Yintoni isitofu sokugonya se-mRNA
Isitofu sokugonya se-mRNA sidlulisela i-RNA kwiiseli zomzimba ukuze ziveze kwaye zivelise ii-antigens zeprotheyini emva kohlengahlengiso olufanelekileyo kwi-vitro, ngaloo ndlela ikhokelela umzimba ukuba uvelise impendulo yokuzikhusela kwi-antigen, ngaloo ndlela ukwandisa amandla omzimba omzimba.[1,3].
Umzobo 1: Umzobo ocwangcisiweyo wesiphumo sokutofa ngokuthe ngqo kwesitofu sokugonya se-mRNA [2]
Ukuhlelwa kogonyo lwe-mRNA
Ugonyo lwe-mRNA lwahlulwe lwaba ziindidi ezimbini:ayiphindaphindiyomRNA kunyeukuzikhulisaI-mRNA: i-mRNA yokwandisa i-self-amplifying mRNA ayifaki i-antigen ekujoliswe kuyo kuphela, kodwa iphinda ifake ikhowudi yokuphindaphinda eyenza i-intracellular RNA ikhulise kunye neprotheyini yokubonisa indlela.Izitofu zokugonya ze-mRNA ezingaphindaphindiyo zifakela kuphela ii-antigens ekujoliswe kuzo kwaye ziqulethe i-5'and 3'imimandla engaguqulelwanga (UTR).Banikezela ngovuselelo oluphangaleleyo lokuziqhelanisa kunye nokhuseleko lwangaphakathi, oluzezi kwi-situ antigen expression kunye nokuhanjiswa komqondiso wengozi, kwaye banezicelo ezilandelayo[2,3]
●Inokubonelela ngovuselelo oluphangaleleyo lokuziqhelanisa kunye nokhuseleko lwangaphakathi, oluzezi kwi-situ antigen expression kunye nokuhanjiswa komqondiso wengozi.
●Inokuphembelela "i-balanced" impendulo yomzimba, kuquka i-humoral kunye ne-cell effect kunye nememori yomzimba
●Inokudibanisa ii-antigens ezahlukeneyo ngaphandle kokunyusa ubunzima bokwenziwa kwesitofu sokugonya
● Ukuphuculwa okuqhubekayo kwamandla omzimba kunokufezekiswa ngokugonywa ngokuphindaphindiweyo, kwaye akukho okanye akukho mpendulo encinci yokuzivikela kumthwali.
● Izitofu zokugonya ezizinzile ze-mRNA zinokwenza kube lula ukuthuthwa kunye nokugcinwa kwezitofu zokugonya
Umzobo 2: Idayagram ecwangcisiweyo yesitofu sokugonya se-mRNA kunye ne-antigen expression mechanism [4]
Iimpawu zogonyo lwe-mRNA
Xa kuthelekiswa nezitofu zokugonya zemveli, izitofu zokugonya ze-mRNA zineenkqubo zokuvelisa ezilula, isantya sophuhliso olukhawulezayo, akukho mfuneko yenkcubeko yeeseli, kunye nexabiso eliphantsi.Xa kuthelekiswa nezitofu ze-DNA, izitofu zokugonya ze-mRNA azidingi ukungena kwi-nucleus kwaye akukho mngcipheko wokudibanisa kwi-genome yokusingatha.Ubomi besiqingatha bunokulungiswa ngokuguqulwa.
