Vaccinia Virus Capping Enzyme

Vaccinia nje capping enzyme sitere na recombinant E. coli nje na-ebu mkpụrụ ndụ ihe nketa maka Vaccinia capping enzyme.Enzyme otu a bụ nke akụkụ abụọ (D1 na D12) nwere ọrụ enzymatic atọ (RNA triphosphatase na guanylyltransferase site na ngalaba D1 na guanine methyltransferase site na ngalaba D12).Vaccinia virus Capping Enzyme dị irè iji kpalite nhazi okpu okpu, nke nwere ike itinye kpọmkwem usoro okpu 7-methylguanylate (m7Gppp, Cap 0) na njedebe 5′ nke RNA.Nhazi okpu (Cap 0) na-arụ ọrụ dị mkpa na nkwụsi ike mRNA, mbugharị na ntụgharị n'ime eukaryotes.Capping RNA site na mmeghachi omume enzymatic bụ ụzọ dị irè ma dị mfe nke nwere ike melite nkwụsi ike na ntụgharị RNA maka ntụgharị in vitro, ntụgharị na microinjection.

Ngwa

Vaccinia Nje Virus Capping Enzyme (10 U/μL)

Ihe nchekwa 10 × Capping

Ọnọdụ nchekwa

-25～- 15℃ maka nchekwa (Zezere freze-thaw cycles ugboro ugboro)

Nchekwa nchekwa

20mM Tris-HCl (pH 8.0), 100 mM NaCl,

1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.

Nkọwa nkeji

A kọwapụtara otu nkeji nje Vaccinia Capping Enzyme dị ka ọnụọgụ enzyme achọrọ iji tinye 10pmol nke GTP n'ime transcript 80nt n'ime elekere 1 na 37°C.

Njikwa ogo

Exonuclease:10U nke Vaccinia virus Capping Enzyme na 1μg λ-Hind III na-agbari DNA na 37 ℃ maka awa 16 anaghị eweta mmebi ọ bụla dịka agarose gel electrophoresis siri kpebie.

Endonuclease:10U nke Vaccinia virus Capping Enzyme nwere 1μg λDNA na 37 ℃ maka awa 16 anaghị eweta mbibi ọ bụla dịka agarose gel electrophoresis siri kpebie.

Nickase:10U nke Vaccinia virus Capping Enzyme nwere 1 μg pBR322 na 37 ℃ maka awa 16 anaghị ebute mmebi dị ka agarose gel electrophoresis siri kpebie.

RNase:10U nke Vaccinia virus Capping Enzyme nwere 1.6μg MS2 RNA maka awa 4 na 37 ℃ anaghị amịpụta mbibi dịka agarose gel electrophoresis siri kpebie.

1.coli DNA:A na-enyocha 10U nke nje Vaccinia Capping Enzyme maka ọnụnọ nke E. coli genomic DNA site na iji TaqMan qPCR na primers kpọmkwem maka E. coli 16S rRNA locus.Ihe mmetọ DNA nke E. coli bụ≤1 E. coli genome.

2.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. Capping Protocol (olu mmeghachi omume: 20 μL)

Usoro a na-adaba na mmeghachi omume capping nke 10μg RNA (≥100 nt) na enwere ike ịbawanye ya dịka ihe nyocha chọrọ.

I) Jikọta 10μg RNA na H2O na-enweghị Nuclease na tube microfuge 1.5 ml na olu ikpeazụ nke 15.0 µL.* 10 × Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH 8.0)

2) Kpoo ọkụ na 65 ℃ maka nkeji 5 na ice bat maka nkeji 5.

3) Tinye ihe ndị a n'usoro akọwapụtara

* 10 × Capping Buffer: 0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH8.0)

4) Tinye na 37 Celsius C maka nkeji 30, RNA bụzi capped na njikere maka ngwa ala.

2.5′ ọnụ mmeghachi omume labeling (olu mmeghachi omume: 20 μL)

Ezubere ụkpụrụ a iji kpọọ RNA nwere 5′ triphosphate na enwere ike ịbawanye ya dịka achọrọ.A ga-emetụta arụmọrụ nke ntinye labelụ site na oke molar nke RNA: GTP yana ọdịnaya GTP dị na nlele RNA.

1) Gwakọta ego kwesịrị ekwesị nke RNA na H2O na-enweghị Nuclease na tube microfuge 1.5 ml na olu ikpeazụ nke 14.0 µL.

2) Kpoo ọkụ na 65 ℃ maka nkeji 5 na ice bat maka nkeji 5.

3) Tinye ihe ndị a n'usoro akọwapụtara.

** GTP MIX na-ezo aka na GTP na ọnụ ọgụgụ dị nta nke akara.Maka itinye uche nke GTP, rụtụ akaka ndetu 3.

4) Tinye na 37ºC maka nkeji 30, RNA 5′ njedebe ka akara ugbu a ma dị njikere maka mgbada.

Ngwa

1. Capping mRNA tupu ntụgharị ntụgharị asụsụ/in vitro

2. Ịkpọ aha 5' njedebe nke mRNA

Ihe ndetu na ojiji

1.Na-ekpo ọkụ ngwọta nke RNA tupu incubation na Vaccinia Capping Enzyme na-ewepu usoro nke abụọ na 5'njedebe nke transcript.Gbatịkwuo oge na nkeji 60 maka ndenye ederede nwere njedebe 5'nhazi nke ọma.

2. RNA eji maka mmeghachi omume capping kwesịrị ịdị ọcha tupu ejiri ya ma kwụsịtụ na mmiri na-enweghị nuclease.EDTA ekwesịghị ịdị na ngwọta ya ekwesịghị ịbụ nke nnu.

3. Maka ịde akara njedebe 5′, mkpokọta GTP kwesịrị ịdị ihe dị ka ugboro 1-3 karịa mkpokọta molar nke mRNA na mmeghachi omume.

4. Mpịakọta nke usoro mmeghachi omume nwere ike ịbawanye ma ọ bụ gbadaa dịka n'ezie.