Qhov tshuaj tiv thaiv mRNA yog dab tsi
Cov tshuaj tiv thaiv mRNA hloov RNA mus rau cov hlwb ntawm lub cev los qhia thiab tsim cov protein ntau antigens tom qab muaj kev hloov pauv hauv vitro, yog li ua rau lub cev tsim cov tshuaj tiv thaiv kab mob tiv thaiv antigen, yog li nthuav lub cev tiv thaiv kab mob.[1,3].
Daim duab 1: Schematic daim duab ntawm cov nyhuv ntawm kev txhaj tshuaj mRNA ncaj qha [2]
Kev faib tawm ntawm cov tshuaj tiv thaiv mRNA
Cov tshuaj tiv thaiv mRNA tau muab faib ua ob hom:tsis rov ua duamRNA thiabtus kheej amplifyingmRNA: self-amplifying mRNA tsis tsuas yog encodes lub hom phiaj antigen, tab sis kuj encodes replication uas enables intracellular RNA amplification thiab protein qhia mechanism.Cov tshuaj tiv thaiv mRNA uas tsis yog replicating tsuas yog encode lub hom phiaj antigens thiab muaj 5' thiab 3'untranslated regions (UTR).Lawv muab kev txhawb nqa ntawm kev yoog raws thiab kev tiv thaiv hauv lub cev, uas yog nyob rau hauv situ antigen qhia thiab kev phom sij kis tau tus mob, thiab muaj cov ntawv thov hauv qab no Nta[2,3]
● Muaj peev xwm muab kev txhawb nqa ntawm kev yoog raws thiab kev tiv thaiv hauv lub cev, uas yog nyob rau hauv situ antigen qhia thiab kev phom sij kis tau tus mob
● Muaj peev xwm ua kom lub cev tiv thaiv kab mob "sib npaug", suav nrog kev lom zem thiab cellular effectors thiab tiv thaiv kev nco
● tuaj yeem ua ke sib txawv antigens yam tsis muaj qhov nyuaj ntawm kev tsim tshuaj tiv thaiv
● Kev txhim kho tsis tu ncua ntawm kev tiv thaiv kab mob tuaj yeem ua tiav los ntawm kev txhaj tshuaj ntau dua, thiab tsis muaj lossis tsis muaj kev tiv thaiv me me rau tus neeg nqa khoom.
● Cov tshuaj tiv thaiv kub kub mRNA tuaj yeem yooj yim rau kev thauj mus los thiab khaws cov tshuaj tiv thaiv
Daim duab 2: Daim duab kos duab ntawm mRNA tshuaj tiv thaiv thiab nws cov tshuaj tiv thaiv antigen [4]
Cov yam ntxwv ntawm cov tshuaj tiv thaiv mRNA
Piv nrog rau cov tshuaj tiv thaiv ib txwm muaj, cov tshuaj tiv thaiv mRNA muaj cov txheej txheem tsim khoom yooj yim, kev loj hlob sai, tsis tas yuav muaj kab lis kev cai ntawm tes, thiab tus nqi qis.Piv nrog rau cov tshuaj tiv thaiv DNA, cov tshuaj tiv thaiv mRNA tsis tas yuav nkag mus rau hauv lub nucleus thiab tsis muaj kev pheej hmoo ntawm kev koom ua ke rau hauv cov tswv tsev genome.Ib nrab-lub neej tuaj yeem hloov kho los ntawm kev hloov kho.
