mRNA Cap2'-O-Methyltransferase

Ko te mRNA Cap 2' -O-methyltransferase i ahu mai i te riaka E. coli recombinant e kawe ana i te ira mo te vaccinia mRNA Cap 2 ´-O-Methyltransferase.Ka tāpirihia e tēnei whākōkī he rōpū methyl ki te tūnga 2'-O o te nucleotide tuatahi e pātata ana ki te hanganga potae i te pito 5' o te RNA. Ka whakamahia e te whākōkī te S-adenosylmethionine (SAM) hei kaituku methyl ki te methylate RNA kapi (potae). -0) ka puta he hanganga potae- 1.

Ka taea e te hanganga Cap1 te whakanui ake i te pai o te whakamaoritanga, te whakapai ake i te whakahuatanga o te mRNA i roto i nga whakamatautau whakawhiti me te microinjection. Ko tenei enzyme e tino hiahia ana ki te RNA me te potae m7GpppN hei taputapu.Kaore e taea te whakamahi RNA me te pN, ppN, pppN, GpppN ranei i te mutunga 5'.Ka taea te whakarite i te RNA kapi ma te whakamaaramatanga in vitro ma te whakamahi i te tairitenga potae, ma te taapiri enzymatic ranei ma te whakamahi i te Vaccinia Capping Enzyme.

Waehanga

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

10×Capping Reaction Buffer

Rokiroki

-25 ～- 15 ℃ mo te rokiroki （ A ape i nga huringa whakatio-rewa tonu）

Pupuri putunga

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.

Te whakamaramatanga o te waeine

Kotahi te waeine kua tautuhia ko te nui o te whaariki e hiahiatia ana ki te methylate 10 pmoles o te 80 nt capted RNA tuhinga i roto i te 1 haora i te 37°C.

Nga whakamatautau mana kounga

Exonuclease:50U o te mRNA Cap 2 ´ -O-Methyltransferase me te 1μg λ-Hind III te keri DNA i te 37 ℃ mo te 16 haora karekau he paheketanga ka whakatauhia e te electrophoresis gel agarose.

Endonuclease: 50 U o te mRNA Cap 2 ´ -O-Methyltransferase me te 1 μg λDNA i te 37 ℃ mo nga haora 16 karekau he paheketanga ka whakatauhia e te electrophoresis gel agarose.

Nickase: 50U o te mRNA Cap 2 ´ -O-Methyltransferase me te 1μg pBR322 i te 37 ℃ mo nga haora 16 karekau he paheketanga ka whakatauhia e te electrophoresis gel agarose.

RNase: 50U o te mRNA Cap 2 ´ -O-Methyltransferase me te 1.6μg MS2 RNA mo nga haora 4 i te 37 ℃ karekau he whakahekenga e whakatauhia ana e te electrophoresis gel agarose.

E. coli DNA: 50U o te mRNA Cap 2 ´ -O-Methyltransferase kua tirotirohia mo te aroaro oE. coli DNA genomic e whakamahi ana i te TaqMan qPCR me nga tohu tuatahi mo teE. coli 16S rRNA waahi.Ko teE. coli ko te poke DNA ira he =1E. coli genome.

huakita Endotoxin: LAL-whakamātautau, e ai ki te Hainamana Pharmacopoeia IV 2020 putanga, te tikanga whakamatautau tepe gel, ture whanui (1143).Ko te ihirangi endotoxin huakita kia =10 EU/mg.

Pūnaha tauhohenga me nga tikanga

1. Whakakotahitia te nui e tika ana o Capped RNA me te RNase-kore H2O ki roto i te ngongo microfuge 1.5 mL ki te 16.0 µL te rōrahi whakamutunga.

2. Whakawerahia i te 65 ℃ mo te 5 meneti ka whai i te kaukau hukapapa mo te 5 meneti.

3. Tāpirihia nga waahanga e whai ake nei i roto i te raupapa kua tohua (mo te methylation o te RNA Capped

iti iho i te 10

*10× Kaipupuri Tauhohenga Kape : 500 mM Tris-HCl(pH 8.0, 25 ℃), 50 mM KCl, 10 mM MgCl2 、 10 mM DTT.

4. Whakanuia ki te 37 ℃ mo te 1 haora (e taunakitia ana kia 2 haora te heki mo te kongakonga whainga iti iho i te 200 nt).

Nga tono

Hei whakapai ake i te korero mRNA i te wa o te microinjection me nga whakamatautau whakawhiti.

Nga korero mo te whakamahi

1. I mua i te tauhohenga, me purea te RNA ka memeha ki roto i te wai kore karihi, kaua nga otinga katoa e whai EDTA me nga katote.

2. E taunaki ana kia whakamahana te tauira RNA ki te 65 ℃ mo te 5 meneti i mua i te tauhohenga ki te tango i te hanganga tuarua i te 5'mutunga o te tuhinga.Ka taea te whakaroa atu ki te 10 meneti mo te hangahanga 5 ´ -whakapapa.