Identification Of The Acid Stability Sites And The Efficient Expression Decisive Region Of O Type FMDV Capsid Antigen

Foot-and-mouth disease (Foot and mouth disease, FMD) is a Zoonotic diseases caused by thefoot-and-mouth disease virus (Foot and mouth disease virus, FMDV). It can infect cloven-hoofedanimals and seriously reduce the quality of animal products, even bring a serious impact on theinternational trade of the national livestock products and related products.At present, the prevention ofFMD is mainly by inactivated vaccine, but with the continuous improvement of the epidemic preventionstandards and the biosafety considerations, traditional vaccines has exposed a series of questions.Therefore, the development of a new type genetic engineering vaccine is extremely important.This study aims to identify the acid stability sites of capsid antigen and develop FMDV subunitvaccines by changing these sites which could improve the expression level of the capsid antigen byBombyx mori baculovirorus expression system. Then O/ON/CHA/strain FMDV was neutralizedwith pH7.6Tris after acidulated by the PH6.0PBS for1hour and then the virus were screened by theneutral red plaque method.Two acid resistance monoclonal strains were picked out and named as S-Aand S-B. The P1gene sequence of S-A and S-B were compared with the unacidificated virus, the resultshowed that four mutations were found, of which the amino acids mutations D86H of VP2and N17D ofVP1in protein P1were found of in both strains. The other two mutations in VP4and VP3did notchange the amino acid sequence. The codon optimized P12A2B3C gene was synthesized whose twomissense mutation sites were replaced, then it was connected to the baculovirus transfer vectorpVL-1393and named as pVL-P12A2B3C.The linearized Bm-BacPAK6DNA and pVL-P12A2B3Cwere cotransfected with Bombyx mori BmN cells to obtaine recombinant virus Bm-P12A2B3C.Theresult of immunofluorescence determination show that recombinant viruses can express exogenous genecorrectly. The harvested silkworm lymph blood from fifth instar silkworm larvaes which were infectedby recombinant virus were detected by double antibody sandwich ELISA assay. The target protein canbe successfully detected and the result of ELISA was positive when it was1:640diluted, its retentiontime was significantly improved too.For the further study of the efficient expression decisive region of O type FMDV capsid antigen,the previous build pVL-P12A2B3C of Asiaâ… /HNK/CHA/05which could highly express capsidantigen was used as the skeleton, five major antigenic sites of O type FMDV were used to replace thesites of the skeleton whith four form combinations:1.the full-length Asiaâ… VP1was replaced by that ofO type VP1;2.13amino acids of VP2and full-length VP1were replaced;3.three amino acids of VP3and full-length VP1were replaced;4.all three amino acids in the VP1,VP2and VP3were replaced.Andthen empty capsid antigen was expressed in the BmN cell and silkworm with the previous method. Thechimeric antigen can be successfully expressed, the result of ELISA was positive when it were1:320diluted. The expression level of chimeric antigen had decreased seriously after the replace of VP1gene.It indicated that VP1may be the main region which effected the efficient expression of O type FMDV capsid antigen.