LV15034B - Modified hbv core nano-containers as universal platform for exhibiting biological material - Google Patents
Modified hbv core nano-containers as universal platform for exhibiting biological material Download PDFInfo
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- LV15034B LV15034B LVP-14-06A LV140006A LV15034B LV 15034 B LV15034 B LV 15034B LV 140006 A LV140006 A LV 140006A LV 15034 B LV15034 B LV 15034B
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IZGUDROJUMA APRAKSTSDESCRIPTION OF THE INVENTION
Tehnikas jomaTechnical field
Izgudrojums attiecas uz molekulāro bioloģiju, gēnu un proteīnu inženieriju, biotehnoloģiju, imunoloģiju, nanotehnoloģiju un biomedicīnu, konkrēti uz paņēmieniem, kas ļauj mērķtiecīgi pievienot izvēlētos proteīnus, peptīdus un/vai oligonukleotīdus pie hepatīta B vīrusa (HBV) core (HBe) pilna garuma proteīna un izvietot tos uz HBe veidoto vīrusiem līdzīgo daļiņu kā nanokonteineru virsmas. 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 purposefully attaching selected proteins, peptides and / or oligonucleotides to the full length hepatitis B virus (HBV) core protein (HBe) and HBe. place them on the surface of HBe-generated viral-like particles as nanocontainers. The invention is applicable to the development of vaccine, gene and drug therapy and diagnostic preparations.
Tehnikas līmenisState of the art
Rekombinantas vīrusiem līdzīgās daļiņas (VLD) ir pašasociēties spējīgi vīrusu struktūras elementi, kas tiek iegūti ar gēnu un proteīnu inženierijas tehnoloģijām un aktīvi izmantoti mūsdienu nanotehnoloģijā un biomedicīnā kā nanokonteineri ar sveša bioloģiskā materiāla ekspozīcijas un piegādes spējām (1,2).Recombinant virus-like particles (VLDs) are self-associating viral structural elements obtained by genetic and protein engineering and actively used in modern nanotechnology and biomedicine as nanocontainers with foreign biological material exposure and delivery capabilities (1,2).
No VLD veidot spējīgiem proteīniem īpaša vieta pieder hepatīta B vīrusa (HBV) core (HBe) proteīnam, kas bija ne tikai viens no pirmajiem VLD objektiem (3-5), bet apliecināja sevi kā vistolerantākais pret proteīninženierisko iejaukšanos struktūrā un svešu sekvenču iebūvēšanu un pakošanu (1,2,6-8). HBe proteīnam piemīt unikāla universalitāte augsti efektīvas ekspresijas jomā - gan homologās (dažādu augstāko eikariotu šūnās), gan arī plaša spektra heterologās (baktēriju, raugu, kukaiņu un augu Šūnās) ekspresijas sistēmās (8).Of the VLD-capable proteins, a special place belongs to the hepatitis B virus (HBV) core (HBe) protein, which was not only one of the first VLD targets (3-5), but also proved to be most tolerant of protein engineering in the structure and foreign sequence insertion and packaging. (1,2,6-8). The HBe protein has a unique versatility in highly efficient expression, both in homologous (various higher eukaryotic cells) as well as in a wide variety of heterologous (bacterial, yeast, insect and plant) expression systems (8).
Šādas HBe VLD universalitātes pamatā ir HBe proteīna neparastā, uz α-domēniem balstīta telpiskā struktūra (9). HBe proteīna garums ir 183 aminoskābju atlikumi (HBV genotipam A 185) un tas ir būvēts no diviem neatkarīgiem domēniem: (a) N-terminālā kapsīdas veidojošāThis versatility of HBe VLDs is based on the unusual spatial structure of the HBe protein based on α-domains (9). The HBe protein has 183 amino acid residues (HBV for genotype A 185) and is constructed from two independent domains: (a) the N-terminal capsid-forming
-2pašasociācijas (self-assembly, SA) domēna no N-gala līdz 140. aminoskābju atlikumam un (b) bāziskā, ar arginīniem bagāta C-terminālā protamīn-līdzīga apgabala - C-terminālā (CTD) jeb nukleīnskābes domēna (10). īsākais rekombinantā HBc proteīna variants, kas ir vēl spējīgs uz pašasociāciju, ir N-gala 140 aminoskābju atlikumu garš variants (11,12). HBc proteīna SA domēns satur vairākus variablus un konservatīvus rajonus, kas sastāda attiecīgi imunoloģiskos Bšūnu epitopus un strukturālos elementus, kamēr CTD domēns pieder pie visaugstāk konservētiem HBc rajoniem (13,14).-2 self-assembly (SA) domain from the N-terminus to the 140th amino acid residue; and (b) a basic arginine-rich C-terminal protamine-like domain, the C-terminal (CTD) or nucleic acid domain (10). the shortest variant of recombinant HBc protein still capable of self-association is the long version of the N-terminal 140 amino acid residues (11,12). 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 belongs to the most conserved HBc regions (13,14).
