CZ299417B6 - Zpusob prípravy živého Birnaviru, kterým je virusinfekcní choroby bursy, plasmid a zpusob prípravyvakcíny - Google Patents
Zpusob prípravy živého Birnaviru, kterým je virusinfekcní choroby bursy, plasmid a zpusob prípravyvakcíny Download PDFInfo
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- CZ299417B6 CZ299417B6 CZ0074299A CZ74299A CZ299417B6 CZ 299417 B6 CZ299417 B6 CZ 299417B6 CZ 0074299 A CZ0074299 A CZ 0074299A CZ 74299 A CZ74299 A CZ 74299A CZ 299417 B6 CZ299417 B6 CZ 299417B6
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Abstract
Zpusob prípravy živého Birnaviru, kterým je virusinfekcní choroby bursy, zahrnuje následující kroky: prípravu jedné nebo více cDNA genomových segmentu A a B viru infekcní choroby bursy, transkripci této jedné nebo techto více cDNA za vzniku syntetických RNA transkriptu, kde tyto RNA transkripty jsou pozitivními RNA transkripty uvedených segmentu A a B, transfekci hostitelských bunek temito syntetickými RNA transkripty, inkubaci techto hostitelských bunek v kultivacním médiu a izolaci živého infekcního viru infekcní choroby bursy z uvedeného kultivacního média.
Description
Oblast techniky
Virus infekční choroby bursy (IBDV), člen rodiny Bimaviridae, je kauzativní činitel imunosupresivních chorob mladých kuřat.
Dosavadní stav techniky
Virus infekční choroby bursy (IBDV), člen rodiny Bimaviridae, je kauzativním činitelem imunosupresivních chorob mladých kuřat (Kibenge, F.S.B., a kol., J.Gen. Vir., 69, 1757-1775 (1988)).
Infekční onemocnění bursy (IBD) nebo Gumboro nemoc jsou charakterizovány destrukcí lymfoidních folikulů ve Fabriciově burse. V plně přístupném hejnu kuřat o stáří 3-6 týdnů způsobí klinická choroba těžkou imunosupresi a je odpovědná za ztráty kvůli zhoršenému růstu, sníženou efektivitu krmení a smrt. Citlivá kuřata mladší než 3 týdny nevykazují viditelné klinické znaky choroby, ale mají patrnou infekci charakterizovanou výrazným poškozením bursy.
Virus, který je spojený se symptomy choroby, se nazývá virus infekční choroby bursy (IBDV). IBDV je patogen s velkým ekonomickým významem z hlediska národního i světového drůbežářského průmyslu. Způsobuje silnou imunodeficienci u mladých kuřat destrukcí prekurzorů B buněk tvořících protilátky ve Fabriciově burse. Imunosuprese způsobí zvýšenou citlivost k jiným chorobám a ovlivňuje účinnost očkování proti Newcastle chorobě, Markově chorobě a virům infekční bronchitidy.
Jsou známy dva serotypy IBDV. Viry serotypu I jsou patogenní pro kuřata, zatímco viry serotypu II infikují kuřata a krocany. Infekce krocanů nemá v současnosti klinický význam.
IBDV patří ke skupině virů nazývaných Bimaviridae, která obsahuje i další dvousegmentové RNA viry, jako virus infekční nekrózy pankreatu (ryby), tellina virus a virus ústřic (mlži) a virus X octomilky (ovocná muška). Tyto viry obsahují vysokomolekulární (MW) genom tvořený dvouvláknovou RNA.
Kapsida IBDV vím se skládá z několika strukturních proteinů. Bylo popsáno až devět struktur proteinů, ale je dokázáno, že některé z nich mohou mít vztah prekurzor-produkt (Kibenge, F.S.B., a kol., J. Gen. Virol., 69, 1757-1775 (1988)). Označení a molekulové hmotnosti virových proteinů (VP) jsou uvedeny níže.
| Virový protein | Molekulová hmotnost |
| VP1 | 90 kDa |
| VP2 | 41 kDa |
| VP3 | 32 kDa |
| VP4 | 28 kDa |
| VP5 | 17 kDa |
V genomu IBDV byly identifikovány dva segmenty dvouvláknové RNA. Genom IBDV je tvořen dvěma segmenty dvouvláknové RNA, které jsou dlouhé od 2827 (segment B) do 3261 (segment A) párů nukleotidových bází (Mundt, E. a kol., Virology, 209, 10-18 (1995)). Větší segment A kóduje polyprotein, který je štěpen autoproteolýzou na maturovanou formu virových proteinů VP2, VP3 a VP4 (Hudson, P.J. a kol., Nucleic Acids Res., 14, 5001-5012 (1986). VP2 a VP3 jsou hlavní strukturní virové proteiny. VP2 je hlavní hostitelský ochranný imunogen IBDV a obsahuje antigenní oblast zodpovědnou za indukci neutralizačních protilátek (Azad, a kol., Virology, 161, 145-152 (1987)). Druhý otevřený čtecí rámec (ORF), předcházející a částečně se pře-1 CZ 299417 B6 krývající s polyproteinovým genem, kóduje protein (VP5) neznámé funkce, který je přítomný v infikovaných buňkách IBDV (Mundt, E., a kol., J. Gen. Virol, 76, 437-443, (1995)). Menší fragment B kóduje VP1, 90-kDa polyfunkční protein s polymerázovou a čepičkovou enzymovou aktivitou (Spies, U., a kol., Vir Res., 8, 127-140 (1987); Spies, U., a kol., J. Gen. Virol., 71, 9775 981 (1990)).
Bylo demonstrováno, že VP2 protein je důležitý hostitelský ochranný imunogen IBDV a že obsahuje antigenní oblast zodpovědnou za indukci neutralizačních protilátek. Oblast obsahující neutralizační místo je značně závislá na konformaci. VP3 protein byl považován za skupinově ío specifický antigen, protože je rozpoznáván monoklonálními protilátkami působícími proti VP3 z kmenů viru obou serotypů I a II. VP4 protein se jeví jako virově kódovaná proteáza, která se účastní zpracování polyproteinového prekurzoru proteinů VP2, VP3 a VP4.
Ačkoliv byly publikovány nukleotidové sekvence pro genomové segmenty A a B různých kmenů
IBDV, až v poslední době byly kompletně určeny oba 5'- a 3'-nekódující úseky obou segmentů. 5'-nekódující oblast IBDV segmentů A a B obsahuje shodné úseky 32 nukleotidů, zatímco 3'nekódující koncové úseky obou segmentů jsou si nepodobné, ale jsou zachované mezi kmeny viru IBDV stejného serotypů (Mundt, E. a kol, Virology, 209, 10-18 (1995)). Tyto konce by mohly obsahovat úseky důležité při sbalování a při regulaci genové exprese viru IBDV, jak je to prokázáno u jiných dsRNA virů, jako jsou reoviry savců a rostlin a rotaviry (Anzola, a kol., Proč. Nati. Acad. Sci. USA, 84, 8301-8305 (1987); Zou, S., a kol., Virology, 186, 377-388 (1992); Gorzigiia, M.I, a kol., Proč. Nati. Acad. Sci. USA, 89, 5784-5788 (1992)).
V posledních letech bylo vyrobeno množství infekčních zvířecích RNA virů zklonované cDNA použitím transkriptů produkovaných DNA-dependentní RNA polymerázou (Boyer, J.C., a kol, Virology, 198, 415-426 (1994)). Například polioviry, +řetězcový RNA virus; chřipkový virus, segmentovaný -řetězcový RNA virus; virus vztekliny, nesegmentovaný -řetězcový RNA virus; byly všechny získány zklonované cDNA jejich genomu (van der Werf, S., a kol.. Proč. Nati. Acad. Sci. USA, 83, 2330-2334 (1986); Enami, M., a kol., proč. Nati. Acad. Sci. USA, 87,
3 802-3 805 (1990); Schnell, M.J., a kol., EMBO J., 13, 4195—4205 (1994)). U reovirů bylo prokázáno, že transfekce buněk kombinací ssRNA, dsRNA a in vitro přeloženého reovirů produkuje vytvořený infekční reovirus, pokud je systém doplněn pomocným virem z rozdílného serotypů (Roner, M.R., a kol., Virology, 179, 845-852 (1990)). Avšak doposud není žádná zpráva o získání infekčního viru tvořeného segmentovanou dsRNA pouze ze syntetické RNA.
Podstata vynálezu
Způsob přípravy živého Bimaviru, kterým je virus infekční choroby bursy (IBDV), podle vynále40 zu zahrnuje následující kroky:
přípravu jedné nebo více cDNA genomových segmentů A a B viru infekční choroby bursy, transkripci této jedné nebo těchto více cDNA za vzniku syntetických RNA transkriptů, kde tyto RNA transkripty jsou pozitivními RNA transkripty uvedených segmentů A a B, transfekci hostitelských buněk těmito syntetickými RNA transkripty, inkubaci těchto hostitelských buněk v kultivačním médiu a izolaci živého infekčního viru infekční choroby bursy z uvedeného kultivačního média.
Tento vynález se tedy týká viru infekční choroby bursy, který způsobuje Gumboro nemoc mladých kuřat. Konkrétně se tento vynález vztahuje k metodě pro konstrukci viru infekční choroby bursy (IBDV) použitím syntetických transkriptů odvozených z klonované cDNA. Předkládaný vynález usnadní studium regulace exprese genu virů, patogeneze a design nových typů živých a inaktivovaných vakcín.
-2CZ 299417 B6
Za účelem vyvinutí reverzního genetického systému pro 1BDV byly zkonstruovány tři nezávislé klony úplné cDNA, které obsahují segment A serotypů I kmene D78 nebo serotypů II kmene 23/82 a segment B serotypů I kmene P2. Syntetická RNA segmentů A a B byla produkována in vitro transkripcí z linearizovaných plasmidů T7 RNA polymerázou. Transkripty těchto segmentů, buď neošetřené nebo ošetřené DNasou nebo RNasou, byly hodnoceny z hlediska výstavby infekčních virů transfekci buněk Věro.
