José Antonio Agüero-Fernández1† and José Pérez-Casal2 MOLECULAR CLONING AND EXPRESSION OF GENE FRAGMENTS FROM COMPUTATIONALLY PREDICTED Mycoplasma gallisepticum SURFACE ANTIGENIC PROTEINS José Antonio Agüero-Fernández1† and José Pérez-Casal2 1Microbial Genomics and Bioinformatics Laboratory, Molecular Biology Group. National Centre for Animal and Plant Health (CENSA). San José de las Lajas. Mayabeque. Cuba 2Bacterial Vaccine Development Research Group. Vaccine and Infectious Disease Organization - International Vaccine Centre (VIDO-InterVac). University of Saskatchewan. Canada †E-mail: jaaguero@censa.edu.cu

INTRODUCTION Mycoplasma gallisepticum (MG) is an avian pathogen. Vaccines provide some protection against disease and lessen the economic burden to producers, but almost universally, they fail to prevent colonization, and disease outbreaks still occur at unacceptable frequencies. The most economically significant mycoplasma pathogen of poultry. Is involved in Chronic Respiratory Disease (CRD). Considerable economic loses to poultry production due to downgrading of carcasses and decreased egg production efficiency, in addition to the costs of disease treatment and control. Mycoplasma gallisepticum (MG) is an avian pathogen. Member of the class Mollicutes. Minute size and total lack of cell wall. MGB-Lab

INTRODUCTION Their attachment to the target cells in birds is thought to be prerequisite for infection and disease and is achieved by lipoproteins anchored and/or exposed in mycoplasma surface. It is known that proteins that are exposed on the bacterial surface are targets for the humoral immune response in the infected animals and it is a fact that mycoplasma surface proteins are highly immunogenic and generate a strong antibody response. Identification of MG surface proteins is therefore crucial to be able to develop diagnostic methods based on serology, to understand the exact contribution of each of these molecules in promoting and maintaining a successful infection in the avian host and consequently, for the construction of new recombinant vaccines. Data emerging from recent studies indicate that although some putative cytadhesins like VlhA family, GapA. PvpA or cytadhesin-related molecules like CrmA have been reported, there are several of these components that have yet to be identified, defined and characterized. MGB-Lab

N° of CDs with Unknown Functions INTRODUCTION Experimentally the identification of these antigens involves an extensive trial and error system. Both new genomic technologies and bioinformatics offer remarkable opportunities in vaccine developmental research. The sequencing of MG genome opened the gates to extensively explore the whole genome derived proteomes of this avian mycoplasma specie. Genome Total Predicted CDs N° of CDs with Unknown Functions Mycoplasma gallisepticum RLow, Accesion Nº NC_004829.2 763 273 MGB-Lab

OBJECTIVES General objective To obtain and characterize, computationally identified putative MG surface antigens. Particular objectives To identify in silico putative MG surface antigens. To obtain, by recombinant technology, identified antigens. To experimentally evaluate the sub-cellular localization, as well as immunogenic properties of predicted proteins. MGB-Lab

Computational identification of surface antigens METHODOLOGY Computational identification of surface antigens 763 CDs from MG strain RLow reference genome, Accesion Number NC_004829.2 Prioritization of selected proteins considering as main criteria citadhesin, surface localization and antigenic properties (in this order) Primer design using VectorNTI Advance™ 11.0 MGB-Lab

METHODOLOGY Obtainment of antigens via recombinant DNA technology Immuno-characterization and evaluation of sub-cellular localization of obtained proteins PCR Amplification and cloning in pQE30 Transformation and expression in E. coli ISM612 Protein solubilización with SDS 1% Protein purification by IMAC Immuno-recognition of recombinant proteins with a policlonal antibody vs. MG Rlow by Western-Blott and ELISA Immuno-recognition of recombinant proteins with a policlonal antibody vs. MG Rlow membrane proteins by ELISA MGB-Lab

Obtainment of antigens via recombinant DNA technology RESULTS Obtainment of antigens via recombinant DNA technology Amplification of genes of predicted MG surface antigens Expression of recombinant genes Western Blott (after 12% SDS-PAGE ) with MAb vs. Poly-His Tag, of total cells after IPTG induction of E. coli ISM612 cultures of recombinant clones. Expression was observed in 15 out of 24 recombinant constructions (12 different genes). 250 kDa 130 kDa 100 kDa 70 kDa 35 kDa 25 kDa 15 kDa 55 kDa Agarose gel electrophoresis (1% ) of PCR amplification products. 92 genes, out of 97, were succesfully amplified. MGB-Lab

RESULTS Immuno-characterization and evaluation of sub-cellular localization of obtained proteins Recognition of recombinant proteins by PAb vs. MG (Rlow) total cells 250 kDa 130 kDa 100 kDa 70 kDa 35 kDa 25 kDa 15 kDa 55 kDa GapA 8 6 2 3 4 5 7 CrmA CrmC pQE30 1 MWM A GapA 1 2 3 4 5 6 7 CrmA CrmC Mgc2 8 MWM 250 kDa 130 kDa 100 kDa 70 kDa 35 kDa 25 kDa 15 kDa 55 kDa B C Western Blott of IPTG induced cultures, with PAb vs. MG (Rlow) total cells. Western Blott of purified proteins with PAb vs. MG (Rlow) total cells. ELISA of purified proteins with PAb vs. MG (Rlow) total cells. Seven recombinant products (from both, known and unknown proteins) were recognized by Western Blott with the Ab vs. MG. Ten recombinant products (from both, known and unknown proteins) were recognized by ELISA with the Ab vs. MG. The reaction of product 3 is as strong as the reaction of GapA and CrmA. Protein 8 also generate strong reaction. MGB-Lab

RESULTS Immuno-characterization and evaluation of sub-cellular localization of obtained proteins Recognition of recombinant proteins by PAb vs. MG (Rlow) membrane proteins ELISA of purified proteins with PAb vs. MG (Rlow) membrane proteins. All proteins are recognized by ELISA with the Ab vs. MG-MP. The level of recognition of protein 3 is the higher among all MGB-Lab

FINAL REMARKS Seventeen genes that encode for proteins identified as Mycoplasma gallisepticum surface antigens were cloned. Twelve recombinant proteins were obtained in soluble form and with high purity. Polipeptides conserve their antigenic properties after solubilization and purification procedures. At least 7 of identified proteins are immunogenic and the immunogenicity of one of them is, at least, as high as the observed for GapA and CrmA. The potential surface localization and the already proven immunogenicity of these proteins, make some of them good candidates to be tested in studies for development of vaccine or diagnostic systems. MGB-Lab

RECOMENDATIONS To confirm the cellular localization of these proteins. To evaluate the role in the pathogenicity of them. To determine the protective ability of the selected proteins. MGB-Lab

ACKNOWLEDGEMENTS To Dr. Andrew Potter for his selfless help and support. This work was funded by a grant from the Vaccine and Infectious Disease Organization - International Vaccine Center (VIDO-InterVac). MGB-Lab