HBc pašasociācijas process tiek ievadīts ar spontānu HBc proteīna dimēru izveidošanos (15), kas spēj izveidot divus ikosaedru izomorfus no 120 un 90 HBc dimēriem ar triangulācijas skaitļiem T=4 un T=3 un attiecīgi 35 un 32 nm diametru (16,17). HBc daļiņu T=4 izomorfa augstas izšķirtspējas telpiskā struktūra tika iegūta ar elektronu kriomikroskopijas (9,18,19) un rentgenstaru kristalogrāfijas (20) palīdzību. HBc daļiņu T=3 izomorfa struktūra tika rekonstruēta vēlāk aprēķinu ceļā (21).The HBc self-association process is introduced by the spontaneous formation of HBc protein dimers (15), capable of forming two icosahedral isomorphs of 120 and 90 HBc dimers with triangulation numbers T = 4 and T = 3 and 35 and 32 nm respectively (16,17). The high resolution spatial structure of the T = 4 isomorph of HBc particles was obtained by electron cryomicroscopy (9,18,19) and X-ray crystallography (20). The T = 3 isomorph structure of HBc particles was reconstructed later by calculation (21).
HBc proteīna SA domēna pašasociācijas spēja, reizē ar augsto kapacitāti svešu sekvenču akceptēšanā, tiek plaši izmantota himēro VLD izveidošanai uz HBc pamata, turklāt kā uz pilna garuma, tā ari uz dažādiem C-termināli īsināto HBc proteīna variantiem (1,2,6-8). Vispiemērotākā vieta svešu sekvenču iestarpināšanai himērajās HBc VLD ir visvairāk uz ārpusi izvirzītā HBc izauguma - α-spirāļu veidotās „matadatas” - galiņi (20). HBc „matadatu” galiņos ir novietoti HBc aminoskābju atlikumi 76-81, kas veido t.s. major immunodominant region jeb HBc imunodominanto rajonu (6-8). 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 aprobežojas ar tradicionālo Lys-Cys ķīmisko sašūšanu un, tādejādi, ar lizīna atlikuma ievadīšanu HBc struktūrā mutācijas ceļā (22).The self-association ability of the HBc protein SA domain, together with its high capacity for accepting foreign sequences, is widely used to generate chimeric VLDs based on HBc, in addition to full-length and various C-terminal truncated HBc protein variants (1,2,6-8 ). The most suitable site for inserting foreign sequences into the chimeric HBc VLDs is the tip of the most protruding HBc outgrowth - hairpin formed by α-helices (20). The HBc "hairpin" ends contain the residues 76-81 of HBc, constituting m.p. major immunodominant region or HBc immunodominant region (6-8). 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 variant is far more versatile and technological than the first, its use is limited to traditional chemical cross-linking of Lys-Cys and, thus, the introduction of lysine residue into the structure of HBc by mutation (22).
