LV15033B

LV15033B - Method for attaching selected material to nano-particles of hbv core protein

Info

Publication number: LV15033B
Application number: LVP-14-05A
Authority: LV
Inventors: Regīna Renhofa; Andris Kazāks; Andris DIŠLERS; Ludmila JACKEVIČA; Velta Ose-Klinklāva; Pauls PUMPĒNS
Original assignee: Latvijas Biomedicīnas Pētījumu Un Studiju Centrs, App
Priority date: 2014-01-13
Filing date: 2014-01-13
Publication date: 2015-12-20
Also published as: LV15033A

Abstract

Invention relates to molecular biology and biomedicine. It describes a method for production of nano-containers of modified hepatitis B virus. The method provides for substitution of four selected the most outwardly extended residues of amino acids of the surface of HBc particles by residues of lysine.

Description

Izgudrojums attiecas uz molekulāro bioloģiju, gēnu un proteīnu inženieriju, biotehnoloģiju, imunoloģiju, nanotehnoloģiju un biomedicīnu, konkrēti uz paņēmieniem tādu modificētu hepatīta B vīrusa core nanokonteineru iegūšanai, kas ļauj mērķtiecīgi novietot izvēlētos peptīdus un/vai oligonukleotīdus uz šādu nanokonteineru virsmas, neiespaidojot to attīrīšanas kvalitāti un iepakošanas spējas. Izgudrojums ir izmantojams vakcīnu, gēnu un zāļu terapijas un diagnostikas preparātu izveidošanai.The present invention relates to molecular biology, genetic and protein engineering, biotechnology, immunology, nanotechnology and biomedicine, in particular to methods for obtaining modified hepatitis B virus core nanocontainers which allow targeted targeting of quality peptides and / or oligonucleotides to such nanocontainers. and packing capabilities. The invention is applicable to the development of vaccine, gene and drug therapy and diagnostic preparations.

Tehnikas līmenisState of the art

Hepatīta B vīrusa (HBV) core (HBc) proteīna veidotās vīrusiem līdzīgās daļiņas (VLD) pieder pie veiksmīgākajiem un visvairāk pētītiem adresēto nanokonteineru modeļiem, kas tiek iegūti heterologās ekspresijas veidā (dažādu baktēriju, raugu, kukaiņu un augu šūnās) un spēj nodrošināt bioloģiskā materiāla ekspozīcijas un piegādes funkciju (1).Hepatitis B virus (HBV) core (HBc) protein-like virus-like particles (VLDs) are among the most successful and most studied models of addressed nanocontainers, obtained by heterologous expression (in various bacteria, yeast, insect and plant cells) and capable of providing biological material. exposure and delivery function (1).

HBV gēns C kodē HBc monomēra proteīnu p21, kas inficētajos cilvēka hepatocītos pašasociējas ikosaedriskajos HBV nukleokapsīdos jeb HBc daļiņās, un iepako HBV DNS genomu un HBV polimerāzi (2), kā arī, iespējams, proteīnkināzi (3). Pašasociācijas process tiek ievadīts ar spontānu p21 proteīna dimēru izveidošanu (4), kas tālāk veido divus ikosaedru izomorfus ar 35 un 32 nm diametru (3,5). HBc daļiņu augstas izšķirtspējas telpiskā struktūra tika noteikta, pateicoties HBc proteīna spējai pašasociēties pēc HBV gēna C ekspresijas E.coli šūnās (6-9). Šādas rekombinantas HBc daļiņas satur divusThe HBV gene C encodes the HBc monomeric protein p21, which self-associates in infected human hepatocytes with icosahedral HBV nucleocapsids or HBc particles, and packaging the HBV DNA genome and HBV polymerase (2), and possibly protein kinase (3). The self-association process is introduced by the spontaneous formation of p21 protein dimers (4), which further form two icosahedral isomorphs of 35 and 32 nm in diameter (3,5). The high-resolution spatial structure of HBc particles was determined by the ability of the HBc protein to self-associate upon expression of HBV gene C in E.coli cells (6-9). Such recombinant HBc particles contain two

-2ikosaedriskus izomorfus ar triangulācijas skaitļiem T=4 un T=3 (10), kas sastāv attiecīgi no 240 un 180 HBc monomēriem (10,11). HBc daļiņu T=4 izomorfa telpiskā struktūra tika atšifrēta ar elektronu kriomikroskopiju (10,12,13) un rentgenstaru kristalogrāfiju [14], bet T=3 izomorfa struktūra tika rekonstruēta aprēķinu ceļā (11). Pierādīts, ka rekombinantās HBc daļiņas neatšķiras no HBc daļiņām no inficēto pacientu hepatocītiem, tajā skaitā pēc T=4 un T=3 izomorfu daudzuma attiecības (15). Tajā pašā laikā ir atrastas atšķirības RNSvai natīvo DNS- saturošo HBc daļiņu virsmas struktūrā (16). Pierādīts, ka natīvajos HBV virionos ir pārstāvēti tikai HBc daļiņu T=4 izomorfi (15,16).-2 picosahedral isomorphs with triangulation numbers T = 4 and T = 3 (10) consisting of 240 and 180 HBc monomers (10.11), respectively. The spatial structure of the T = 4 isomorph of HBc particles was deciphered by electron cryomicroscopy (10,12,13) and X-ray crystallography [14], while the T = 3 isomorph structure was reconstructed by calculation (11). Recombinant HBc particles have been shown to be indistinguishable from HBc particles in hepatocytes from infected patients, including the ratio of T = 4 to T = 3 isomorphs (15). At the same time, differences have been found in the surface structure of RNA or native DNA-containing HBc particles (16). Only HBc particle T = 4 isomorphs have been shown to be present in native HBV virions (15,16).

Bez E.coli šūnām, HBc proteīns spēj efektīvi sintezēties un pašasociēties par HBc VLD tādās jaudīgās heterologās ekspresijas sistēmās kā baktērijās B.subtilis (17) un S.typhimurium (18), raugos S.cerevisiae (19,20) un P.pastoris (21,22), kukaiņu šūnās (2325) un augos (26-28).In addition to E.coli cells, the HBc protein is able to efficiently synthesize and self-associate with HBc VLDs in high-potent heterologous expression systems such as B.subtilis (17) and S.typhimurium (18), S.cerevisiae (19,20) and P.pastoris. (21,22), insect cells (2325), and plants (26-28).

HBc proteīna garums ir 183 aminoskābes (retāk, 185) un tas ir uzbūvēts no diviem neatkarīgiem domēniem: N-terminālā pašasociācijas (self-assembly, SA) domēna 1-140 un protamīnam līdzīga, ar arginīniem bagāta C-terminālā domēna (CTD) 150-183 (29). Abi domēni ir nodalīti ar „eņģes” peptīdu 141-149 (30), kas veic morfogēno funkciju un regulē nukleīnskābes iepakošanu (30,31). HBc proteīna SA domēns satur vairākus variablus un konservatīvus rajonus, kas sastāda attiecīgi imunoloģiskos B-šūnu epitopus un strukturālos elementus, kamēr CTD domēns un „eņģes” peptīds pieder pie visaugstāk konservētiem HBc rajoniem (32-34).The HBc protein is 183 amino acids (less common, 185) and is constructed from two independent domains: the N-terminal self-assembly (SA) domain 1-140 and the protamine-like C-terminal domain rich in arginines 150 -183 (29). Both domains are separated by a "hinge" peptide 141-149 (30), which performs morphogenetic function and regulates nucleic acid packaging (30,31). The SA domain of the HBc protein contains several variable and conserved regions, constituting immunologic B-cell epitopes and structural elements, whereas the CTD domain and hinge peptide belong to the most conserved HBc regions (32-34).

SA domēns 1-140 ir nepieciešamas un pietiekams HBc proteīna pašasociācijai - CTD domēna nošķelšana neatceļ HBc pašasociāciju (35-37) un neiespaido HBc proteīna spēju veidot T=4 izomorfus, bet turpmāka „eņģes” peptīda 141-149 saīsināšana noved pie T=3 izomorfu skaitliskās dominances pār T=4 izomorfiem (38,39). SA domēna pašasociācijasSA domain 1-140 is necessary and sufficient for self-association of the HBc protein - Cleavage of the CTD domain does not abrogate the self-association of HBc (35-37) and does not affect the ability of the HBc protein to form T = 4 isomorphs, but further truncation of hinge peptide 141-149 leads to T = 3 numerical dominance of isomorphs over T = 4 isomorphs (38,39). SA domain self-associations

-3spēja, reizē ar augsto kapacitāti svešu sekvenču akceptēšanā, tiek plaši izmantota himēro VLD izveidošanai uz HBc pamata (1,40,41), turklāt kā uz pilna gamma, tā ari uz Ctermināli īsināto HBc proteīna veidiem (42,43).The ability, along with its high capacity for accepting foreign sequences, is widely used to generate chimeric VLDs based on HBc (1,40,41), in addition to both full-gamma and Cterminally truncated HBc proteins (42,43).