Předloženým vynálezem bylo ukázáno, že umělé transkripty odvozené z klonované DNA odpovídající úplnému genomu segmentované dsRNA živočišného viru mohou vést k vytvoření repli10 kace-schopného viru. Obnovení infekčního viru po transfekci buněk syntetickou +RNA odvozenou z klonované cDNA viru s dsRNA genomem (IBDV) uzavírá hledání reverzního infekčního systému pro RNA viry. Rada vynálezů vystavěla infekční živočišné RNA viry z Honované cDNA (Boyer, J.C., a kol., Virology, 198, 415-426 (1994)). Van der Werf a kol. byli první, kteří vytvořili poliovirus, +řetězcový RNA virus, použitím syntetické RNA vyprodukované T7 RNA polymerázou z nakloňovaného templátu cDNA (van der Werf, S., a kol., Proč. Nati. Acad. Sci. USA, 83, 2330-2334 (1986)). Později Enami a kol. izolovali chřipkový virus, segmentovaný řetězcový RNA virus (Enami, M., a kol.. Proč. Nati. Acad. Sci. USA, 87, 3802-3805 (1990)); a Chnell a kol. vytvořili virus vztekliny, nesegmentovaný -řetězcový RNA virus, z klonovaných cDNA jejich genomu (Schnell, M.J., a kol., EMBO J., 13, 4195-4205 (1994)). Roner a kol. vyvi20 nuli infekční metodu pro segmentovaný dsRNA reovirus spočívající v transfekci buněk kombinací syntetických ssRNA, dsRNA, in vitro transkribovaných produktů reovirů a doplnění pomocným virem rozdílného serotypů (Roner, M.R., a kol., Virology, 179, 845-852 (1990)). Výsledný virus byl rozlišen od pomocného viru plakovým testem. Avšak v tomto systému bylo užití pomocného viru nutné. Na rozdíl od toho nyní popsaný reverzní genetický systém pro IBDV nevyžaduje pomocný virus nebo virové proteiny. Transfekce buněk +RNA obou segmentů úspěšně vytvořila infekční vir (IBDV). Původ přidaných jednoho nebo čtyř nukleotidů na 3'konci části A nebyl zjištěn. Avšak toto nezabránilo replikaci virové dsRNA. Podobné efekty byly pozorovány u +řetězcových RNA virů v různých studiích (Boyer, J.C., a kol., Virology, 198, 415 426 (1994)).
Transfekce +RNA obou segmentů do téže buňky byla nezbytná pro úspěšnou produkci IBDV. Transfekované RNA obou segmentů musely být přeloženy buněčným translačním aparátem. Polyprotein segmentu A byl pravděpodobně transformován do proteinů VP2, VP3 a VP4, které tvoří virový kapsid. Translatovaný protein VP1 segmentu B pravděpodobně fungoval jako RNA dependentní RNA polymeráza a vytvořil -řetězce podle syntetických +řetězců obou segmentů a následně vznikla výsledná dsRNA. Dobos nedávno oznámil, že in vitro transkripce virionovou RNA-dependentní RNA polymerázou infekčního viru nekrózy pankreatu (IPNV), modelový virus čeledi Birnaviridae, začíná z VP1 a potom přes nesouměrný semikonzervativní vytěsňovací mechanizmus vede k syntéze pouze +řetězců během replikace virového genomu (Dobos, P.,
Virology, 208, 10—25 (1995)). Současný způsob ukazuje, že se syntéza —řetězců provádí na +řetězcích. Jestli výsledná transkribovaná -řetězcová RNA slouží jako templát pro transkripci +řetězce, zůstává předmětem dalšího výzkumu.
K potvrzení, že infekční IBDV obsažený v supematantech z transfeko váných buněk byl opravdu odvozen ze syntetického transkriptů, byla vytvořena umělá chiméra obsahující segment A kmene serotypů II a segment B kmene serotypů I. Sekvenační analýza ověřila tuto genomovou kombinaci. Výsledky také naznačují, že terminální sekvenční motivy popsané Mundtem a Mullerem jsou pravděpodobně odpovědné za replikaci, třídění a sbalování virového genomu (Mundt, E. a kol., Virology, 209, 10-18 (1995). Přítomnost serotypově specifických terminálních sekvencí obvykle nezabrání celkové replikaci segmentu A serotypů II působením RNA-dependentní RNA polymerázy VP1 segmentu B ze serotypů I. Schopnost vytvořit rekombinantní viry významně pomůže při analýze konkrétních přesných funkcí serotypově specifických a serotypově nespecifických terminálních sekvencí.
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Obnovení infekčního IBDV ukazuje, že pouze +řetězcové RNA obou segmentů jsou schopné iniciovat replikaci dsRNA. Takže výsledky jsou ve shodě se základními rysy replikací reovirů a rotavirů., kde +řetězcové RNA slouží jako templát pro syntézu progenních -řetězců za vzniku dsRNA (Schonberg, M., a kol., Proč. Nati. Acad. Sci. Patton, J.T., Vir Res., 6, 217-233 (1986);
Chen, D, a kol., J. Virol., 68, 7030-7039 (1994)). Nicméně semikonzervativní mechanismus vytěsňování řetězců navržený Spiesem a kol. a Dobosem nemůže být vyloučen (Spies, U., a kol., Vir Res., 8, 127-140 (1987); Dobos, P., Virology, 208, 10-25 (1995)). Vývoj reverzního genetického systému pro IBDV hodně usnadní budoucí studium exprese genů, patogeneze a pomůže v navrhování nových generací živých a inaktivovaných IBDV vakcín.
Jak je použito v současné aplikaci, termín „syntetické“ v označení nukleových kyselin vyjadřuje, že nukleová kyselina je vytvořena člověkem, na rozdíl od nukleové kyseliny přírodního původu. Termín nepředpokládá omezení ve způsobu přípravy, který může být chemický či biologický, pokud způsob zhotovení zahrnuje zásah člověka.
Termín „cDNA“ zahrnuje jakékoliv cDNA obsahující segmenty A a B a 5' a 3' nekódované oblasti segmentů A a B.
Termín „infekční“, jak je použito u virů, označuje, že virus má schopnost reprodukce. Virus může být patogenní nebo nepatogenní a stále infekční.
Předložený vynález popisuje způsob pro tvoření viru infekčního onemocnění bursy použitím syntetické transkripce RNA. Tento způsob může být použit ke studiu regulace exprese virového genu, patogeneze a pro navržení nové generace živých nebo inaktivovaných IBDV vakcín.
Předložený vynález zahrnuje rekombinantní vektor obsahující alespoň jednu kopii cDNA ve shodě s předloženým vynálezem. Rekombinantní vektor může také nést další nezbytné sekvence jako sekvenci pro kontrolu exprese, značky, amplifikační geny, signální sekvence, promotory apod., jak je obecně známo. Použitelnými vektory pro tento účel jsou plasmidy a viry, jako bakuloviry, herpes viry (HVT) a viry neštovic, například virus drůbežích neštovic apod.
Také je zde uvedena hostitelská buňka transformovaná rekombinantním vektorem předloženého vynálezu nebo hostitelská buňka transfekovaná syntetickou RNA předloženého vynálezu. Hostitelská buňka může být eukaryotická nebo prokaryotická. Vhodné příklady jsou E. coli, hmyzí buněčná linie Sf-9, kuřecí embryonální fibroblastová buňka, kuřecí embryonální ledvinová buňka, Věro buňka kočkodana apod.
Částí tohoto vynálezu je také IBDV drůbeží vakcína obsahující množství rekombinantně produkovaných virů nebo částí virů, které navozuje ochranu drůbeže, kde je virus inaktivován nebo modifikován tak, že není virulentní.
Virus může být inaktivován chemicky nebo fyzikálně. Chemická inaktivace viru může být provedena například enzymy, formaldehydem, β-propiolaktonem, ethyleniminem nebo deriváty těchto látek či pomocí organického rozpouštědla (např. halogenovaného uhlovodíku) a nebo detergentu. Pokud je to nutné, inaktivující látka může být po inaktivaci viru zneutralizována. Fyzikální inaktivace může být provedena působením záření na virus, jako je UV záření, rentgenové záření nebo γ záření.
Virus může být oslaben známými metodami zahrnujícími sériové pasážování, deleci sekvencí nukleových kyselin a místně specifickou mutagenezu buď před nebo po produkci infekčního viru, aby byl vytvořen virus, který si zachovává dostatečnou antigenicitu, ale který má redukovanou virulenci.
Fyziologicky akceptovatelné nosiče pro vakcinaci drůbeže jsou obecně známé a není třeba je zde dále popisovat. Kromě toho, že nosič musí být fyziologicky akceptovatelný drůbeží, nesmí inter-4CZ 299417 B6 ferovat s imunologickou odpovědí vyvolanou vakcínou a/nebo s expresí jejího polypeptidového produktu.
Ve vakcíně jsou přítomna i další aditiva, jako pomocné látky a stabilizátory, a to v obvyklém množství. Nej lepší pomocné látky jsou hydroxid hlinitý, fosforečnan hlinitý, rostlinné a živočišné oleje apod., které jsou podávány s vakcínou v množství schopném podpořit imunitní odpověď proti IBDV. Množství pomocných látek přidávaných k vakcíně se bude měnit v závislosti na povaze pomocné látky, nejčastěji v rozsahu od 0,1 do 100 násobku hmotnosti IBDV, spíše však v rozmezí od 1 do 10 násobku hmotnosti IBDV.
