reoviridae family. It was spread byCulicoides. The mortality of sheep was as high as80%. BTV was firstly discovered in1876inSouth Africa. In China, it was firstly discovered in1979. It has made serious harm to sheepindustry. In our previous study, the MAb4H7prepared by VP7protein expressed by prokaryoticexpression system had shown the group specific blocking effect. Based on this, MAb4H7wasused to identification of the VP7protein competitive inhibitory group specific epitope.The conformational epitopes of BTV VP7were screened with the MAb4H7by phagedisplay technology, the details were specified on Ph.D.TMPhage Display Libraries. Chose20clone from the phage and analyzed the nucleotide. The results showed that the epitope of MAb4H7was decided by four regions (aa34-aa35, aa175-aa177, aa185-aa186and aa278-aa279).These regions are the competitive inhibitory group specific epitope of VP7protein.Methods4pairs of mutant primers were designed according to the recombinant plasmidpMAL-VP7with amino acids GI which were located in the position of34to35were changed toSK, FQG which were located in the position of175to177were changed to KDS, YL whichwere located in the position of185to186were changed to KN, WH which were located in theposition of278to279were changed to KR. Indirect ELISA analysis reacted with the MAb4H7and the IDEXX’s MAb demonstrated that the conformational eptiope of VP7protein wereconsist of175FQG177、185YL186�'�278WH279, and ELISA analysis reacted with the MAb1G2confirmed that the site-direct mutation did not change the conformation eptiope of VP7protein.Site-direct mutations based on these regions were further confirmed that the175and186amino acids were critical residues of the epitope. These eptiopes among different serotype ofBTV shown high conservation. so they could used to indentify BTV.Currently, serodiagnosis methods to VP7protein has been one of main means to diagnoseBT. The results of this study had great significance to develop and improve the serologicdiagnostic method.