Galvenā CTD domēna funkcija ir pregenomiskās, 3,5 kb garās HBV mRNS iepakošana ar tālāku tās pārvēršanu par daļēji divpavedienu HBV DNS (44). Šim procesam ir nepieciešami sekojoši ar HBc daļiņām saistītie soļi: HBc piesaistīšanās pie kodola (45), disociācija un re-asociācija (46) un fosforilēšana un defosforilēšana (47-51), kam seko HBc daļiņu nobriešana, apvalkošana un izkļūšana no šūnas (2,52). CTD domēna nukleīnskābes saistošie saiti ir lokalizēti četros arginīna blokos (53). Būdams kustīgs bez izteiktas stingras otrējās struktūras, CTD domēns var parādīties kā HBc daļiņu iekšienē (54), tā arī uz to ārējās virsmas (55,56). Pēc jaunākiem datiem, būtiska CTD daļa ir eksponēta uz nenogatavojošos RNS-saturošo HBc daļiņu virsmas, kamēr nobriedušās DNS-saturošās daļiņās CTD ir noslēpts daļiņu iekšienē (57).The primary function of the CTD domain is the packaging of the pregenomic 3.5 kb HBV mRNA by further converting it into partially double stranded HBV DNA (44). This process requires the following HBc particle-related steps: HBc nuclear binding (45), dissociation and re-association (46), and phosphorylation and dephosphorylation (47-51), followed by HBc particle maturation, enveloping, and cell depletion (2). , 52). The nucleic acid binding site of the CTD domain is localized in four arginine blocks (53). Being mobile without a distinctly rigid secondary structure, the CTD domain can appear both on the inside (54) of the HBc particles and on their outer surface (55,56). According to recent data, a substantial portion of CTD is exposed on the surface of unripe RNA-containing HBc particles, whereas in mature DNA-containing particles, CTD is hidden within the particles (57).

HBc, kā nanotehnoloģijās objekta, nanokonteineru funkcija tiek realizēta ar svešā bioloģiskā materiāla iepakošanu rekombinantajās HBc daļiņās in vivo (58) un in vitro (59). Līdzīgi natīvajām HBc daļiņām, kas selektīvi pako pregenomisko HBV mRNS (44), rekombinantās HBc VLD dod priekšroku vienpavediena RNS pakošanai, turklāt CTD rajona eliminēšana bloķē RNS pakošanu E.coli (35,36,60) un kukaiņu (37) šūnās. Tomēr HBc VLD piemīt zināma afinitāte arī pret DNS pakošanu (61), kaut arī zemāka par RNS pakošanas efektivitāti (62). In vitro, HBc VLD pako imunostimulējošās sekvences (ISS) CpG (63-66) vai citus īsus oligonukleotīdus (67), vienpavediena RNS, neatkarīgi no HBc VLD fosforilēšanas līmeņa (68,69), un mazākā mērā vienpavediena DNS un vēl mazāk divpavediena DNS (68), citus polianjonus (poliglutamātu un poliakrilskābi) un polikatjonus (polilizīnu un polietilēnimīnu) (68), kā arī magnētiskās nanodaļiņas (70).The function of HBc as an object in nanotechnology is achieved by packaging foreign biological material into recombinant HBc particles in vivo (58) and in vitro (59). Similar to native HBc particles that selectively package pregenomic HBV mRNA (44), recombinant HBc VLDs prefer single-stranded RNA packaging, with the elimination of the CTD region blocking RNA packaging in E.coli (35,36,60) and insect (37) cells. However, HBc VLD also has some affinity for DNA packaging (61), although lower than RNA packaging efficiency (62). In vitro, HBc VLD encodes immunostimulatory sequences (ISS) CpG (63-66) or other short oligonucleotides (67), single-stranded RNA, regardless of the level of phosphorylation of HBc VLD (68.69), and to a lesser degree single-stranded DNA and even less double-stranded DNA. (68), other polyanions (polyglutamate and polyacrylic acid) and polycations (polylysine and polyethyleneimine) (68), and magnetic nanoparticles (70).

-4HBc, kā nanotehnoloģijas objekta, ekspozīcijas un piegādes funkcija tiek realizēta ar svešu sekvenču izvietošanu uz HBc VLD virsmas. Šim mērķim parasti izvēlas visvairāk uz ārpusi izvirzīto HBc izaugumu - α-spirālo „matadatu” - galus, kam atbilst HBc aminoskābju atlikumu pozīcijas 76-81 ar tur novietoto galveno HBc imunodominances rajonu, t.s. MIR (major immunodominant regiori) jeb epitopa el rajonu (1,32,40). Pēc savas eksponēšanas pakāpes, nākošie aiz MIR ir HBc N-gals un aminoskābju atlikumu rajons 127-133 (t.s. epitops e2), tomēr MIR ir absolūtais līderis izveidoto ekspozīcijas un piegādes konstrukciju skaitā. Pastāv divi galvenie svešu sekvenču izvietošanas paņēmieni uz HBc VLD virsmas: ar (a) sapludināto gēnu konstruēšanu, t.i., rekombinanto HBc gēnu izveidošanu ar attiecīgajā HBc gēna vietā iebūvētām svešos proteīnu fragmentus kodējošām sekvencēm un (b) ķīmisku svešā bioloģiskā materiāla piešūšanu pie izveidotām HBc VLD. Kaut arī otrais variants ir daudz universālāks un tehnoloģiskāks par pirmo, tā izmantošana ir aizmetņa līmenī (71).The exposure and delivery function of -4HBc as a nanotechnology object is accomplished by the placement of foreign sequences on the surface of HBc VLD. For this purpose, the ends of the most protruding HBc outgrowths, the α-helical "hairpin", are usually selected which correspond to the residues 76-81 of the HBc amino acids with the major HBc immunodominant region located there, i.e. MIR (major immunodominant regiori) or epitope el region (1,32,40). In terms of their degree of exposure, the ones behind MIR are the N-terminus of HBc and the amino acid residue region 127-133 (i.e. epitope e2), yet MIR is the absolute leader in the number of exposure and delivery constructs created. There are two major techniques for introducing foreign sequences on the surface of HBc VLDs: (a) constructing the fused genes, i.e., creating recombinant HBc genes with sequences encoding foreign protein fragments inserted at the appropriate HBc gene site, and (b) chemically stitching foreign . Although the second option is far more universal and technological than the first, its use is at the cutting edge. (71)

Izgudrojuma izpaušanaDisclosure of the Invention

Izgudrojums attiecas uz jauna HBc ekspozīcijas un piegādes modeļa izveidošanu, kas ļautu svešā bioloģiskā materiāla (proteīnu un peptīdu, nukleīnskābju fragmentu) izvietošanu HBc daļiņu optimālajos, uz āru maksimāli izvirzītajos HBc proteīna rajonos, t.s. „matadatu” galos. Šim nolūkam izvēlētas četras pozīcijas uz HBc virsmas, kas ir maksimāli izvirzītas uz HBc-virsmas un kas atbilst aminoskābju atlikumiem Asn75, Glu77, Pro79 un Ile80, un ar gēnu inženierijas tehniku veikta to individuāla nomaiņa pret lizīna atlikumu - ar Četru modificēto HBc variantu iegūšanu. Jauniegūto HBV core proteīna veidoto nanodaļiņu «matadatu” posmu aminoskābju sekvences un to telpiskās struktūrasThe invention relates to the development of a novel HBc exposure and delivery model that allows the placement of foreign biological material (proteins and peptides, nucleic acid fragments) in the optimal, outwardly protruding regions of HBc proteins, i.e. Matadata ends. For this purpose, four positions on the HBc surface, which are maximally raised on the HBc surface and corresponding to the amino acid residues Asn75, Glu77, Pro79 and Ile80, have been selected and their individual substitution with lysine residues has been made by four engineered HBc variants. Amino acid sequences and spatial structures of newly acquired HBV core protein nanoparticle hairpin

-5attēlotas atbilstoši 1. un 2. zīmējumā. Ieviestās oriģinālo aminoskābju nomaiņas uz lizīnu (K) izceltas un pasvītrotas. Izrādījās, ka visos četros gadījumos saglabājās augsts proteīna ekspresijas līmenis Escherichia coli biomasā un tā spēja pašsavākties ikosaedriskās struktūrās, morfoloģiski neatšķiramās no oriģinālām HBc daļiņām.-5 shown in Figures 1 and 2 respectively. The introduced replacement amino acids for lysine (K) are highlighted and underlined. Protein expression in Escherichia coli biomass was found to be high in all four cases and was able to self-assemble within the icosahedral structure, morphologically indistinguishable from the original HBc particles.

