Ente ea mRNA ke eng
Ente ea mRNA e fetisetsa RNA liseleng tsa 'mele ho hlahisa le ho hlahisa li-antigen tsa protheine ka mor'a liphetoho tse nepahetseng tsa in vitro, ka tsela eo e lebisa' mele ho hlahisa karabelo ea 'mele ea ho itšireletsa mafung khahlanong le antigen, kahoo e atolosa matla a' mele a ho itšireletsa mafung.[1,3].
Setšoantšo sa 1: Setšoantšo sa moralo oa phello ea ente e tobileng ea ente ea mRNA [2]
Sehlopha sa liente tsa mRNA
Liente tsa mRNA li arotsoe ka mefuta e 'meli:e sa qollangmRNA leboiketsisomRNA: ho ikemela mRNA ha e kenye feela antigen e lebisitsoeng, empa e boetse e kenyelletsa phetisetso e nolofalletsang ho holisa RNA ea intracellular le mokhoa oa ho hlahisa liprotheine.Liente tse sa ikatiseng tsa mRNA li kenyelletsa li-antigen tse lebisitsoeng feela 'me li na le libaka tse 5' le 3' tse sa fetoletsoeng (UTR).Ba fana ka khothatso e felletseng ea ho ikamahanya le maemo le boits'ireletso ba tlhaho, e leng in situ antigen expression le phetiso ea matšoao a kotsi, 'me ba na le lits'ebetso tse latelang[2,3]
●E ka fana ka khothatso e felletseng ea ho ikamahanya le maemo le boits'ireletso ba tlhaho, e leng in situ antigen expression le phetiso ea matšoao a kotsi.
●E ka etsa hore 'mele o be o leka-lekaneng' karabelo ea 'mele ea ho itšireletsa mafung, ho kenyeletsoa li-humoral le li-cellular effect le mohopolo oa 'mele oa ho itšireletsa mafung
●E ka kopanya li-antigen tse fapaneng ntle le ho eketsa ho rarahana ha tlhahiso ea ente
● Ntlafatso e tsoelang pele ea matla a 'mele ea ho itšireletsa mafung e ka finyelloa ka ho entoa khafetsa, 'me ha ho na tšireletso ea 'mele ea ho itšireletsa mafung kapa e fokolang ho mojari.
●Meento ea mocheso e tsitsitseng ea mRNA e ka nolofatsa lipalangoang le polokelo ea liente
Setšoantšo sa 2: Sets'oants'o sa moralo oa ente ea mRNA le mochini oa eona oa polelo ea antigen [4]
Likarolo tsa liente tsa mRNA
Ha ho bapisoa le liente tsa setso, liente tsa mRNA li na le mekhoa e bonolo ea tlhahiso, lebelo la nts'etsopele e potlakileng, ha ho hlokahale setso sa lisele, le theko e tlaase.Ha ho bapisoa le liente tsa DNA, liente tsa mRNA ha li hloke ho kena khubung 'me ha ho kotsi ea ho kopanngoa ho genome e amohelang.Halofo ea bophelo e ka fetoloa ka ho fetoloa.
