DK2899267T3 - Fremgangsmåde til fremstilling in-vitro af rotavirus-dobbeltlagede viruslignende partikler - Google Patents
Fremgangsmåde til fremstilling in-vitro af rotavirus-dobbeltlagede viruslignende partikler Download PDFInfo
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- DK2899267T3 DK2899267T3 DK13838213.0T DK13838213T DK2899267T3 DK 2899267 T3 DK2899267 T3 DK 2899267T3 DK 13838213 T DK13838213 T DK 13838213T DK 2899267 T3 DK2899267 T3 DK 2899267T3
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Claims (8)
1. Fremgangsmåde til fremstilling af en dobbeltlaget viruslignende partikel af rotavirus omfattende VP2-protein og VP6-protein, hvor fremgangsmåden omfatter de følgende trin: a) at eksprimere VP6 i en opløselig form i et prokaryotisk ekspressionssystem, og at oprense VP6'et, hvor det oprensede VP6-protein bibeholder dets korrekte konformation og foreligger i form af en trimer; b) co-samling af det oprensede VP6 med VP2 i en ikke-partikelformet tilstand for at danne en dobbeltlaget partikel 2/6-VLP.
2. Fremgangsmåde ifølge krav 1, hvor VP6-proteinet fremstilles ved hjælp af de følgende trin: 1) at eksprimere et rotavirus VP6-protein i E. colr, 2) at tilsætte polyethylenimin (PEI) eller en analog deraf, eller en divalent eller trivalent metalion, til lyseringssupernatanten omfattende VP6-proteinet, med henblik på at udfælde nukleinsyrer og nogle uønskede proteiner, hvor koncentrationen af PEI ligger mellem 0,05 % og 0,2 %, fortrinsvis 0,1 %; hvor metalionen omfatter, men ikke er begrænset til Mn2+, Mg2+, Ca2+, Zn2+ and Al3+, fortrinsvis Mn2+ og Ca2+, mest foretrukket Ca2+; koncentrationen Ca2+ ligger mellem 10 og 80mM, fortrinsvis mellem 15 og 50mM, mest foretrukket 20mM; 3) at udføre centrifugering, og at udsætte supernatanten for udsaltning og kromatografisk oprensning.
3. Fremgangsmåde ifølge krav 2, hvor udsaltningen udføres under anvendelse af mættet ammoniumsulfat.
4. Fremgangsmåde ifølge krav 2, hvor kromatografien omfatter hydrofobisk in-teraktionskromatografi og ionbytningskromatografi, fortrinsvis hydrofobisk in-teraktionskromatografi, mest foretrukket kromatografien ved hjælp af Phenyl HP (GE) under betingelsen af 3M NaCI, hvor nukleinsyrer passerer igennem søjlen, VP6-protein elueres under betingelsen af 2M NaCI, og det uønskede protein elueres under betingelsen af ingen salte.
5. Fremgangsmåde ifølge krav 1, hvor VP2-proteinet er VP2-protein i en ikke-partikelformet tilstand, der fremstilles ved hjælp af de følgende trin: 1) at eksprimere rotavirus VP2-protein i E. coir, 2) at tilsætte polyethylenimin (PEI) til lyseringssupernatanten indeholdende VP2-proteinet, hvor koncentrationen af PEI ligger mellem 0,05 % og 1 %, fortrinsvis mellem 0,3 % og 0,5 %, mest foretrukket 0,5 %; 3) at udføre centrifugering, og at oprense supernatanten ved udsaltning og kromatografi.
6. Fremgangsmåde ifølge krav 5, hvor udsaltningen udføres under anvendelse af ammoniumsulfat.
7. Fremgangsmåde ifølge krav 5, hvor kromatografien omfatter ionbytningskromatografi og hydrofobisk interaktionskromatografi, fortrinsvis kationbyt-ningskromatografi, mest foretrukket kromatografien ved hjælp af SP FF (GE) under betingelsen af Tris-HCI (pH 8,0), hvor nukleinsyrer og nogle uønskede proteiner passerer igennem søjlen eller elueres under betingelsen af 150 mM NaCI, og VP2-protein elueres under betingelsen af 500 mM NaCI.
8. Fremgangsmåde ifølge et af kravene 1-7, hvor samlingen af den dobbelt-lagede viruslignende partikel 2/6-VLP omfatter, at blande VP6- og VP2-prote-iner, derefter at erstatte bufferen med en samlingsbuffer for at danne en dobbeltlaget viruslignende partikel, hvor: a) VP2 og VP6 er oprensede proteiner, som ikke samles til partikler og har en renhed på over 95 %; b) samlingsbufferen er en phosphatbuffer, en MES-buffer eller en citratbuffer, med en pH-værdi på mellem 3,0 og 7,0, fortrinsvis mellem pH 4,0 og pH 6,4, og mest foretrukket 6,4; c) samlingsbufferen indeholder 0-2M saltioner, fortrinsvis NaCI, mere foretrukket bufferen indeholder 150mM-2M NaCI, mest foretrukket bufferen indeholder 300mM NaCI; d) masseforholdet mellem VP2 og VP6 ligger mellem 1:1 og 1:10, fortrinsvis, forholdet ligger mellem 1:2 og 1:3, og mest foretrukket, forholdet er 1:2.6.
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| CN201210350365.8A CN103667199B (zh) | 2012-09-20 | 2012-09-20 | 体外制备轮状病毒双层类病毒颗粒的方法 |
| PCT/CN2013/077736 WO2014044067A1 (zh) | 2012-09-20 | 2013-06-24 | 体外制备轮状病毒双层类病毒颗粒的方法 |
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| US10548970B2 (en) * | 2015-10-05 | 2020-02-04 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | Human rotavirus G9P[6] strain and use as a vaccine |
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