Vakcína podle tohoto vynálezu může také obsahovat různé stabilizátory. Je možné použít jakýkoliv vhodný stabilizátor, který obsahuje sacharidy např. sorbitol, mannitol, škrob, sacharózu, dextrin nebo glukózu, rovněž může být použito proteinů např. albumin nebo kasein. Jako vhodný pufr se jeví alkalický fosforečnan apod. Stabilizátor je obzvláště výhodný, pokud je suchá vakcí15 na připravena lyofilizací.
Vakcína může být podána jakýmkoliv známým způsobem očkování drůbeže např. nazálně, oftalmicky, injekčně, pitnou vodou, potravou, expozicí apod. Výhodné je podat vakcínu hromadně, umístěním vakcíny do pitné vody nebo rozprášením do prostředí zvířat. Pokud je podávána injekcí, je upřednostňováno parenterální podávání. Parenterální podávání tak, jak je zde užito, znamená podání intravenosní, intramuskulámí nebo intraperitoneální injekcí.
Vakcína podle tohoto vynálezu je podána drůbeži k prevenci IBVD kdykoliv před nebo po vylíhnutí. Je obzvláště vhodné pokud je vakcína podána před narozením a následovně ještě v 6 týd25 nech. Drůbež je definována tak, že zahrnuje, avšak není tím nijak limitována, kuřata, kohouty, slepice, brojlery, pulardy, kvočny, nosnice, krocany a kachny.
Vakcína může být uchována ve sterilní nádobě ve formě jednotky nebo jiného množství. Výhodné je uchovávat ji při teplotě pod -20 °C nebo ještě lépe pod -70 °C. Vakcína je rozmražena před použitím a může být ihned potom opět zmražena. Pro podávání rekombinantně vyrobeného viru drůbeži může být suspendována v nosiči v množství od 104 do 107 pfu/ml, výhodněji od 105 do 106 pfu/ml nosiče, kterým může být např. fyziologický roztok. Inaktivovaná vakcína může obsahovat antigeny ekvivalentní 104 až 107 pfu/ml rozpuštěné v nosiči. Jsou použity i jiné nosiče, které jsou obecně známé. Příklady farmaceuticky akceptovatelných nosičů jsou rozpouštědla a inertní farmaceutické nosiče, které jsou obvykle užívány. Je výhodné, aby byly nosič nebo rozpouštědlo využitelné pro skupinové podání. Avšak nosič nebo rozpouštědlo by měly být také slučitelné s jinými způsoby podávání jako injekcí, očními kapkami, nosními kapkami, apod.
Vynález může být také použit k produkci kombinovaných vakcín s IBDV. IBDV může být kom40 binován s antigenním materiálem viru Newcastle choroby, bronchiálního viru, Reo viru, Adeno viru a/nebo Markova viru.
Předchozí provedení tohoto vynálezu jsou dále popsaná v následujících příkladech. Avšak tento vynález není omezen příklady a varianty budou odborníkům zřejmé bez odchýlení se od rozsahu současného vynálezu.
Podrobný popis obrázků na výkresech
Obrázek 1 je schématický diagram cDNA použitých pro syntézu +ssRNA IBDV T7 RNA polymerázou. Konstrukt pUC19FLAD78 obsahuje cDNA segmentu A IBDV kmene D78 a rekombinantní plasmid pUC18FFA23 obsahuje úplnou cDNA segmentu A IBDV kmene 23/82. Segment A IBDV kóduje polyprotein (VP2-VP4-VP3) a nedávno identifikovaný VP5 protein. Plasmid pUC18FFBP2 obsahuje cDNA segmentu B kmene P2, který kóduje RNA-dependentní RNA polymerázu (VP1). Specifické sekvence viru jsou podtržené a T7 promotorova sekvence je psána
-5CZ 299417 B6 kurzívou. Restrikční místa jsou vytištěna tučně a označena. Rozštěpená místa linearizovaných plasmidů jsou označena vertikálními šipkami a směr transkripce je označen horizontální šipkou.
Obrázek 2 ukazuje agarový gel s analýzou produktů transkripce, které byly použity pro transfekci buněk Věro. Syntetické RNA transkribované in vitro použitím T7 RNA polymerázy a linearizovaného plasmidů pUC19FLAD78 (pruhy 2, 4 a 6) obsahujícího cDNA segmentu A IBDV kmene D78 a pUC18FLBP2 (pruhy 1, 3 a 5) obsahujícího cDNA segmentu B kmene P2. Po skončení transkripce byly reakění směsi ošetřeny DNasou (pruhy 1 a 2), RNasou (pruhy 3 a 4) nebo neošetřeny (pruhy 5 a 6). Dva pí reakčních produktů byly analyzovány na 1 % agarovém gelu.
ío Lambda DNA, štěpená Hind ΠΙ/EcoR I, byla použita jako markér (pruh M).
Obrázek 3 zobrazuje srovnání nukleotidové sekvence klonovaných RT-PCR fragmentů ze segmentů A a B IBDV kmene 23A/P2B (napsáno tučně) se známou sekvencí segmentů A a B serotypů II kmene 23/82 a serotypů I kmene P. Nukleotidová identita je označena dvojtečkou.
Obrázek 4 ukazuje DNA sekvenci pUC18FLA23.
Obrázek 5 ukazuje DNA sekvenci pUC19FLAD78.
Obrázek 6 ukazuje DNA sekvenci pUC18FLBP2.
Příklady provedení vynálezu
Příklad 1: Viry a buňky
Dva kmeny serotypů I IBDV, oslabený P2 kmen z Německa a vakcína kmene D78 (Intervet International), a jeden kmen serotypů II, nepatogenní 23/82 kmen, byly namnoženy v embryonál30 nich buňkách kuřat (CEC) a vyčištěny (Mundt, E. a kol., Virology, 209, 10-18 (1995); Vakharia, V.N., a kol., Vir Res., 31, 265-273 (1994)). Buňky Věro byly pěstovány v médiu Ml99 doplněném 5 % fetálního telecího séra (FCS) a použity pro transfekění experimenty. Další propagace získaného viru a imunofluorescenční studie byly prováděny ve Věro buňkách (Mundt, E., a kol., J. Gen. Virol., 76, 437^143, (1995)). Pro plakový test byly připraveny a použity monovrstvy sekundárních CEC (Muller, H, a kol.. Vir Res., 4, 297-309 (1986)).
Příklad 2: Konstrukce kompletních cDNA klonů IBDV genomu
Nezávisle byly připraveny kompletní cDNA klony segmentů A a B viru IBDV. cDNA klony obsahující úplný úsek kódující RNA segmentu A kmene D78 byly připraveny použitím standardních klonovacích postupů a metod (Vakharia, V.N., a kol., Vir Res., 31, 265-273 (1994)). Při srovnání terminálních sekvencí kmene D78 s nedávno publikovanými terminálními sekvencemi dalších IBDV kmenů (Mundt, E. a kol., Virology, 209, 10—18 (1995)) bylo zjištěno, že D78 cDNA klony postrádají prvních 17 a posledních 10 nukleotidů na 5'-, resp. 3'-koncích. Proto bylo pro konstrukci úplného cDNA klonu segmentu A použito dvou základních párů primem (A5-D78, A5-IPD78 a A3-IPD78), které byly syntetizovány a použity pro PCR amplifikaci (Tabulka 1). DNA segmenty byly amplifikovány podle protokolu dodavatele (New England Biolabs) použitím „Deep Vent Polymerace“ (vysoce přesná termofilní DNA polymeráza). Ampli50 fikované fragmenty byly klonovány do EcoRI místa vektoru pCRII (Invitrogen Corp.), čímž byly získány plasmidy pCRD78A5' a pCRD78A3'. Všechny plasmidy byly rozštěpeny EcoR I a Sal I a výsledné fragmenty byly ligovány do pUCl9 obsahujícího EcoR I, aby byl získán plasmid pLICl 9FLAD78 (SEQ ID NOS:27 a 29), který nyní obsahuje úplnou cDNA kopii segmentu A kódujícího všechny strukturní proteiny (VP2, VP4 a VP3, SEQ ID N0:30), stejně jako nestruk55 tumí VP5 protein (SEQ ID N0:28) (Obr. 1).
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Dva základní páry primem (A5-23, A5IP23 a A3'-23, A3-IP23, tabulka 1) byly použity pro reverzní transkripci (RT) virové genomové dsRNA kmene 23/82 použitím „SuperScript RT II“ (RNA dependentní DNA polymeráza se sníženou RNasovou H aktivitou, GIBCO/BRL). RT reakční produkty byly čištěny extrakcí fenolem/chloroformem a vysráženy ethanolem. K získání dvou cDNA fragmentů ohraničených základními páry primem A5-23, A5-IP23 a A3-23, A31P23 byly reakční produkty RT amplifikovány pomocí PCR použitím „Deep Vent polymerázy“. RT a PCR byly provedeny podle dodavatelského protokolu. Výsledné PCR fragmenty byly ligovány s tupými konci Srna I rozštěpeného pUC18 vektoru, aby byly získány pUC23A' a ío pUC23A3'. 3'-konec segmentu A obsažený v plasmidů pUC23A3' byl ligován do Hind IIl-BstB I rozštěpeného plasmidů pUC23A5', aby byla vytvořena úplná cDNA segmentu A kmene 23/82. Výsledný plasmid byl označen pUC18FLA23 (SEQ ID NOS:31 a 33) (Obr. 1) a kóduje strukturní proteiny VP2, VP3 a VP4 (SEQ ID N0:32) a nestruktumí protein VP5 (SEQ ID N0:34).