Letlapa la 1: Melemo le bofokoli ba liente tsa mRNA
|
| Molemo | Khaello |
| ente ea mRNA | Patlisiso e potlakileng le nts'etsopele, tlhahiso ea ente e nka matsatsi a 40 feela | Hlahisa karabelo e sa hlokahaleng ea 'mele ea ho itšireletsa mafung
|
| mRNA ho hloka botsitso tlas'a maemo a fisioloji, ho bonolo ho e theola | E ke ke ea kenella ka har'a genome ho qoba liphetoho tse ka bang teng tsa kalafo
| |
| Ha ho hlokahale lets'oao la sebaka sa nyutlelie, mongolo | Ho sebetsa hantle ha nyutlelie ya polokeho ho sa ntse ho tla netefatswa
|
Setšoantšo sa 3: Chate e phallang ea tlhahiso le litokisetso tsa ente ea mRNA [4]
Foregene Viral RNA Isolation kit
RT-qPCR Easy (Mohato o le Mong)
Maano a ntlafetseng a ho lokisa liente tsa mRNA
Ka lebaka la botsitso bo fokolang ba mRNA ka boeona, ho senyeha habonolo ke li-nuclease tsa lisele, ts'ebetso e tlase ea ho kena ha lisele, le matla a tlase a phetolelo, likoli tsena li fokotsa ts'ebeliso ea liente tsa mRNA.Bokhoni ba ho fetolela le bona bo phetha karolo ea bohlokoa haholo.Likoloi tsa ho tsamaisa li ka aroloa ka li-vectors tsa kokoana-hloko le li-vector tse se nang kokoana-hloko (ho kenyeletsoa liposomes, non-liposomes, virus, nanoparticles, joalo-joalo).Ka hona, ho hlokahala mehato e nepahetseng ea ntlafatso.Se latelang ke leano la ntlafatso ea meriana bakeng sa ho itokisetsa mRNA[2]
1 Synthesize cap analogues kapa sebelisa capping enzymes ho tsitsisa mRNA le ho eketsa protheine phetolelo ka ho tlama ho eukaryotic translation initiation factor 4E (EIF4E)
2 Fetola likarolo sebakeng sa 5'-untranslated (UTR) le 3′-UTR ho tsitsisa mRNA le ho eketsa phetolelo ea protheine.
3 Ho eketsa Poly(A) mohatla ho ka tsitsisa mRNA le ho eketsa phetolelo ea protheine
4 Li-nucleosides tse fetotsoeng ho fokotsa ts'ebetso ea tlhaho ea 'mele ea ho itšireletsa mafung le ho eketsa phetolelo
5 Kalafo e nang le RNase III le tlhoekiso e potlakileng ea protheine liquid chromatography (FPLC) e ka fokotsa ts'ebetso ea 'mele ea ho itšireletsa mafung le ho eketsa phetolelo.
6 Ntlafatsa tatelano kapa likhodone ho eketsa phetolelo
7 Tšebelisano-'moho ea lintlha tsa qalo ea phetolelo le mekhoa e meng ea ho fetola phetolelo le ho itšireletsa mafung
Setšoantšo sa 4: tlhahiso ea in vitro transcription (IVT) mRNA le ts'ebetso ea kopano [5]
Tokiso e kholo ea plasmid DNA
Ho hloekisoa ha plasmid DNA haholo-holo ho tlosa litšila tse kang RNA, DNA endotoxin e bulehileng-selika-likoe, liprotheine tse amohelang le ho amohela nucleic acid, 'me hangata e fetola plasmid e recombinant ho E. coli.E. coli e belisoa ka bongata bo boholo, ebe karohano ea metsi a tiileng, 'me e bokelle E. coli.Joale E. coli e tlas'a alkaline lysis, centrifugal solid-liquid karohano le ho hlakisoa ha microfiltration ka mor'a lysis, ultrafiltration le mahloriso ka mor'a ho hlakisoa, ebe joale ho hloekisoa ha chromatographic.
Ho hloekisa DNA ea plasmid:
Foregene General Plasmid Mini Kit
【1】苗鹤凡, 郭勇, 江新香.mRNA疫苗研究进展及挑战[J].免疫学杂志, 2016(05):446-449.
【2】Pardi N , Hogan MJ , Porter FW , et al.Liente tsa mRNA - nako e ncha ea thuto ea ente[J].Maikutlo a Tlhaho ka Ho sibolla Lithethefatsi, 2018.
【3】Kramps T., Elbers K. (2017) Selelekela sa Liente tsa RNA.Ho: Kramps T., Elbers K. (eds) Liente tsa RNA.Methods in Molecular Biology, vol 1499. Humana Press, New York, NY.
【4】Maruggi G , Zhang C , Li J , et al.mRNA e le Phetoho ea Thekenoloji bakeng sa Ntlafatso ea Moento ho Laola Mafu a Tšoaetso[J].Phekolo ea Limolek'hule, 2019.
【5】Sergio Linares-Fernández, Céline Lacroix, ,Tailoring mRNA Vaccine to Balance Innate/Adaptive Immune Response,Trends in Molecular Medicine,Volume 26, Issue 3,2020,Maqephe 311-323.
Nako ea poso: Aug-05-2021
