Uyini umgomo we-mRNA
Umuthi wokugomela i-mRNA udlulisela i-RNA emangqamuzaneni omzimba ukuze iveze futhi ikhiqize ama-antigen amaprotheni ngemuva kokuguqulwa okufanelekile kwe-vitro, ngaleyo ndlela iholele umzimba ukuthi ukhiqize impendulo yokuzivikela ngokumelene ne-antigen, ngaleyo ndlela andise amandla omzimba omzimba.[1,3].
Umfanekiso 1: Umdwebo wohlelo lomphumela womjovo oqondile womgomo we-mRNA [2]
Ukuhlukaniswa kwemithi yokugoma ye-mRNA
Imithi yokugoma ye-mRNA ihlukaniswe yaba izinhlobo ezimbili:engaphindaphindimRNA futhiezikhulisayoI-mRNA: i-mRNA ezikhulisayo ayigcini nje ngokuhlanganisa i-antigen eqondiwe, kodwa futhi ibhala ngekhodi ukuphindaphinda okuvumela ukukhulisa i-RNA ye-intracellular kanye nendlela yokuveza amaprotheni.Imithi yokugoma ye-mRNA engaphindi ifake ikhodi kuphela ama-antigen aqondiwe futhi iqukethe izifunda ezingu-5'ne-3' ezingahumushiwe (UTR).Banikeza ukukhuthazwa okuphelele kokuzivumelanisa nezimo nokuvikeleka okungokwemvelo, okuyi-in situ antigen expression kanye nokudluliselwa kwesignali yengozi, futhi babe nezinhlelo zokusebenza ezilandelayo Izici[2,3]
●Inganikeza ukugqugquzelwa okuphelele kokuzivumelanisa nezimo nokuvikeleka okungokwemvelo, okuyi-in situ antigen expression kanye nokudluliswa kwesignali yengozi.
●Ingakwazi ukunxenxa ukusabela kwamasosha omzimba "okulinganiselayo", okuhlanganisa ama-humoral namaselula kanye nenkumbulo yokuzivikela komzimba
●Ingakwazi ukuhlanganisa ama-antigen ahlukene ngaphandle kokukhulisa inkimbinkimbi yokwakhiwa komgomo
●Ukuthuthukiswa okuqhubekayo kwamandla omzimba kungafinyelelwa ngokugonywa okuphindaphindiwe, futhi akukho noma ukusabela okuncane kwamasosha omzimba kumthwali.
●Imithi yokugoma ye-mRNA yokushisa ingenza kube lula ukuthuthwa nokugcinwa kwemithi yokugoma
Umfanekiso 2: Umdwebo ohleliwe womgomo we-mRNA kanye nendlela yawo yokubonisa i-antigen [4]
Izici zomgomo we-mRNA
Uma kuqhathaniswa nemithi yokugoma yendabuko, imigomo ye-mRNA inezinqubo zokukhiqiza ezilula, isivinini sokukhula ngokushesha, asikho isidingo sosikompilo lwamaseli, kanye nezindleko eziphansi.Uma kuqhathaniswa nemithi yokugoma ye-DNA, imigomo ye-mRNA ayidingi ukungena ku-nucleus futhi ayikho ingozi yokuhlanganiswa ku-genome yokusingatha.I-half-life ingalungiswa ngokuguqulwa.