-3Izgudrojuma izpaušana-3 Disclosure of Invention
Izgudrojums attiecas uz jauna HBc nanokonteineru ekspozīcijas un piegādes modeļa izveidošanu, kas ļauj 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 izvēlēta ķīmiskas piešūšanas shēma, kas nodrošina sveša bioloģiskā materiāla piešūšanu pie diviem HBc «matadatas” sastāvā maksimāli uz āru izvirzītajiem Glu77 un Asp78 aminoskābju atlikumiem. Izvēlētā ķīmiskās piešūšanas shēma neprasa HBc proteīna sekvences modifikāciju ar jaunu aminoskābju atlikumu ievadīšanu un ļauj izmantot visas nemodificētā HBc proteīna priekšrocības: (a) augstu ekspresijas līmeni baktēriju E.coli un rauga P.pastoris šūnās, (b) iespēju pielietot agrāk izstrādātas efektīvas attīrīšanas un nanokonteineru pako sanas shēmas.The invention relates to the development of a novel HBc nanocontainer exposure and delivery model that allows the placement of foreign biological material (proteins and peptides, nucleic acid fragments) in the optimal, outwardly projecting regions of the HBc protein, i.e. At the ends of the "hairpin". For this purpose, a chemical sewing scheme has been chosen that provides sewage of foreign biological material to the two protruding Glu77 and Asp78 amino acid residues in the HBc «hairpin». The chemical stitching scheme selected does not require modification of the HBc protein sequence with the introduction of new amino acid residues and allows all the benefits of unmodified HBc protein to be exploited: (a) high expression levels in E.coli and P. pastoris cells, (b) nanocontainer packing schemes.
Realizējot piedāvātās svešā bioloģiskā materiāla piešūšanas shēmas, HBc VLD pakļaujas agrāk izstrādātajām pakošanas shēmām un tādejādi ļauj izmantot izveidotos tehnoloģiskos risinājumus par HBc VLD pakošanu ar bioloģisko: nukleīnskābju un proteīnu materiālu (24).By implementing the proposed foreign biological stitching schemes, HBc VLD follows the previously developed packaging schemes and thus allows the application of developed technological solutions for HBc VLD packaging with biological: nucleic acid and protein material (24).
Realizējot piedāvāto ķīmiskas piešūšanas shēmu pie Glu77 un Asp78 atlikumiem, HBc VLD uzrāda efektīvu bioloģiskā materiāla pievienošanu. Pateicoties optimāli izvēlētām uz āru izvirzītām piešūšanas vietām, piešūtais bioloģiskais materiāls tiek eksponēts uz HBc VLD virsmas.By implementing the proposed chemical stitching scheme for Glu77 and Asp78 residues, HBc VLD exhibits efficient addition of biological material. Thanks to the optimum selection of protruding sutures, the sewn biological material is exposed on the surface of HBc VLD.
Atkarībā no piešūtā bioloģiskā materiāla, piemēram, proteīnu, peptīdu vai oligonukleotīdu, īpašībām un nanokonteineru pildījuma, šādi HBc nanokonteineri, kuru pamatā ir dabīgais, savā sekvencē nepārveidotais HBc proteīns kā nesējs, var tikt izmantoti kā vakcīnu prototipi vai kā gēnu un zāļu terapijas līdzekļi, kas ir adresēti uz noteiktām šūnām vai orgāniem un izmantoti tādejādi profilaktiskiem, diagnostiskiem vai terapeitiskiem mērķiem.Depending on the properties of the sewn biological material, such as proteins, peptides or oligonucleotides, and the filling of the nanocontainers, such HBc nanocontainers based on native, unmodified HBc protein as a carrier can be used as vaccine prototypes or as gene and drug therapy products addressed to specific cells or organs and used for preventive, diagnostic or therapeutic purposes.
-4Eksperimentālā daļa-4Experimental part
Eksperiments ietver sevī HBV core pilna garuma proteīna veidoto rekombinanto daļiņu iegūšanu ar tālāko atbrīvošanos no to dabiskā pildījuma - tukšo daļiņu iegūšanu, imunomodulātorā oligonukleotīda (sekvenci CpG saturoša) iepakošanu un HBV preSl sekvenci nesoša peptīda ķīmisku piešūšanu pie daļiņu virsmas. Ķīmiskai piešūšanai tika izvēlētas t.s. „matadatu”-HBc daļiņu izvirzījumu aminoskābes - 77. glutamīnskābe (E) un 78. asparagīnskābe (D), kuras abas savās sānu virknēs satur reaģēt spējīgas karboksilgrupas. 1. zīmējumā ar izceltām mērķa aminoskābēm parādītas «matadatu” aminoskābju sekvence un tās telpiskā struktūra.The experiment involves the production of recombinant particles of HBV core full-length protein with further release of their natural filler - blank particles, packaging of an immunomodulatory oligonucleotide (containing CpG sequence) and chemical stitching of the HBV preSl sequence-carrying peptide to the surface of the particles. Chemical stitches were selected e.g. 'Hairpin' -HBc particle protrusions amino acids 77 glutamic acid (E) and 78 aspartic acid (D), both of which contain reactive carboxyl groups in their side chains. Figure 1, with highlighted target amino acids, shows the amino acid sequence of the hairpin and its spatial structure.
