Vaccinia Virus Capping Enzyme
Vaccinia virus capping enzyme e nkiloe ho mofuta o kopaneng oa E. coli o jara liphatsa tsa lefutso tsa enzyme ea Vaccinia capping.Enzyme ena e le 'ngoe e entsoe ka li-subunits tse peli (D1 le D12) mme e na le mesebetsi e meraro ea enzymatic (RNA triphosphatase le guanylyltransferase ke karoloana ea D1 le guanine methyltransferase ka karoloana ea D12).Vaccinia virus Capping Enzyme e sebetsa hantle ho etsa hore ho thehoe sebopeho sa cap, se ka kopanyang ka ho khetheha sebopeho sa cap ea 7-methylguanylate (m7Gppp, Cap 0) pheletsong ea 5' ea RNA.Sebopeho sa cap (Cap 0) se phetha karolo ea bohlokoa ho ts'ebetsong ea mRNA, ho tsamaisa le ho fetolela ho eukaryotes.Capping RNA ka karabelo ea enzymatic ke mokhoa o sebetsang o bonolo o ka ntlafatsang haholo botsitso le phetolelo ea RNA bakeng sa in vitro transcription, transfection, le microinjection.
Likaroloana
Vaccinia Virus Capping Enzyme (10 U/μL)
10 × Capping Buffer
Maemo a 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), 100 mM NaCl,
1mM DTT, 0. 1mM EDTA, 0. 1% Triton X- 100, 50% glycerol.
Tlhaloso ea Yuniti
Yuniti e le 'ngoe ea Vaccinia virus Capping Enzyme e hlalosoa e le palo ea enzyme e hlokahalang ho kenyelletsa 10pmol ea GTP ho sengoloa sa 80nt ka hora e le 1 ho 37°C.
Taolo ea Boleng
Exonuclease:10U ea Vaccinia kokoana-hloko Capping Enzyme e nang le 1μg λ-Hind III cheka DNA ho 37 ℃ bakeng sa lihora tse 16 ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.
Endonuclease:10U ea Vaccinia Vaccinia Capping Enzyme e nang le 1μg λDNA ho 37℃ bakeng sa lihora tse 16 ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.
Nickase:10U ea Vaccinia Vaccinia Capping Enzyme e nang le 1 μg pBR322 ho 37 ℃ bakeng sa lihora tse 16 ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.
RNase:10U ea Vaccinia kokoana-hloko Capping Enzyme e nang le 1.6μg MS2 RNA bakeng sa lihora tse 4 ho 37 ℃ ha e fane ka ho senyeha ho ea ka qeto ea agarose gel electrophoresis.
1.coli DNA:10U of Vaccinia virus Capping Enzyme e hlahlojoa boteng ba E. coli genomic DNA e sebelisa TaqMan qPCR e nang le li-primers tse khethehileng bakeng sa locus ea E. coli 16S rRNA.Tšilafalo ea E. coli genomic DNA ke≤1 E. coli genome.
2.Baktheria Endotoxin: Tlhahlobo 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. Capping Protocol (bolumo ea karabelo: 20 μL)
Ts'ebetso ena e sebetsa ho karabelo ea capping ea 10μg RNA (≥100 nt) mme e ka fokotsoa ho latela litlhoko tsa liteko.
I) Kopanya 10μg RNA le H2O e se nang Nuclease ka har'a tube ea microfuge ea 1.5 ml ho bophahamo ba ho qetela ba 15.0 µL.*10×Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH 8.0)
2) Chesa ka 65 ℃ bakeng sa metsotso e 5 e lateloa ke ho hlatsoa leqhoa ka metsotso e 5.
3) Kenya likarolo tse latelang ka tatellano e boletsoeng
| Cmohanyetsi | Volume |
| Denatured RNA (≤10μg, bolelele≥100 nt) | 15 μL |
| 10×Capping Buffer* | 2 μL |
| GTP (10 mM) | 1 μL |
| SAM (2 mM) | 1 μL |
| Vaccinia virus Capping Enzyme (10U/μL) | 1 μL |
*10×Capping Buffer:0.5 M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25℃, pH8.0)
4) Eketsa ka 37°C ka metsotso e 30, RNA e se e koetsoe 'me e se e loketse ho sebelisoa.
2. 5′ terminal karabelo ya ho ngola (bolumo ea karabelo: 20 μL)
Protocol ena e etselitsoe ho tšoaea RNA e nang le 5' triphosphate 'me e ka eketsoa ho latela litlhoko.Bokhoni ba ho kenyelletsa li-label bo tla angoa ke tekanyo ea molar ea RNA: GTP, hammoho le litaba tsa GTP ho lisampole tsa RNA.
1) Kopanya palo e loketseng ea RNA le H2O e se nang Nuclease ka har'a tšupu ea microfuge ea 1.5 ml ho bophahamo ba molumo oa ho qetela oa 14.0 µL.
2) Chesa ka 65 ℃ bakeng sa metsotso e 5 e lateloa ke ho hlatsoa leqhoa ka metsotso e 5.
3) Kenya likarolo tse latelang ka tatellano e boletsoeng.
| Cmohanyetsi | Volume |
| RNA e hlophisitsoeng | 14 μL |
| 10 × Capping Buffer | 2 μL |
| Motsoako oa GTP** | 2 μL |
| SAM (2 mM) | 1 μL |
| Vaccinia virus Capping Enzyme (10U/μL) | 1 μL |
** GTP MIX e bua ka GTP le palo e nyane ea matšoao.Bakeng sa khatello ea GTP, shebaho Hlokomela 3.
4) Emisa ka 37°C ka metsotso e 30, RNA 5′ qetellong e se e ngotsoe 'me e loketse ho theohela tlase.
Lisebelisoa
1. Ho tlatsa mRNA pele ho litlhahlobo tsa phetolelo/phetolelo ea in vitro
2. Ho ngola 5' pheletso ea mRNA
Lintlha mabapi le tšebeliso
1.Ho futhumatsa tharollo ea RNA pele ho ho kenngoa ka Vaccinia Capping Enzyme ho tlosa sebopeho sa bobeli karolong ea 5' ea sengoloa.Eketsa nako ho isa ho metsotso e 60 bakeng sa lingoliloeng tse nang le 5'ends tse tsebahalang haholo.
2. RNA e sebelisetsoang capping reactions e lokela ho hloekisoa pele e sebelisoa le ho emisoa metsing a se nang nuclease.EDTA ha ea lokela ho ba teng 'me tharollo e lokela ho ba ntle le letsoai.
3. Bakeng sa ho ngola 5' pheletso, kakaretso ea GTP e lokela ho ba makhetlo a 1-3 ho feta molar concentration ea mRNA karabelong.
4. Molumo oa tsamaiso ea karabelo e ka fokotsoa kapa ea theoha ho ea ka ea sebele.