Visi četri modificētie HBc varianti: HBc-K75, HBc-K77, HBc-K79 un HBc-K80 pakļaujas agrāk izstrādātajām attīrīšanas shēmām un tādejādi ļauj izmantot agrāk apgūtos tehnoloģiskos risinājumus par HBc VLD attīrīšanu (72).All four modified HBc variants: HBc-K75, HBc-K77, HBc-K79 and HBc-K80 follow the previously developed purification schemes and thus allow the application of prior technology solutions for HBc VLD purification (72).

Lai varētu VLD izmantot kā nanokonteinerus mērķtiecīgai izvēlētā bioloģiskā materiāla iepildīšanai, ir nepieciešams atbrīvoties no to dabiskā pildījuma, kas izveidojas ekspresijas procesā. Viens no iespējamiem risinājumiem ir daļiņu pilnīga izjaukšana ar tai sekojošu pakošanu un rekonstrukciju no attīrīta proteīna. Iepriekš esam izstrādājuši citu risinājumu, kad HBc VLD dabiskais pildījums tika izšķelts ar sārma hidrolīzi un tika iegūtas tukšās daļiņas, morfoloģiski līdzīgas dabiskajām, un tika izstrādāta efektīva pakošanas metode (59). Izrādījās, ka visas četras jauniegūtās VLD ar uz lizīnu nomainītajām aminoskābēm arī var tikt atbrīvotas no dabiskā iekšējā nukleīnskābju pildījuma ar sārma hidrolīzi. Rezultātā var iegūt tukšas daļiņas, kuras spēj pakot mērķtiecīgi izvēlētu materiālu. Visus četrus modificētos HBc VLD produktus izdalījām no biomasas saskaņā ar agrāk aprakstīto metodi (72). Sagatavošana sārma hidrolīzei notika gēlfiltrējot caur Sephacryl HR S300 kolonu (1,5x60 cm) ar 0,1 M nātrija karbonāta, 2 mM DTT šķīdumu (3ml/h/frakciju). Apvienotā materiāla dialīzē pret 0,1 M nātrija ortofosfata, 0,65M nātrija hlorīda šķīdumu, pH 12 pie 37°C 18h, neitralizēšanas dialīzē pret tādu pašu šķīdumu ar pH 7,8 (pakošanas un refoldinga buferšķīdums), izgulsnēšanās ar amonija sulfātu un, visbeidzot, frakcionēšanas uz Sepharose CL 4B kolonas (2x60 cm) 20mM Tris-HCl, 5mM EDTA, 0,65 M NaCl pH 7,8 buferšķīdumā iegūst tukšas VLD HBc-K75, HBc-K77, HBc-K79,In order to use VLDs as nanocontainers for purposeful filling of selected biological material, it is necessary to dispose of their natural fill formed during the expression process. One possible solution is to completely disintegrate the particles with subsequent packaging and reconstruction from the purified protein. We have previously developed another solution whereby the natural filler of HBc VLD was digested by alkali hydrolysis to yield blank particles morphologically similar to natural and an efficient packaging method was developed (59). It turned out that all four of the newly acquired VLDs with lysine-substituted amino acids could also be freed from the natural internal filling of nucleic acids by alkaline hydrolysis. As a result, empty particles can be obtained which are capable of packing purposefully selected material. All four modified HBc VLD products were isolated from biomass according to the method previously described (72). Preparation for alkaline hydrolysis was by gel filtration through a Sephacryl HR S300 column (1.5 x 60 cm) with 0.1 M sodium carbonate, 2 mM DTT solution (3 ml / h / fraction). Dialysis of the pooled material against 0.1 M sodium orthophosphate, 0.65M sodium chloride solution, pH 12 at 37 ° C for 18h, neutralization dialysis against the same solution, pH 7.8 (packaging and refolding buffer), precipitation with ammonium sulfate and, finally, fractionation on a Sepharose CL 4B column (2x60 cm) yields blank VLD HBc-K75, HBc-K77, HBc-K79 in 20mM Tris-HCl, 5mM EDTA, 0.65M NaCl pH 7.8 buffer,

-6HBc-K80 ar iznākumu līdz 5mg no lg ņemtās biomasas. Tukšās daļiņas tika raksturotas ar UV- adsorbcijas spektriem (attiecība A280/A260) un elektronu mikroskopā (EM). UVadsorbcijas spektri tika uzņemti uz NanoDrop ND-100 iekārtas (3.zTmējums). Ar vertikālajām līnijām iezīmēti mērījumi pie 260nm un 280 nm. Dabisko pildījumu saturošu kapsīdu spektru pārstāv HBc-K79 (A) ar attiecību A260/A280=l,45, bet tukšajām daļiņām HBc-K79 (B) un HBc-K80 (C) ir raksturīgs proteīnu spektrs ar adsorbcijas maksimumu pie 280 nm un attiecību A280/A260 attiecīgi 1,41 (B) un 1,33 (C). 4. zīmējumā parādīti elektonmikroskopijas attēli visiem četriem jaunizveidoto un no iekšējā satura atbrīvoto kapsīdu veidiem. Tātad, pakošanai un ārējai ķīmiskai „dekorēšanai”-piešūšanai sagatavotie materiāli saglabā savu nanodaļiņu struktūru.-6HBc-K80 with a yield of up to 5 mg of lg biomass. Blank particles were characterized by UV-adsorption spectra (A280 / A260 ratio) and electron microscope (EM). UV absorption spectra were recorded on a NanoDrop ND-100 (Dimension 3). Vertical lines mark measurements at 260nm and 280nm. The spectrum of naturally occurring capsules is represented by HBc-K79 (A) with a ratio of A260 / A280 = 1.45, while the empty particles HBc-K79 (B) and HBc-K80 (C) are characterized by a protein spectrum with adsorption peaks at 280 nm and A280 / A260 1.41 (B) and 1.33 (C) respectively. Figure 4 shows electron microscopy images for each of the four newly developed and internally released capsule types. Thus, materials prepared for packaging and external chemical "decorating" - retaining - retain their nanoparticulate structure.

Nukleīnskābes vai oligodezoksinukleotīdus (ODN) iepakojām jauniegūtajās tukšajās daļiņās saskaņā ar iepriekš izstrādāto paņēmienu (59). Pakošanas un ķīmiskās piešūšanas procesus raksturojām ar UV-spektriem, EM un elektroforēzi natīvajos agarozes gēlos (TAE, pH 8,3) ar etīdija bromīdu. Ķīmisko piešūšanu pie modifikācijā iegūtā lizīna -NEb grupas parādījām piešujot aptamēru sgc8 (73) 5’-Fam-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTT TTT-Thiol(C3)-3’, izmantojot Sulfo-KMUS reaģentu un tam atbilstošu protokolu (Pierce). Elektroforēze natīvajā 0,7% agarozes gēlā (5. zīmējums) ilustrē izmaiņas, kuras radās daļiņu pakošanas vai „dekorēšanas” dēļ. Tukšās daļiņas eksperimenta apstākļos elektriskā laukā praktiski neizkustētos no parauga uznešanas kabatas un nebūtu redzamas, jo nesatur nukleīnskābi. Divdesmit nukleobāzes gara oligodezoksinukleotīda 5’Fluo-TCC ATG ACG TTC CTG ACG TT-3’ iepakošana rezultējās ar pakoto daļiņu HBc-K75 x ODN mazāku kustīgumu (5. zīmējums - 3) salīdzinot ar dabiski pildītajām daļiņām HBc-K75 VLD (5. zīmējums 1), bet papildus oligodezoksinukleotīda piešūšana pie daļiņām caur lizīna aminogrupāmThe nucleic acid or oligodeoxynucleotides (ODN) were packaged in the newly obtained blank according to the method previously developed (59). Packing and chemical stitching processes were characterized by UV spectra, EM and electrophoresis on native agarose gels (TAE, pH 8.3) with ethidium bromide. Chemical cross-linking to the lysine -NEb group obtained by modification was demonstrated by suturing the aptamer sgc8 (73) 5'-Fam-ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GAT TTT TTT-Thiol (C3) -3 ' KMUS reagent and protocol (Pierce). Electrophoresis in native 0.7% agarose gel (Figure 5) illustrates the changes caused by particle packaging or "decorating". Under the experimental conditions, the empty particles would practically not move out of the sample pocket in an electric field and would not be visible because they do not contain nucleic acid. Packaging of the twenty nucleobase-length oligodeoxynucleotide 5'Fluo-TCC ATG ACG TTC CTG ACG TT-3 'resulted in reduced mobility of the packed particle HBc-K75 x ODN (Figure 5 - 3) compared to naturally charged particles HBc-K75 VLD (Figure 5). 1), but addition of the oligodeoxynucleotide to the particles via lysine amino groups