Raugā Pichia pastoris ekspresētās HBV core daļiņas attīrītas pēc iepriekš izstrādātās shēmas (23). Escherichia coli ekspresētās HBV core daļiņas tika iegūtas saskaņā ar iepriekš izstrādāto paņēmienu (24). Tukšo bakteriālās izcelsmes HBV core daļiņu iegūšana un to pakošana ar ODN tika veikta saskaņā ar iepriekš izstrādāto shēmu (25). Tukšo rauga izcelsmes HBV core daļiņu iegūšanai tika veikta materiāla sagatavošana sārma hidrolīzei bromatogrāfejot to uz Sephacryl HR S300 kolonas 0,1 M nātrija karbonātā ar 2mM DTT (ap 50 mg VLD 2-3 ml 7M urinvielas uznests uz kolonas 1,5x65 cm; elūcijas ātrums 3 ml/h/frakciju). Materiāla elūcija tika pārbaudīta frakciju paraugus elektroforēzējot natīvā agarozes gēlā TAE buferšķīdumā (Lermentas). Frakciju ar VLD saturs tika apvienots (15-20 ml), iepildīts dialīzes tūbā (Sigma), ievietots traukā ar 0,1 M nātrija ortofosfatu, 0,65 M nātrija hlorīdu, pH 12 un veikta sārma hidrolīze no ārpuses maisot 37°C 18h. Tālāk dialīzes tūba ar VLD tiek ievietota buferšķīdumā 0,1 M nātrija hidrogenfosfats, 0,65 M nātrija hlorīds, pH 7,8, pēc lh šis buferšķīdums tiek apmainīts pret svaigu un pēc 3h dialīzes tūbas saturs tiek savākts un izgulsnēts ar 60% (no piesātinājuma) amonija sulfātu (0,45g/ml). Nogulšņu materiāls tiek šķīdināts buferšķīdumā 20mM Tris-HCl, 5 mM EDTA, 0,65 M NaCl, pH 7,8 (tas ir arī pakošanas un refoldinga buferšķīdums) frakcionēts uz Sepharose CL4B kolonas (2x60 cm) šajā buferšķīdumā. Uzglabāšanai materiāls tiek izgulsnēts ar amonija sulfātu.HBV core particles expressed in Pichia pastoris were purified according to a previously developed scheme (23). HBV core particles expressed in Escherichia coli were obtained according to a previously developed method (24). The production of the empty bacterial HBV core particles and their packaging with ODN was carried out according to the previously developed scheme (25). Preparation of the yeast-derived HBV core particles was performed by alkali hydrolysis by chromatography on a Sephacryl HR S300 column in 0.1 M sodium carbonate with 2mM DTT (about 50 mg VLD 2-3 mL of 7M urea applied to the column 1.5x65 cm; elution rate) 3 ml / h / fraction). The elution of the material was tested by electrophoresis of the fractional samples in native agarose gel in TAE buffer (Lermentas). The VLD fractions were pooled (15-20 mL), filled into dialysis tube (Sigma), placed in a vessel with 0.1 M sodium orthophosphate, 0.65 M sodium chloride, pH 12, and subjected to alkaline external hydrolysis at 37 ° C for 18h. . Next, the dialysis tube with VLD is placed in buffer 0.1 M sodium hydrogen phosphate, 0.65 M sodium chloride, pH 7.8, after 1 h this buffer is exchanged for fresh and after 3 h dialysis the tube contents are collected and precipitated with 60% ( saturated) ammonium sulfate (0.45g / ml). The precipitate material is dissolved in a buffer solution of 20mM Tris-HCl, 5mM EDTA, 0.65M NaCl, pH 7.8 (also a packing and refolding buffer) fractionated on a Sepharose CL4B column (2x60cm) in this buffer. The material is precipitated with ammonium sulphate for storage.
Oligodezoksinukleotīda iepakošana tika veikta saskaņā ar iepriekš izstrādāto paņēmienu (25).The oligodeoxynucleotide was packaged according to the procedure previously developed (25).
