Vaccinia Virus Capping Enzyme

I-Vaccinia virus capping enzyme ithathwe kwi-recombinant E. coli strain ithwala ijene ye-Vaccinia capping enzyme.Le enzyme enye yenziwe ngamacandelo amabini (i-D1 kunye ne-D12) kwaye inemisebenzi emithathu ye-enzymatic (i-RNA triphosphatase kunye ne-guanylyltransferase yi-subunit ye-D1 kunye ne-guanine methyltransferase yi-subunit ye-D12).I-Vaccinia virus Capping Enzyme iyasebenza ekwenzeni ukubunjwa kwe-cap structure, enokuthi ifake ngokukodwa i-7-methylguanylate cap structure (m7Gppp, Cap 0) ukuya kwi-5' ekupheleni kwe-RNA.Isakhiwo seCap (i-Cap 0) idlala indima ebalulekileyo ekuzinziseni kwe-mRNA, ukuthutha kunye nokuguqulelwa kwe-eukaryotes.I-Capping RNA ngokusabela kwe-enzymatic yindlela esebenzayo nelula enokuphucula ngokubonakalayo uzinzo kunye nokuguqulelwa kwe-RNA kushicilelo lwe-in vitro, ukudluliselwa, kunye ne-microinjection.

Amacandelo

Vaccinia Virus Capping Enzyme (10 U/μL)

10×I-Capping Buffer

Iimeko zokugcina

-25～- 15℃ yokugcina (Kuphephe ukuphinda kunyibilike imijikelo yokunyibilikisa)

Isithinteli sogcino

20 mM Tris-HCl (pH 8.0), 100 mM NaCl,

1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.

Inkcazelo yeyunithi

Iyunithi enye yeVaccinia virus Capping Enzyme ichazwa njengobungakanani be-enzyme efunekayo ukuze kufakwe i-10pmol ye-GTP kwi-80nt transcript kwiyure e-1 kuma-37°C.

Ulawulo lwemeko

I-Exonuclease:I-10U yeVaccinia virus Capping Enzyme ene-1μg λ-Hind III yokwetyisa i-DNA kwi-37 ℃ kwiiyure ze-16 ivelisa akukho kuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.

I-Endonuclease:I-10U yeVaccinia virus Capping Enzyme ene-1μg λDNA ku-37℃ kwiiyure ze-16 ivelisa akukho kuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.

I-Nickase:I-10U yeVaccinia virus Capping Enzyme ene-1 μg pBR322 ku-37 ℃ kwiiyure ezili-16 ivelisa akukho kuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.

RNase:I-10U yeVaccinia virus Capping Enzyme ene-1.6μg MS2 RNA kwiiyure ezi-4 kwi-37℃ ayivelisi ukuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.

1.coli DNA:I-10U yeVaccinia virus Capping Enzyme ijongiwe ubukho be-E. coli genomic DNA isebenzisa i-TaqMan qPCR eneziqalo ezikhethekileyo ze-E. coli 16S rRNA locus.Ungcoliseko lwe-E. coli genomic DNA yi≤1 E. coli genome.

2.Intsholongwane I-Endotoxin: Uvavanyo lwe-LAL, ngokwe-Chinese Pharmacopoeia IV 2020 edition, indlela yokuvavanya umda we-gel, umthetho jikelele (1143).Umxholo we-endotoxin yebhaktheriya kufuneka ube ≤10 EU/mg.

Inkqubo yokusabela kunye neemeko

1. I-Capping Protocol (umthamo wokuphendula: 20 μL)

Le nkqubo isebenzayo kwi-capping reaction ye-10μg RNA (≥100 nt) kwaye inokunyuswa ngokweemfuno zovavanyo.

I) Hlanganisa i-10μg RNA kunye ne-Nuclease-free H2O kwi-tube ye-microfuge ye-1.5 ml ukuya kumthamo wokugqibela we-15.0 µL.*10×Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)

2) Ukufudumala kwi-65 ℃ imizuzu emi-5 ilandelwa yi-ice bath imizuzu emi-5.

3) Yongeza amacandelo alandelayo ngokulandelelana okuchaziweyo

*10×Capping Buffer:0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH8.0)

4) Fudumeza kwi-37 ° C kwimizuzu engama-30, i-RNA ngoku ivaliwe kwaye ilungele izicelo ezisezantsi.

2. 5′ terminal ukuleyibhela impendulo (umthamo wokuphendula: 20 μL)

Le ndlela yomthetho yenzelwe ukulebhelisha i-RNA equlethe i-5′ triphosphate kwaye inokunyuswa ngokweemfuno.Ukusebenza kakuhle kokufakwa kwelebula kuya kuba nefuthe kwi-molar ratio ye-RNA: GTP, kunye nomxholo we-GTP kwiisampuli ze-RNA.

1) Dibanisa isixa esifanelekileyo se-RNA kunye ne-Nuclease-free H2O kwityhubhu ye-microfuge eyi-1.5 ml ukuya kumthamo wokugqibela we-14.0 µL.

2) Fudumeza kwi-65 ℃ imizuzu emi-5 elandelwa yibhafu ye-ice imizuzu emi-5.

3)Yongeza la malungu alandelayo ngolandelelwano oluchaziweyo.

** I-GTP MIX ibhekisa kwi-GTP kunye nenani elincinci labamakishi.Ngoxinaniso lwe-GTP, bhekisakwiNqaku 3.

4) Fudumeza kuma-37°C kangangemizuzu engama-30, isiphelo se-RNA 5′ ngoku sibhalwe kwaye silungele ukuya ezantsi.

Usetyenziso

1. Ukufaka i-mRNA phambi kovavanyo loguqulo/ukuguqulelwa kwe-in vitro

2. Ukuleyibhelishwa 5′ ekupheleni kwe-mRNA

Amanqaku okusetyenziswa

1.Ukufudumeza isisombululo se-RNA ngaphambi kokufukanyelwa kunye ne-Vaccinia Capping Enzyme kususa isakhiwo sesibini kwi-5'end of transcript.Yandisa ixesha ukuya kwimizuzu engama-60 ngokushicilelweyo kunye ne-5'ends ezakhiwe kakhulu.

2. I-RNA esetyenziselwa i-capping reactions kufuneka ihlanjululwe ngaphambi kokusetyenziswa kwaye inqunyanyiswe emanzini angenayo i-nuclease.I-EDTA kufuneka ingabikho kwaye isisombululo kufuneka singabi natyuwa.

3. Ukulebula i-5′ ekupheleni, i-concentration ye-GTP iyonke kufuneka ibe malunga ne-1-3 amaxesha e-molar concentration ye-mRNA kwi-reaction.

4. Umthamo wenkqubo yokusabela unokunyuswa okanye uthotywe ngokwenyani.