-7noveda pie ātrāk migrējošu daļiņu HBc-K75 x sgc-8 izveidošanās (5. zīmējums - 2). Jāatzīmē, ka elektroforēze natīvajos agarozes gēlos vislabāk raksturo daļiņu kopējo lādiņu, un var novērot, ka jaunizveidotās lizīnus nesošas daļiņas kustās elektriskajā laukā lēnāk par oriģinālajām, jo lizīnu brīvās NH2 grupas pie pH 8,3 samazina daļiņu kustīgumu pret anodu (izoelektriskais punkts šīm aminoskābēm pl=9,74). Nukleīnskābju klātbūtne kā daļiņu iekšējais pildījums, vai kā mūsu gadījumā - piešūtais materiāls, spēcīgi palielina daļiņu kopējo negatīvo lādiņu un kustīgumu.-7 led to the formation of the faster migrating particles HBc-K75 x sgc-8 (Figure 5 - 2). It should be noted that electrophoresis in native agarose gels best describes the particle total charge, and the newly formed lysine-bearing particles move more slowly than the original in the electric field, since lysine-free NH2 groups at pH 8.3 reduce particle mobility to the anode (isoelectric point pl = 9.74). The presence of nucleic acids, either as the internal charge of the particles or, in our case, the sewn-in material, strongly increases the overall negative charge and mobility of the particles.

Literatūrā ir parādīts, ka šis aptamērs specifiski saistās pie T šūnām (73). Tas apstiprinājās arī mūsu pētījumā ar Jurkat šūnām, kad izmantojot plūsmas citometru (6. zīmējums) un fluorescento mikroskopu (7. zīmējums), novērojām efektīvu ķīmiskās piešūšanas produkta HBc-K75 x sgc8 mijiedarbību ar Jurkat (T limfocītu) šūnām lh pie +37°C (6. zīmējums 3 un 7A zīmējums), atšķirībā no atbilstošām kontrolēm - ar FITC iezīmētām daļiņām HBcK75-FITC (6. zīmējums - 2 un 7B zīmējums) un pašām Jurkat šūnām barotnē (6. zīmējums -1).The literature has shown that this aptamer binds specifically to T cells (73). This was also confirmed in our study on Jurkat cells, where we observed an efficient interaction of the chemical staining product HBc-K75 x sgc8 with Jurkat (T lymphocytes) cells lh at + 37 ° using a flow cytometer (Figure 6) and a fluorescent microscope (Figure 7). C (Figure 6, Figure 3 and Figure 7A), as opposed to appropriate controls, with FITC-labeled HBcK75-FITC (Figure 6 - Figure 2 and Figure 7B) and Jurkat cells themselves in the medium (Figure 6 -1).

Tādejādi pierādījās, ka visi četri modificētie HBc varianti var tikt izmantoti kā nanokonteineri ar tādu pašu efektivitāti kā natīvās HBc VLD. Modificētie HBc varianti pakļaujas agrāk izstrādātajām pakošanas shēmām, un ļauj izmantot agrāk izstrādātos tehnoloģiskos risinājumus par HBc VLD pakošanu ar nukleīnskābju un proteīnu materiālu (59), kā arī ar magnētiskajām nanodaļiņām (70).Thus, it has been shown that all four modified HBc variants can be used as nanocontainers with the same efficiency as native HBc VLDs. Modified HBc variants follow the previously developed packaging schemes and allow the use of previously developed technological solutions for HBc VLD packaging with nucleic acid and protein materials (59) as well as magnetic nanoparticles (70).

Realizējot ķīmiskas piešūšanas shēmas pie lizīna atlikumiem, visi četri modificētie HBc varianti uzrāda efektīvu bioloģiskā materiāla pievienošanu. Pateicoties optimāli izvēlētām modifikācijas (lizīna apmaiņu) vietām, piešūtais materiāls tiek eksponēts uz modificēto HBc daļiņu virsmas.All four modified HBc variants exhibit efficient biological addition by chemical stitching schemes at lysine residues. Thanks to optimally selected modification (lysine exchange) sites, the sewn material is exposed on the surface of the modified HBc particles.

-8Atkarībā no piešūtā bioloģiskā materiāla, piemēram, peptīdu vai oligonukleotīdu, īpašībām un nanokonteineru pildījuma, šādi modificētie HBc nanokonteineri var tikt izmantoti kā vakcīnu prototipi vai kā gēnu un zāļu terapijas līdzekļi, kas ir adresēti uz noteiktām šūnām vai orgāniem un turpmāk izmantoti profilaktiskiem, diagnostiskiem vai terapeitiskiem mērķiem.-8Depending on the properties of the sewn biological material, such as peptides or oligonucleotides, and nanocontainer fillings, such modified HBc nanocontainers can be used as vaccine prototypes or as gene and drug therapies, targeting specific cells or organs and further used for prophylactic, diagnostic purposes. or for therapeutic purposes.

-9Citetas literatūras saraksts-9Citatory References

1. Pushko P, Pumpens P, Grens E. Development of Virus-Like Particle Technology from Small Highly Symmetric to Large Complex Virus-Like Particle Structures. Intervirology. 2013;56(3): 141-165.1. Pushko P, Pumpens P, Grens E. Development of Virus-Like Particle Technology from Small Highly Symmetric to Large Complex Virus-Like Particle Structures. Intervirology. 2013; 56 (3): 141-165.

2. Kann, M. and Gerlich, W.H., Replication of hepatitis B virus, in Molecular Medicine of Virai Hepatitis,Harrison, T. J. and Zuckerman, A. J., Eds., John Wiley & Sons, New York, 1997, pp. 63—87.2. Kann, M. and Gerlich, W.H., Replication of Hepatitis B Virus, in Molecular Medicine of Virai Hepatitis, Harrison, T.J. and Zuckerman, A.J., Eds., John Wiley & Sons, New York, 1997, p. 63-87.

3. Gerlich WH, Goldmann U, Miiller R, Stibbe W, Wolff W. Specificity and localization of the hepatitis B virus-associated protein kinase. J Virol. 1982 Jun;42(3):761-766.3. Gerlich WH, Goldmann U, Miiller R, Stibbe W, Wolff W. Specificity and localization of hepatitis B virus-associated protein kinase. J Virol. 1982 Jun; 42 (3): 761-766.

4. Zhou S, Standring DN. Hepatitis B virus capsid pārticies are assembled from core-protein dimer precursors. Proc Nati Acad Sci USA. 1992 Nov 1 ;89(21): 10046-10050.4. Zhou S, Standring DN. Hepatitis B virus capsid affluent are assembled from core-protein dimer precursors. Proc Nati Acad Sci USA. 1992 Nov 1; 89 (21): 10046-10050.

5. Cohen BJ, Richmond JE. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature. 1982 Apr l5;296(5858):677-679.5. Cohen BJ, Richmond JE. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature. 1982 Apr 15; 296 (5858): 677-679.

6. Pasek M, Goto T, Gilbert W, Zink B, Schaller H, MacKay P, Leadbetter G, Murray K. Hepatitis B virus genes and their expression in E. coli. Nature. 1979 Dec 6;282(5739):575 -579.6. Pasek M, Goto T, Gilbert W, Zink B, Schaller H, MacKay P, Leadbetter G, Murray K. Hepatitis B virus genes and their expression in E. coli. Nature. 1979 Dec 6; 282 (5739): 575 -579.

7. Edman JC, Hallevvell RA, Valenzuela P, Goodman HM, Rutter WJ. Synthesis of hepatitis B surface and core antigens inE. coli. Nature. 1981 Jun 11;291(5815):503-506.7. Edman JC, Hallevvell RA, Valenzuela P, Goodman HM, Rutter WJ. Synthesis of hepatitis B surface and core antigens inE. coli. Nature. Jun 11, 1981; 291 (5815): 503-506.