-5Pakošanai tika ņemts 63 nukleotīdus garš CpG sekvenci saturošs (TCC ATG ACG TTC CTG-5Packaged 63 nucleotides long CpG containing (TCC ATG ACG TTC CTG
AAT AAT)3 oligonukleotīds.AAT AAT) 3 oligonucleotide.
Maisījumu no 4 mg tukšo rauga izcelsmes VLD (830 pmol) 3 ml pakošanas-refoldinga buferšķīdumā un 0,lml ODN ūdenī dializē lh pret 7 M urinvielu ar 0,5 M nātrija hlorīda, dialīzi turpina pret pakošanas-refoldinga buferškīdumu ar buferšķīdumā maiņu; reakcijas maisījumu frakcionē uz Sepharose CL-2B kolonas un pakoto VLD materiālu izgulsnē ar amonija sulfātu. Visi produkti tika raksturoti natīvās agarozes un poliakrilamīda elektroforēzes sistēmās, arī ar UV-spektriem un eletronmikroskopijā (EM). Vīrusiem līdzīgo daļiņu saglabāšanās veikto procesu rezultātā ir droši pierādīta ar elektronmikroskopiju (2. zīmējums): (A) „tukšas” HBV core VLD, (B) HBV core VLD x ODN un (C) HBV core VLD x ODN x preSl(K21-47). UVspektri (3. zīmējums) uzņemti uz NanoDrop ND-1000 iekārtas. Vertikālās līnijas iezīmē adsorbcijas vērtības pie 260 nm un 280 nm. No iekšējā dabiskā satura atbrīvotajām VLD ir proteīnam raksturīgs UV-spektrs ar attiecību A260/A280 = 0,75 (3. zīmējums - A), bet pēc ODN iepakošanas tas atgūst dabiski pildīto daļiņu spektra raksturu ar attiecību A260/A280= 1,13 (3. zīmējums - B). Pierādījās, ka iepriekš izstrādātais bakteriālas izcelsmes (producētas Escherichia coīi) tukšo HBV core daļiņu iegūšanas un izvēlētā materiāla iepakošanas paņēmiens (25) ir pilnībā kompetents arī priekš raugā Pichia pastoris producētām HBV core daļiņām, kuras nes eikariotiem raksturīgās fosforilētās aminoskābes (23).A mixture of 4 mg of blank yeast VLD (830 pmol) in 3 ml packing-refolding buffer and 0.1 ml ODN in water is dialyzed against lh to 7 M urea in 0.5 M sodium chloride, dialysis is performed against packing-refolding buffer with buffer exchange; the reaction mixture is fractionated on a Sepharose CL-2B column and the packed VLD material is precipitated with ammonium sulfate. All products were characterized in native agarose and polyacrylamide electrophoresis systems, including UV spectra and electron microscopy (EM). The retention of virus-like particles as a result of the processes performed has been safely demonstrated by electron microscopy (Figure 2): (A) "blank" HBV core VLD, (B) HBV core VLD x ODN and (C) HBV core VLD x ODN x preSl (K21- 47). UV spectra (Figure 3) taken on the NanoDrop ND-1000. The vertical lines plot the adsorption values at 260 nm and 280 nm. The VLDs released from the intrinsic natural content have a protein-specific UV spectrum at a ratio of A260 / A280 = 0.75 (Figure 3-A), but after ODN packaging it regains the nature of the naturally-charged particle spectrum at a ratio of A260 / A280 = 1.13 ( Figure 3 - B). The previously developed method of obtaining HBV core particles of bacterial origin (produced by Escherichia coii) and packaging of selected material (25) is fully competent also for HBV core particles produced by Pichia pastoris, which carry eukaryotic-specific phosphorylated amino acids (23).