K přípravě cDNA klonů segmentu Β P2 kmene byly podle publikovaných sekvencí vytvořeny dva základní páry primerů (B5-P2, B5-IPP2 a B3-P2, B3-IPP2) a použity pro RT-PCR amplifikaci (Tab. 1). Použitím genomu dsRNA jako templátu byly syntetizovány cDNA fragmenty a amplifikovány podle protokolu výrobce (Perkin-Elmer Cetus). Amplifikované fragmenty byly zatupeny a vligovány do Srna I rozštěpeného pBS vektoru (Stratagen), aby byly získány klony pBSP2B5' a pBSP2B3'. Ke konstrukci úplného klonu segmentu B byl nakloňován 5'-koncový fragment z plasmidů pBSP2B3' mezi EcoR I a Pst I místa vektoru pUC18, čímž byl získán pUCP2B5'. Potom byl 3 '-konec fragmentu z plasmidů pUCP2B3' vligován mezi unikátní Bgl 11 a Pst I místa plasmidů pUCP2B5' a tak byl získán úplný plasmid pUC18FLBP2 (SEQ ID N0:25), který kóduje VP1 protein (SEQ ID N0:26) (Obr. 1). Ke kompletní sekvenaci plasmidů pUC18FLBP2, pUC18FLA23 a pUC19FLAD78 byl použit DNA sekvenační systém „SEQUENASE“ (U. S. Biochem.) a sekvenační data byla analyzována použitím software „DNAS1S“ (Pharmacia) nebo „PC/Gene“ (Intelligenetics). Celistvost úplných konstruktů byla testována in vitro transkripcí a translací ve spojeném systému lysátu retikulocytů s T7 RNA polymerázou (Promega).
Příklad 3; Transkripce a transfekce syntetických RNA
Plasmidy pUC19FLAD78, pUC18FLA23 a pUC18FLBP2 byly štěpeny enzymy BsrG I, Nsi I, respektive Pst I (Obr. 1) a použity jako templáty pro transkripci in vitro T7 RNA polymerasou (Promega). Stručně, k restrikčním směsím bylo přidáno 0,5 % SDS (výsledná koncentrace) a plasmidy byly inkubovány s proteasou K (0,5 mg/ml) jednu hodinu při 37 °C. Linearizované DNA templáty (3 pg) byly získány srážením etanolem a po oddělení byly přidány k transkripční reakční směsi (50 pl) obsahující 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl2, 2 mM spermidin, 0,5 mM ATP, CTP a UTP od každého, 0,1 mM GTP, 0,25 mM analog čepičky [m7G(5') PPP(5') G], 120 jednotek „RNasinu“ (ribonukleásový inhibitor), 150 jednotek T7 RNA polymerasy (Promega) a byly inkubovány při 37 °C jednu hodinu. Syntetické RNA transkripty byly vyčištěny extrakcí fenolem/chloroformem a srážením etanolem. Jako kontroly byly ještě před čištěním produkty transkripce ošetřeny DNasou nebo RNasou (Promega).
Věro buňky byly pěstovány do nárůstu 80 % v 60 mm misce a jednou promyty fosforečnanem pufrovaným fyziologickým roztokem (PBS). Tři ml „OPTI-MEM I“ (médium se sníženým obsahem séra obsahující HEPES pufr, hydrogenuhličitan sodný, hypoxantin, thymin, pyruvát sodný, L-glutamin, stopové prvky, růstové faktory a fenolovou červeň; od firmy GIBCO/BRL) byly přidány k monovrstvě a buňky byly inkubovány při 37 °C jednu hodinu v CO2 inkubátoru. Současně bylo 0,15 ml „OPTI-MEM I“ inkubováno 45 minut v polystyrénové zkumavce při pokojové teplotě s 1,25 pg činidla „Lipofectin“(V-[l-(2,3-dioleyloxy)propyl]-Ar,/V,/V-trimethylammonium-chlorid a dioleoylfosfatidylethanolamin, GIBCO/BRL). Syntetické RNA transkripty obou segmentů byly opět suspendovány v 0,15 ml vody obsahující diethylpyrokarbonát a přidány ke směsi OPTI-MEM-Lipofectin. Tato směs byla pomalu míchána a inkubována 5 minut v ledu. Po
-7 CZ 299417 B6 odstranění „OPTI-MEM“ z monovrstvy v 60 mm miskách a nahrazení čerstvými 1,5 ml „OPTIMEM“ byla směs obsahující nukleové kyseliny přidána po kapkách k Věro buňkám a jemně promíchána. Po dvou hodinách inkubace při 37 °C byla směs nahrazena médiem M 199 [CaCfi (bezvodý), Fe(No3)3. 9H2O, KCI, MgSO4 (bezvodý). NaCl, NaH2PO4H2O, NaHCCfi, L-Alanin,
L-Arginin HCI, L-aspartámová kyselina, L-Cystein HCI H2O, L-Cystein 2HC1, L-Glutamová kyselina, L-Glutamin, Glycin, L—Histidin HCI H2O, L-Hydroxyprolin, L-Isoleucin, L-Leucin, L-Lysin HCI, L-Methionin, L-Fenylalanin, L-Prolin, L-Serin, L-Threonin, L-Tryptofan, LTyrosin 2Na 2H2O, L-Valin, Alfa tokoferol PO4 Na2, kyselina askorbová, Biotin, Kalciferol, DKalcium pantotenát, Cholin chlorid, Kyselina listová, I-Inositol, Menandion NaHSO3.3H2O, ío Niacin, Nikotin amid, Paraaminobenzoová kyselina, Pyridoxin HCI, Riboflavin, Thiamin HCI, Acetát Vitamínu A, síran Adeninu, kyselina adenylová, ATP, Na2, Cholesterol, 2-deoxy-D-ribosa, D-glukosa, Glutathion, Guanin HCI, Hypoxantin sodný. Fenolová červeň sodná, ribosa, bezvodý octan sodný, thymin, tween 80, Uráčil a xantin sodný; od Mediatech, lne.], které obsahovalo 5 % FCS (bez promytí buněk) a buňky byly dále inkubovány při 37 °C v žádoucích časo15 vých intervalech.
Identifikace vyrobeného IBDV
CEC byly nakaženy přefiltrovaným supematantem (0,2 pm) z Věro buněk tranfekovaných trans20 kripty pUC18FLA23 a pUC18FLP2B. 16 hodin po infekci byly izolovány neporušené buněčné nukleové kyseliny (Mundt, E. a kol., Virology, 209, 10-18 (1995)). Primery byly navrženy podle publikovaných sekvencí a RT-PCR fragmenty byly amplifikovány, nakloňovány a sekvenovány (Mundt, E. a kol., Virology, 209, 10-18 (1995)). Sekvenační data byla analyzována použitím software „DNASIS“.
Imunofluorescence
Věro buňky vypěstované na krycím sklíčku do 80 % byly infikovány supematanty získanými z transfekovaných Věro buněk (po zamražení-rozmražení) a inkubovány při 37 °C dva dny. Buňky byly potom promyty, fixovány acetonem a inkubovány s polyklonálním králičím anti-lBDV sérem. Po promytí byly buňky inkubovány s fluoresceinem značenou kozí proti—králičí protilátkou (Kirkegaard &Perry Lab.) a zkoumány fluorescenčním mikroskopem.
Plakový test
Monovrstvy sekundárních CEC, pěstované na 60 mm misce, byly naočkovány supematanty z transfekovaných Věro buněk. Po jedné hodině infekce byly buňky jednou promyty PBS a převrstveny 0,8% čistým Agarem (Difco) obsahujícím 10% tryptosového fosfátového živného roztoku, 2% FCS, 0,112% NaHCCfi, 103 jednotek penicilinu, 103 pg/ml streptomycinu,
0,25 pg/ml fungizonu, 0,005 % neutrální červeně, 0,0015 % fenolové červeně. Buňky byly inkubovány při 37 °C 2 až 3 dny, dokud se nedaly plaky pozorovat a počítat (Muller, H., a kol., Vir Res., 4, 297-309(1986)).
Konstrukce úplných cDNA klonů IBDV genomu
K vyvinutí reverzního genetického systému pro dsRNA virus IBDV byly zkonstruovány dva nezávislé cDNA klony, které obsahovaly segment A kmene D78 a segment B kmene P2 (Obr. 1) Každý plasmid kódoval buď prekurzor strukturních proteinů (VP2, VP4, VP3) a VP5 nebo jenom VP1 protein (RNA-dependentní RNA polymeráza). Plasmid pUC18FLBP2 po štěpení Pst I a transkripci in vitro pomocí T7 RNA polymerázy poskytoval RNA obsahující správné 5'- a 3konce. Plasmid pUC19FLAD78 po štěpení B srG I a trankripci poskytoval RNA obsahující správný 5'-konec, ale s připojenými čtyřmi nukleotidy na 3'-konci. Spojená transkripce a translace zmíněných plasmidů v králičím systému retikulocytů poskytly proteinové produkty, které byly přesně zprocesovány a komigrovaly s markerovými IBDV proteiny po rozdělení v SDS55 polyakrylamidovém gelu a autoradiografii (data nejsou ukázána).
-8CZ 299417 B6
Transkripce, transfekce a produkce infekčního viru.