8. Borisova GP, Pumpen PP, Bychko W, Pushko PM, Kalis IaV, Dishler AV, Gren EJ, Tsibinogin W, Kukaine RA: Structure and expression in Escherichia coli celis of the core antigen gene of the human hepatitis B virus (HBV). (Article in Russian) Doki Akad Nauk SSSR 1984;279:1245-1249.8. Borisova GP, Pumpen PP, Bychko W, Pushko PM, Kalis IaV, Dishler AV, Gren EJ, Tsibinogin W, Kukaine RA: Structure and expression in the Escherichia coli cell line of the human hepatitis B virus (HBV) . (Article in Russian) Doki Akad Nauk SSSR 1984; 279: 1245-1249.

9. Nassal M. Total Chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization. Gene. 1988 Jun 30;66(2):279-294.9. Nassal M. Total chemical synthesis of a gene for hepatitis B virus core protein and its functional characterization. Gene. 1988 Jun 30; 66 (2): 279-294.

10. Crowther RA, Kiselev NA, Bottcher B, Berriman JA, Borisova GP, Ose V, Pumpens P. Threedimensional structure of hepatitis B virus core pārticies determined by electron cryomicroscopy. Celi. 1994 Jun 17;77(6):943-950.10. Crowther RA, Kiselev NA, Bottcher B, Berriman JA, Borisova GP, Ose V, Pumpens P. Three-dimensional structure of hepatitis B virus core confirmed by electron cryomicroscopy. Celi. 1994 Jun 17; 77 (6): 943-950.

11. Roseman AM, Borschukova O, Berriman JA, Wynne SA, Pumpens P, Crowther RA. Structures of Hepatitis B Virus Cores Presenting a Modei Epitope and Their Complexes with Antibodies. J Mol Biol. 2012 Oct 12;423(l):63-78. doi: 10.1016/j.jmb.2012.06.032. Epub 2012 Jun 28.11. Roseman AM, Borschukova O, Berriman JA, Wynne SA, Pumpens P, Crowther RA. Structures of Hepatitis B Virus Cores Presenting a Modei Epitope and Their Complexes with Antibodies. J Mol Biol. 2012 Oct 12; 423 (l): 63-78. doi: 10.1016 / j.jmb.2012.06.032. Epub 2012 Jun 28.

12. Bottcher B, Wynne SA, Crowther RA. Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature. 1997 Mar 6;386(6620):88-91.12. Bottcher B, Wynne SA, Crowther RA. Determination of fold of core protein of hepatitis B virus by electron cryomicroscopy. Nature. 1997 Mar 6; 386 (6620): 88-91.

13. Conway JF, Cheng N, Zlotnick A, Wingfield PT, Stahl SJ, Steven AC. Visualization of a 4-helix bundle inthe hepatitis B virus capsid by cryo-electron microscopy. Nature. 1997 Mar 6;3 86(6620):9194.13. Conway JF, Cheng N, Zlotnick A, Wingfield PT, Stahl SJ, Steven AC. Visualization of a 4-helix bundle inthe hepatitis B virus capsid by cryo-electron microscopy. Nature. 1997 Mar 6; 3 86 (6620): 9194.

14. Wynne SA, Crowther RA, Leslie AG. The crystal structure of the human hepatitis B virus capsid. Mol Celi. 1999 Jun;3(6):771-780.14. Wynne SA, Crowther RA, Leslie AG. The crystal structure of human hepatitis B virus capsid. Mol Celi. 1999 Jun; 3 (6): 771-780.

15. Kenney JM, von Bonsdorff CH, Nassal M, Fuller SD. Evolutionary conservation in the hepatitis B virus core structure: comparison of human and duck cores. Structure. 1995 Oct 15;3(10): 1009-1019.15. Kenney JM, von Bonsdorff CH, Nassal M, Fuller SD. Evolutionary conservation in the hepatitis B virus core structure: a comparison of human and duck cores. Structure. 1995 Oct 15; 3 (10): 1009-1019.

16. Roseman AM, Berriman JA, Wynne SA, Butler PJ, Crowther RA. A structural modei for maturation ofthe hepatitis B virus core. Proc Nati Acad Sci USA. 2005 Nov l;102(44):15821-15826. Epub 2005 Oct 24.16. Roseman AM, Berriman JA, Wynne SA, Butler PJ, Crowther RA. A structural modei for maturation ofthe hepatitis B virus core. Proc Nati Acad Sci USA. 2005 Nov l; 102 (44): 15821-15826. Epub 2005 Oct 24.

17. Hardy K, Stahl S, Kiipper H. Production in B. subtilis of hepatitis B core antigen and a major antigen of foot and mouth disease virus. Nature. 1981 Oct 8;293(5832):481-483.17. Hardy K, Stahl S, Kiipper H. Production in B. subtilis of hepatitis B core antigen and major foot and mouth disease virus. Nature. 8 Oct 1981; 293 (5832): 481-483.

18. Schodel F, Milich DR, Will H. Hepatitis B virus nucleocapsid/pre-S2 fusion proteīns expressed in attenuated Salmonella for oral vaccination. J Immunol. 1990 Dec 15; 145(12):4317-4321.18. Schodel F, Milich DR, Will H. Hepatitis B virus nucleocapsid / pre-S2 fusion protein expressed in attenuated Salmonella for oral vaccination. J Immunol. December 15, 1990; 145 (12): 4317-4321.

19. Kniskem PJ, Hagopian A, Montgomery DL, Burke P, Dunn NR, Hofinann KJ, Miller WJ, Ellis RW. Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae. Gene. 1986;46(1):135-141.19. Kniskem PJ, Hagopian A, Montgomery DL, Burke P, Dunn NR, Hofinann KJ, Miller WJ, Ellis RW. Unusually high-level expression of a foreign gene (hepatitis B virus core antigen) in Saccharomyces cerevisiae. Gene. 1986; 46 (1): 135-141.

20. Miyanohara A, Imamura T, Araki M, Sugavvara K, Ohtomo N, Matsubara K. Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons. J Virol. 1986 Jul;59(l): 176-180.20. Miyanohara A, Imamura T, Araki M, Sugavvara K, Ohtomo N, Matsubara K. Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons. J Virol. 1986 Jul; 59 (l): 176-180.

21. Rolland D, Gauthier M, Dugua JM, Foumier C, Delpech L, Watelet B, Letoumeur O, Amaud M, Jolivet M. Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size-exclusion chromatography and ultracentrifugation. J Chromatogr B Biomed Sci Appl. 2001 Mar 25;753(l):51-65.21. Rolland D, Gauthier M, Dugua JM, Foumier C, Delpech L, Watelet B, Letoumeur O, Amaud M, Jolivet M. Purification of recombinant HBc antigen expressed in Escherichia coli and Pichia pastoris: comparison of size exclusion chromatography and ultracentrifugation . J Chromatogr B Biomed Sci Appl. 2001 Mar 25; 753 (l): 51-65.

-1022. Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Highly efficient production of phosphorylated hepatitis B core pārticies in yeast Piciņa pastoris. Protein Expr Purif. 2011 Feb;75(2):218-224.-1022. Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Highly efficient production of phosphorylated hepatitis B core flourishes in yeast Picina pastoris. Protein Expr Purif. 2011 Feb; 75 (2): 218-224.

23. Takehara K, Ireland D, Bishop DH. Co-expression of the hepatitis B surface and core antigens using baculovirus multiple expression vectors. J Gen Virol. 1988 Nov;69 (Pt 11):2763-77.23. Takehara K, Ireland D, Bishop DH. Co-expression of hepatitis B surface and core antigens using baculovirus multiple expression vectors. J Gen Virol. 1988 Nov; 69 (Pt 11): 2763-77.

24. Lanford RE, Notvall L. Expression of hepatitis B virus core and precore antigens in insect celis and characterization of a core-associated kinase activity. Virology. 1990 May;176(l):222-233.24. Lanford RE, Notvall L. Expression of hepatitis B virus core and precore antigen in insect cell and characterization of core-associated kinase activity. Virology. 1990 May; 176 (l): 222-233.

25. Hilditch CM, Rogers LJ, Bishop DH. Physicochemical analysis of the hepatitis B virus core antigen produced by a baculovirus expression vector. J Gen Virol. 1990 Nov;71 ( Pt 11):2755-2759.25. Hilditch CM, Rogers LJ, Bishop DH. Physicochemical analysis of the hepatitis B virus core antigen produced by a baculovirus expression vector. J Gen Virol. 1990 Nov; 71 (Pt 11): 2755-2759.

26. Tsuda S, Yoshioka K, Tanaka T, Iwata A, Yoshikawa A, Watanabe Y, Okada Y. Application of the human hepatitis B virus core antigen from transgenic tobacco plants for serological diagnosis. Vox Sang. 1998;74(3):148-155.26. Tsuda S, Yoshioka K, Tanaka T, Iwata A, Yoshikawa A, Watanabe Y, Okada Y. Application of human hepatitis B virus core antigen from transgenic tobacco plants for serological diagnosis. Vox Sang. 1998; 74 (3): 148-155.

