mRNA Cap2'-O-Methyltransferase

I-mRNA Cap 2' -O-methyltransferase yafunyanwa kwi-recombinant E. coli strain ethwala ijini yokugonya i-mRNA Cap 2 ´-O-Methyltransferase.Le enzyme yongeza iqela le-methyl kwindawo ye-2'-O ye-nucleotide yokuqala esecaleni kwesakhiwo se-cap kwi-5'ekupheleni kwe-RNA. I-enzyme isebenzisa i-S-adenosylmethionine (SAM) njenge-methyl donor kwi-methylate capped RNA (cap -0) okubangela i-cap- 1 isakhiwo.

Isakhiwo se-Cap1 sinokunyusa ukusebenza kakuhle kokuguqulela, ukuphucula ukubonakaliswa kwe-mRNA kwi-transfection kunye ne-microinjection experiments.Le enzyme idinga ngokukodwa i-RNA kunye ne-m7GpppN cap njenge-substrate.Ayinakusebenzisa i-RNA nge-pN, i-ppN, i-pppN okanye i-GpppN ekupheleni kwe-5'.I-Capped RNA inokulungiswa nge-in vitro transcription kusetyenziswa i-cap analog okanye nge-enzymatic capping kusetyenziswa i-Vaccinia Capping Enzyme.

Amacandelo

mRNA Cap 2'-O-Methyltransferase (50U/μL)

10× I-Capping Reaction Buffer

Ugcino

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

Isithinteli sogcino

20 mM Tris-HCl（pH 8.0,25℃), 100 mM NaCl, 1 mM DTT, 0. 1 mM EDTA, 0. 1% Triton X- 100, 50% glycerol.

Inkcazo yeyunithi

Iyunithi enye ichazwa njengenani le-enzyme efunekayo kwi-methylate i-10 pmoles ye-80 nt capped RNA transcript kwiyure ye-1 kwi-37 ° C.

Iimvavanyo zolawulo lomgangatho

I-Exonuclease:50U ye-mRNA Cap 2 ´ -O-Methyltransferase ene-1μg λ-Hind III yokugaya i-DNA kwi-37 ℃ kwiiyure ze-16 ayiniki ukuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.

I-Endonuclease: I-50 U ye-mRNA Cap 2 ´ -O-Methyltransferase ene-1 μg λDNA kwi-37℃ kwiiyure ze-16 ayiniki ukuthotywa njengoko kunqunywe yi-agarose gel electrophoresis.

I-Nickase: 50U ye-mRNA Cap 2 ´ -O-Methyltransferase ene-1μg pBR322 ku-37 ℃ kwiiyure ze-16 ayiniki ukuthotywa njengoko kumiselwe yi-agarose gel electrophoresis.

RNase: I-50U ye-mRNA Cap 2 ´ -O-Methyltransferase ene-1.6μg MS2 RNA kwiiyure ze-4 kwi-37℃ ayiniki ukuthotywa njengoko kunqunywe yi-agarose gel electrophoresis.

E. coli DNA: 50U ye-mRNA Cap 2 ´ -O-Methyltransferase ihlolwe ubukho beE. coli genomic DNA usebenzisa TaqMan qPCR kunye primers ezikhethekileyoE. coli 16S rRNA indawo.IE. coli ungcoliseko lwe-DNA ye-genomic yi =1E. coli igenome.

Intsholongwane 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. Dibanisa isixa esifanelekileyo se-Capped RNA kunye ne-RNase-free H2O kwi-tube ye-microfuge ye-1.5 mL ukuya kumthamo wokugqibela we-16.0 µL.

2. Fudumeza nge-65℃ imizuzu emi-5 elandelwa yibhafu yomkhenkce imizuzu emi-5.

3. Yongeza la macandelo alandelayo ngolandelelwano oluchaziweyo (kwi-methylation ye-Capped RNA

ngaphantsi kwe-10

*10× I-Capping Reaction Buffer : 500 mM Tris-HCl(pH 8.0, 25℃), 50 mM KCl, 10 mM MgCl2 , 10 mM DTT.

4. Fukamisa nge-37℃ ngeyure e-1 (iiyure ezi-2 zokufukamela ziyacetyiswa kwiqhekeza ekujoliswe kulo elingaphantsi kwe-200 nt).

Usetyenziso

Ukuphucula ukubonakaliswa kwe-mRNA ngexesha le-microinjection kunye novavanyo lokudluliselwa.

Amanqaku okusetyenziswa

1. Ngaphambi kokuphendula, i-RNA kufuneka ihlanjululwe kwaye ichithwe emanzini angenayo i-nuclease, zonke izisombululo akufanele ziqulethe nayiphi na i-EDTA kunye ne-ion.

2. Kunconywa ukutshisa isampula ye-RNA kwi-65 ℃ kwi-5 min ngaphambi kokuphendula ukususa isakhiwo sesibini kwi-5'end of transcript.Inokwandiswa ukuya kwimizuzu eli-10 kulwakhiwo oluntsonkothileyo lwe-5 ´ -terminal.