Table 1: Qhov zoo thiab qhov tsis zoo ntawm cov tshuaj tiv thaiv mRNA
|
| Qhov zoo | Ua tsis tau |
| tshuaj tiv thaiv mRNA | Kev tshawb fawb thiab kev loj hlob sai, kev tsim tshuaj tiv thaiv tsuas yog siv sijhawm 40 hnub | Ua rau lub cev tsis muaj zog
|
| mRNA instability nyob rau hauv physiological tej yam kev mob, yooj yim rau degrade | Yuav tsis koom ua ke rau hauv genome kom tsis txhob muaj kev hloov pauv hloov tau
| |
| Tsis xav tau ib qho teeb meem nuclear localization, transcription | Qhov kev ua tau zoo ntawm kev nyab xeeb nuclear tseem yuav raug txheeb xyuas
|
Daim duab 3: Flow daim ntawv ntawm mRNA tshuaj tiv thaiv thiab kev npaj [4]
Foregene Viral RNA Isolation Kit
RT-qPCR Yooj Yim (Ib kauj ruam)
Cov tswv yim txhim kho rau kev npaj cov tshuaj tiv thaiv mRNA
Vim tias tsis muaj kev ruaj ntseg ntawm mRNA nws tus kheej, yooj yim degradation los ntawm nucleases hauv cov ntaub so ntswg, tsis muaj cell nkag efficiency, thiab kev txhais lus tsis zoo, cov teeb meem no txwv tsis pub siv cov tshuaj tiv thaiv mRNA.Kev txhais lus ua tau zoo kuj ua lub luag haujlwm tseem ceeb heev.Cov tsheb thauj khoom tuaj yeem muab faib ua cov kab mob kab mob thiab cov kab mob uas tsis yog kab mob (xws li liposomes, tsis yog liposomes, kab mob, nanoparticles, thiab lwm yam).Yog li ntawd, yuav tsum muaj kev txhim kho kom zoo.Cov hauv qab no yog lub tswv yim txhim kho tshuaj rau kev npaj mRNA[2]
1 Synthesize cap analogs lossis siv capping enzymes kom ruaj khov mRNA thiab nce protein txhais lus los ntawm kev khi rau eukaryotic translation pib qhov tseem ceeb 4E (EIF4E)
2 Kho cov ntsiab lus hauv cheeb tsam 5′-tsis txhais lus (UTR) thiab 3′-UTR kom ruaj khov mRNA thiab nce protein ntau txhais
3 Ntxiv Poly(A) tus Tsov tus tw tuaj yeem txhim kho mRNA thiab ua kom cov protein ntau dua
4 Hloov kho nucleosides kom txo qis hauv lub cev tiv thaiv kab mob thiab nce kev txhais lus
5 Kev kho mob nrog RNase III thiab ceev ceev protein kua chromatography (FPLC) purification tuaj yeem txo qis kev tiv thaiv kab mob thiab nce kev txhais lus.
6 Txhim kho cov kab ke los yog codons kom muaj kev txhais lus ntau ntxiv
7 Kev sib koom ua ke ntawm kev pib txhais lus thiab lwm txoj hauv kev los hloov kev txhais lus thiab kev tiv thaiv kab mob
Daim duab 4: Hauv vitro transcription (IVT) mRNA ntau lawm thiab txheej txheem sib dhos [5]
Kev npaj loj ntawm plasmid DNA
Plasmid DNA purification tsuas yog tshem tawm cov kab mob xws li RNA, qhib lub voj voog DNA endotoxin, cov protein ntau thiab tus tswv tsev nucleic acid, thiab feem ntau hloov cov plasmid recombinant rau hauv E. coli.E. coli undergoes siab fermentation, ces khoom-kua sib cais, thiab sau ntawm E. coli.Lub E. coli yog ces raug rau alkaline lysis, centrifugal solid-kua sib cais thiab microfiltration clarification tom qab lysis, ultrafiltration thiab concentration tom qab clarification, thiab ces chromatographic purification.
Purification ntawm plasmid DNA:
Foregene General Plasmid Mini Cov Khoom Siv
【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.mRNA tshuaj tiv thaiv — lub sijhawm tshiab hauv tshuaj tiv thaiv [J].Nature Reviews Drug Discovery, 2018.
【3】Kramps T., Elbers K. (2017) Taw qhia rau RNA Vaccines.Hauv: Kramps T., Elbers K. (eds) RNA Vaccines.Methods in Molecular Biology, vol 1499. Humana Press, New York, NY.
【4】Maruggi G, Zhang C, Li J, et al.mRNA ua Kev Hloov Kho Tshuab rau Kev Txhim Kho Tshuaj Tiv Thaiv Kab Mob Sib Kis [J].Kev Kho Mob Molecular, 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, Nplooj 311-323.
Post lub sij hawm: Aug-05-2021
