Epitope Prediction And Prokaryotic Expression Of Genes Of TM-1 & Mgc3 Of Mycoplasma Gallisepticum HS Strain

Mycoplasma gallisepticum is the aetiologic agent of chronic respiratory disease inchickens.The diseases caused by MG in chickens break out around the world, whichmainly led to high unhealthy chicken rate and egg production of layer dropped, feedconversion efficiency reduced. Infection of MG don't cause large scale death of chickendirectly but it lead to heavier economy loss in chicken firm indirectly all of the world.At present, the main of normal methods for MG-diagnosis, such as the isolation andidentification of thallus culture, serum plate agglutination (SPA), polymerase chainreaction (PCR) and enzyme linked immunosorbent assay(ELISA), are limited in spotapplication because of their time-consuming, complicated manipulation or requitingexpensive instruments. The development of rapid diagnosis for MG has become one ofthe problems need to be solved urgently. Gold-immunochromatographic assay (GICA) isconvenient, rapid and direct-viewing. However, the preparation of monoclonal antibody(McAb) against the different epitopes is the prerequisite of GICA. The result of this studywill provide strong theoretical support for the development of the detceted method GICAand ELISA and the preparation of genetically engineering vaccine. The main contents andresults of research are summarized as following:1. Cloning of TM-1 and mgc3 genes coding the protective antigen of MycoplasmagallisepticumAccording to published TM-1 and the mgc3 gene sequences MG in Genbank, primerswith restriction sites were designed and synthesized. The expectant fragments wereamplified from strain HS by common PCR.The sequence analysis showed that TM-landthe mgc3 genes consisted of 819 base pair(bp) and 3186 base pair respectively.2. Sequence analysis and epitope prediction of TM-1 and mgc3 by biologicalsoftware.The comparison of the TM-1 gene sequence amplified from MG-HS strain to othermycoplasma revealed that their homology were up to 99% with MG-S6 strain. The repeat sequences of TM-1 gene exists as the 103～134bp and 135～166bp in MG-HS strain. Itwas predicted that this epitope have strong antigenically by secondary structure analysis.A striking homology of DNA sequences between MG-HS and MG-R is up to 91% and93 % the sequence of TM-1 gene. It is confirmed that there are three gene sequenceswith high homology up to 99% by blasting of the entire mgc3 gene sequence betweenMG-HS and MG-R. Moreover there are three sequences present homology in front 1100bpof mgc3 gene between MG-HS and MG-S6, their homology is 99%, 98% and 91%respectively. So the 1000-3186bp sequences of mgc3 gene have been analysised andpredictied by software, we found four epitope regions, they are 142～1440bp,1704～2169bp,2165～2521bp and 2507～2835bp respectively. It is prognosed that they arestronge hydrophilic, surface accessible, antigenic. There are 10 TGA codons encodingtryptophan in mgc3 gene sequence.3. The expression of MG-HS TM-1 and epitopes of mgc3 in E.coil BL21 (DE3) andthe identification of biologic activity of the expressed proteinThe fragments of TM-1 and mgc3-P1, ngc3-P1 were subcloned into prokaryoticexpressing vector pGEX-KG and expressed induced by IPTG in E. coli B121. Analyzed bySDS-PAGE and western blotting, 29Ku, MGC3-P1 and MGC3-P2 fusion proteinspresented immunogenicity with 55 KDa,37 KDa and 44KDa proteins respectively.