Modificētas Lopbarības pupiņas raibuma vīrusam līdzīgās daļiņas vakcīnu konstruēšanai
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Latvijas Universitāte
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lav
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Maģistra darba mērķis ir izveidot CCMV CP VLP konstrukcijas E.coli, kas paredzētas efektīvākai ķīmisko epitopu pievienošanai uz VLP virsmas un lielākai daļiņu stabilitātei. Maģistra darbs izstrādāts no 2016. gada septembra līdz 2018. gada maijam Latvijas Biomedicīnas pētījumu un studiju centrā, Augu virisoloģijas laboratorijā Dr. biol. Andra Zeltiņa un Dr. biol. Inas Baļķes vadībā. Pētījuma objekts ir lopbarības pupiņas raibumu vīruss – CCMV. Maģistra darba laikā tika izveidotas divpadsmit CCMV CP ekspresijas konstrukcijas – pET42-CCMV-G3SKG3, pET4-CCMV-G3SKG3-Ntt830, pET42-CCMV-G3SKG3-Ctt830, pET42-CCMV-SS, pET42-CCMV-Ntt830-SS, pET42-CCMV-Ctt830-SS, pET42-∆24CCMV-SS, pET42-∆24CCMV-Ntt830-SS, pET42-∆24CCMV-Ctt830-SS, pET42-CCMV-G3SKG3-SS, pET42-CCMV-G3SKG3-Ntt830-SS, pET42-CCMV-G3SKG3-Ctt830-SS. Ekspresijas pārbaudes analīze uzrādīja, ka visi CP varianti ekspresējas E.coli un ir šķīstoši. CCMV-G3SKG3, CCMV-G3SKG3-Ntt830, CCMV-SS, CCMV-Ntt830-SS, ∆24CCMV-SS, CCMV-G3SKG3-SS, CCMV-G3SKG3-Ntt830-SS veidoja T=3 simetrijas VLP, taču CCMV-Ctt830-SS veidoja filamentozas daļiņas. Stabilitātes eksperimenti liecināja, ka visstabilākā konstrukcija ir CCMV-G3SKG3-SS, kas uzglabāta 15 mM KHPO4 pH 5.5 un 0.25M NaCl buferī, kā arī SS konstrukcijas ir izturīgākas pret lielākām sāls koncentrācijām. CCMV-wt, CCMV-Ntt830, CCMV-SS, CCMV-Ntt830-SS, izjaucot un savācot ar oligonukleotīdu G-10, pie mazākas G-10 koncentrācijas veido filamentozas daļiņas, taču pie lielākas formējas ikozahederālās daļiņās. CCMV-G3SKG3-SS var izmantot ķīmiskai epitopu pievienošanai uz VLP virsmas ieviestajam lizīnam. Atslēgas vārdi: lopbarības pupiņas raibumu vīruss, apvalka proteīns, tetanusa toksīns, VLP, sāls stabils, G-10
The aim of my masters work was to construate CCMC CP VLP’s in E.coli for more efficient chemical coupling on the surface of VLP, and enhance stability. My research has been worked out from September 2016 to May 2018 at Plant Virology group at Latvian Biomedical Research and Study Centre under supervision of Dr. biol. Andris Zeltiņš and Dr. biol. Ina Baļķe. The object of my research was cowpea chlorotic mottle virus – CCMV. Twelve CCMV CP expression constructions was made during my masters work - pET42-CCMV-G3SKG3, pET4-CCMV-G3SKG3-Ntt830, pET42-CCMV-G3SKG3-Ctt830, pET42-CCMV-SS, pET42-CCMV-Ntt830-SS, pET42-CCMV-Ctt830-SS, pET42-∆24CCMV-SS, pET42-∆24CCMV-Ntt830-SS, pET42-∆24CCMV-Ctt830-SS, pET42-CCMV-G3SKG3-SS, pET42-CCMV-G3SKG3-Ntt830-SS, pET42-CCMV-G3SKG3-Ctt830-SS. Expression analysis revealed that all CP constructs were soluble and expressed in E.coli. CCMV-G3SKG3, CCMV-G3SKG3-Ntt830, CCMV-SS, CCMV-Ntt830-SS, ∆24CCMV-SS, CCMV-G3SKG3-SS, CCMV-G3SKG3-Ntt830-SS have T=3 symmetry, but CCMV-Ctt830-SS forms filaments. Stability experiments suggested that CCMV-G3SKG3-SS, which was kept in 15 mM KHPO4 pH 5.5 and 0.25M NaCl buffer, is the most stabile construcion of all; also – SS konstructions were more stabile in higher salt concentrations. Disassembly and reassembly of CCMV-wt, CCMV-Ntt830, CCMV-SS, CCMV-Ntt830-SS VLP’s with oligonucleotide G-10 produced filaments in decreased G-10 concentrations, but formed icosahedral particles in increased G-10 concentrations. CCMV-G3SKG3-SS can be used for coupling reactions on VLP surface due to inserted lysine. Key words: cowpea chlorotic mottle virus, coat protein, tetanus toxin, VLP, salt stabile, G-10.
The aim of my masters work was to construate CCMC CP VLP’s in E.coli for more efficient chemical coupling on the surface of VLP, and enhance stability. My research has been worked out from September 2016 to May 2018 at Plant Virology group at Latvian Biomedical Research and Study Centre under supervision of Dr. biol. Andris Zeltiņš and Dr. biol. Ina Baļķe. The object of my research was cowpea chlorotic mottle virus – CCMV. Twelve CCMV CP expression constructions was made during my masters work - pET42-CCMV-G3SKG3, pET4-CCMV-G3SKG3-Ntt830, pET42-CCMV-G3SKG3-Ctt830, pET42-CCMV-SS, pET42-CCMV-Ntt830-SS, pET42-CCMV-Ctt830-SS, pET42-∆24CCMV-SS, pET42-∆24CCMV-Ntt830-SS, pET42-∆24CCMV-Ctt830-SS, pET42-CCMV-G3SKG3-SS, pET42-CCMV-G3SKG3-Ntt830-SS, pET42-CCMV-G3SKG3-Ctt830-SS. Expression analysis revealed that all CP constructs were soluble and expressed in E.coli. CCMV-G3SKG3, CCMV-G3SKG3-Ntt830, CCMV-SS, CCMV-Ntt830-SS, ∆24CCMV-SS, CCMV-G3SKG3-SS, CCMV-G3SKG3-Ntt830-SS have T=3 symmetry, but CCMV-Ctt830-SS forms filaments. Stability experiments suggested that CCMV-G3SKG3-SS, which was kept in 15 mM KHPO4 pH 5.5 and 0.25M NaCl buffer, is the most stabile construcion of all; also – SS konstructions were more stabile in higher salt concentrations. Disassembly and reassembly of CCMV-wt, CCMV-Ntt830, CCMV-SS, CCMV-Ntt830-SS VLP’s with oligonucleotide G-10 produced filaments in decreased G-10 concentrations, but formed icosahedral particles in increased G-10 concentrations. CCMV-G3SKG3-SS can be used for coupling reactions on VLP surface due to inserted lysine. Key words: cowpea chlorotic mottle virus, coat protein, tetanus toxin, VLP, salt stabile, G-10.