+transkripty IBDV segmentů A a B byly syntetizovány separátně in vitro T7 RNA polymerázou použitím linearizovaných plasmidů obsahujících úplné cDNA jako templátů (Obr. 2). Ačkoliv v neutrálním gelu byly pozorovány dva druhy RNA transkriptů pro segmenty B (pruhy 1 a 5), dělení vzorků v denaturačním gelu poskytlo jen jeden transkripčně-specifický pruh (data nejsou poskytnuta). Za účelem ukázat, že +RNA transkripty obou segmentů jsou potřebné pro produkci infekčního viru, byly transkripční směsi inkubovány s různými nukleázami, jak je ukázáno na ío obr. 2. Syntetické RNA po ošetření produktů transkripce DNasou (pruhy 1+2), RNasou (pruhy 3+4) nebo bez ošetření (pruhy 5+6) byly použity pro transfekci Věro buněk. Jako kontrola byl použit pouze Lipofectin. Pět dní po transfekci byl jen v buňkách Věro transfekovaných kombinací neošetřených nebo Dnasou ošetřených produktů transkripce viditelný cytopatický efekt (CPE), ale ne u RNasou ošetřené transkripční směsi nebo zdánlivě-transfekované kontroly. Navíc nebyl detekován žádný CPE, když byly Věro buňky transfekovány RNA pouze segmentu A nebo B (data nejsou poskytnuta). Tyto výsledky demonstrují replikaci IBDV následující po transfekci Věro buněk +ssRNA obou segmentů IBDV. K ověření, že činitel způsobující CPE ve Věro buňkách byl opravdu IBDV, byly transfekované Věro buňky zmraženy-rozmraženy a supematant byl vyčištěn odstředěním a použit pro infekci CEC nebo Věro buněk. CEC infikované super20 natantem získaným z Věro transfekovaných buněk neošetřených nebo Dnasou ošetřených transkripčních směsí vykazovaly CPE v prvním dnu po naočkování (Tab. 2). Avšak žádné CPE nemohly být objeveny ani po pěti dnech v CEC infikovaných supematantem z VĚRO buněk transfekovaných RNasou ošetřených transkřípěních směsí, neošetřenou transkripční směsí segmentu A, respektive B a simulované transfekovaných Věro buněk. Podobně, když byly Věro buňky infikovány na krycích sklíčkách stejnými supematanty, jak je popsáno výše, a zkoušeny imunofluorescencí prováděnou po dvou dnech, pouze supematanty získané z transfekovaných Věro buněk neošetřených nebo DNasou ošetřených transkripčních směsí poskytly pozitivní imunofluorescenční signál (Tabulka 2).
Obnovení transfekovaného viru
K. určení časového bodu obnovení infekčního viru byly Věro buňky transfekovány rekombinantními RNA vzniklými transkripcemi segmentů A a B. Po 4, 8, 16, 24, 36 a 48 hodinách od transfekce byl supernatant podroben zkoušce, zda obsahuje transfekovaný virus pomocí infekčního a plakového testu, jak je ukázáno v tabulce 3. Naše výsledky ukazují, že vir může být obnoven již 36 hodin po transfekci. Virový titr byl 2,3xl02 pfu/ml, a klesal u vzorků získaných později než 48 hodin po transfekci.
Vytvoření chimérických virů
K. ověření, že +ssRNA obou segmentů IBDV jsou dostatečné pro obnovení infekčních virů, byl vytvořen chimérický IBDV vir. Plasmid pUC18FLA23 obsahující úplnou sekvenci segmentu A serotypů kmene II byl linearizován štěpením Nsi I a ssRNA byla syntetizována in vitro použitím T7 RNA polymerázy. SsRNA transkript vykazuje správný 5'-konce, ale obsahuje jeden přidaný zbytek na 3'-konci (Obr. 1). Věro buňky byly transfekovány ssRNA segmentu A serotypů II kmene 23/82 a ssRNA segmentu B serotypů I kmene P2. Pět dní po transfekci, když byl CPE zřejmý, byl supernatant vyčištěn (po zmražení-rozmražení) a použit pro infekci CEC. Po druhé pasáži v CEC byla genomová RNA viru analyzována RT-PCR a sekvenací PCR produktů. Bylo nutné, aby příměry pro segment A amplifikovaly pouze segment A získaný ze serotypů kmene II.
Primer segmentu B se váže na sekvence obou serotypů. Amplifikované fragmenty byly klonovány a sekvenovány. Získaná sekvence segmentu A vykazovala dokonalý pár se známou sekvencí segmentu A serotypů II kmene 23/82, zatímco sekvence segmentu B vykazovala kompletní homologii k publikované sekvenci segmentu B serotypů I kmene P2 (Obr. 3).
-9Tabulka 1.
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- 10CZ 299417 B6
Tabulka 2. Vytváření infekčního IBDV ze syntetických RNA segmentů A a B
Transfekovaný materiál CPE Imunofluorescence
| ssRNA A+B, DNasou ošetřeny | + | 4- |
| ssRNA A+B, RNasou ošetřeny | - | - |
| ssRNA A+B, neošetřeny | 4- | 4* |
| ssRNA A, neošetřeny | - | - |
| ssRNA B, neošetřeny | - | - |
| Pouze lipofektin | - | - |
Věro buňky byly transfekovány syntetickými RNA segmentů A a B získanými z transkripčních reakcí, které byly neošetřeny nebo ošetřeny DNasou nebo RNasou. Po pěti dnech byly supematanty spojeny, vyčištěny odstředěním a analyzovány, zda obsahují virus. Infekčnost obnoveného viru byla určena v CEC zjišťováním cytopatického efektu (CPE) 1 až 2 dny po naočkování. Βρει o cifita obnoveného viru byla určena imunofluorescenčním barvením infikovaných Věro buněk králičím anti-IBDV sérem.
Tabulka 3. Obnovení Viru v různých časech po transfekci.
Čas v hodinách po transfekci
CPE Imunofluorescence pfu/ml
| 4 | - | - | 0 |
| 8 | - | - | 0 |
| 16 | - | - | 0 |
| 24 | - | 0 | |
| 36 | + | + | 2,3 x 10* |
| 48 | + | + | 6,0 x 10’ |
Věro buňky byly transfekovány syntetickými RNA segmentů A a B jak bylo již popsáno. Infekčnost a specifita obnoveného viru byla zjištěna pomocí CPE v CEC a imunofluorescencí v buň20 kách Věro. Mono vrstvy sekundárních CEC byly použity pro plakový test po naočkování buněk supernatantem získaným z transfekovaných buněk Věro. Přibližný titr viru byl počítán jako počet jednotek vytvořených plaků na ml (pfu/ml).
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DÉLKA: 33 PÁRŮ BÁZÍ TYP: NUKLEOVÁ KYSELINA ŘETĚZEC: JEDNODUCHÝ TYPOLOGIE: LINEÁRNÍ
TYP MOLEKULY: DNA
POPIS SEKVENCE: SEQ ID NO:22:
CGATCTGCTG CAGGGGGCCC CCGCAGGCGA AGG
INFORMACE PRO SEQ ID NO:23:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 24 PÁRŮ BA'ŽÍ TYP: NUKLEOVÁ KYSELINA
ŘETĚZEC: JEDNODUCHÝ
TYPOLOGIE: LINEÁRNÍ TYP MOLEKULY: DNA POPIS SEKVENCE: SEQ IDNO:23:
CTTGAGACTC TTGTTCTCTA CTCC
INFORMACE PRO SEQ ID NO:24:
SEKVENČNÍ CHARAKTERISTIKY 30 DÉLKA: 19 PÁRŮ BÁZÍ
TYP: NUKLEOVÁ KYSELINA ŘETĚZEC: JEDNODUCHÝ TYPOLOGIE: LINEÁRNÍ TYP MOLEKULY: DNA
POPIS SEKVENCE: SEQ ID NO:24:
ATACAGCAA GATCTCGGG
INFORMACE PRO SEQ ID NO:25:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 2827 PÁRŮ BÁZÍ TYP: NUKLEOVÁ KYSELINA ŘETĚZEC: JEDNODUCHÝ
TYPOLOGIE: KRUHOVÁ
TYP MOLEKULY: cDNA ZNAKY: JMÉNO/KLÍČ: CDS UMÍSTĚNÍ: 112..2745
POPIS SEKVENCE: SEQ ID NO:25:
- 18CZ 299417 B6
GGATACGATG GGTCTOACCC TCTGGGAGTC ACGAATTAAC
