Enzyme ya Vaccinia
Imisemburo ya virusi ya Vaccinia ikomoka kuri recombinant E. coli amoko atwara ingirabuzimafatizo ya Vaccinia.Iyi misemburo imwe igizwe na subunits ebyiri (D1 na D12) kandi ifite ibikorwa bitatu byimikorere (RNA triphosphatase na guanylyltransferase na D1 subunit na guanine methyltransferase na D12 subunit).Virusi ya Vaccinia Capping Enzyme ifite akamaro kanini muguhindura imiterere yimiterere ya cap, ishobora guhuza cyane na 7-methylguanylate cap cap (m7Gppp, Cap 0) kumpera ya 5 ′ ya RNA.Imiterere ya cap (Cap 0) igira uruhare runini muguhindura mRNA, gutwara no guhindura muri eukaryote.Gufata RNA ukoresheje enzymatique nuburyo bwiza kandi bworoshye bushobora kunoza cyane ituze noguhindura RNA kugirango wandike vitro, kwanduza, na microinjection.
Ibigize
Enzyme yo gufata virusi ya Vaccinia (10 U / μL)
10 × Gufata Buffer
Imiterere yo kubika
-25 ～ - 15 ℃ kubika （Irinde gusubiramo inshuro nyinshi gukonjesha haw
Ububiko
20 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Igisobanuro
Igice kimwe cya virusi ya Vaccinia Capping Enzyme isobanurwa nkubunini bwa enzyme isabwa kugirango winjize 10pmol ya GTP mumyandikire ya 80nt mumasaha 1 kuri 37 ° C.
Kugenzura ubuziranenge
Exonuclease:10U ya virusi ya Vaccinia Capping Enzyme hamwe na 1μg λ-Hind III igogora ADN kuri 37 ℃ mu masaha 16 nta musaruro utanga nkuko byagenwe na agarose gel electrophorei.
Endonuc Please:10U ya virusi ya Vaccinia Capping Enzyme hamwe na 1μg λDNA kuri 37 ℃ kumasaha 16 nta musaruro utanga nkuko byagenwe na agarose gel electrophorei.
Nickase:10U ya virusi ya Vaccinia Capping Enzyme hamwe na 1 μg pBR322 kuri 37 ℃ kumasaha 16 nta musaruro utanga nkuko byagenwe na agarose gel electrophorei.
RNase:10U ya virusi ya Vaccinia Capping Enzyme hamwe na 1.6μg MS2 RNA mu masaha 4 kuri 37 ℃ nta musaruro ugabanuka nkuko byagenwe na agarose gel electrophorei.
1.ADN ya coli:10U ya virusi ya Vaccinia Capping Enzyme isuzumwa kugirango haboneke ADN genomic E. coli ikoresheje TaqMan qPCR hamwe na primers yihariye ya E. coli 16S rRNA.E. coli genomic ADN yanduye ni ≤1 E. geni genome.
2.Indwara ya bagiteri Endotoxin: Ikizamini cya LAL, ukurikije Pharmacopoeia yo mu Bushinwa IV 2020, uburyo bwo gupima gel, uburyo rusange (1143).Indwara ya bagiteri igomba kuba ≤10 EU / mg.
Sisitemu yo kwitwara hamwe nibisabwa
1. Gufata Protokole (ingano ya reaction: 20 μL)
Ubu buryo burakoreshwa kuri capping reaction ya 10μg RNA (≥100 nt) kandi irashobora kwaguka ukurikije ibyifuzo byubushakashatsi.
I) Huza 10μg RNA na Nuclease-H2O idafite umuyoboro wa microfuge ya 1.5 kugeza mubunini bwa 15.0 µL.* 10 × Gufata Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH 8.0)
2) Shyushya kuri 65 ℃ muminota 5 ukurikirwa no kwiyuhagira urubura muminota 5.
3) Ongeramo ibice bikurikira murutonde rwerekanwe
| CIbigize | Volume |
| RNA yamaganwe (≤10μg, uburebure≥100 nt) | 15 μL |
| 10 × Gufata Buffer * | 2 μL |
| GTP (10 mM) | 1 μL |
| SAM (2 mM) | 1 μL |
| Virusi ya Vaccinia Capping Enzyme (10U / μL) | 1 μL |
* 10 × Gufata Buffer ： 0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH8.0)
4) Kwiyongera kuri 37 ° C muminota 30, RNA irafunzwe kandi yiteguye kubisabwa hasi.
2. 5 ′ terminal Ikimenyetso (ingano ya reaction: 20 μL)
Porotokole yagenewe kuranga RNA irimo 5´ triphosifate kandi irashobora gupimwa ukurikije ibisabwa.Imikorere ya label yinjizwamo izaterwa nigipimo cya mara ya RNA: GTP, kimwe nibirimo GTP mubitegererezo bya RNA.
1) Huza ingano ikwiye ya RNA na Nuclease idafite H2O mumiyoboro ya microfuge ya ml 1.5 kugeza mubunini bwa 14.0 µL.
2) Shyushya kuri 65 ℃ muminota 5 ukurikirwa no kwiyuhagira urubura muminota 5.
3) Ongeramo ibice bikurikira murutonde rwerekanwe.
| CIbigize | Volume |
| RNA | 14 μL |
| 10 × Gufata Buffer | 2 μL |
| GTP ivanga ** | 2 μL |
| SAM (2 mM) | 1 μL |
| Virusi ya Vaccinia Capping Enzyme (10U / μL) | 1 μL |
** GTP MIX bivuga GTP numubare muto wibimenyetso.Kubijyanye na GTP, rebaKuri Icyitonderwa 3.
4) Kwiyongera kuri 37 ° C muminota 30, RNA 5 ′ iherezo ryanditseho kandi ryiteguye kumanuka
Porogaramu
1. Gufata mRNA mbere yubusobanuro bwibisobanuro / mubisobanuro bya vitro
2. Kwandika 5´ iherezo rya mRNA
Inyandiko zikoreshwa
1.Gushyushya igisubizo cya RNA mbere yubushakashatsi hamwe na Vaccinia Capping Enzyme ikuraho imiterere ya kabiri kuri 5´end ya transcript.Ongera umwanya kuminota 60 kubitabo byanditse hamwe bizwi cyane byubatswe 5´ends.
2. RNA ikoreshwa mugukata reaction igomba kwezwa mbere yo kuyikoresha no guhagarikwa mumazi adafite nuclease.EDTA ntigomba kuba ihari kandi igisubizo kigomba kuba kitarimo umunyu.
3. Kugirango ushireho 5´ iherezo, igiteranyo cya GTP kigomba kuba hafi inshuro 1-3 ubunini bwa mRNA muri reaction.
4. Ingano ya sisitemu ya reaction irashobora kugabanywa cyangwa kumanuka ukurikije nyirizina.