Ithebula 1: Izinzuzo kanye nokubi kwemigomo ye-mRNA
|
| Inzuzo | Ukushoda |
| umgomo we-mRNA | Ucwaningo olusheshayo nokuthuthukiswa, ukukhiqizwa komgomo kuthatha izinsuku ezingama-40 kuphela | Qalisa impendulo yokuzivikela komzimba engadingekile
|
| Ukungazinzi kwe-mRNA ngaphansi kwezimo zomzimba, kulula ukuyehlisa | Ngeke ihlanganiswe ku-genome ukuze igweme ukuguqulwa okungenzeka kokwelapha
| |
| Asikho isidingo sanoma iyiphi isignali yendawo yenuzi, ukuloba | Ukusebenza kwe-nuclear yokuphepha kusazoqinisekiswa
|
Umfanekiso 3: Ishadi eligelezayo lokukhiqizwa nokulungiswa komgomo we-mRNA [4]
Ikhithi ye-Foregene Viral RNA Isolation
I-RT-qPCR Easy (Isinyathelo esisodwa)
Amasu athuthukisiwe okulungiswa kwemigomo ye-mRNA
Ngenxa yokungaqini kahle kwe-mRNA ngokwayo, ukuwohloka kalula kwama-nuclease ezicutshini, ukusebenza kahle kokungena kwamaseli, kanye nokusebenza kahle okuphansi kokuhumusha, lezi zinkinga zikhawulela ukusetshenziswa kwemithi yokugomela i-mRNA.Ukusebenza kahle kokuhumusha nakho kudlala indima ebaluleke kakhulu.Izimoto zokulethwa zingahlukaniswa zibe ama-viral vectors kanye nama-non-viral vectors (kufaka phakathi ama-liposomes, ama-non-liposomes, amagciwane, ama-nanoparticles, njll.).Ngakho-ke, izinyathelo zokuthuthukisa ezifanele ziyadingeka.Okulandelayo isu lokuthuthukisa ekhemisi lokulungiselela i-mRNA[2]
1 Hlanganisa ama-analogue we-cap noma usebenzise ama-enzyme e-capping ukuze uzinzise i-mRNA futhi wandise ukuhunyushwa kwamaprotheni ngokubophezela ku-eukaryotic translation initiation factor 4E (EIF4E)
2 Lungisa izakhi endaweni ye-5′-untranslated (UTR) kanye ne-3′-UTR ukuze uzinze i-mRNA futhi ukhulise ukuhumusha kwamaprotheni
3 Ukungeza i-Poly(A) umsila kunganzinzisa i-mRNA futhi kwandise ukuhunyushwa kwamaprotheni
4 Ama-nucleosides aguquliwe ukuze kuncishiswe ukusebenza kwamasosha omzimba ngaphakathi futhi andise ukuhumusha
5 Ukwelashwa nge-RNase III kanye nokuhlanza kwe-protein liquid chromatography (FPLC) esheshayo kunganciphisa ukusebenza kokuzivikela komzimba futhi kwandise ukuhumusha.
6 Lungiselela ukulandelana noma amakhodoni ukuze ukhuphule ukuhumusha
7 Ukulethwa ngokubambisana kwezici zokuqalisa ukuhumusha nezinye izindlela zokushintsha ukuhumusha kanye nokuzivikela komzimba
Umfanekiso 4: Ukukhiqizwa kwe-in vitro transcription (IVT) mRNA kanye nenqubo yokuhlanganisa [5]
Ukulungiswa okukhulu kwe-plasmid DNA
Ukuhlanzwa kwe-Plasmid DNA kususa ikakhulukazi ukungcola okufana ne-RNA, i-DNA endotoxin eyindilinga evulekile, iphrotheni ephethe kanye ne-nucleic acid ephethe, futhi ngokuvamile iguqula i-plasmid recombinant ibe i-E. coli.I-E. coli ivutshelwa ukuminyana okuphezulu, bese ihlukaniswa noketshezi oluqinile, kanye nokuqoqwa kwe-E. coli.I-E. coli ibe isingaphansi kwe-alkaline lysis, ukuhlukaniswa kwe-centrifugal okuqinile-uketshezi kanye nokucaciswa kwe-microfiltration ngemva kwe-lysis, i-ultrafiltration kanye nokugxila ngemva kokucaciswa, bese kuba ukuhlanzwa kwe-chromatographic.
Ukuhlanzwa kwe-plasmid DNA:
I-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.Imithi yokugomela i-mRNA — inkathi entsha ekugomeni[J].Ukubuyekeza Imvelo Ukutholwa Kwezidakamizwa, 2018.
【3】I-Kramps T., Elbers K. (2017) Isingeniso Semijovo ye-RNA.Ku: Kramps T., Elbers K. (eds) Imijovo ye-RNA.Izindlela ze-Molecular Biology, vol 1499. Humana Press, New York, NY.
【4】UMaruggi G, Zhang C, Li J, et al.I-mRNA Njengobuchwepheshe Oguqulayo Bokuthuthukiswa Komgomo Wokulawula Izifo Ezithathelwanayo[J].I-Molecular Therapy, 2019.
【5】USergio Linares-Fernández, Céline Lacroix, ,Ukuhlanganisa Umuthi Wokugoma we-mRNA Ukuze Ulinganise Impendulo Yomzimba Ngayo/Eguquguqukayo,Okuthrendayo Kwemithi Yemolekyuli,Umqulu 26, Ukukhishwa 3,2020,Amakhasi 311-323.
Isikhathi sokuthumela: Aug-05-2021