GTGGCTACTA GGGGCGATAC
CCGCCGCTGG CCGCCACGTT AGTGGCTCCT CTTCTTGATG ATTCTGCCAC C ATG AGT 117 Met Ser
| GAC | ATT | TTC | AAC | AGT | CCA | CAG | GCG | CGA | AGC | ACG | ATC | TCA | GCA | GCG | TTC | 165 |
| Asp | Xle | Pho | Asn | Ser | Pro | Gin | Ala | Arg | Ser | Thr | Ile | Ser | Ala | Ala | Phe | |
| s | 10 | 15 |
| GGC ATA Gly Ile | AAG CCT | ACT Thr | GCT Ala | GGA CAA GAC GTG GAA GAA CTC | TTG ATC | CCT Pro | 213 | |||||||||
| i»ys | Pro | Gly Gin 25 | Asp | Val | Glu Olu 30 | Leu | Leu | Ile | ||||||||
| 20 | ||||||||||||||||
| AAA | GTT | TGG | GTG | CCA | CCT | GAG | GAT | CCG | CTT | GCC | AGC | CCT | AGT | CGA | CTG | 261 |
| bys | Val | Trp | Val | Pro | Pro | Glu | Aap | Pro | Leu | Ala | Ser | Pro | Ser | Arg | Leu | |
| 35 | 40 | 45 | 50 | |||||||||||||
| GCA | AAG | TTC | CTC | AGA | GAG | AAC | GGC | TAC | AAA | GTT | TTG | CAG | CCA | CGG | TCT | 309 |
| Ala | Lys | Phe | Leu | Arg | Glu | Asn | Gly | Tyr | Lys | Val | Leu | Gin | Pro | Arg | Ser | |
| 55 | 60 | 65 | ||||||||||||||
| CTG | CCC | GAG | AAT | GAG | GAG | TAT | GAG | ACC | GAC | CAA | ATA | CTC | CCA | GAC | TTA | 357 |
| Leu | Pro | Glu | Asn | GlU | GlU | Tyr | Glu | Thr | Asp | Gin | Ile | Leu | Pro | Asp | Leu | |
| 70 | 75 | 00 | ||||||||||||||
| GCA | TGG | ATG | CGA | CAG | ATA | GAA | GGG | GCT | GTT | TTA | AAA | CCC | ACT | CTA | TCT | 405 |
| Ala | Trp | Met | Arg | Gin | Ile | Glu | Gly | Ala | Val | Leu | Lye | Pro | Thr | Leu | Ser | |
| 85 | 90 | 95 | ||||||||||||||
| CTC | CCT | ATT | GGA | GAT | CAG | GAG | TAC | TTC | CCA | AAG | TAC | TAC | CCA | ACA | CAT | 453 |
| Leu | Pro | llo | Gly | Asp | Gin | Glu | Tyr | Phe | Pro | Lys | Tyr | Tyr | Pro | Thr | His | |
| 100 | 105 | 110 | ||||||||||||||
| CGC | CCT | AGC | AAG | GAG | AAG | CCC | AAT | GCG | TAC | CCG | CCA | GAC | ATC | GCA | CTA | 501 |
| Arg | Pro | Ser | Lya | Glu | Lys | Pro | Asn | Ala | Tyr | Pro | Pro | Asp | Xle | Ala | Leu | |
| 115 | 120 | 125 | 130 |
- 19CZ 299417 B6
| CTC | AAG | CAG | ATG | ATT | TAC | CTG | TTT | CTC | CAG | GTT | CCA | GAG | GCC | AAC | GAG | 549 |
| Leu | Lys | Gin | Met | lle 135 | Tyr | Leu | Phe | Leu | Gin 140 | Val | Pro | Olu | Ala | Asn 145 | Glu | |
| GGC | CTA | AAG | GKT | GAA | GTA | ACC | CTC | TTG | ACC | CAA | AAC | ATA | AGG | GAC | AAG | S97 |
| Oly | Leu | Lys | Asp 150 | Glu | Val | Thr | Leu | Leu 155 | Thr | Gin | Asn | Ile | Arg 160 | A|sp | Lys | |
| GCC | TAT | GGA | AGT | GGG | ACC | TAC | ATG | GGA | CAA | GCA | AAT | CGA | CTT | GTG | GCC | 645 |
| Ala | Tyr | Gly 165 | Ser | Gly | Thr | Tyr | Met 170 | Gly | Gin | Ala | Asn | Arg 175 | Leu | Val | Ala | |
| ATO | AAG | GAG | GTC | GCC | ACT | GGA | AGA | AAC | CCA | AAC | AAG | GAT | CCT | CTA | AAG | 693 |
| Met | Lys 180 | Glu | Val | Ala | Thr | Gly 185 | Arg | Asn | Pro | Asn | Lys 190 | Asp | Pro | Leu | Lys | |
| CTT | GGG | TAC | ACT | TTT | GAG | AGC | ATC | GCG | CAG | CTA | CTT | GAC | ATC | ACA | CTA | 741 |
| Leu 195 | Gly | Tyr | Thr | Phe | Glu 200 | Ser | Ile | Ala | Gin | Leu 205 | Leu | Asp | Ile | Thr | Leu 210 | |
| CCG | GTA | GGC | CCA | CCC | GGT | GAG | GAT | GAC | AAG | CCC | TGG | GTG | CCA | CTC | ACA | 789 |
| Pra | val | Gly | Pro | Pro 215 | Gly | Glu | Asp Asp | Lys 220 | Pro | Trp | Val | Pro | Leu 225 | Thr | ||
| AGA | GTG | CCG | TCA | CGG | ATG | TTG | GTG | CTG | ACG | GGA | GAC | GTA | GAT | GGC | GAC | 837 |
| Arg | Val | Pro | Ser 230 | Arg | Met | Leu | Val | Leu 235 | Thr | Gly | Asp | Val | Asp 240 | Gly | Asp | |
| TTT | GAG | GTT | GAA | GAT | TAC | CTT | CCC | AAA | ATC | AAC | CTC | AAG | TCA | TCA | AGT | 885 |
| Phe | Glu | Val 245 | Olu | Asp | Tyr | Leu | Pro 250 | Lye | Ile | Asn | Leu | Lys 255 | Ser | Ser | Ser | |
| GGA | CTA | CCA | TAT | GTA | GGT | CGC | ACC | AAA | GGA | GAG | ACA | ATT | GGC | GAG | ATG | 933 |
| Gly | Leu 260 | Pro | Tyr | Val | Gly | Arg 265 | Thr | Lys | Gly | GlU | Thr 270 | Ile | Gly | GlU | Mat | |
| ΛΤΑ | GCT | ATC | TCA | AAC | CAG | TTT | CTC | AGA | GAG | CTA | TCA | ACA | CTG | TTG | AAG | 981 |
| Ile 27S | Ala | Ilo | Ser | Asn | Gin 280 | Phe | Leu | Arg | Glu | Leu 205 | Ser | Thr | Leu | Leu | Lys 290 | |
| CAA | GGT | GCA | GGG | ACA | AAG | GGG | TCA | AAC | AAG | AAG | AAG | CTA | CTC | AGC | ATG | 1029 |
| Gin | Gly | Ala | Gly | Thr 295 | Lys | Gly | Ser | Asn | Lys 300 | Lys | Lys | Leu | Leu | Ser 305 | Met | |
| TTA | AGT | GAC | TAT | TGG | TAC | TTA | TCA | TGC | GGG | CTT | TTG | TTT | CCA | AAG | GCT | 1077 |
| Leu | Ser | Asp | Tyr 310 | Trp | Tyr | Leu | Ser | Cys 315 | Gly | Leu | Leu | Phe | Pro 320 | Lys | Ala | |
| GAA | AGG | TAC | GAC | AAA | AGT | ACA | TGG | CTC | ACC | AAG | ACC | CGG | AAC | ATA | TGG | 1125 |
| GlU | Arg | Tyr | Asp | Lys | Ser | Thr | Trp | Leu | Thr | Lys | Thr | Arg | Asn | Ile | Trp |
325 330 335
-20CZ 299417 B6
| TCA GCT CCA | TCC CCA ACA Ser Pro Thr | CAC His 345 | CTC Leu | ATG ATC TCT ATG ATC ACC TGG | CCC Pro | 1173 | ||||||||||
| Ser Ala 340 | Pro | Met | Ile Ser Met 350 | Ile Thr Trp | ||||||||||||
| GTG | ATG | TCC | AAC | AGC | CCA | AAT | AAC | GTG | TTG | AAC | ATT | GAA | GGG | TGT | CCA | 1221 |
| Val | Mot | Ser | Asn | Ser | Pro | Asn | Asn | Val | Leu | Asm | Ile | Olu | Gly | Cys | Pro | |
| 355 | 360 | 365 | 370 | |||||||||||||
| TCA | CTC | TAC | AAA | TTC | AAC | CCG | TTC | AGA | GGA | GGG | TTG | AAC | AGG | ATC | GTC | 1269 |
| Ser | Leu | Tyr | Lys | Phe | Asn | Pro | Phe | Arg | Gly | Gly | Leu | Afin | Arg | Ile | val | |
| 375 | 380 | 385 | ||||||||||||||
| GAG | TGG | ATA | TTG | GCC | CCG | GAA | GAA | CCC | AAG | GCT | CTT | GTA | TAT | GCG | GAC | 1317 |
| Glu | Trp | Ile | Leu | Ala | Pro | GlU | Glu | Pro | Lye | Ala | Leu | Val | Tyr | Ala | Asp | |
| 390 | 395 | 400 | ||||||||||||||
| AAC | ATA | TAC | ATT | GTC | CAC | TCA | AAC | ACG | TGG | TAC | TCA | ATT | GAC | CTA | GAG | 1365 |
| Asn | Ile | Tyr | Ile | Val | His | Ser | Asn | Thr | Trp | Tyr | Ser | Ile | Asp | Leu | Glu | |
| 405 | 410 | 415 | ||||||||||||||
| AAG | GGT | GAG | GCA | AAC | TGC | ACT | CGC | CAA | CAC | ATG | CAA | GCC | GCA | ATG | TAC | 1413 |
| Lys | Gly | Glu | Ala | Asn | Cys | Thr | Arg | Gin | His | Met | Gin | Ala | Ala | Met | Tyr | |
| 420 | 425 | 430 | ||||||||||||||
| TAC | ATA | CTC | ACC | AGA | GGG | TGG | TCA | GAC | AAC | GGC | GAC | CCA | ATG | TTC | AAT | 1461 |
| Tyr | Ile | Leu | Thr | Arg | Gly | Trp | Ser | Aep | Asn | Gly Asp | Pro | Met | Phe | Asn | ||
