Naslov
Izolacija i karakterizacija monoklonskih antitijela specifičnih na m74 genski produkt mišjeg citomegalovirusa
(Isolation and characterization of monoclonal antibodies specific for m74 gene product of mouse cytomegalovirus)

Autori
Skenderi, Faruk

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, magistarski rad

Fakultet
Medicinski fakultet

Mjesto
Sarajevo

Datum
01.01

Godina
2011

Stranica
84

Mentor
Jonjić, Stipan

Ključne riječi
citomegalovirus; m74 gen; glikoprotein O (gO); monoklonska antitijela

Sažetak
Cytomegalovirus (CMV) is present in 40-90% of human population and is an important pathogen that causes disease in immune immature or immunocompromised host. It is the main viral cause of congenital malformations, including hearing loss and mental retardation in children. Vaccine and therapy are priorities of the WHO. Glycoprotein O is a product of the MCMV m74 gene and homologue of the gene product of HCMV UL74. It is a part of the viral surface protein complex gH/gL/gO, whose role in infection is not completely understood. Data from different studies are controversial and the role of this protein in infection has yet to be defined and clarified in order to define its suitability for the development of targeted therapies or vaccines. Monoclonal antibodies are produced by distinct clone of B lymphocytes or hybridoma. They are powerful tool in research, diagnostics and therapy. The development of monoclonal antibodies (mAb) to MCMV glycoprotein O would allow the application of multiple research mAb-dependant methods, which would facilitate research of gO its protein complexes. The aim of this research was to generate and characterize several monoclonal antibodies to different epitopes of MCMV glycoprotein O, which will allow in vitro and in vivo studies on the role of MCMV glycoprotein O in infection. We used BALB/c mouse strain, several strains of MCMV, including the wild type and mutants, then, the plasmids carrying the MCMV m74 gene, tagged with HA molecular marker, several types of mouse and human cells, and we produced a new type of transient transfected cells which expressed recombinant gO. Methods used in the study included in vivo work with animals, technology of hybridoma production, ELISA, western blot, flow cytometry, immunofluorescence microscopy, neutralization assays, transfection and transformation of cells, restriction analysis of DNA and others. Immunization of mice with inactivated virions gave rise to immune response to structural proteins, including the gO. We successfully isolated spleen from immunized mice, and using hybridoma technology with Sp2/O cells immortalized activated mouse B lymphocytes. We selected hybridomas secreting Abs specific for MCMV antigens by screening using ELISA and flow cytometry. By gene transfection we created a transient transfectant that expressed recombinant gO tagged with HA molecular marker. It was used for selection of hybridomas secreting specific anti-gO antibodies, among the earlier selected MCMV specific hybridomas. We selected several gO specific hybridomas and characterized them by Western blot and neutralization assays. Neutralization assays indicated that gO-specific antibodies are not able to neutralize in vitro infection of cells. In conclusion, we isolated and characterized six new monoclonal antibodies specific to mouse cytomegalovirus glycoprotein O. Their application is further in vitro and in vivo studies will provide elucidation of the role of gO and its protein complex gH/gL/gO in infection.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

Napomena
The development of targeted therapies or vaccines. Monoclonal antibodies are produced by distinct clone of B lymphocytes or hybridoma. They are powerful tool in research, diagnostics and therapy. The development of monoclonal antibodies (mAb) to MCMV glycoprotein O would allow the application of multiple research mAb-dependant methods, which would facilitate research of gO its protein complexes. The aim of this research was to generate and characterize several monoclonal antibodies to different epitopes of MCMV glycoprotein O, which will allow in vitro and in vivo studies on the role of MCMV glycoprotein O in infection. We used BALB/c mouse strain, several strains of MCMV, including the wild type and mutants, then, the plasmids carrying the MCMV m74 gene, tagged with HA molecular marker, several types of mouse and human cells, and we produced a new type of transient transfected cells which expressed recombinant gO. Methods used in the study included in vivo work with animals, technology of hybridoma production, ELISA, western blot, flow cytometry, immunofluorescence microscopy, neutralization assays, transfection and transformation of cells, restriction analysis of DNA and others. Immunization of mice with inactivated virions gave rise to immune response to structural proteins, including the gO. We successfully isolated spleen from immunized mice, and using hybridoma technology with Sp2/O cells immortalized activated mouse B lymphocytes. We selected hybridomas secreting Abs specific for MCMV antigens by screening using ELISA and flow cytometry. By gene transfection we created a transient transfectant that expressed recombinant gO tagged with HA molecular marker. It was used for selection of hybridomas secreting specific anti-gO antibodies, among the earlier selected MCMV specific hybridomas. We selected several gO specific hybridomas and characterized them by Western blot and neutralization assays. Neutralization assays indicated that gO-specific antibodies are not able to neutralize in vitro infection of cells. In conclusion, we isolated and characterized six new monoclonal antibodies specific to mouse cytomegalovirus glycoprotein O. Their application is further in vitro and in vivo studies will provide elucidation of the role of gO and its protein complex gH/gL/gO in infection.

POVEZANOST RADA