1. 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 †

2. 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.
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3. 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.
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4. 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_
763
273
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5. 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.
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6. Computational identification of surface antigens
METHODOLOGY
Computational identification of surface antigens
763 CDs from MG strain RLow reference genome, Accesion Number NC_
Prioritization of selected proteins considering as main criteria citadhesin, surface localization and antigenic properties
(in this order)
Primer design using VectorNTI Advance™ 11.0
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7. 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
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8. 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.
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9. 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.
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10. 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
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11. 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.
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12. 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.
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13. 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).
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