| 435 | 440 | 445 | 450 | |||||||||||||
| CAA | ACA | TGG | GCC | ACC | TTT | GCC | ATG | AAC | ATT | GCC | CCT | GCT | CTA | GTG | GTG | 1509 |
| Gin | Thr | Trp | Ala | Thr | Phe | Ala | Met | Asn | Ile | Ala | Pro | Ala | Leu | Val | Val | |
| 455 | ·. | 460 | 465 | |||||||||||||
| GAC | TCA | TCG | TGC | CTG | ATA | ATG | AAC | CTG | CAA | ATT | AAG | ACC | TAT | GGT | CAA | 1557 |
| Asp | Set | Ser | Cys | Leu | Ile | Met | Asn | Leu | Gin | Ile | Lys | Thr | Tyr | c?iy | Gin | |
| 470 | 475 | 400 | ||||||||||||||
| GGC | AGC | GGG | AAT | GCA | GCC | ACG | TTC | ATC | AAC | AAC | CAC | CTC | TTG | AGC | ACA | 1605 |
| Gly | Ser | Gly | Asn | Ala | Ala | Thr | Phe | Ile | Asn | Asn | His | Leu | Leu | Ser | Thr | |
| 405 | 490 | 495 | ||||||||||||||
| CTA | GTG | CTT | GAC | CAG | TGG | AAC | CTG | ATG | AGA | CAG | CCC | AGA | CCA | GAC | AGC | 1653 |
| Leu | Val | Leu | Asp | Gin | Trp | Asn | Leu | Met | Arg | Gin | Pro | Arg | Pro | Asp | Ser | |
| 500 | 505 | 510 | ||||||||||||||
| GAG | GAG | TTC | AAA | TCA | ATT | GAG | GAC | AAG | CTA | GGT | ATC | AAC | TTT | AAG | ATT | 1701 |
| Olu | GlU | Phe | Lys | Ser | Ile | GlU | Asp | Lya | Leu | Gly | Ile | Asn | Phe | Lys | Ile | |
| 515 | 520 | 525 | 530 | |||||||||||||
| GAG | AGG | TCC | ATT | GAT | GAT | ATC | AGG | GGC | AAG | CTG | AGA | CAG | CTI’ | GTC | CTC | 1749 |
| Glu | Arg | Ser | Ile | Asp | Asp | Ile | Arg | Gly | Lys | Leu | Arg | Gin | Leu | Val | Leu | |
| 535 | 540 | S4S |
-21 CZ 299417 B6
| crr | GCA | CAA | CCA | GGG | TAC | CTG | AGT | GGG | GGG | GTT | GAA | CCA | GAA | CAA | TCC | 1797 |
| Leu | Ala | Gin | Pro 550 | Gly | Tyr | Leu | Ser | Gly Gly 555 | Val | Glu | Pro | Glu 560 | Gin | Ser | ||
| AGC | CCA | ACT | GTT | GAG | CTT | GAC | CTA | CTA | GGG | TGG | TCA | GCT | ACA | TAC | AGC | 1845 |
| Ser | Pro | Thr 565 | val | Glu | Leu | Asp | Leu 570 | Leu | Gly | Trp | Ser | Ala 575 | Thr | Tyr | Ser | |
| AAA | GAT | CTC | GGG | ATC | TAT | GTG | CCG | GTG | CTT | GAC | AAG | GAA | CGC | CTA | TTT | 1893 |
| Lya | Asp 580 | hen | Gly | Ile | Tyr | Val 585 | Pro | Val | Leu | Asp | Lys 590 | Glu | Arg | Leu | Phe | |
| TGT | TCT | GCT | GCG | TAT | CCC | AAG | GGA | GTA | GAG | AAC | AAG | AGT | CTC | AAG | TCC | 1941 |
| Cye 595 | Ser | Ala | Ala | Tyr | Pro 600 | Lya | Gly | Val | Glu | Asn 605 | Lya | Ser | Leu | Lys | Ser 610 | |
| ΑΛΑ | GTC | GGG | ATC | GAG | CAG | GCA | TAC | AAG | GTA | GTC | AGG | TAT | GAG | GCG | TTG | 1989 |
| Lys | Val | Gly | llo | Glu 615 | Gin | Ala | Tyr | Lys | Val 620 | Val | Arg | Tyr | Glu | Ala 625 | Leu | |
| AGG | TTG | GTA | GGT | GGT | TGG | AAC | TAC | CCA | CTC | CTG | AAC | AAA | GCC | TGC | AAG | 2037 |
| Arg | Leu | Val | Gly 630 | Gly | Trp | Asn | Tyr | Pro 635 | Leu | Leu | Asn | Lys | Ala 640 | Cye | Lys | |
| AAT | AAC | GCA | GGC | GCC | GCT | CGG | CGG | CAT | CTG | GAG | GCC | AAG | GGG | TTC | CCA | 2085 |
| Asn | Asn | Ala 645 | Gly | Ala | Ala | Arg | Arg 650 | His | Leu | Glu | Ala | Lys 655 | Gly | Phe | Pro | |
| CTC | GAC | GAG | TTC | CTA | GCC | GAG | TGG | TCT | GAG | CTG | TCA | GAG | TTC | GGT | GAG | 2133 |
| neu | Asp 660 | GlU | Phe | Leu | Ala | Glu 665 | Trp | Ser | Glu | Leu | ser 670 | GlU | Phe | Gly | GlU | |
| GCC | TTC | GAA | GGC | TTC | ÁAT | ATC | AAG | CTG | ACC | GTA | ACA | TCT | GAG | AGC | CTA | 2181 |
| Kin 675 | Phe | GlU | Gly | Phe | Asn 680 | Ile | Lys | Leu | Thr | Val 685 | Thr | Ser | Glu | Ber | Leu 690 | |
| GCC | GAA | CTG | AAC | AAG | CCA | GTA | CCC | CCC | AAG | CCC | CCA | AAT | GTC | AAC | AGA | 2229 |
| Ala | Glu | Leu | Asn | Lys 695 | Pro | Val | Pro | Pro | Lys 700 | Pro | Pro | Asn | Val | Asn 70S | Arg | |
| CCA | GTC | AAC | ACT | GGG | GGA | CTC | AAG | GCA | GTC | AGC | AAC | GCC | CTC | AAG | ACC | 2277 |
| Pro | Val | Asn | Thr 710 | Giy | Gly | Leu | Lya | Ala 715 | Val | Ser | Asn | Ala | Leu 720 | Lys | Thr | |
| GGT | CGG | TAC | AGG | AAC | GAA | GCC | GGA | CTG | AGT | GGT | CTC | GTC | CTT | CTA | GCC | 2325 |
| Gly | Arg | Tyr 725 | Arg | Asn | Glu | Ala | Gly 730 | Leu | Sar | Oly | Leu | Val 735 | Leu | Leu | Ala | |
| ACA | GCA | AGA | AGC | CGT | CTG | CAA | GAT | GCA | GTT | AAG | GCC | AAG | GCA | GAA | GCC | 2373 |
| Thr | Ala 740 | Arg | Ser | Arg | Leu | Gin 745 | Asp | Ala | Val | Lys | Ala 750 | Lya | Ala | Glu | Ala |
-22CZ 299417 B6
| GAG Glu 755 | AAA CTC CAC | AAG TCC AAG CCA GAC GAC CCC GAT GCA GAC TGG TTC | 2421 | |||||||||||||
| bys | heu Hře | Lys Ser Lys 760 | Pro | Asp | Asp Pro Asp 765 | Ala | Asp | Trp | Phe 770 | |||||||
| GAA | AGA | TCA | GAA | ACT | CTG | TCA | GAC | CTT | CTG | GAG | AAA | GCC | GAC | ATC | GCC | 2469 |
| Glu | Arg | Ser | GlU | Thr | Leu | Ser | Asp | Leu | Leu | Glu | Lys | Ala | Asp | Ile | Ala | |
| 775 | 780 | 785 | ||||||||||||||
| AGC | AAG | GTC | GCC | CAC | TCA | GCA | CTC | GTG | GAA | ACA | AGC | GAC | GCC | CTT | GAA | 2517 |
| Ser | Lys | Val | Ala | His | ser | Ala | Leu | Val | Glu | Thr | ser | A8p | Ala | Leu | Glu | |
| 790 | 795 | 800 | ||||||||||||||
| GCA | GTT | CAG | TCG | ACT | TCC | GTG | TAC | ACC | CCC | AAG | TAC | CCA | GAA | GTC | AAG | 2565 |
| Ala | Val | Gin | ser | Thr | Ser | Val | Tyr | Thr | Pro | Lys | Tyr | Pro | GlU | Val | Lys | |
| 805 | 810 | 815 | ||||||||||||||
| AAC | CCA | CAG | ACC | GCC | TCC | AAC | CCC | GTT | GTT | GGG | CTC | CAC | CTG | CCC | GCC | 2613 |
| Aan | Pro | Gin | Thr | Ala | Ser | Asn | Pro | Val | Val | Gly | Leu | His | Leu | Pro | Ala | |
| 820 | 825 | 830 | ||||||||||||||
| AAG | AGA | GCC | ACC | GGT | GTC | CAG | GCC | GCT | CTT | GTG | GGA | GCA | GGA | ACG | AGC | 2661 |
| Lys | Arg | Ala | Thr | Gly | Val | Gin | Ala | Ala | Leu | Leu | Gly | Ala | Gly | Thr | Ser | |
| 035 | 040 | 845 | 850 | |||||||||||||
| AGA | CCA | ATG | GGG | ATG | GAG | GCC | CCA | ACA | CGG | TCC | AAG | AAC | GCC | GTG | AAA | 2709 |
| Arg | Pro | Met | Gly | Met | Glu | Ala | Pro | Thr | Arg | Ser | Lys | Asn | Ala | Val | Lye | |
| 855 | 860 | 065 | ||||||||||||||
| ATG | GCC | AAA | CGG | CGG | CAA | CGC | CAA | AAG | GAG | AGC | CGC | TAKCAGCCAT | 2755 | |||
| Met | Ala | Lys | Arg | Arg | Gin | Arg | Gin | Lys | Glu | Ser | Arg |
870 075
GATGGGAACC ACTCAAGAAG AGGACACTAA TCCCAGACCC CGTATCCCCG GCCTTCGCCT 2015
GCGGGGGCCC CC 2027
INFORMACE PRO SEQ ID NO:26:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 878 AMINOKYSELIN TYP: AMINOKYSELINA TYPOLOGIE: LINEÁRNÍ
