Synthesis of paramyxoviridae nucleoproteins in yeast Saccharomyces cerevisiae and their application in viral diagnostics ; Paramyxoviridae šeimos virusų nukleokapsidės baltymų sintezė mielėse Saccharomyces cerevisiae ir jų panaudojimas diagnostikai

Jurga

Sujets

• Biochemistry
• Diagnostics
• Synthesis

Informations

• Publié par: vilnius_university
• Publié le: 01 janvier 2011
• Nombre de lectures: 29
• Poids de l'ouvrage: 1 Mo

Extrait

VILNIUSNIVERSITY






MindaugasJuozapaitis

SYNTHESISF PARAMYXOVIRIDAEUCLEOPROTEINSNEAST
SACCHAROMYCESCEREVISIAENDHEIRPPLICATIONNIRAL
DIAGNOSTICS


Summaryofdoctoraldissertation
Physicalsciences,biochemistry(04P)









Vilnius,011ThisstudyhasbeencarriedoutattheLaboratoryofEukaryouteGeneticEngineeringof
theInstituteofBiotechnology,VilniusUniversity,during20042010.

Dissertationismaintainedbuextern.

Scientificconsultant:
Prof.dr.Habil.KstutisSasnauskas(InstituteofBiotechnology,VilniusUniversity,
physicalscience,biochemistry–04P).

Evaluationoardfissertationfiochemistryrend:

Chairman:
Prof.dr.VirginijusŠikšnys(InstituteofBiotechnology,VilniusUniversity,physical
sciences,biochemistry–04P).
Members:
Prof.dr.habil.AniolasSruoga(VytautasMagnusUniversity,biomedicalsciences,
ecologyandenviromentalscience–03B);
Dr. Giedrius Vilkaitis (Institute of Biotechnology, Vilnius University, physical
sciences,biochemistry–04P);
Dr. Arnas Lagunaviius (Thermo Fisher Scientific, physical sciences,
biochemistry–04P);
Dr.AušraRažanskien (InstituteofBiotechnology,VilniusUniversity,physical
sciences,biochemistry–04P).
Officialopponents:
Dr. Arvydas Kanopka (Institute of Biotechnology, Vilnius University, physical
sciences,biochemistry–04P);
Dr. Artras Jakubauskas (VUH Santariški Klinikos, Center of Hematology,
OnkologyandTransfuzion,physicalsciences,biochemistry–04P).

th
Thedissertationdefencwilltakepalceat10a.m.on20 ofJuly,2011attheInstituteof
Biotechnology,VilniusUniversity,Graiino8,VilniusLT02241,Lithuania.

The dissertation is available at the Library of Institute of Biotechnology, Vilnius
University,andattheLibraryofVilniusUniversity.






VILNIAUSNIVERSITETAS




MindaugasJuozapaitis

PARAMYXOVIRIDAEEIMOSIRUSNUKLEOKAPSIDALTYM
SINTEZMIELE SACCHAROMYCESCEREVISIAER
PANAUDOJIMASIAGNOSTIKAI



Daktarodisertacija
Fiziniaimokslai,biochemija(04P)






Vilnius,011
Disertacija rengta 20042010 metais Vilniaus universiteto Biotechnologijos instituto
Eukariotgeninžinerijosskyriuje.

Disertacijaginamaeksternu.

Moksliniskonsultantas:
Prof. habil. dr. Kstutis Sasnauskas (VU Biotechnologijos institutas, fiziniai mokslai,
biochemija–04P)

Disertacijainamailniausniversitetoiochemijosoksloryptiesaryboje:

Pirmininkas:
Prof.dr.VirginijusŠikšnys(VUBiotechnologijosinstitutas,fiziniaimokslai,
biochemija–04P)
Nariai:
Prof.habil.dr.AniolasSruoga(VytautoDidžiojouniversitetas,biomedicinos
mokslai,ekologijairaplinkotyra–03B);
Dr. Giedrius Vilkaitis (VU Biotechnologijos institutas, fiziniai mokslai,
biochemija–04P);
Dr. Arnas Lagunaviius (Thermo Fisher Scientific, fiziniai mokslai,
biochemija–04P);
Dr. Aušra Ražanskien (VU Biotechnologijos institutas, fiziniai mokslai,
biochemija–04P).
Oponentai:
Dr. Arvydas Kanopka (VU Biotechnologijos institutas, fiziniai mokslai,
biochemija–04P);
Dr.ArtrasJakubauskas(VULSantariškiklinik Hematologijos,onkologijos
irtransfuziologijoscentras,fiziniaimokslai,biochemija–04P).

DisertacijabusginamaviešameBiochemijosmokslokryptiesposdyje2011m.birželio
mn.20d.10val.VUBiotechnologijosinstitutokonferencijsalje.
Adresas:Graiino8,VilniusLT02241,Lietuva.

Disertacijossantraukaišsiuntinta2011m.__mn.__d.
Disertacij galima peržirti VU Biotechnologijos instituto ir Vilniaus Universiteto
bibliotekose.







