The ostrich mycoplasma Ms02 Partial genome assembly, bioinformatic analysis and the development of three DNA vaccines Marliz Strydom Thesis presented in fulfillment of the requirements for the degree of Master of Science (Biochemistry) At the University of Stellenbosch Supervisor: Dr. A. Botes Co-Supervisor: Prof. D.U. Bellstedt Department of Biochemistry University of Stellenbosch March 2013 Stellenbosch University http://scholar.sun.ac.za II Declaration By submitting this thesis/dissertation electronically, I declare that the entirety of the work contained therein is my own, original work, that I am the sole author thereof (save to the extent explicitly stated otherwise), that reproduction and publication thereof by Stellenbosch University will not infringe any third party rights and that I have not previously in its entirety or in part submitted it for obtaining any qualification. Date: March 2013 Copyright © 2013 Stellenbosch University All rights reserved Stellenbosch University http://scholar.sun.ac.za III Abstract The South African ostrich industry is under enormous threats due to diseases contracted by the ostriches. H5N2 virus (avian influenza) outbreaks the past two years have resulted in thousands of ostriches having to be culled. However, the more silent respiratory infectious agents of ostriches are the three ostrich-specific mycoplasmas. Named Ms01, Ms02, and Ms03, these three mycoplasmas are responsible for dramatic production losses each year, due to their intrusive nature and the fact that no vaccines are currently available to prevent mycoplasma infections in ostriches. The use of antibiotics does not eradicate the disease completely, but only alleviates symptoms. The ostrich industry commissioned investigations into the development of three specific vaccines using the relatively novel approach of DNA vaccination. The concept of DNA vaccine development is based on the availability of complete genome sequences of the pathogen against which the vaccine is to be developed. This is necessary in order to identify vaccine candidate genes through comparative genomic studies. The Ms02 genome has previously been sequenced, resulting in 28 large contiguous sequences. This thesis used the technique of Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) to attempt assembly of these 28 contiguous sequences. The number was reduced to 14 large contiguous sequences, which were then subjected to repetitive sequence analysis and open reading frame analysis. Bioinformatic software was also used to predict the origin of replication. The extent of repeats in the Ms02 genome is illustrated, as well as the problems with genome assembly when dealing with repetitive-rich and A+T-rich genomes as those of mycoplasmas. Previous studies determined the mycoplasma oppA gene to be a good vaccine candidate gene, due to its cytadherent properties. This thesis describes the development of three DNA vaccines containing the Ms02 oppA gene, and a preliminary attempt to prove expression of one of these vaccines in a cell culture-based system. The DNA vaccine vectors pCI-neo, VR1012, and VR1020 were chosen for the vaccine development. The Ms02 oppA gene was also cloned into the prokaryotic expression vector pGEX-4T-1 in order to express the OppA protein for purification. The purified protein may be used in future serological tests in ostrich vaccination trials. In this study the protein was used to elicit anti-OppA rabbit antibodies, which were used to attempt detection of the pCI-neo-driven OppA protein expression in an MDA cell line in a transfection study. However, preliminary findings could not detect expression, but did indicate that the currently used colorimetric western blot technique may not be sensitive enough. It is suggested that different cell lines need to be investigated. Further optimisations are also required to decrease the observed non-specific binding. Stellenbosch University http://scholar.sun.ac.za IV Opsomming Die Suid-Afrikaanse volstruisbedryf is onder geweldige druk vanweë siektes wat die volstruise bedreig. Die epidemie van die H5N2 virus (voëlgriep) in die afgelope twee jaar het veroorsaak dat duisende volstruise van kant gemaak moes word. Daar is egter nog ‘n bedreiging wat tot geweldige produksie verliese lei elke jaar: die respiratoriese infeksies wat versoorsaak word deur die drie volstruis mikoplasmas, genoem Ms01, Ms02 en Ms03. Geen entstowwe is tans beskibaar om die infeksies te voorkom nie, en behandeling met behulp van antibiotikas is nie effektief in die genesing van infeksie nie, maar help net om die simptome te verlig. Weens die erns van die saak, het die Suid-Afrikaanse volstruisbedryf ‘n ondersoek geloods na die ontwikkeling van enstowwe teen elkeen van die drie volstruis