Ķīmisko piešūšanu starp VLD sastāvā ietilpstošo asparagīnskābes un/vai glutamīnskābes karboksilgrupām -COOH un pievienojamā proteīna lizīna -NH2 grupām veica saskaņā ar standarta protokolu „EDC and Sulfo-NHS” (Pierce), izmantojot N-(3-dimetilaminopropil)-N’etilkarbodiimīdu (Fluka) un N-hidroksisulfosukcinimīdu (Aldrich) MOPS (Sigma) saturošā buferšķīdumā. Tika piešūts proteīns, kas pēc sekvences atbilst HBV preSl posmam no 21. līdz 47. aminoskābei, un kas N-termināli papildināts ar lizīnupreSl(K21-47):Chemical stitching between the carboxyl groups of aspartic acid and / or glutamic acid -COOH in the VLD and the lysine -NH 2 groups of the protein to be added was performed according to the standard protocol "EDC and Sulfo-NHS" (Pierce) using N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide (Fluka) and N-hydroxysulfosuccinimide (Aldrich) in MOPS (Sigma) containing buffer. A protein corresponding to amino acid residues 21-47 of the preSl sequence of HBV was sewn together and added at the N-terminus with lysineupreSl (K21-47):
KPLGFFPDHQLDPAFRANTANPDWDFNP.KPLGFFPDHQLDPAFRANTANPDWDFNP.
-6Starpprodukta, kā ari galaprodukta izdalīšana tika veikta uz īsas Sepharose CL-6B kolonas (1x10 cm) iepriekšminētajā MOPS buferšķīdumā.-6The isolation of the intermediate as well as the final product was performed on a short column of Sepharose CL-6B (1x10 cm) in the above MOPS buffer.
īpaša uzmanība tika pievērsta ķīmiskās piešūšanas produkta pierādīšanai. HBV core proteīna pagarināšanos par 28 aminoskābēm daļiņās HBV core VLD x ODN x preSl(K21-47) ar piešūto peptīdu (4. zīmējums - C) attiecībā pret proteīnu no HBV core VLD (4. zīmējums - A) parāda elektroforēze 15% poliakrilamīda gēlā un Westem-blots ar peļu monoklonālām anti-core antivielām 14G3 (27). Kā papildus kontrole tika ņemtas VLD, kas ģenētiski pagarinātas ar 37 aminoskābju garu proteīnu no HBV core preSl (4. zīmējums - K). Šādas daļiņas, kas tika iegūtas, iestarpinot preSl sekvenci starp 78. un 79. aminoskābi core proteīna imunodominantajā rajonā, lai ari spēja eksponēt iecerēto epitopu (šeit - preSl sekvenci), tomēr izrādījās neizturīgas pret iekšējā dabiskā pildījuma nomaiņas procedūru. Pēdējais apstāklis arī rosināja mūs izstrādāt šeit piedāvāto paņēmienu potenciālā epitopa-antigēna piešūšanai pie VLD ar nomainītu dabisko pildījumu pret imunostimulatoro CpG-oligonukleotīdu.special attention was given to the demonstration of the chemical sewing product. HBV core protein elongation by 28 amino acids in HBV core VLD x ODN x preSl (K21-47) with stitched peptide (Figure 4 - C) versus protein from HBV core VLD (Figure 4 - A) is shown by 15% polyacrylamide gel and Westem blots with mouse monoclonal anti-core antibody 14G3 (27). VLDs genetically extended with the 37 amino acid long protein from HBV core preSl were taken as additional controls (Figure 4 - K). Such particles, obtained by inserting the preSl sequence between amino acids 78 and 79 in the immunodomain region of the core protein, although able to exhibit the intended epitope (herein referred to as the preSl sequence), nevertheless proved to be resistant to the internal natural charge replacement procedure. The latter circumstance also prompted us to develop a method for sewing a potential epitope-antigen on a VLD with an altered natural filling against the immunostimulatory CpG-oligonucleotide.
Pārliecinošs piešūtā peptīda pierādījums tika iegūts arī konkurējošā ELISA (5-2 zīmējums) ar MA 18/7 tipa antivielām. Šīs monoklonālās antivielas specifiski atpazīst īsu, piecu aminoskābju sekvenci DPAFR(26), kas iekļauta piešūtajā peptīdā. Ķīmiski sintezēts preSl(21-47) peptīds tika sorbēts uz Maxisorp (Nunc) platēm no šķīduma lOug/ml nātrija karbonātā. Plates tika līdzsvarotas ar 0,1% BSA PBS buferšķīdumā. Antivielas tika titrētas ar atšķaidīšanas soli 2 inkubējot lh pie 37°C, pēc skalošanas inkubējot ar anti-peļu IgG-peroksidāzes konjugātu un attīstot ar o-fenilendiamīnu (Sigma). No 5-1 zīmējumā redzams, ka pie atšķaidījuma 50 tūkstoš reizes mērijuma vērtība ir 0,8-1,0 OB (492nm) vienības.Conclusive ELISA (Figure 5-2) with MA 18/7 antibodies was also used to provide convincing evidence of the sewn peptide. These monoclonal antibodies specifically recognize the short, five-amino acid sequence of DPAFR (26) contained in the sewn peptide. Chemically synthesized preSl (21-47) peptide was sorbed onto Maxisorp (Nunc) plates from a solution of 10ug / ml sodium carbonate. Plates were equilibrated with 0.1% BSA in PBS buffer. Antibodies were titrated with dilution step 2 by incubating lh at 37 ° C, incubating with anti-mouse IgG-peroxidase conjugate and developing with o-phenylenediamine (Sigma). Figure 5-1 shows that at dilution 50,000 times the measurement value is 0.8-1.0 OB (492nm) units.
