Jauna pieeja audzēju antigēnu mikročipu izgatavošanā
Date
2009
Authors
Endzeliņš, Edgars
Journal Title
Journal ISSN
Volume Title
Publisher
Latvijas Universitāte
Abstract
Ļaundabīgo audzēju antigēnu mikročipu tehnoloģija paver jaunas iespējas izstrādāt uz autoantivielu noteikšanu balstītus neinvazīvus vēža skrīninga un/vai diagnostikas testus. Līdz šim mūsu laboratorijā projekta "Seroloģiska ļaundabīgo audzēju diagnostikas testa izstrādāšana" ietvaros tika izstrādāti un testēti T7 fāgu displeja audzēju antigēnu mikročipi, kuru galvenos trūkumus raksturo tas, ka rekombinanto proteīnu skaits T7 fāga kapsīdā ir ļoti variabls, kā arī T7 fāga proteīnu klātbūtne var dot viltus pozitīvus signālus, ja testējamais serums satur augsta titra antivielas pret T7 fāgu vai E. coli antigēniem. Bakalaura darba mērķis bija izstrādāt jaunu pieeju antigēnu mikročipu konstruēšanā, kas ļautu uz čipa printēt no T7 displeja vektora nošķeltus tīrus antigēnus ar Strep-II-Tag iezīmi C galā, kam vajadzētu padarīt šos antigēnus viegli attīrāmus un kvantitējamus.
Bakalaura darba ietvaros uz Strep-II-Tag iezīmi saturoša T7 vektora bāzes tika konstruēts T7 displeja vektors ar 3C proteāzes šķelšanas saita insertu. Šajā vektorā klonējot 12 dažādu audzēju antigēnu kDNS fragmentus, tika izveidots rekombinato fāgu panelis, kas tālāk tika izmantots gan rekombinato proteīnu šķelšanas metodikas izstrādāšanai, gan rekombinato fāgu – antigēnu mikročipu izgatavošanai. Imunoblota un mikročipu analīzes parādīja, ka fāgi uz savas virsmas ekspresē attiecīgos audzēju antigēnus ar Strep-II-Tag iezīmi, 3C proteāze ar augstu efektivitāti atšķeļ rekombinantos proteīnus, tomēr atšķeltie antigēni nebija detektējami ar StrepMAB antivielām pret C gala Strep-II-Tag iezīmi. Mikročipu testi liecināja, ka 3C proteāze degradē vai modificē Strep-II-Tag iezīmi, kā rezultātā antigēni nevar tikt attīrīti ar Strep-Taktīna kolonnu vai atpazīti ar antivielām pret Strep-II-Tag.
Bakalaura darbs tika izstrādāts Latvijas Biomedicīnas pētījumu un studiju centrā vēža molekulārās ģenētikas darba grupā, grupas vadītājas Dr. biol. Aijas Linē vadībā laikā no 2008. līdz 2009. gadam, 5. un 6. studiju semestrī.
Atslēgvārdi: 3C proteāze, audzēju antigēni, ļaundabīgie audzēji, proteīnu mikročipi, T7 vektors
Antigen microarray technology opens up new opportunities for the development of non-inavsive tests for the diagnosis and/or screening of cancer. Up to now within the framework of project „Development of the novel, non-invasive diagnostic test for the early detection of cancer” T7 phage-displayed tumor antigen microarrays were developed and tested. Major disadvantages of such microarrays are highly variable copy number of recombinant proteins in T7 capside as well as false positive signals resulting from high-titre antibodies against T7 phage and E. coli antigens present in human sera. The objective of this bachelor’s paper was to develop a novel approach for the construction of antigen microarrays that would allow cleaving the recombinant Strep-II-Tagged proteins from the surface of the phage and using the purified proteins for the production of antigen microarrays. Within the framework of bachelor’s paper 3C protease’s cleavage site containing T7 display vector was constructed on the basis of the previously created Strep-II-Tag containing T7 display vector. cDNA fragments of 12 known tumour antigens were cloned in the obtained vector and the panel of the recombinant phages was used for the elaboration of the cleavage methodology and for the production of phage displayed antigen microarrays. Western blot and microarray analysis demonstrated that the phages display the respective Strep-II-Tagged proteins on their surface, the recombinant proteins can be cleaved of by 3C protease with high efficiency, however the cleaved antigens were undetectable by StrepMAB antibodies against C-terminal Strep-II-Tag label. Tests with microarrays indicated that 3C protease is capable of modifying or degrading Strep-II-Tag label that do not allow purification of antigens by using Strep-Tactin column or the detection with antibodies against Strep-II-Tag. The bachelor’s paper was done in the Latvian Biomedical Research and Study Center, in the Laboratory of Molecular Genetics of Cancer under the supervision of Dr. biol. Aija Linē during the 2008 and 2009 year, during the 5rd and 6th semester. Keywords: 3C protease, cancer, protein microarrays, T7 phage vector, tumor antigens
Antigen microarray technology opens up new opportunities for the development of non-inavsive tests for the diagnosis and/or screening of cancer. Up to now within the framework of project „Development of the novel, non-invasive diagnostic test for the early detection of cancer” T7 phage-displayed tumor antigen microarrays were developed and tested. Major disadvantages of such microarrays are highly variable copy number of recombinant proteins in T7 capside as well as false positive signals resulting from high-titre antibodies against T7 phage and E. coli antigens present in human sera. The objective of this bachelor’s paper was to develop a novel approach for the construction of antigen microarrays that would allow cleaving the recombinant Strep-II-Tagged proteins from the surface of the phage and using the purified proteins for the production of antigen microarrays. Within the framework of bachelor’s paper 3C protease’s cleavage site containing T7 display vector was constructed on the basis of the previously created Strep-II-Tag containing T7 display vector. cDNA fragments of 12 known tumour antigens were cloned in the obtained vector and the panel of the recombinant phages was used for the elaboration of the cleavage methodology and for the production of phage displayed antigen microarrays. Western blot and microarray analysis demonstrated that the phages display the respective Strep-II-Tagged proteins on their surface, the recombinant proteins can be cleaved of by 3C protease with high efficiency, however the cleaved antigens were undetectable by StrepMAB antibodies against C-terminal Strep-II-Tag label. Tests with microarrays indicated that 3C protease is capable of modifying or degrading Strep-II-Tag label that do not allow purification of antigens by using Strep-Tactin column or the detection with antibodies against Strep-II-Tag. The bachelor’s paper was done in the Latvian Biomedical Research and Study Center, in the Laboratory of Molecular Genetics of Cancer under the supervision of Dr. biol. Aija Linē during the 2008 and 2009 year, during the 5rd and 6th semester. Keywords: 3C protease, cancer, protein microarrays, T7 phage vector, tumor antigens
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Bioloģija