Smiltsērkšķu marafivīrusa kodēto proteīnu klonēšana, ekspresija un raksturošana
Date
2022
Authors
Ludviga, Rebeka
Journal Title
Journal ISSN
Volume Title
Publisher
Latvijas Universitāte
Abstract
Smiltsērķšu marafivīruss (SBuMV) ir iepriekš neraksturots, smiltsērkšķus inficējošs vīruss. Tam ir divas atklātās translācijas fāzes (ORF), kur ORF1 kodē poliproteīnu, kas satur četrus proteīnu domēnus, bet ORF2 kodē apvalka proteīnu (CP). Šajā darbā tika izveidotas ekspresijas plazmīdas SBuMV kodētajiem proteīniem, taču E. coli sistēmā izdevās ekspresēt un attīrīt tikai abas CP formas un proteāzes (PRO) domēnu. Tika pārbaudīta PRO aktivitāte, PRO šķelšanas saits un verificēta PRO deubikvitināzes (DUB) aktivitāte. SBuMV mažorais CP E. coli spēj pašsavākties vīrusam līdzīgajās daļinās (VLP), bet minorais CP veido agregātus. Koekspresijas rezultātā, ja abas CP formas ekspresē no vienas plazmīdas, VLP pašsavākšanās ir traucēta, bet, ja abas CP formas ekspresē no dažādām plazmīdām, tad VLP veidošanās tiek ietekmēta minimāli. PRO un CP tika kristalizēti, taču neizdevās iegūt 3D struktūras. Tādēļ tās tika vizualizētas, izmantojot AlphaFold 2.0 programmu.
Sea buckthorn marafivirus (SBuMV) is a previously not described sea buckthorn infecting virus. It has two open reading frames (ORF) where ORF1 codes a polyprotein, which contains four protein domains, but ORF2 codes coat protein (CP). SBuMV coded protein expression plasmids were created in this work, however it was only possible to express and purify in E. coli system both CP forms and the protease (PRO). PRO activity, PRO cleavage site and PRO deubiquitinase activity was tested in this work. SBuMV major CP can self-assemble in E. coli in virus-like particles (VLP), but minor CP aggregates. Using coexpression, if both CP forms are expressed from one plasmid, self-assembly is disturbed, but if they are expressed from two different plasmids, VLP assembly is minimally disturbed. PRO and CP were crystallized, however it wasn’t possible to acquire their 3D structures. For this reason they were visualized with AlphaFold 2.0 programme.
Sea buckthorn marafivirus (SBuMV) is a previously not described sea buckthorn infecting virus. It has two open reading frames (ORF) where ORF1 codes a polyprotein, which contains four protein domains, but ORF2 codes coat protein (CP). SBuMV coded protein expression plasmids were created in this work, however it was only possible to express and purify in E. coli system both CP forms and the protease (PRO). PRO activity, PRO cleavage site and PRO deubiquitinase activity was tested in this work. SBuMV major CP can self-assemble in E. coli in virus-like particles (VLP), but minor CP aggregates. Using coexpression, if both CP forms are expressed from one plasmid, self-assembly is disturbed, but if they are expressed from two different plasmids, VLP assembly is minimally disturbed. PRO and CP were crystallized, however it wasn’t possible to acquire their 3D structures. For this reason they were visualized with AlphaFold 2.0 programme.
Description
Keywords
Bioloģija , Marafivirus , E. coli ekspresijas sistēma , 3D struktūra , nestrukturālie proteīni , apvalka proteīns