mRNA Cap2'-O-Methyltransferase

mRNA Cap 2'-O-methyltransferase sitere na nje E. coli recombinant nke na-ebu mkpụrụ ndụ ihe nketa maka vaccinia mRNA Cap 2 ′-O-Methyltransferase.Enzyme a na-agbakwụnye otu methyl na 2'-O ọnọdụ nke mbụ nucleotide dị n'akụkụ ihe okpu okpu na 5'njedebe nke RNA. Enzyme na-eji S-adenosylmethionine (SAM) dị ka onye na-enye onyinye methyl na methylate capped RNA (okpu). -0) na-ebute okpu-1 nhazi.

Nhazi Cap1 nwere ike ime ka ntụgharị asụsụ dịkwuo mma, na-eme ka nkwupụta nke mRNA dị na ntụgharị na nnwale microinjection. Enzyme a chọrọ kpọmkwem RNA nwere okpu m7GpppN dị ka mkpụrụ.Ọ nweghị ike iji RNA jiri pN, ppN, pppN ma ọ bụ GpppN na njedebe 5′.Enwere ike ịkwado RNA mkpuchi site na ntụgharị in vitro site na iji okpu okpu ma ọ bụ site na capping enzymatic site na iji Vaccinia Capping Enzyme.

Ngwa

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

10 × Capping Reaction Buffer

Nchekwa

-25 ～- 15℃ maka nchekwa (zere ugboro ugboro ifriizi-thaw cycles)

Nchekwa nchekwa

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.

Nkọwa nkeji

A kọwapụtara otu nkeji dị ka ọnụọgụ enzyme achọrọ maka methylate 10 pmoles nke 80 nt capped transcript RNA na 1 hour na 37°C.

Nyocha njikwa mma

Exonuclease: 50U nke mRNA Cap 2 '-O-Methyltransferase na 1μg λ-Hind III na-agbari DNA na 37 ℃ maka awa 16 anaghị eweta mmebi dị ka agarose gel electrophoresis kpebisiri ike.

Endonuclease: 50 U nke mRNA Cap 2 '-O-Methyltransferase na 1 μg λDNA na 37 ℃ maka awa 16 anaghị eweta mmebi dị ka agarose gel electrophoresis kpebisiri ike.

Nickase: 50U nke mRNA Cap 2 '-O-Methyltransferase na 1μg pBR322 na 37 ℃ maka awa 16 anaghị eweta mmebi dị ka agarose gel electrophoresis kpebisiri ike.

RNase: 50U nke mRNA Cap 2 '-O-Methyltransferase na 1.6μg MS2 RNA maka awa 4 na 37 ℃ anaghị eweta mbibi dị ka agarose gel electrophoresis kpebisiri ike.

E. coli DNA: 50U nke mRNA Cap 2 '-O-Methyltransferase na-enyocha maka ọnụnọ nkeE. coli DNA genomic na-eji TaqMan qPCR nwere primers kpọmkwem makaE. coli 16S rRNA mpaghara.NkeE. coli mmetọ DNA nke genomic bụ =1E. coli genome.

Ọrịa nje Endotoxin: LAL-ule, dị ka Chinese Pharmacopoeia IV 2020 mbipụta, gel ókè ule usoro, izugbe iwu (1143).Ọdịnaya endotoxin nke nje bacteria kwesịrị ịbụ =10 EU/mg.

Usoro mmeghachi omume na ọnọdụ

1. Gwakọta ego kwesịrị ekwesị nke Capped RNA na H2O na-enweghị RNase n'ime tube microfuge 1.5 ml na olu ikpeazụ nke 16.0 µL.

2. Kpoo ọkụ na 65 ℃ maka nkeji 5 na-esote ice-bath maka nkeji 5.

3. Tinye ihe ndị a n'usoro akọwapụtara (maka methylation nke Capped RNA

erughị 10

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

4. Incubate na 37 ℃ maka 1 hour (2 awa incubation na-atụ aro maka iche iberibe ihe na-erughị 200 nt).

Ngwa

Iji kwalite okwu mRNA n'oge nnwale microinjection na mbufe.

Ihe ndetu na ojiji

1. Tupu mmeghachi omume, RNA kwesịrị ịdị ọcha ma gbazee na mmiri na-enweghị nuclease, ngwọta niile ekwesịghị inwe EDTA na ions ọ bụla.

2. A na-atụ aro ka ikpo ọkụ RNA ihe atụ na 65 ℃ maka 5 min tupu mmeghachi omume wepụ usoro nke abụọ na 5'end nke transcript.Enwere ike ịgbatị ya ruo nkeji 10 maka nhazi 5 ′-terminal siri ike.