Konkurējošai ELISAi (zīmējums 5-2), katrā plates iedobē tika iepildīti 50 μΐ VLD ar piešūto peptīdu VLD x ODN x preSl(K21-47) atšķaidījumi, arī ar soli 2, sākot no 0,2 mg/ml, kam sekoja pa 50 μΐ iepriekš 25 tūkstošus reizes atšķaidītas antivielas MA 18/7. Rezultātā antivielas atšķaidījās 50 tūkstošus reizes, bet OB (492 nm) mērījumi pakāpeniski sasniedza plānoto vērtību, parādot, ka piešūšanas produkts efektīvi konkurē ar uzsorbēto peptīdu par saistību ar antivielām.For competitive ELISA (Figure 5-2), 50 μΐ dilutions of VLD with stitched peptide VLD x ODN x preSl (K21-47) were added to each well of the plate, also in step 2 starting at 0.2 mg / ml followed by 50 μΐ MA 25/7 diluted previously 25,000 times. As a result, the antibodies diluted 50,000-fold, but OB (492 nm) measurements gradually reached their target value, showing that the stitching product competed effectively with the absorbed peptide for binding to the antibodies.
-ΊLV 15034-ΊLV 15034
Tādejādi, mūsu piedāvātais paņēmiens iecerētā epitopa, aprīkota ar lizīnu, piešūšanai pie VLD core caur mijiedarbību ar „matadatās” izvirzītajām aminoskābēm, izveidojot jaunu peptīdsaiti starp -COOH un NH2- grupām, ļauj iegūt tādas core proteīna daļiņas, vienalga - bakteriālas vai rauga izcelsmes, kurām var nomainīt dabisko pildījumu uz iecerēto ODN, un kuras uz virsmas nes iecerēto peptīda sekvenci.Thus, our proposed method of ligating a proposed epitope equipped with lysine to the VLD core through interaction with the "hairpin" raised amino acids, by creating a new peptide linkage between -COOH and NH 2 , allows the production of core protein particles, either bacterial or yeast. , which can be replaced by a natural filler on the intended ODN, and which carry the intended peptide sequence on the surface.
-8Citetas literatūras saraksts-8Citatory References
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12. 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.0075938.12. 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.
13. Pumpens P, Grens E, Nassal M. Molecular epidemiology and immunology of hepatitis B virus infection - an update. Intervirology. 2002;45(4-6):218-232.13. Pumpens P, Grens E, Nassal M. Molecular epidemiology and immunology of hepatitis B virus infection - an update. Intervirology. 2002; 45 (4-6): 218-232.
14. Chain BM, Myers R. Variability and conservation in hepatitis B virus core protein. BMC Microbiol. 2005 May 27;5:33.14. Chain BM, Myers R. Variability and conservation in hepatitis B virus core protein. BMC Microbiol. 2005 May 27; 5: 33.
15. 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): 10046-10050.15. 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.
16. Gerlich WH, Goldmann U, Mtiller R, Stibbe W, Wolff W. Specificity and localization of the hepatitis B virus-associated protein kinase. J Virol. 1982 Jun;42(3):761-766.16. Gerlich WH, Goldmann U, Mtiller R, Stibbe W, Wolff W. Specificity and localization of hepatitis B virus-associated protein kinase. J Virol. 1982 Jun; 42 (3): 761-766.
17. Cohen BJ, Richmond JE. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature. 1982 Apr 15;296(5858):677-679.17. Cohen BJ, Richmond JE. Electron microscopy of hepatitis B core antigen synthesized in E. coli. Nature. 15 Apr 1982; 296 (5858): 677-679.
