Alurar rigakafin cutar Enzyme

Kwayar cutar Vaccinia capping enzyme ta samo asali ne daga recombinant E. coli iri yana ɗauke da kwayoyin halitta don maganin Vaccinia capping enzyme.Wannan enzyme guda ɗaya ya ƙunshi sassan biyu (D1 da D12) kuma yana da ayyukan enzymatic guda uku (RNA triphosphatase da guanylyltransferase ta sashin D1 da guanine methyltransferase ta sashin D12).Kwayar cutar ta Vaccinia Capping Enzyme tana da tasiri don haɓaka samuwar tsarin hula, wanda zai iya haɗa tsarin hula na 7-methylguanylate musamman (m7Gppp, Cap 0) zuwa ƙarshen 5′ na RNA.Tsarin Cap (Cap 0) yana taka muhimmiyar rawa a daidaitawar mRNA, sufuri da fassarar cikin eukaryotes.Capping RNA ta hanyar amsawar enzymatic hanya ce mai inganci kuma mai sauƙi wacce zata iya haɓaka kwanciyar hankali da fassarar RNA don kwafin in vitro, canzawa, da microinjection.

Abubuwan da aka gyara

Enzyme Capping Virus (10 U/μL)

10 × Capping Buffer

Yanayin ajiya

-25～- 15℃ don ajiya (A guji maimaita daskare-narke hawan keke)

Ma'ajiyar ajiya

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

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

Ma'anar Naúrar

Raka'a ɗaya na ƙwayar cuta ta Vaccinia Capping Enzyme an bayyana shi azaman adadin enzyme da ake buƙata don haɗa 10pmol na GTP a cikin kwafin 80nt a cikin awa 1 a 37°C.

Kula da inganci

Exonuclease:10U na ƙwayar cuta ta Vaccinia Capping Enzyme tare da 1μg λ-Hind III narke DNA a 37 ℃ na awanni 16 ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

Endonuclease:10U na kwayar cutar Vaccinia Capping Enzyme tare da 1μg λDNA a 37 ℃ na awanni 16 ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

Nickase:10U na kwayar cutar Vaccinia Capping Enzyme tare da 1 μg pBR322 a 37 ℃ na sa'o'i 16 ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

RNase:10U na kwayar cutar Vaccinia Capping Enzyme tare da 1.6μg MS2 RNA na tsawon awanni 4 a 37℃ ba ya haifar da lalacewa kamar yadda aka ƙaddara ta agarose gel electrophoresis.

1.coli DNA:10U na ƙwayar cuta ta Vaccinia Capping Enzyme an duba shi don kasancewar E. coli genomic DNA ta amfani da TaqMan qPCR tare da abubuwan da suka dace don E. coli 16S rRNA locus.E. coli kwayoyin halittar DNA gurbatawa shine≤1 E. coli genome.

2.Kwayoyin cuta Endotoxin: LAL-gwajin, bisa ga Pharmacopoeia IV 2020 na Sinanci, hanyar gwajin iyaka ta gel, ƙa'ida ta gaba ɗaya (1143).Ya kamata abun cikin endotoxin na ƙwayoyin cuta ya zama ≤10 EU/mg.

Tsarin amsawa da yanayi

1. Yarjejeniyar Capping (ƙarar amsawa: 20 μL)

Wannan hanya ta dace da yanayin capping na 10μg RNA (≥100 nt) kuma ana iya haɓaka ta gwargwadon buƙatun gwaji.

I) Haɗa 10μg RNA da H2O mara Nuclease a cikin bututun microfuge na 1.5 ml zuwa ƙarar ƙarshe na 15.0 µL.* 10 × Capping Buffer: 0.5M Tris-HCl, 50 mM KCl, 10 mM MgCl2, 10 mM DTT, (25 ℃, pH 8.0)

2) Gasa a 65 ℃ na minti 5 sannan a yi wanka na kankara na minti 5.

3) Ƙara abubuwan da ke gaba a cikin tsari da aka ƙayyade

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

4) Sanya a 37 ° C na minti 30, RNA yanzu an rufe shi kuma yana shirye don aikace-aikacen ƙasa.

2.5' Tashar amsa alamar alama (girman amsa: 20 μL)

An tsara wannan yarjejeniya don yiwa RNA alama mai ɗauke da 5'triphosphate kuma ana iya haɓaka ta gwargwadon buƙatu.Ingancin haɗa alamar alamar za a yi tasiri ta hanyar molar rabo na RNA: GTP, da kuma abun ciki na GTP a cikin samfuran RNA.

1) Haɗa daidai adadin RNA da H2O mara Nuclease a cikin bututun microfuge 1.5 ml zuwa ƙarar ƙarshe na 14.0 µL.

2) Gasa a 65 ℃ na minti 5 sannan a yi wanka na kankara na minti 5.

3) Ƙara abubuwan da ke gaba a cikin tsari da aka ƙayyade.

** GTP MIX yana nufin GTP da ƙananan adadin alamomi.Don maida hankali na GTP, komazuwa Note 3.

4) Sanya a 37°C na mintuna 30, RNA 5′ ƙarshen yanzu an yiwa alama kuma a shirye don ƙasa.

Aikace-aikace

1. Capping mRNA kafin fassarar fassarar/fassarar in vitro

2. Lakabi 5' ƙarshen mRNA

Bayanan kula akan amfani

1.Dumama maganin RNA kafin shiryawa tare da Vaccinia Capping Enzyme yana cire tsarin na biyu akan 5'karshen rubutun.Tsawaita lokaci zuwa mintuna 60 don kwafi tare da sanannen ingantaccen tsarin 5'ends.

2. RNA da aka yi amfani da shi don ɗaukar halayen ya kamata a tsarkake kafin amfani kuma a dakatar da shi a cikin ruwan da ba shi da ƙwayar cuta.EDTA bai kamata ya kasance ba kuma maganin ya kamata ya kasance ba tare da gishiri ba.

3. Don yin lakabin ƙarshen 5′, jimlar tattarawar GTP yakamata ya kasance kusan sau 1-3 na molar taro na mRNA a cikin martani.

4. Za'a iya haɓaka ƙarar tsarin amsawa sama ko ƙasa bisa ga ainihin.