Itheyibhile 1: Izinto eziluncedo kunye nezingeloncedo kugonyo lwe-mRNA
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| Inzuzo | Ukusilela |
| isitofu sokugonya se-mRNA | Uphando olukhawulezileyo kunye nophuhliso, ukuveliswa kwesitofu sokugonya kuthatha iintsuku ezingama-40 kuphela | Qalisa impendulo engafanelekanga yokhuselo lomzimba
|
| Ukungazinzi kwe-mRNA phantsi kweemeko ze-physiological, kulula ukuthotywa | Ayizukudibanisa kwi-genome ukunqanda utshintsho olunokwenzeka lonyango
| |
| Akukho sidingo salo naluphi na uphawu lwenyukliya, ushicilelo | Ukusebenza kokhuseleko lwenyukliya kusaza kuqinisekiswa
|
Umzobo 3: Itshati equkuqelayo yemveliso yogonyo lwe-mRNA kunye nolungiselelo [4]
Ikhithi ye-Foregene Viral RNA Isolation
I-RT-qPCR Easy (Inyathelo elinye)
Ukuphuculwa kwezicwangciso zokulungiselela izitofu zokugonya ze-mRNA
Ngenxa yozinzo olulambathayo lwe-mRNA ngokwayo, ukuthotywa lula ngama-nucleases kwizicubu, ukusebenza kakuhle kokungena kweeseli, kunye nokusebenza okuphantsi kokuguqulela, ezi ziphene zinciphisa ukusetyenziswa kwezitofu zokugonya i-mRNA.Ukuchaneka koguqulo nako kudlala indima ebaluleke kakhulu.Izithuthi zokuhambisa zingahlulwa zibe yi-viral vectors kunye ne-non-viral vectors (kubandakanywa i-liposomes, i-non-liposomes, i-virus, i-nanoparticles, njl.).Ngoko ke, amanyathelo okuphucula afanelekileyo ayafuneka.Oku kulandelayo sisicwangciso sokuphucula i-pharmacological ukulungiselela i-mRNA[2]
1 Yenza i-cap analogs okanye usebenzise i-capping enzymes ukuzinzisa i-mRNA kunye nokwandisa iprotheyini yokuguqulela ngokubophelela kwi-eukaryotic translation initiation factor 4E (EIF4E)
2 Lungisa izinto kwingingqi ye-5′-engaguqulelwanga (UTR) kunye ne-3′-UTR ukuzinzisa i-mRNA kunye nokwandisa ukuguqulelwa kweprotheyini
I-3 Ukongeza i-Poly(A) umsila ingazinzisa i-mRNA kwaye yandise iprotein translation
I-4 i-nucleosides elungisiweyo yokunciphisa ukusebenza kwe-immune yemvelo kunye nokwandisa uguqulelo
I-5 Unyango nge-RNase III kunye neprotheyini ekhawulezayo yolwelo lwe-chromatography (FPLC) ukucocwa kunokunciphisa ukusebenza komzimba kunye nokwandisa ukuguqulelwa.
6 Lungiselela ulandelelwano okanye iikhodons ukwandisa inguqulelo
7 Ukuhanjiswa ngokubambisana kwemiba yokuqaliswa koguqulo kunye nezinye iindlela zokutshintsha uguqulo kunye nokungakhuseleki
Umzobo 4: I-in vitro transcription (IVT) imveliso ye-mRNA kunye nenkqubo yokuhlanganisa [5]
Ukulungiswa okukhulu kweplasmid DNA
Ukucocwa kwe-Plasmid DNA kususa ikakhulu izinto ezingcolisayo ezifana ne-RNA, i-endotoxin ye-DNA evulekileyo, i-protein ye-host kunye ne-nucleic acid, kwaye ngokuqhelekileyo iguqula i-plasmid edibeneyo ibe yi-E. coli.I-E. coli ifumana ukuvubela koxinzelelo oluphezulu, emva koko ukwahlulwa kolwelo oluqinileyo, kunye nokuqokelelwa kwe-E. coli.I-E. coli emva koko ixhomekeke kwi-alkaline lysis, i-centrifugal eqinile-ulwelo lokwahlula kunye nokucaciswa kwe-microfiltration emva kwe-lysis, i-ultrafiltration kunye nokugxila emva kokucaciswa, kunye nokuhlanjululwa kwe-chromatographic.
Ukucocwa kweplasmid 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.Ugonyo lwe-mRNA — ixesha elitsha kugonyo[J].Uphononongo lweNdalo ukuFumana kweziyobisi, ngo-2018.
【3】I-Kramps T., Elbers K. (2017) Intshayelelo kwi-RNA Vaccines.Ku: Kramps T., Elbers K. (eds) Izitofu ze-RNA.Iindlela kwi-Biology yeMolekyuli, i-vol 1499. I-Humana Press, eNew York, NY.
【4】UMaruggi G , Zhang C , Li J , et al.I-mRNA njengeTekhnoloji yoTshintsho kuPhuhliso lweSitofu sokuLawula iZifo ezosulelayo[J].Unyango lweMolekyuli, ngo-2019.
【5】USergio Linares-Fernández, uCéline Lacroix, ,UkuLungisa uGonyo lwe-mRNA ukuze ulungelelanise iMpendulo yoMzimba eNgazalwayo/eZiqhelanisayo,IiNdlela zoNyango lweMolekyuli, uMqulu wama-26, ukukhutshwa kwe-3,2020, amaPhepha 311-323.
Ixesha lokuposa: Aug-05-2021