mikoplasmas. Die relatiewe nuwe benadering van DNA-entstof ontwikkeling was die strategiese keuse. Die beginsel van DNA-entstof ontwikkeling berus op die beskikbaarheid van die genoomvolgordes van die siekte-veroorsakende organisme waarteen die enstof ontwikkel word. Geskikte kandidaat entstof gene word so opgespoor met behulp van vergelykende studies met ander beskikbare genome. Die Ms02 genoomvolgorde is voorheen bepaal en word verteenwoordig deur 28 groot geenvolgorde fragmente. Die tegniek van Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR) is gebruik om van die 28 fragmente aan mekaar te las. Die aantal fragmente is verminder na 14 groot geenvolgorde fragmente, wat vervolgens gebruik was om die omvang van herhalende volgordes in die genoom te bepaal, om nuwe leesrame te ondersoek, asook om die oorsprong van DNA replikasie op te spoor met behulp van bioinformatika sagteware. Die omvang van die herhalende aard van die Ms02 genoom word geïllustreer, asook die gepaardgaande probleme met die las van geenvolgorde fragmente wanneer met genome van veelvuldige herhalende volgordes, wat boonop A+T-ryk is, gewerk word, soos die van mikoplasmas. Vorige studies het die mikoplasma oppA geen geïdentifiseer as ‘n geskikte kandidaat entstof geen as gevolg van sy selaanhegting-eienskappe. Hierdie studie behels die invoeging van die Ms02 oppA geen in drie DNA-enstof vektore, naamlik pCI-neo, VR1012, en VR1020, asook die voorlopige poging om bewys van uitdrukking van een van die entstowwe in ‘n selkultuursisteem te bewerkstellig. Die geen is ook gekloneer in die prokariotiese ekspressie vektor pGEX-4T-1, ten einde die Ms02 OppA proteïen te isoleer. Die geïsoleerde proteïen kan in serologiese toetse in toekomstige volstruis enstof proewe gebruik word. In hierdie studie is die proteïen gebruik om konyn teenliggame teen dit op te wek, wat dan gebruik was om vir die pCI-neo-gedrewe ekspressie van die oppA geen te toets in ‘n selkultuur omgewing deur ‘n MDA sellyn te transfekteer. Die voorlopige resultate toon nie ekspressie van die OppA proteïen aan nie, maar het wel uitgelig dat die western blot tegniek wat tans gebruik word, dalk nie sensitief genoeg is nie. Dit kan belowend wees om ander tipes selle te toets. Verdere optimisering is ook nodig om die nie-spesifieke binding wat waargeneem is, te verlaag. Stellenbosch University http://scholar.sun.ac.za V Acknowledgements There are so many people who helped to make this thesis possible. I would like to thank all of you, whom have supported and inspired me. My sincerest gratitude. I thank the following in particular: Dr Annelise Botes, my supervisor, for her support and direction. Prof. Dirk U. Bellstedt, for his leadership and never-ending well of wisdom. Mrs Coral de Villiers, for her help, both emotional and technical. The South African Ostrich Business Chamber for financial support. The Bellstedt laboratory, for interesting times and stimulating conversation, and more recently, the Botes laboratory, for company in solitude (2010-2012). My family, friends and loved-ones, for their encouragement, love, and advice, even when not asked for. Stellenbosch University http://scholar.sun.ac.za VI Abbreviations A adenine ABC ATP-binding cassette AD arbitrary degenerate Ag antigen ampR ampicillin resistance gene APC antigen presenting cell BGH bovine growth hormone BM basal membrane bp base pair(s) BSA bovine serum albumin C cytosine CAT chloramphenicol acetyltransferase CpG cytidine-phosphate-guanosine dATP deoxyadenosine triphosphate dCTP deoxycytidine triphosphate dGTP deoxyguanosine triphosphate DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate dTTP deoxythymidine triphosphate EDTA ethylene diamine tetra-acetic acid di-sodium salt ELISA enzyme-linked immunosorbent assay EMC extracellular matrix components EU European Union G guanine G+C guanine and cytosine GOI gene(s) of interest GST glutathione S-transferase HIV human immunodeficiency virus hCMV human cytomegalovirus HMM hidden Markov models IBV infectious bronchitis virus IDT Integrated DNA Technologies IFN interferon Ig immunoglobulin Stellenbosch University http://scholar.sun.ac.za VII IL interleukin ISS immunostimulatory sequence kanR kanamycin resistance gene kb kilo base pairs kDa kilo dalton LB Luria-Bertani MCS multiple cloning site MG Mycoplasma gallisepticum MHC major histo-compatability complex mRNA messenger ribonucleic acid MS Mycoplasma synoviae NCBI National Center for Biotechnology Information NDV Newcastle Disease virus NKC natural killer cells Opp oligopeptide permease ORF open reading frame ori origin of replication PBS phosphate buffered saline PCR polymerase chain reaction RBS ribosome-binding site RE restriction endonuclease rRNA ribosomal ribonucleic acid SDM site-directed mutagenesis