Vaccinia Virus Capping Enzyme
Vaccinia virus capping enzyme imachokera ku mtundu wina wa E. coli womwe umanyamula majini a Vaccinia capping enzyme.Enzyme imodzi imeneyi imapangidwa ndi ma subunits awiri (D1 ndi D12) ndipo imakhala ndi ntchito zitatu za enzymatic (RNA triphosphatase ndi guanylyltransferase ndi D1 subunit ndi guanine methyltransferase ndi D12 subunit).Vaccinia Virus Capping Enzyme imathandizira kupanga mapangidwe a kapu, omwe amatha kulumikiza kapu ya 7-methylguanylate (m7Gppp, Cap 0) kumapeto kwa RNA.Kapangidwe ka cap (Cap 0) imakhala ndi gawo lofunikira pakukhazikika kwa mRNA, zoyendetsa ndi kumasulira mu eukaryotes.Kulemba RNA ndi enzymatic reaction ndi njira yabwino komanso yosavuta yomwe ingathandize kwambiri kukhazikika ndi kumasulira kwa RNA kwa in vitro transcription, transfection, and microinjection.
Zigawo
Vaccinia Virus Capping Enzyme (10 U/μL)
10 × Capping Buffer
Zosungirako
-25～- 15 ℃ posungira (Pewani kuzizira kobwerezabwereza)
Bafa yosungirako
20 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Tanthauzo la Chigawo
Chigawo chimodzi cha Vaccinia virus Capping Enzyme chimatanthauzidwa ngati kuchuluka kwa enzyme yofunikira kuphatikiza 10pmol ya GTP mu 80nt transcript mu ola limodzi pa 37°C.
Kuwongolera Kwabwino
Exonuclease:10U ya Vaccinia virus Capping Enzyme yokhala ndi 1μg λ-Hind III digest DNA pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
Endonuclease:10U ya Vaccinia Virus Capping Enzyme yokhala ndi 1μg λDNA pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
Nickase:10U ya Vaccinia Virus Capping Enzyme yokhala ndi 1 μg pBR322 pa 37 ℃ kwa maola 16 sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
RNase:10U ya Vaccinia Virus Capping Enzyme yokhala ndi 1.6μg MS2 RNA kwa maola 4 pa 37 ℃ sikutulutsa kuwonongeka komwe kumatsimikiziridwa ndi agarose gel electrophoresis.
1.coli DNA:10U ya Vaccinia virus Capping Enzyme imawunikiridwa ngati pali E. coli genomic DNA pogwiritsa ntchito TaqMan qPCR yokhala ndi zoyambira za E. coli 16S rRNA locus.Kuwonongeka kwa E. coli genomic DNA ndi≤1 E. coli genome.
2.Bakiteriya Endotoxin: Kuyesa kwa LAL, malinga ndi kope la Chinese Pharmacopoeia IV 2020, njira yoyesera malire a gel, malamulo onse (1143).Mabakiteriya endotoxin ayenera kukhala ≤10 EU/mg.
Dongosolo ndi zikhalidwe
1. Capping Protocol (voliyumu yamachitidwe: 20 μL)
Njirayi imagwiranso ntchito ku capping reaction ya 10μg RNA (≥100 nt) ndipo imatha kukulitsidwa molingana ndi zoyeserera.
I) Phatikizani 10μg RNA ndi H2O yopanda Nuclease mu chubu cha microfuge cha 1.5 ml mpaka voliyumu yomaliza ya 15.0 µL.*10×Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)
2) Kutenthetsa pa 65 ℃ kwa mphindi 5 ndikutsatiridwa ndi madzi oundana kwa mphindi zisanu.
3) Onjezani zigawo zotsatirazi mu dongosolo lomwe latchulidwa
| Cwotsutsa | Volume |
| Denatured RNA (≤10μg, kutalika≥100 nt) | 15 μl pa |
| 10 × Capping Buffer * | 2 ml |
| GTP (10 mM) | 1 ml |
| SAM (2 mM) | 1 ml |
| Vaccinia Virus Capping Enzyme (10U/μL) | 1 ml |
*10×Capping Buffer:0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH8.0)
4) Yalirani pa 37 ° C kwa mphindi 30, RNA tsopano yatsekedwa ndipo ikukonzekera ntchito zapansi.
2. 5′ terminal kulemba kachitidwe (volumu yamayankhidwe: 20 μL)
Protocol iyi idapangidwa kuti ilembe RNA yokhala ndi 5' triphosphate ndipo imatha kukulitsidwa malinga ndi zofuna.Kuchita bwino kwa kuphatikizidwa kwa zilembo kudzakhudzidwa ndi chiŵerengero cha molar cha RNA: GTP, komanso zomwe zili mu GTP mu zitsanzo za RNA.
1) Phatikizani kuchuluka koyenera kwa RNA ndi H2O yopanda Nuclease mu chubu cha microfuge cha 1.5 ml mpaka voliyumu yomaliza ya 14.0 µL.
2) Kutenthetsa pa 65 ℃ kwa mphindi 5 ndikutsatiridwa ndi madzi oundana kwa mphindi zisanu.
3) Onjezani zigawo zotsatirazi mu dongosolo lomwe latchulidwa.
| Cwotsutsa | Volume |
| RNA yosinthidwa | 14 μl pa |
| 10 × Capping Buffer | 2 ml |
| GTP mix** | 2 ml |
| SAM (2 mM) | 1 ml |
| Vaccinia virus Capping Enzyme (10U/μL) | 1 ml |
** GTP MIX imatanthauza GTP ndi zolembera zochepa.Kuti mumve zambiri za GTP, onaniku Note 3.
4) Yalirani pa 37°C kwa mphindi 30, mapeto a RNA 5′ tsopano alembedwa ndipo akukonzekera kutsika.
Mapulogalamu
1. Kulemba mRNA musanayese zomasulira/zomasulira mu vitro
2. Kulemba 5' mapeto a mRNA
Zolemba pakugwiritsa ntchito
1.Kutenthetsa yankho la RNA musanayambe kukulitsidwa ndi Vaccinia Capping Enzyme kumachotsa dongosolo lachiwiri pa 5'mapeto a cholembera.Wonjezerani nthawi mpaka mphindi 60 za zolembedwa zodziwika bwino za 5'ends.
2. RNA yogwiritsidwa ntchito popanga ma capping reaction iyenera kuyeretsedwa isanayambe kugwiritsidwa ntchito ndikuyimitsidwa m'madzi opanda nuclease.EDTA sayenera kukhalapo ndipo yankho liyenera kukhala lopanda mchere.
3. Polemba 5'mapeto, chiwerengero chonse cha GTP chiyenera kukhala 1-3 nthawi ya molar ndende ya mRNA mu zomwe zimachitika.
4. Voliyumu ya anachita dongosolo akhoza scaled mmwamba kapena pansi malinga ndi zenizeni.