27. Mechtcheriakova IA, Eldarov MA, Nicholson L, Shanks M, Skryabin KG, Lomonossoff GP. The use of virai vectors to producē hepatitis B virus core pārticies in plants. J Virol Methods. 200627. Mechtcheriakova IA, Eldarov MA, Nicholson L, Shanks M, Skryabin KG, Lomonossoff GP. The use of virai vectors is produced by the hepatitis B virus core being thrived in plants. J Virol Methods. 2006

Jan;131(l):10-15.Jan; 131 (l): 10-15.

28. Huang Z, Santi L, LePore K, Kilboume J, Amtzen CJ, Mason HS. Rapid, high-level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice. Vaccine 2006 Mar 24;24(14):2506-2513.28. Huang Z, Santi L, LePore K, Kilboume J, Amtzen CJ, Mason HS. Rapid, high-level production of hepatitis B core antigen in plant leaf and its immunogenicity in mice. Vaccine 2006 Mar 24; 24 (14): 2506-2513.

29. Bimbaum F, Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol. 1990 Jul;64(7):3319-30.29. Bimbaum F, Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol. 1990 Jul; 64 (7): 3319-30.

30. Seifer M, Standring DN. A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the virai capsid surface. J Virol. 1994 Sep;68(9):5548-5555.30. Seifer M, Standring DN. A protease-sensitive hinge linking the two domains of the hepatitis B virus core protein is exposed on the viral capsid surface. J Virol. 1994 Sep; 68 (9): 5548-5555.

31. Watts NR, Conway JF, Cheng N, Stahl SJ, Belnap DM, Steven AC, Wingfield PT. The morphogenic linker peptide of HB V capsid protein forms a mobile array on the interior surface. EMBO J. 2002 Mar l;21(5):876-884.31. Watts NR, Conway JF, Cheng N, Stahl SJ, Belnap DM, Steven AC, Wingfield PT. The morphogenic linker peptide of HB V capsid protein forms a mobile array on the interior surface. EMBO J. 2002 Mar l; 21 (5): 876-884.

32. Pumpens P, Grens E. HBV core pārticies as a carrier for B cell/T celi epitopes. Intervirology. 2001;44(2-3):98-114.32. Pumpens P, Grens E. HBV core enriched as a carrier for B cell / T cell epitopes. Intervirology. 2001; 44 (2-3): 98-114.

33. Pumpens P, Grens E, Nassal M. Molecular epidemiology and immunology of hepatitis B virus infection - an update. Intervirology. 2002;45(4-6):218-232.33. Pumpens P, Grens E, Nassal M. Molecular epidemiology and immunology of hepatitis B virus infection - an update. Intervirology. 2002; 45 (4-6): 218-232.

34. Chain BM, Myers R. Variability and conservation in hepatitis B virus core protein. BMC Microbiol. 2005 May 27;5:33.34. Chain BM, Myers R. Variability and conservation in hepatitis B virus core protein. BMC Microbiol. 2005 May 27; 5: 33.

35. Borisova GP, Kalis IaV, Pushko PM, Tsibinogin VV, Loseva Via, Ose VP, Stankevich EI, Dreimane Ala, Sniker DIa, Grinstein EE, Pumpen PP, Gren EJ. Genetically engineered mutants of the core antigen of the human hepatitis B virus preserving the ability for native self-assembly. Doki Akad Nauk SSSR. (Article in Russian) 1988;298(6): 1474-1478.35. Borisova GP, Kalis IaV, Pushko PM, Tsibinogin VV, Loseva Via, Ose VP, Stankevich EI, Dreimane Ala, Sniker DIa, Grinstein EE, Pumpen PP, Gren EJ. Genetically engineered mutants of the core antigen of human hepatitis B virus preserving the ability for native self-assembly. Doki Akad Nauk USSR. (Article in Russian) 1988; 298 (6): 1474-1478.

36. Gallina A, Bonelli F, Zentilin L, Rindi G, Muttini M, Milanesi G. A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid pārticies but fails to bind nucleic acids. J Virol. 1989 Nov;63(l 1):4645-4652.36. Gallina A, Bonelli F, Zentilin L, Rindi G, Muttini M, Milanesi G. A recombinant hepatitis B core antigen polypeptide with a protamine-like domain deleted self-assembled into files containing bind nucleic acids. J Virol. 1989 Nov; 63 (11): 4645-4652.

37. Beames B, Lanford RE. Carboxy-terminal truncations of the HBV core protein affect capsid formation and the apparent size of encapsidated HBV RNA. Virology. 1993 Jun;194(2) :597-607.37. Beames B, Lanford RE. Carboxy-terminal truncations of HBV core protein affect capsid formation and apparent size of encapsidated HBV RNA. Virology. 1993 Jun; 194 (2): 597-607.

38. Wingfield PT, Stahl SJ, Williams RW, Steven AC. Hepatitis core antigen produced in Escherichia coli: subunit composition, conformational analysis, and in vitro capsid assembly. Biochemistry. 1995 Apr 18;34(15):4919-4932.38. Wingfield PT, Stahl SJ, Williams RW, Steven AC. Hepatitis core antigen produced in Escherichia coli: subunit composition, conformational analysis, and in vitro capsid assembly. Biochemistry. 1995 Apr 18; 34 (15): 4919-4932.

39. Zlotnick A, Cheng N, Conway JF, Booy FP, Steven AC, Stahl SJ, Wingfield PT. Dimorphism of hepatitis B virus capsids is strongly influenced by the C-terminus of the capsid protein. Biochemistry. 1996 Jun 11;35(23):7412-7421.39. Zlotnick A, Cheng N, Conway JF, Booy FP, Steven AC, Stahl SJ, Wingfield PT. Dimorphism of hepatitis B virus capsids is strongly influenced by the C-terminus of the capsid protein. Biochemistry. 1996 Jun 11; 35 (23): 7412-7421.

40. Pumpens P, Ulrich R, Sasnauskas K, Kazaks A, Ose V, Grens E: Construction of novel vaccines on the basis of the virus-like pārticies: Hepatitis B virus proteīns as vaccine carriers; in Khudyakov Y (ed.): Medicīnai Protein Engineering. Boca Raton, London, New York, CRC Press, Taylor & Francis Group, 2008, pp 205-248.40. Pumpens P, Ulrich R, Sasnauskas K, Kazaks A, Ose V, Grens E: Construction of novel vaccines on the basis of virus-like affluence: Hepatitis B virus protein as vaccine carriers; in Khudyakov Y (ed.): In Medicine Protein Engineering. Boca Raton, London, New York, CRC Press, Taylor & Francis Group, 2008, pp 205-248.

41. Whitacre DC, Lee BO, Milich DR. Use of hepadnavirus core proteīns as vaccine platforms. Expert Rev Vaccines. 2009 Nov;8(l 1):1565-1573.41. Whitacre DC, Lee BO, Milich DR. Use of hepadnavirus core protein as vaccine platforms. Expert Rev Vaccines. 2009 Nov; 8 (l 1): 1565-1573.

42. Skrastina D, Bulavaite A, Sominskaya I, Kovalevska L, Ose V, Priede D, Pumpens P, Sasnauskas K. High immunogenicity of a hydrophilic component of the hepatitis B virus preSl sequence exposed on the surface of three virus-like particle carriers. Vaccine. 2008 Apr 7;26(16):1972-1981. Epub 2008 Feb 26.42. Skrastina D, Bulavaite A, Sominskaya I, Kovalevska L, Ose V, Priede D, Pumpens P, Sasnauskas K. High immunogenicity of a hydrophilic component of hepatitis B virus preSl sequence exposed on surface of three virus-like particle carriers . Vaccine. 2008 Apr 7; 26 (16): 1972-1981. Epub 2008 Feb 26.

43. Sominskaya I, Skrastina D, Dislers A, Vasiljev D, Mihailova M, Ose V, Dreilina D, Pumpens P. Construction and immunological evaluation of multivalent hepatitis B virus (HBV) core virus-like43. Sominskaya I, Skrastina D, Dislers A, Vasilyev D, Mikhailova M, Ose V, Dreilina D, Pumpens P. Construction and immunological evaluation of multivalent hepatitis B virus (HBV) core virus-like

-11particles carrying HBV and HCV epitopes. Clin Vaccine Immunol. 2010 Jun;17(6):1027-1033. Epub 2010 Apr 21.-11particles carrying HBV and HCV epitopes. Clin Vaccine Immunol. 2010 Jun; 17 (6): 1027-1033. Epub 2010 Apr 21

44. Nassal M. The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive virai positive-strand DNA synthesis but not for virus assembly. J Virol. 1992 Jul;66(7):4107-4116.44. Nassal M. The arginine-rich domain of hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol. 1992 Jul; 66 (7): 4107-4116.

