Hepatīta C vīrusa ARFP ekspresija Escherichia coli un īpašību raksturošana
Date
2017
Authors
Andersone, Santa
Journal Title
Journal ISSN
Volume Title
Publisher
Latvijas Universitāte
Abstract
Ar C hepatīta vīrusu inficēto cilvēku skaits pasaulē ir apmēram 170 miljoni. Pret HCV nav vakcīnas, tāpēc tiek veikti pētījumi, kas saistīti ar tās izstrādi. Darba mērķis bija iegūt HCV alternatīvā nolasīšanas rāmja proteīnus divās plazmīdu sistēmās, un optimizēt ekspresijas un attīrīšanas apstākļus augstas kvalitātes proteīnu iegūšanai. Darbā tika izmantoti dažāda garuma un genotipa ARFP un divas klonēšanas un ekspresijas plazmīdas ar atšķirīgu 6xHistag iezīmes novietojumu. Pēc indukcijas ar IPTG apstākļu optimizācijas augstākā ARFP ekspresija E. coli BL21(DE3) šūnās tika iegūta, inducējot 4h pie + 37°C un 6h pie + 30°C ar 0,4 mM IPTG. Pēc ARFP attīrīšanas ar imobilizēto metālafīno hromatogrāfiju tika konstatēts, ka proteīni ir daļēji šķīstoši un nedegradē, tāpēc tos ir plānots izmantot imunoloģiskiem pētījumiem dzīvnieku modelī.
The number of people infected by hepatitis C virus worldwide is about 170 million. There is no suitable vaccine against the HCV, therefore it is important to carry out research that is connected with vaccine development. The aim of the work was to obtain HCV alternative reading frame proteins in two plasmid systems, and to optimize conditions of protein expression and purification to achieve high quality recombinant proteins. In this study ARFP of different length and genotypes in two plasmids with different position of 6xHistag were used. After optimization of IPTG induction conditions, the highest ARFP expression level in E.coli BL21(DE3) cells was achieved with 0,4 mM IPTG concentration and 4h induction at + 37°C and after 6h induction at + 30°C. Purification of ARFP by immobilized metal ion affinity chromatography indicated that proteins were partly soluble, stable, therefore they can be used in immunological studies in mice.
The number of people infected by hepatitis C virus worldwide is about 170 million. There is no suitable vaccine against the HCV, therefore it is important to carry out research that is connected with vaccine development. The aim of the work was to obtain HCV alternative reading frame proteins in two plasmid systems, and to optimize conditions of protein expression and purification to achieve high quality recombinant proteins. In this study ARFP of different length and genotypes in two plasmids with different position of 6xHistag were used. After optimization of IPTG induction conditions, the highest ARFP expression level in E.coli BL21(DE3) cells was achieved with 0,4 mM IPTG concentration and 4h induction at + 37°C and after 6h induction at + 30°C. Purification of ARFP by immobilized metal ion affinity chromatography indicated that proteins were partly soluble, stable, therefore they can be used in immunological studies in mice.
Description
Keywords
Bioloģija , HCV , ARFP , ekspresija , IMAC