mRNA Cap2'-O-Methyltransferase

mRNA Cap 2'-O-methyltransferase na maua mai i le recombinant E. coli strain lea e tauaveina le kenera mo tui mRNA Cap 2'-O-Methyltransferase.O lenei enzyme e faʻaopoopoina se vaega methyl i le tulaga 2'-O o le nucleotide muamua e lata ane i le fausaga o le pulou i le 5'iuga o le RNA. O le enzyme e faʻaaogaina le S-adenosylmethionine (SAM) e avea o se methyl donor e methylate tapuni RNA (cap). -0) i'u ai i se fausaga cap- 1.

O le Cap1 fausaga e mafai ona faʻateleina le faʻaliliuga lelei, faʻaleleia le faʻaalia o le mRNA i le faʻaliliuina ma le microinjection faʻataʻitaʻiga. O lenei enzyme e manaʻomia faapitoa le RNA ma se pulou m7GpppN e fai ma substrate.E le mafai ona fa'aogaina le RNA fa'atasi ma pN, ppN, pppN po'o le GpppN ile pito 5'.E mafai ona saunia le RNA fa'apipi'i e ala ile fa'aliliuga in vitro e fa'aaoga ai le pulou analog po'o le fa'apipi'iina o le enzymatic e fa'aaoga ai le Vaccinia Capping Enzyme.

Vaega

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

10 × Capping Reaction Buffer

Teuina

-25 ～- 15 ℃ mo le teuina （ Aloese mai taamilomilo fa'a'aisa-liu fa'atele)

Taofi puipui

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.

Fa'amatalaga o le iunite

E tasi le iunite o loʻo faʻamatalaina o le aofaʻi o le enzyme e manaʻomia e methylate 10 pmoles o le 80 nt faʻapipiʻi RNA transcript i le 1 itula ile 37°C.

Su'ega fa'atonuga lelei

Exonuclease:50U o le mRNA Cap 2 ´ -O-Methyltransferase ma le 1μg λ-Hind III digest DNA i le 37 ℃ mo le 16 itula e leai se fa'aleagaina e pei ona fuafuaina e le electrophoresis gel agarose.

Endonuclease: 50 U o le mRNA Cap 2 ´ -O-Methyltransferase ma le 1 μg λDNA i le 37 ℃ mo le 16 itula e leai se faʻaleagaina e pei ona fuafuaina e le electrophoresis gel agarose.

Nickase: 50U o le mRNA Cap 2 ´ -O-Methyltransferase ma le 1μg pBR322 i le 37 ℃ mo le 16 itula e leai se faʻaleagaina e pei ona fuafuaina e le electrophoresis gel agarose.

RNase: 50U o le mRNA Cap 2 ´ -O-Methyltransferase ma le 1.6μg MS2 RNA mo le 4 itula i le 37 ℃ e leai se faʻaleagaina e pei ona fuafuaina e le electrophoresis gel agarose.

E. coli DNA: 50U o le mRNA Cap 2 ´ -O-Methyltransferase o loʻo suʻeina mo le i ai oE. coli genomic DNA fa'aaoga TaqMan qPCR fa'atasi ai ma fa'amatalaga fa'apitoa mo leE. coli 16S rRNA nofoaga.O leE. coli genomic DNA contamination o =1E. coli genome.

siama Endotoxin: LAL-suʻega, e tusa ai ma le Chinese Pharmacopoeia IV 2020 lomiga, gel faʻatapulaʻa suʻega metotia, tulafono lautele (1143).O mea e maua i le endotoxin siama e tatau ona = 10 EU/mg.

Faiga ma tulaga

1. Fa'afefiloi le aofa'i talafeagai o le Capped RNA ma le RNase-free H2O i totonu o le 1.5 mL microfuge paipa i se voluma mulimuli o le 16.0 µL.

2. Fa'avela ile 65℃ mo le 5 minute sosoo ai ma le ta'ele aisa mo le 5 minute.

3. Fa'aopoopo vaega nei i le fa'atonuga ua fa'amaonia (mo le methylation o Capped RNA

itiiti ifo i le 10

*10× Fa'amauina Tali Fa'asaga: 500 mM Tris-HCl(pH 8.0, 25℃), 50 mM KCl, 10 mM MgCl2, 10 mM DTT.

4. Fa'asusu ile 37 ℃ mo le 1 itula (2 itula ole fa'aoso e fautuaina mo le vaega fa'atatau ile itiiti ifo ile 200 nt).

Talosaga

Faʻaleleia le faʻamatalaga mRNA i le taimi o suʻega microinjection ma suʻega.

Fa'amatalaga ile fa'aogaina

1. Aʻo leʻi faʻaalia, e tatau ona faʻamamaina le RNA ma faʻamavae i totonu o vai e leai se vai, o vaifofo uma e le tatau ona aofia ai soʻo se EDTA ma ions.

2. E fautuaina e fa'avevela le fa'ata'ita'iga RNA ile 65 ℃ mo le 5 min a'o le'i faia le tali e aveese ai le fausaga lona lua ile pito 5' ole tusitusiga.E mafai ona fa'alautele i le 10 min mo le fa'alavelave lavelave 5´-terminal fausaga.