45. Kann M, Sodeik B, Vlachou A, Gerlich WH, Helenius A. Phosphorylation-dependent binding of hepatitis B virus core pārticies to the nuclear pore complex. J Celi Biol. 1999 Apr 5; 145(1):45-55.45. Kann M, Sodeik B, Vlachou A, Gerlich WH, Helenius A. Phosphorylation-dependent binding of hepatitis B virus core to the nuclear pore complex. J Celi Biol. April 5, 1999; 145 (1): 45-55.

46. Rabe B, Delaleau M, Bischof A, Foss M, Sominskaya I, Pumpens P, Cazenave C, Castroviejo M, Kann M. Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids. PLoS Pathog. 2009 Aug;5(8):el000563. Epub 2009 Aug 28.46. Rabe B, Delaleau M, Bischof A, Foss M, Sominskaya I, Pumpens P, Cazenave C, Castroviejo M, Kann M. Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids. PLoS Pathog. 2009 Aug; 5 (8): el000563. Epub 2009 Aug 28.

47. Machida A, Ohnuma H, Tsuda F, Yoshikawa A, Hoshi Y, Tanaka T, Kishimoto S, Akahane Y, Miyakawa Y, Mayumi M. Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody. J Virol. 1991 Nov;65(l 1):6024-6030.47. Machida A, Ohnuma H, Tsuda F, Yoshikawa A, Hoshi Y, Tanaka T, Kishimoto S, Akahane Y, Miyakawa Y, Mayumi M. Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody. J Virol. 1991 Nov; 65 (11): 6024-6030.

48. Lan YT, Li J, Liao W, Ou J. Roles of the three major phosphorylation sites of hepatitis B virus core protein in virai replication. Virology. 1999 Jul 5;259(2):342-34848. Lan YT, Li J, Liao W, Ou J. Roles of three major phosphorylation sites of hepatitis B virus core protein in viral replication. Virology. 1999 Jul 5; 259 (2): 342-348

49. Le Pogām S, Chua PK, Newman M, Shih C. Exposure of RNA templates and encapsidation of spliced virai RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173). J Virol. 2005 Feb;79(3):1871-1887.49. Le Pogām S, Chua PK, Newman M, Shih C. Exposure of RNA templates and encapsidation of spliced viral RNA are influenced by the arginine-rich domain of human hepatitis B virus core antigen (HBcAg 165-173). J Virol. 2005 Feb; 79 (3): 1871-1887.

50. Perlman DH, Berg EA, O'connor PB, Costello CE, Hu J. Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids. Proc Nati Acad Sci USA. 2005 Jun 21; 102(25):9020-9025. Epub 2005 Jun 10.50. Perlman DH, Berg EA, O'connor PB, Costello CE, Hu J. Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids. Proc Nati Acad Sci USA. Jun 21, 2005; 102 (25): 9020-9025. Epub 2005 Jun 10.

51. Lewellyn EB, Loeb DD. Serine phosphoacceptor sites within the core protein of hepatitis B virus contribute to genome replication pleiotropically. PLoS One. 2011 Feb 15;6(2):el7202.51. Lewellyn EB, Loeb DD. Serine phosphoacceptor sites within the core protein of hepatitis B virus contribute to genome replication pleiotropically. PLoS One. 2011 Feb 15; 6 (2): el7202.

52. Moses SE, Lim Z, Zuckerman MA. Hepatitis B virus infection: pathogenesis, reactivation and management in hematopoietic stem celi transplant recipients. Expert Rev Anti Infect Ther. 2011 Oct;9(10):891-899.52. Moses SE, Lim Z, Zuckerman MA. Hepatitis B virus infection: pathogenesis, reactivation and management in hematopoietic stem cell transplant recipients. Expert Rev Anti Infect Ther. 2011 Oct; 9 (10): 891-899.

53. Hatton T, Zhou S, Standring DN. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a modei for their roles in virai replication. J Virol. 1992 Sep;66(9):5232-41.53. Hatton T, Zhou S, Standring DN. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J Virol. 1992 Sep; 66 (9): 5232-41.

54. Machida A, Ohnuma H, Takai E, Tsuda F, Tanaka T, Naito M, Munekata E, Miyakawa Y, Mayumi M. Antigenic sites on the arginine-rich carboxyl-terminal domain of the capsid protein of hepatitis B virus distinct from hepatitis B core or e antigen. Mol Immunol. 1989 Apr;26(4):413-21.54. Machida A, Ohnuma H, Takai E, Tsuda F, Tanaka T, Naito M, Munekata E, Miyakawa Y, Mayumi M. Antigenic sites on the arginine-rich carboxyl-terminal domain of the capsid protein of hepatitis B virus distinct from hepatitis B core or e antigen. Mol Immunol. 1989 Apr; 26 (4): 413-21.

55. Bundule MA, Bychko VV, Saulitis IuB, Liepinsh EE, Borisova GP, Petrovski IA, Tsibinogin W, Pumpen PP, Gren Eīa. C-terminal polyarginine tract of hepatitis B core antigen is located on the outer capsid surface. (Article in Russian) Doki Akad Nauk SSSR. 1990;312(4):993 -6.55. Bundule MA, Bychko VV, Saulitis IuB, Liepinsh EE, Borisova GP, Petrovski IA, Tsibinogin W, Pumpen PP, Gren Eīa. The C-terminal polyarginine tract of hepatitis B core antigen is located on the outer capsid surface. (Article in Russian) Doki Akad Nauk SSSR. 1990; 312 (4): 993 -6.

56. Vanlandschoot P, Van Houtte F, Serruys B, Leroux-Roels G. The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surfaceexpressed heparan sulfate. J Gen Virol. 2005 Jan;86(Pt 1):75-84.56. Vanlandschoot P, Van Houtte F, Serruys B, Leroux-Roels G. The arginine-rich carboxy-terminal domain of hepatitis B virus core protein mediated attachment of nucleocapsids to cell-surface-expressed heparan sulfate. J Gen Virol. 2005 Jan; 86 (Pt 1): 75-84.

57. Meng D, Hjelm RP, Hu J, Wu J. A theoretical modei for the dynamic structure of hepatitis B nucleocapsid. Biophys J. 2011 Nov 16; 101(10):2476-2484. Epub 2011 Nov 15.57. Meng D, Hjelm RP, Hu J, Wu J. A theoretical model for the dynamic structure of hepatitis B nucleocapsid. Biophys J. 2011 Nov 16; 101 (10): 2476-2484. Epub 2011 Nov 15.

58. Strods, A., Argule, D., Cielens, I., Jackeviča, L. and Renhofa, R. (2012) Expression of GA coat protein-derived mosaic virus-like pārticies in Saccharomyces cerevisiae and packaging in vivo of mRNAs into pārticies. Proc. Latv. Acad. Sci., Section B 66,234—241.58. Strods, A., Argule, D., Cielens, I., Jackevicha, L. and Renhofa, R. (2012) Expression of GA coat protein-derived mosaic virus-like enrichment in Saccharomyces cerevisiae and packaging in vivo of mRNAs. into prosperous. Proc. Latv. Acad. Sci., Section B 66,234-241.

59. Renhofa R, Ozols J, Ose-Klinklāva V, Pumpens P. Paņēmiens izvēlētā bioloģiskā materiāla iepakošanai hepatīta B vīrusa core pilna garumaproteīna kapsīdās. Latvijas patents, LV 14084 B, publicēts 20.04.2010.59. Renhof R, Oak J, Osse-Clinklawa V, Pumpen P. Method for packaging selected biological material in hepatitis B virus core full length protein capsids. Latvian patent, LV 14084 B, published 20.04.2010.

60. Sominskaya I, Skrastina D, Petrovskis I, Dishlers A, Berza I, Mihailova M, Jansons J, Akopjana I, Stahovska I, Dreilina D, Ose V, Pumpens P. A VLP library of C-terminally truncated hepatitis B core proteīns: correlation of RNA encapsidation with a Thl/Th2 switch in the immune responses of mice. PLoS One, 2013 Sep 23;8(9):e75938. doi: 10.1371/joumal.pone.0075938.60. Sominskaya I, Skrastina D, Petrovsky I, Dishlers A, Berza I, Mikhailova M, Jansons J, Akopyan I, Stahovska I, Dreilin D, Osse V, Pumpens P. A VLP library of C-terminally truncated hepatitis B core protein. : correlation of RNA encapsidation with a Thl / Th2 switch in the immune responses of mice. PLoS One, 2013 Sep 23; 8 (9): e75938. doi: 10.1371 / joumal.pone.0075938.