CONTENTS
INTRODUCTION..........................................................................................................7
MATERIALSANDMETHODS......................................................................................11
Reagents,enzymesandkits........................................................................................11
Oligonucleotides11
Bacterial,yeaststrainsandplasmids...........................................................................13
Bacterial,yeastmedia,transformationandgrowingconditions.................................13
DNApreparationandmanipulation............................................................................13
Expressionandpurificationofvirusnucleocapsidlikeparticlesfromyeast...............14
SDSPAGEandWestern...............................................................................................14
Determinationofexpressionlevelofrecombinantproteinsinyeastlysates.............14
Electronmicroscopy....................................................................................................15
IsolationandanalysisofRNA......................................................................................15
Massspectrometricanalysisofrecombinantproteins................................................15
RESULTS...................................................................................................................17
SynthesisofrecombinantSendaivirusNproteininyeastS.cerevisiae.......................17ofhumanparainfluenzavirus1and3Nproteinsinyeast
S.cerevisiae..................................................................................................................21
SynthesisofrecombinanthumanrespiratorysyncytialvirusNproteinsinyeast
S.cerevisiae24
SynthesisofrecombinanthenipahvirusNproteinsinyeastS.cerevisiae...................26ofMenanglevirusNproteininyeastS.cerevisiae..................30
ImmunoreactivityofyeastexpressedhPIV1andhPIV3Nproteinswithhumansera33ityofyeasthRSVNproteinwithhumansera....................35
ImmunoreactivityofyeastexpressedmenanglevirusNprotein................................37
GenerationofmonoclonalantibodiesagainstyeastexpressedhPIV1,hPIV3,NiVand
MenVNproteins.........................................................................................................37
DISCUSSION..............................................................................................................40
CONCLUSIONS..........................................................................................................47
5

PUBLICATIONS..........................................................................................................48
ACKNOWLEDGMENTS...............................................................................................50
REFERENCES.............................................................................................................51
REZIUM ...................................................................................................................57
CURRICULUMVITAE.................................................................................................60





















6

INTRODUCTION
TheParamyxoviridaeincludesomeofthegreatandubiquitousdiseasecausing
virusesofhumansandanimals.Humanrespiratorysyncytialvirus(hRSV)andhuman
parainfluenza virus types 1 and 3 (hPIV1 and hPIV3, respectively) are the most
prevalent and infectious respiratory pathogens which belong to the family
Paramyxoviridae(Chanock,1957;Chanocketal.,1958).Accordingtoestimatesofthe
WorldHealthOrganization,globalhRSVdiseaseburdenis64millioncasesand160.000
deathseveryyear,andinfantstogetherwithyoungchildrenareamongthosewhoare
mostlyaffected.Parainfluenzavirusescauseaspectrumofrespiratoryillnessesandthe
annualestimatedratesofhospitalizationscausedbythesevirusesarefrom15.000to
80.000casesonlyintheUS(WorldHealthOrganization2009).Parainfluenzaviruses
andhumanrespiratorysyncytialvirushavebeenassociatedwitheverykindofupper
andlowerrespiratorytractillnesseswhich,intheirsymptoms,onlyslightlydifferfrom
respiratorydiseasescausedbyotherinfectiouspathogens.Consequently,diagnosisof
theinfectiousagentcausingtherespiratorydiseasebasedonlyonclinicalsymptomsin
mostcasesisimpossible.Forthisalaboratorydiagnosisishighlyrecommended(Hall,
2001;Henrickson,2003).
Nipah,HendraandMenangleviruses(NiV,HeVandMenV,respecively)joina
growinglistofrecentlyidentifiedparamyxovirusesforwhichbatshavebeenimplicated
asthenaturalhost(Chuaetal.,1999;Philbeyetal.,1998;SelveyandSheridan,1995).
Latestreportshaveindicatedthatdeadlyoutbreakscausedbythesevirusesaresparse
andworldwideburdenisnotverysignificant(Eatonetal.,2006;Philbeyetal.,2008).
However,thesenewlyidentifiedparamyxoviruseschangefast,infectwidehostrange
including several terrestrial species in four mammalian orders, cause systemic
infectionsaffectingvariousorgansystemsalsocentralnervoussystem,andNiVand
HeVaredeadlyhumanpathogens(Eatonetal.,2006;Philbeyetal.,2008).Therefore,it
islikelythatwiththelapsoftimeinthechangingclimateerathesevirusesmaybecome
widespreadinfectiouspathogenscausingdeadlyoutbreaksinhumansandlivestock.
Since NiV, HeV and MenV were identified only recently, consequently, knowledge
7
aboutthemisrelativelyscanty.Itislittleknownaboutthebiology,spread,virulence
andpathologyoftheseviruses,alsonoeffectivemeansofdiagnostics,whichwould
helpalottofillexistingknowledgegaps,areavailablebynow(Wong,2010).
ThestudypresentedheredescribestheyeastderivedNproteinsasusefultools
forthedevelopmentofnewdetectionsystemsfortheinfectionscausedbyrespiratory
andzoonoticparamyxoviruses.