18. 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):88-91.18. 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.
19. Conway JF, Cheng N, Zlotnick A, Wingfield PT, Stahl SJ, Steven AC. Visualization of a 4-helix bundle in the hepatitis B virus capsid by ciyo-electron microscopy. Nature. 1997 Mar 6;386(6620):91-94.19. Conway JF, Cheng N, Zlotnick A, Wingfield PT, Stahl SJ, Steven AC. Visualization of a 4-helix bundle in hepatitis B virus capsid by ciyo-electron microscopy. Nature. 1997 Mar 6; 386 (6620): 91-94.
20. Wynne SA, Crowther RA, Leslie AG. The ciystal structure of the human hepatitis B virus capsid. Mol Celi. 1999 Jun;3(6):771-780.20. Wynne SA, Crowther RA, Leslie AG. The cystic structure of the human hepatitis B virus capsid. Mol Celi. 1999 Jun; 3 (6): 771-780.
21. 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 28.21. 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.
22. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kūndig T, Bāchi T, Stomi T, Jennings G, Pumpens P, Renner WA, Bachmann MF. A molecular assembly system that renders antigēns of choice highly repetitive for induction of protective B celi responses. Vaccine. 2002 Aug 19;20(25-26):3104-3112.22. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kūndig T, Bāchi T, Stomi T, Jennings G, Pumpens P, Renner WA, Bachmann MF. A molecular assembly system that renders antigen of choice highly repetitive for induction of protective B cell responses. Vaccine. 2002 Aug 19; 20 (25-26): 3104-3112.
23. Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Highly efficient production of phosphorylated hepatitis B core pārticies in yeast Pichia pastoris. Protein Expr Purif 75,218-224,2011.23. Freivalds J, Dislers A, Ose V, Pumpens P, Tars K, Kazaks A. Highly efficient production of phosphorylated hepatitis B core by yeast Pichia pastoris. Protein Expr Purif 75,218-224,2011.
24. Renhofa R, Ozols J, Ose-Klinklāva V, Pumpens P. Hepatīta B vīrusa core pilna garuma proteīna kapsīdu iegūšanas paņēmiens. Latvijas patents, LV 13958 B, publicēts 20.12.2009.24. Renhof R, Oak J, Osse-Clinklava V, Pumpen P. Method for obtaining full length protein capsules of hepatitis B virus core. Latvian patent, LV 13958 B, published 20.12.2009.
9LV 150349LV 15034
25. Renhofa R.,Ozols J., Ose-Klinklāva V., Pumpēns P. Paņēmiens izvēlētā bioloģiskā materiāla iepakošanai hepatīta vīrusa core pilna garuma proteīna kapsīdās. Latvijas patents, LV 14084 B, publicēts 20.04.2010.25. Renhofa R., Oak J., Ose-Klinklawa V., Pumpen P. Method of Packaging Selected Biological Material in Hepatitis Virus Core Full-Length Protein Capsules. Latvian patent, LV 14084 B, published 20.04.2010.
26. Sominskaya I., Pushko P., Dreilina D., Kozlovskaya T., Pumpen P. Determination of the minimal length of preSl epitope recognized by a monoclonal antibody which inhibits attachment of Hepatitis B virus to hepatocytes. Med.Microbiol Immunol 181,215-226,1992.26. Sominskaya I., Pushko P., Dreilina D., Kozlovskaya T., Pumpen P. Determination of the minimal length of the preSl epitope recognized by a monoclonal antibody which inhibits the attachment of hepatitis B virus to hepatocytes. Med.Microbiol Immunol 181,215-226,1992.
27. Bichko V., Schodel F., Nassal M., Gren E., Berzinsh L, Borisova G., Miska S., Peterson DL., Gren E., Pushko P., Will H. Epitopes recognized by antibodies to denatured core protein of hepatitis B virus. Mol Immunol, 30(3): 221-31, 1993.27. Bichko V., Schodel F., Nassal M., Gren E., Berzinsh L., Borisova G., Miska S., Peterson D.L., Gren E., Pushko P., Will H. Epitopes recognized by antibodies to denatured core protein of hepatitis B virus. Mol Immunol, 30 (3): 221-31, 1993.
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