61. Hatton T, Zhou S, Standring DN. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a modei for their roles in virai replication. J Virol. 1992 Sep;66(9):5232-41.61. Hatton T, Zhou S, Standring DN. RNA- and DNA-binding activities in hepatitis B virus capsid protein: a model for their roles in viral replication. J Virol. 1992 Sep; 66 (9): 5232-41.

62. Dhason MS, Wang JC, Hagan MF, Zlotnick A. Differential assembly of Hepatitis B Virus core protein on single- and double-stranded nucleic acid suggest the dsDNA-filled core is spring-loaded. Virology. 2012 Aug 15;430(l ):20-9. Epub 2012 May 16.62. Dhason MS, Wang JC, Hagan MF, Zlotnick A. Differential assembly of Hepatitis B Virus core protein on single- and double-stranded nucleic acid suggest a dsDNA-filled core is spring-loaded. Virology. 2012 Aug 15; 430 (l): 20-9. Epub 2012 May 16.

63. Stomi T, Ruedl C, Schwarz K, Schwendener RA, Renner WA, Bachmann MF. Nonmethylated CG motife packaged into virus-like pārticies inducē protective cytotoxic T celi responses in the absence of systemic side effects. J Immunol. 2004 Feb 1; 172(3): 1777-85.63. Stomi T, Ruedl C, Schwarz K, Schwendener RA, Renner WA, Bachmann MF. Nonmethylated CG motife packaged into virus-like affluent induces protective cytotoxic T cell responses in the absence of systemic side effects. J Immunol. 2004 Feb 1; 172 (3): 1777-85.

-1264. Song S, Wang Y, Zhang Y, Wang F, He Y, Ren D, Guo Y, Sun S. Augmented induction of CD8+ cytotoxic T-cell response and antitumor effect by DCs pulsed with virus-like pārticies packaging with CpG. Cancer Lett. 2007 Oct 18;256(l):90-l00. Epub 2007 Jul 25.-1264. Song S, Wang Y, Zhang Y, Wang F, He Y, Ren D, Guo Y, Sun S. Augmented induction of CD8 + cytotoxic T-cell response and antitumor effect by DCs pulsed with virus-like food packaging with CpG. Cancer Lett. 2007 Oct 18; 256 (l): 90-100. Epub 2007 Jul 25.

65. Kazaks A, Balmaks R, Voronkova T, Ose V, Pumpens P. Melanoma vaccine candidates from chimeric hepatitis B core virus-like pārticies carrying a tumor-associated MAGE-3 epitope. Biotechnol J. 2008 Nov;3(l 1):1429-36.65. Kazaks A, Balmaks R, Voronkova T, Ose V, Pumpens P. Melanoma vaccine candidates from chimeric hepatitis B core virus-like proliferators carrying a tumor-associated MAGE-3 epitope. Biotechnol J. 2008 Nov; 3 (l 1): 1429-36.

66. Song S, Zhang K, You H, Wang J, Wang Z, Yan C, Liu F. Significant anti-tumour activity of adoptively transferred T celis elicited by intratumoral dendritic celi vaccine injection through enhancing the ratio of CD8(+) T cell/regulatory T celis in tumour. Clin Exp Immunol. 2010 Oct; 162(1):75-83. doi: 10.111 l/j,1365-2249.2010.04226.x.66. Song S, Zhang K, You H, Wang J, Wang Z, Yan C, Liu F. Significant anti-tumor activity of adoptively transferred T cell elicited by intratumoral dendritic cell vaccine injection through enhancing CD8 (+) T cell / regulatory T celis in tumour. Clin Exp Immunol. Oct 2010; 162 (1): 75-83. doi: 10.111 l / j, 1365-2249.2010.04226.x.

67. Cooper A, Shaul Y. Recombinant virai capsids as an efficient vehicle of oligonucleotide delivery into celis. Biochem Biophys Res Commun. 2005 Feb 25;327(4):1094-9.67. Cooper A, Shaul Y. Recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into the cell. Biochem Biophys Res Commun. 2005 Feb 25; 327 (4): 1094-9.

68. Newman M, Chua PK, Tang FM, Su PY, Shih C. Testing an electrostatic interaction hypothesis of hepatitis B virus capsid stability by using an in vitro capsid disassembly/reassembly system. J Virol.68. Newman M, Chua PK, Tang FM, Su PY, Shih C. Testing an electrostatic interaction hypothesis of hepatitis B virus capsid stability by using an in vitro capsid disassembly / reassembly system. J Virol.

2009 Oct;83(20):10616-26. Epub 2009 Aug 5.2009 Oct; 83 (20): 10616-26. Epub 2009 Aug 5.

69. Porterfield JZ, Dhason MS, Loeb DD, Nassal M, Stray SJ, Zlotnick A. Full-length hepatitis B virus core protein packages virai and heterologous RNA with similarly high Ievels of cooperativity. J Virol.69. Porterfield JZ, Dhason MS, Loeb DD, Nassal M, Stray SJ, Zlotnick A. Full-length hepatitis B virus core protein packages in viral and heterologous RNA with similarly high levels of cooperativity. J Virol.

2010 Jul;84(14):7174-84. Epub 2010 Apr 28.2010 Jul; 84 (14): 7174-84. Epub 2010 Apr 28

70. Renhofa R, Dišlers A, Ose-Klinklāva V, Ozols J, Pumpēns P. Magnētisko nanodaļiņu iepakošana HBV core proteīna veidotajās kapsīdās. Latvijas patents, LV 14304 B, publicēts 20.05.2011.70. Renhof R, Dysler A, Osse-Clinklava V, Oak J, Pumpen P. Packaging of Magnetic Nanoparticles in HBV Core Protein Capsules. Latvian patent, LV 14304 B, published 20.05.2011.

71. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kundig T, Bāchi T, Stomi T, Jennings G, Pumpens P, Renner WA, Bachmaim MF. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B celi responses. Vaccine. 2002 Aug 19;20(25-26):31043112.71. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kundig T, Bāchi T, Stomi T, Jennings G, Pumpens P, Renner WA, Bachmaim MF. A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses. Vaccine. 2002 Aug 19; 20 (25-26): 31043112.

72. Renhofa R, Ozols J, Ose-Klinklāva V, Pumpēns P.Hepatīta B vīrusa core pilna garuma proteīna kapsīdu iegūšanas paņēmiens. Latvijas patents, LV 13958 B, publicēts 20.12.2009.72. Renhof R, Oak J, Osse-Clinklava V, Pumpen P. Method for obtaining full length protein capsids of hepatitis B virus core. Latvian patent, LV 13958 B, published 20.12.2009.

73. Shangguan D., Li Y., Tang Z., Cao Z.Ch., Chen H.W., Mallikaratchy P., Sefan K., Yang Ch.J., Tan W. Aptamers evolved from live celis as effective molecular probes for cancer study. Proc.Natl,Acad.Sci.USA, 2006 Aug 8;103(32):l 1838-11843.73. Shangguan D., Li Y., Tang Z., Cao Z.Ch., Chen H.W., Mallikaratchy P., Sefan K., Yang Ch.J., Tan W. Aptamers evolved from live cell as effective molecular probes for cancer study. Proc.Natl, Acad.Sci.USA, 8 Aug 2006; 103 (32): l 1838-11843.

Claims

1. A method of depositing selected biological material on the surface of modified hepatitis B core nanocontainers made with the full length (183 amino acid residues) core protein of the hepatitis B virus, with chemical stitching technology, wherein the modification is as follows:

a. asparagine residue 75 (Asn75) replaced by lysine residue,

b. glutamic acid residue 77 (Glu77) replaced by lysine residue,

c. proline residue 79 (Pro79) replaced by lysine residue,

d. isoleucine residue 80 (Ile80) replaced by lysine residue.

The method of claim 1, wherein the expression of the modified HBc proteins in E.coli cells yields HBc virus-like particles.

Method according to claims 1 and 2, characterized in that after the internal filler has been cleaved with the alkaline solution, the structure of the modified HBc virus-like particles is retained.

Method according to any one of claims 1 to 3, characterized in that the biological modified material, ribonucleic acid, oligodeoxynucleotides or deoxynucleic acids, can be packaged in the empty modified HBc virus-like particles.

Method according to any one of claims 1 to 4, characterized in that the biological material is chemically sewn onto the surface of the modified HBc particles by lysine residues.