Vaccinia Virus Capping Enzyme
Ko Vaccinia huaketo capping enzyme i ahu mai i te recombinant E. coli strain e mau ana i nga ira mo te Vaccinia capping enzyme.Ko tenei enzyme kotahi e rua nga waahanga iti (D1 me D12) e toru nga mahi enzymatic (RNA triphosphatase me te guanylyltransferase e te D1 subunit me te guanine methyltransferase e te D12 subunit).Ka whai hua te Vaccinia virus Capping Enzyme ki te whakakorikori i te hanganga o te potae, ka taea te taapiri i te hanganga potae 7-methylguanylate (m7Gppp, Cap 0) ki te pito 5′ o te RNA.Ko te hanganga potae (Cap 0) he mahi nui ki te whakapumautanga mRNA, te kawe me te whakamaoritanga i roto i nga eukaryotes.Ko te kapi i te RNA ma te tauhohenga enzymatic he tikanga whai hua me te ngawari ka taea te whakapai ake i te pumau me te whakamaoritanga o te RNA mo te whakamaoritanga in vitro, te whakawhiti, me te microinjection.
Waehanga
Vaccinia Virus Capping Enzyme (10 U/μL)
10×Taupiri Kaipupuri
Nga tikanga rokiroki
-25～- 15 ℃ mo te rokiroki （ A ape i nga huringa whakatio-rewa tonu）
Pupuri putunga
20 mM Tris-HCl (pH 8.0), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Whakamaramatanga Waeine
Kotahi te waeine o Vaccinia virus Capping Enzyme kua tautuhia ko te nui o te whākōkī e hiahiatia ana hei whakauru i te 10pmol o te GTP ki te tuhinga 80nt i roto i te 1 haora i te 37°C.
Mana Kounga
Exonuclease:10U o Vaccinia huaketo Capping Enzyme 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:10U o Vaccinia virus Capping Enzyme me te 1μg λDNA i te 37 ℃ mo te 16 haora karekau he paheketanga ka whakatauhia e te electrophoresis gel agarose.
Nickase:10U o Vaccinia virus Capping Enzyme me te 1 μg pBR322 i te 37 ℃ mo te 16 haora karekau he paheketanga ka whakatauhia e te electrophoresis gel agarose.
RNase:10U o Vaccinia huaketo Capping Enzyme me te 1.6μg MS2 RNA mo te 4 haora i te 37 ℃ karekau he kino e whakatauhia ana e te electrophoresis gel agarose.
1.coli DNA:Ko te 10U o Vaccinia virus Capping Enzyme ka tirotirohia mo te aroaro o te E. coli genomic DNA ma te whakamahi i te TaqMan qPCR me nga tohu tuatahi mo te waahi E. coli 16S rRNA.Ko te E. coli genomic DNA contamination is≤1 E. coli genome.
2.huakita Endotoxin: LAL-whakamatautau, e ai ki te Hainamana Pharmacopoeia IV 2020 whakaputanga, tikanga whakamatautau tepe gel, ture whanui (1143).Ko te ihirangi endotoxin huakita kia ≤10 EU/mg.
Pūnaha tauhohenga me nga tikanga
1. Kawa Whakakapi (rahi tauhohenga: 20 μL)
Ko tenei tikanga e pa ana ki te tauhohenga kapi o te 10μg RNA (≥100 nt) ka taea te whakanui ake i runga i nga tono whakamatautau.
I) Whakakotahitia te 10μg RNA me te Nuclease-free H2O ki roto i te ngongo microfuge 1.5 ml ki te rōrahi whakamutunga o te 15.0 µL.*10×Tuapapa Whakapiri: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)
2) Whakawerahia i te 65 ℃ mo te 5 meneti ka whai i te pati hukapapa mo te 5 meneti.
3) Tāpirihia nga waahanga e whai ake nei i roto i te raupapa kua tohua
| Component | Volume |
| RNA kua whakatara (≤10μg, roa≥100 nt) | 15 μL |
| 10×Tuapapa Whakapiri* | 2 μL |
| GTP (10 mM) | 1 μL |
| SAM (2 mM) | 1 μL |
| Vaccinia huaketo Capping Enzyme (10U/μL) | 1 μL |
*10×Tuapapa Whakapiri:0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH8.0)
4) Whakanuia ki te 37°C mo te 30 meneti, kua kati te RNA, kua rite mo nga tono whakararo.
2. 5′ kāpeka tauhohenga tapanga (rōrahi tauhohenga: 20 μL)
I hoahoatia tenei kawa ki te tapanga i te RNA kei roto he 5' triphosphate ka taea te whakanui ake i runga i nga hiahia.Ko te kaha o te kaporeihana tapanga ka awehia e te owehenga molar o RNA: GTP, me te ihirangi GTP i roto i nga tauira RNA.
1) Whakakotahitia te rahinga tika o te RNA me te H2O kore-Nuclease i roto i te ngongo microfuge 1.5 ml ki te 14.0 µL te rōrahi whakamutunga.
2) Whakawerahia i te 65 ℃ mo te 5 meneti ka whai i te pati hukapapa mo te 5 meneti.
3) Tāpirihia nga waahanga e whai ake nei i roto i te raupapa kua tohua.
| Component | Volume |
| RNA kua whakatara | 14 μL |
| 10×Taupiri Kaipupuri | 2 μL |
| GTP ranunga** | 2 μL |
| SAM (2 mM) | 1 μL |
| Vaccinia huaketo Capping Enzyme (10U/μL) | 1 μL |
** E tohu ana a GTP MIX ki te GTP me te iti o nga tohu.Mo te kukū o te GTP, tirohiaki Note 3.
4) Whakanuia ki te 37°C mo te 30 meneti, kua tapaina te mutunga RNA 5′ kua reri mo te awa
Nga tono
1. Whakakapi i te mRNA i mua i te whakamaoritanga whakamaoritanga/whakamaori in vitro
2. Te tapanga 5' mutunga o te mRNA
Nga korero mo te whakamahi
1.Ko te whakamahana i te otinga o te RNA i mua i te hekitanga me te Vaccinia Capping Enzyme ka tangohia te hanganga tuarua i te pito 5' o te tuhinga.Whakaroahia te wa ki te 60 meneti mo nga tuhinga e mohiotia ana he 5'munga tino hanganga.
2. Ko te RNA e whakamahia ana mo te kapi tauhohenga me purea i mua i te whakamahi me te whakatarewa ki te wai kore karihi.Kaua te EDTA e noho, me kore he tote te otinga.
3. Mo te tapanga i te mutunga 5', ko te tapeke o te kukū o te GTP kia 1-3 taima ki te kukū molar o te mRNA i te tauhohenga.
4. Ko te rōrahi o te pūnaha tauhohenga ka taea te whakanui ake, te heke iho ranei i runga i te tikanga.
