mRNA Cap2'-O-Methyltransferase

mRNA Cap 2' -O-methyltransferase e nkiloe ho mofuta o kopaneng oa E. coli o jereng lefutso la ente ea mRNA Cap 2 ´-O-Methyltransferase.Enzyme ena e eketsa sehlopha sa methyl sebakeng sa 2'-O sa nucleotide ea pele e haufi le sebopeho sa cap ho 5'qetellong ea RNA. Enzyme e sebelisa S-adenosylmethionine (SAM) e le mofani oa methyl ho methylate capped RNA (cap. -0) ho fella ka cap- 1 sebopeho.

Sebopeho sa Cap1 se ka eketsa bokhoni ba ho fetolela, ho ntlafatsa polelo ea mRNA litekong tsa transfection le microinjection.Enzyme ena e hloka ka ho khetheha RNA e nang le cap m7GpppN e le substrate.Ha e khone ho sebelisa RNA le pN, ppN, pppN kapa GpppN qetellong ea 5′.Capped RNA e kanna ea lokisoa ka mongolo oa in vitro o sebelisa cap analog kapa ka enzymatic capping o sebelisa Vaccinia Capping Enzyme.

Likaroloana

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

10 × Capping Reaction Buffer

Polokelo

-25 ～- 15℃ bakeng sa polokelo (Qoba makhetlo a mangata a ho qhibiliha a leqhoa)

Sebaka sa polokelo

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.

Tlhaloso ea yuniti

Yuniti e le 'ngoe e hlalosoa e le palo ea enzyme e hlokahalang ho methylate 10 pmoles ea 80 nt capped RNA transcript ka hora e le 1 ho 37 ° C.

Litlhahlobo tsa taolo ea boleng

Exonuclease:50U ea mRNA Cap 2 ´ -O-Methyltransferase e nang le 1μg λ-Hind III cheka DNA ka 37 ℃ bakeng sa lihora tse 16 ha e fane ka ho senyeha ho ea ka agarose gel electrophoresis.

Endonuclease: 50 U ea mRNA Cap 2 ´ -O-Methyltransferase e nang le 1 μg λDNA ho 37℃ bakeng sa lihora tsa 16 ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.

Nickase: 50U ea mRNA Cap 2 ´ -O-Methyltransferase e nang le 1μg pBR322 ho 37 ℃ bakeng sa lihora tsa 16 ha e fane ka ho senyeha ha ho khethoa ke agarose gel electrophoresis.

RNase: 50U ea mRNA Cap 2 ´ -O-Methyltransferase e nang le 1.6μg MS2 RNA bakeng sa lihora tsa 4 ho 37℃ ha e fane ka ho senyeha ho ea ka agarose gel electrophoresis.

E. coli DNA: 50U ea mRNA Cap 2 ´ -O-Methyltransferase e hlahlojoa bakeng sa boteng baE. coli DNA ea genomic e sebelisang TaqMan qPCR e nang le li-primers tse khethehileng bakeng saE. coli Sebaka sa 16S rRNA.TheE. coli tšilafalo ea DNA ea genomic ke =1E. coli genome.

Baktheria Endotoxin: Teko ea LAL, ho latela khatiso ea China Pharmacopoeia IV 2020, mokhoa oa tlhahlobo ea moeli oa gel, molao o akaretsang (1143).Likahare tsa endotoxin ea baktheria e lokela ho ba =10 EU/mg.

Mokhoa oa ho itšoara le maemo

1. Kopanya palo e loketseng ea Capped RNA le RNase-free H2O ka har'a tube ea microfuge ea 1.5 mL ho ea ho molumo oa ho qetela oa 16.0 µL.

2. Chesa ka likhato tse 65 ℃ metsotso e 5 e lateloe ke ho hlapa ka leqhoa metsotso e 5.

3. Kenya likarolo tse latelang ka tatellano e boletsoeng (bakeng sa methylation ea Capped RNA

ka tlase ho 10

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

4. Eketsa ka 37 ℃ bakeng sa hora ea 1 (lihora tse 2 tsa ho qhoqhoa li buelloa bakeng sa sekhechana sa sepheo se ka tlase ho 200 nt).

Lisebelisoa

Ho ntlafatsa polelo ea mRNA nakong ea liteko tsa microinjection le transfection.

Lintlha mabapi le tšebeliso

1. Pele ho karabelo, RNA e lokela ho hloekisoa le ho qhibiliha metsing a se nang nuclease, litharollo tsohle ha lia lokela ho ba le EDTA le li-ion.

2. Ho khothalletsoa ho futhumatsa sampuli ea RNA ho 65 ℃ bakeng sa 5 min pele ho karabelo ho tlosa sebopeho sa bobeli ho 5'qetellong ea sengoloa.E ka eketsoa ho metsotso e 10 bakeng sa sebopeho se rarahaneng sa 5 ´ -terminal.