University of Jyväskylä | JYX Digital Repository

  • English  | Give feedback |
    • suomi
    • English
 
  • Login
JavaScript is disabled for your browser. Some features of this site may not work without it.
View Item 
  • JYX
  • Opinnäytteet
  • Pro gradu -tutkielmat
  • View Item
JYX > Opinnäytteet > Pro gradu -tutkielmat > View Item

Immunodetection of VP11, the minor capsid protein of thermophilic bacteriophage P23-77

Thumbnail
View/Open
1.3 Mb

Downloads:  
Show download detailsHide download details  
Authors
Moilanen, Anni
Date
2015
Discipline
Solu- ja molekyylibiologiaCell and molecular biology

 
VP11 on termofiilisen bakteriofagin P23-77 kapsidiproteiini ja se muodostaa ikosahedraalisen kapsidirakenteen yhdessä pääkapsidiproteiinien, virusproteiini (VP)16 ja VP17, kanssa. VP11 tarkkaa paikkaa ja toimintaa kapsidissa ei tiedetä. Aiemmissa tutkimuksissa VP11 on arveltu sijaitsevan joko kapsidikuoren alapuolella tai kapsidin ulkopinnalla piikkirakenteissa. Tutkimuksen tavoitteena oli optimoida VP11 havaitsemisessa käytettävä western blot –menetelmä, arvioida VP11 määrää kapsidissa ja tutkia VP11 sijaintia vasta-ainevärjäyksen ja transmissio elektronimikroskopian (TEM) avulla. Western blot –menetelmän optimoinnissa käytettiin puoli kuivaa (engl. semi-dry) menetelmää vaihtelemalla vasta-ainevärjäysolosuhteita. VP11 määrää kapsidissa analysoitiin denaturoivan polyakryyliamidigeelin juovien intensiteetteihin avulla. P23-77 kasvatettiin isäntäbakteeri Thermus thermophiluksessa, jota seurasi kaksivaiheinen viruspuhdistus. Puhdistettu virus vasta-aineleimattiin ja kuvattiin TEMilla. Western blot –menetelmän optimointi onnistui, menetelmä antoi tarkan signaalin optimaalisten vasta-ainevärjäysolosuhteiden löydyttyä. Lisäksi western blot –tulokset viittasivat siihen että VP11 on rakenteeltaan dimeeri. VP11 kopiomäärän arvioitiin olevan 147, joka viittaa siihen että VP11 ei ole osallisena viruksen kapsidin piikkirakenteissa. Alustavat TEM kokeet tukevat sitä että VP11 ei sijaitse kapsidin ulkopinnalla. Saatujen tulosten perusteella VP11 sijaitsee viruskapsidin sisäpuolella dimeeri-rakenteena. Proteiinin sijainnin ja muiden ominaisuuksien vuoksi VP11 saattaa olla hyödyllinen tulevaisuuden bioalan sovelluksissa. ...
 
VP11 is a minor capsid protein of the thermophilic bacteriophage P23-77 and comprises the icosahedral capsid mainly with two major capsid proteins (MCPs) virion protein (VP) 16 and VP17. The exact localization and function of VP11 is unknown. Yet, previous studies suggests that VP11 is located either between the protein capsid and the internal lipid membrane or occupies the capsid vertices on the outer side of the capsid. The aims of the study were to optimize a western blot method for the detection of VP11, estimate the copy number of VP11 in the virus particle and study the localization of VP11 by using immunolabelling and transmission electron microscopy (TEM). Western blots were performed with purified virus samples in a semi-dry transfer system with varying immunolabelling conditions. The copy number of VP11 was analyzed based on band intensities in denaturing polyacrylamide gels. P23-77 was cultured by using Thermus thermophilus as a host and purified by rate zonal and equilibrium centrifugation. Purified virus particles were immunolabelled for TEM. The optimization of the western blot method succeeded, specific signals were received after suitable immunolabelling conditions were found. In addition, the western blot results imply that VP11 exists as a dimer in the P23-77 virion. The copy number of VP11 was estimated to be approximately 147 copies. Both findings indicate that VP11 is not the penton protein occupying the capsid vertices. Supporting this theory, preliminary studies with TEM suggest that VP11 does not exist on the outer side of the capsid. Rather, VP11 is located underneath the protein shell as a dimer. Due to its proposed location and other qualities, VP11 might have potential in future biochemical applications. ...
 
Keywords
virukset kapsidi bakteriofagit
URI

http://urn.fi/URN:NBN:fi:jyu-201602101524

Metadata
Show full item record
Collections
  • Pro gradu -tutkielmat [24518]

Related items

Showing items with similar title or keywords.

  • Something old, something new : exploring membrane-containing bacteriophages 

    Mäntynen, Sari (University of Jyväskylä, 2016)
  • The Minor Capsid Protein VP11 of Thermophilic Bacteriophage P23-77 Facilitates Virus Assembly by Using Lipid-Protein Interactions 

    Pawlowski, Alice; Moilanen, Anni M.; Rissanen, Ilona; Määttä, Juha A. E.; Hytönen, Vesa P.; Ihalainen, Janne; Bamford, Jaana (American Society for Microbiology, 2015)
    Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membranecontaining double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic ...
  • Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77 

    Rissanen, Ilona; Pawlowski, Alice; Harlos, Karl; Grimes, Jonathan; Stuart, David; Bamford, Jaana (International Union of Crystallography (IUCr), 2012)
    Members of the diverse double--barrel lineage of viruses are identified by the conserved structure of their major coat protein. New members of this lineage have been discovered based on structural analysis and we are ...
  • Thermus bacteriophage P23-77 : key member of a novel, but ancient family of viruses from extreme environments 

    Pawlowski, Alice (University of Jyväskylä, 2015)
  • Small things matter : of phages and antibiotic resistance conferring plasmids 

    Mattila, Sari (University of Jyväskylä, 2016)
    Viruses and plasmids are small units of genetic material dependent on cells either transiently or continuously. Intriguingly, stories of these small entities intertwine in antibiotic resistance crisis. Horizontal gene ...
  • Browse materials
  • Browse materials
  • Articles
  • Conferences and seminars
  • Electronic books
  • Historical maps
  • Journals
  • Tunes and musical notes
  • Photographs
  • Presentations and posters
  • Publication series
  • Research reports
  • Research data
  • Study materials
  • Theses

Browse

All of JYXCollection listBy Issue DateAuthorsSubjectsPublished inDepartmentDiscipline

My Account

Login

Statistics

View Usage Statistics
  • How to publish in JYX?
  • Self-archiving
  • Publish Your Thesis Online
  • Publishing Your Dissertation
  • Publication services

Open Science at the JYU
 
Data Protection Description

Accessibility Statement

Unless otherwise specified, publicly available JYX metadata (excluding abstracts) may be freely reused under the CC0 waiver.
Open Science Centre