TYP MOLEKULY: protein POPIS SEKVENCE: SEQ ID NO:26:
Met ser Asp Ile Phe Aan Ser Pro Gin Ala Arg Ser Thr Ile Ser Ala 1 S 10 15
Ala Phe Gly Ile Lys Pro Thr Ala Gly Gin Asp Val Glu Glu Leu Leu
-23 CZ 299417 B6
25 30
| Ile | Pro | Lya 35 | Val | Trp | Val | Pro | Pro 40 | Glu | Asp | Pro | Leu | Ala 45 | Ser | Pro | Ser |
| Arg | Leu 50 | Ala | Lys | Phe | Leu | Arg 55 | Glu | Aan | Gly Tyr | Lys 60 | Val | Leu | Gin | Pro | |
| Arg 65 | Ser | Leu | Pro | Glu | Asn 70 | Úlu | Glu | Tyr | Glu | Thr 75 | Asp | Gin | Ile | Leu | Pro 80 |
| Asp | Leu | Ala | Trp | Met 85 | Arg | Gin | Ile | Glu | Gly 90 | Ala | Val | Leu | Lya | Pro 95 | Thr |
| Leu | Ser | Leu | Pro 100 | Xle | Gly | Asp | Gin | Glu 105 | Tyr | Phe | Pro | Lys | Tyr 110 | Tyr | Pro |
| Thr | His | Arg 115 | Pro | Ser | Lys | Glu | Lya 120 | Pro | Asn | Ala | Tyr | Pro 125 | Pro | ASp | Ile |
| Ala | Leu 130 | Leu | Lys | Gin | Met | Ile 135 | Tyr | Leu | Phe | Leu | Gin 140 | Val | Pro | Glu | Ala |
| Aan 14S | Glu | Gly | Leu | Lya | Asp 150 | Glu | Val | Thr | Leu | Leu 155 | Thr | Gin | Asn | Ile | Arg 160 |
| Αβρ | Lys | Ala | Tyr | Gly 165 | Ser | Gly | Thr | Tyr | Met 170 | Gly | Gin | Ala | Asn | Arg 175 | Leu |
| Val | Ala | Met | Lys 180 | Glu | Val | Ala | Thr | Gly Arg 185 | Asn | Pro | Asn | Lya 190 | Aap | Pro | |
| Leu | Lya | Leu 195 | Gly Tyr | Thr | Phe | Glu 200 | Ser | Ile | Ala | Gin | Leu 205 | Leu | Asp | Ile | |
| Thr | Leu 210 | Pro | Val | Gly | Pro | Pro 215 | Gly | Glu | Asp | Aap | Lys 220 | Pro | Trp | Val | Pro |
| Lou 225 | Thr | Arg | Val | Pro | Ser 230 | Arg | Met | Leu | Val | Leu 235 | Thr | Gly | Asp | Val | Asp 240 |
| Oly | Aap | Phe | Glu | Val 245 | Glu | Asp | Tyr | Leu | Pro 250 | Lys | Ile | Aan | Leu | Lya 255 | Ser |
| Ser | Ser | ciy | Leu 260 | Pro | Tyr | Val | Gly | Arg 265 | Thr | Lys | Gly | Glu | Thr 270 | Ile | Gly |
| Olu | Met | Ile 275 | Ala | Ile | Ser | Asn | Gin 280 | Phe | Leu | Arg | Glu | Leu 285 | Ser | Thr | Leu |
| Leu | Lys | Gin | Gly | Ala | Gly | Thr | Lys | Gly | Ser | Asn | Lya | Lys | Lys | Leu | Leu |
-24CZ 299417 B6
290 295 300
| Ser 3 OS | Met | Leu | Ser | Asp | Tyr 310 | Trp | Tyr | Leu | Ser | Cys 315 | Gly | Leu | Leu | Phe | Pro 320 |
| Lyn | Ala | Glu | Arg | Tyr 325 | Asp | Lys | Ser | Thr | Trp 330 | Leu | Thr | Lys | Thr | Arg 335 | Asn |
| Ile | Trp | Ser | Ala 340 | Pro | Ser | Pro | Thr | His 345 | Leu | Met | Ile | Ber | Met 350 | Ile | Thr |
| Trp | Pro | Val 355 | Met | Ser | Asn | Ser | Pro 360 | Asn | Asn | Val | Leu | Asn 36S | Ile | Glu | Gly |
| Cye | Pro 370 | Ser | Leu | Tyr | Lys | Phe 375 | Asn | Pro | Phe | Arg | Gly Oly 380 | Leu | Asn | Arg | |
| Ile 3Θ5 | Val | Glu | Trp | Ile | Leu 390 | Ala | Pro | Glu | Glu | Pro 39S | Lys | Ala | Leu | Val | Tyr 400 |
| Ala | Aap | Asn | Ile | Tyr 405 | Ile | Val | His | Ser | Asn 410 | Thr | Trp | Tyr | Ser | Ile 415 | Asp |
| Leu | Olu | Lys | Gly 420 | Glu | Ala | Asn | Cys | Thr 425 | Arg | Gin | His | Mat | Gin 430 | Ala | Ala |
| Met | Tyr | Tyr 435 | Ile | Leu | Thr | Arg | Gly 440 | Trp | Ser | Asp | Asn | Gly 445 | Asp | Pro | Met |
| Phe | Aíjo 450 | Gin | Thr | Trp | Ala | Thr 455 | Ptie | Ala | Met | Asn | Ile 460 | Ala | Pro | Ala | Leu |
| val 465 | Val | Aop | Ser | Ser | Cys 470 | Leu | Ile | Met | Asn | Leu 475 | Gin | Ile | Lys | Thr | Tyr 480 |
| oly | om | Gly | Ser | Gly 405 | Asn | Ala | Ala | Thr | Phe 490 | Ile | Asn | Asn | His | Leu 495 | Leu |
| Ser | Thr | LOU | val 500 | Leu | Asp | Gin | Trp | Asn 505 | Leu | Met | Arg | Gin | Pro 510 | Arg | Pro |
| Aap | Ser | Glu 515 | Glu | Phe | Lys | Ser | Ile 520 | Glu | Asp | bys | Leu | Oly 525 | Ile | Asn | Phe |
| Lys | Ile 530 | GlU | Arg | Ser | Ile | Asp 535 | Asp | Ile | Arg | Gly | Lys 540 | Leu | Arg | Gin | Leu |
| Val 545 | Leu | beu | Ala | Gin | Pro 550 | Gly | Tyr | Leu | Ser | Oly 555 | Gly | Val | Glu | Pro | Glu 560 |
| Gin | Ser | Ser | Pro | Thr | Val | Glu | Leu | Asp | Leu | Leu | Gly | Trp | Ser | Ala | Thr |
-25CZ 299417 B6
565 570 57S
| Tyr | ser | Lys | Asp 580 | Leu | Gly | Ile | Tyr | Val 585 | Pro | Val | Leu | Asp | Lys 590 | Glu | Arg |
| Leu | Phe | Cys S95 | Ser | Ala | Ala | Tyr | Pro 600 | Lya | Gly | Val | Glu | Asn 605 | Lys | Ser | Leu |
| Lys | Ser 610 | Lya | Val | Gly | Ile | Glu 615 | Gin | Ala | Tyr | Lys | Val 620 | Val | Arg | Tyr | Glu |
| Ala 625 | Leu | Arg | Leu | Val | Gly 630 | Gly | Trp | Aen | Tyr | Pro 635 | Leu | Leu | Asn | Lys | Ala 640 |
| Cya | Lys | Asn | Asn | Ala 645 | Gly | Ala | Ala | Arg | Arg 650 | His | Leu | Glu | Ala | Lys 655 | Gly |
| Phe | Pro | Leu | Asp 660 | Glu | Phe | Leu | Ala | Glu 665 | Trp | Ser | Glu | Leu | Ser 670 | Glu | Phe |
| Oly | GIU | Ala 67S | Phe | Glu | Gly | Phe | Asn 680 | Ile | Lys | Leu | Thr | Val 685 | Thr | Ser | Glu |
| Ser | Leu 690 | Ala | Glu | Leu | Aen | Lys 695 | Pro | Val | Pro | Pro | Lys 700 | Pro | Pro | Asn | Val |
| Asn 705 | Arg | Pro | Val | Asn | Thr 710 | Gly | Gly | Leu | Lys | Ala 715 | Val | Ser | Asn | Ala | Leu 720 |
| Lys | Thr | Gly | Arg | Tyr 725 | Arg | Afin | Glu | Ala | Gly 730 | Leu | Ser | Gly | Leu | Val 735 | Leu |
| Leu | Ala | Thr | Ala 740 | Arg | Ser | Arg | Leu | Gin 745 | Aep | Ala | Val | Lys | Ala 750 | Lys | Ala |
| Glu | Ala | Glu 755 | Lys | Leu | His | Lys | Ber 760 | Lys | Pro | Asp | Asp | Pro 765 | Asp | Ala | Asp |
| Trp | Phe 770 | Glu | Arg | Ser | Glu | Thr 775 | Leu | Ser | Asp | Leu | Leu 780 | GIU | Lys | Ala | Asp |
| Ile 705 | Al a | Ser | Lya | val | Ala 790 | His | Ser | Ala | Leu | Val 795 | Glu | Thr | Ser | Aep | Ala 800 |
| Leu | GlU | Ala | Val | Gin 805 | ser | Thr | ser | val | Tyr 8l0 | Thr | Pro | Lys | Tyr | Pro 015 | Glu |
| Val | Lys | Asn | Pro 820 | Gin | Thr | Ala | Ser | Asn 825 | Pro | Val | Val | Gly | Leu 830 | His | Leu |
| .Pro | Ala | Lys | Arg | Ala | Thr | Gly | Val | Gin | Ala | Ala | Leu | Leu | Gly | Ala | Gly |
835 840 845
Thr ser Arg Pro Met Gly Met Glu Ala Pro Thr Arg Ser Lys Asn Ala 850 8SS 860
Val Lye Met Ala Lys Arg Arg Gin Arg Gin bys Glu Ser Arg 865 870 075
-26CZ 299417 B6
INFORMACE PRO SEQ IDNO:27:
SEKVENČNÍ CHARAKTERISTIKY
DÉLKA: 3261 PÁRŮ BÁZÍ
TYP: NUKLEOVÁ KYSELINA ŘETĚZEC: JEDNODUCHÝ TYPOLOGIE: KRUHOVÁ ío TYP MOLEKULY: cDNA
ZNAKY: JMÉNO/KLÍČ: CDS
UMÍSTĚNÍ: 97..531
POPIS SEKVENCE: SEQ ID NO:27:
GGATACGATC GGTCTGACCC CGGGGGAGTC ACCCGGOGAC AGGCCGTCAA GGCCTTGTTC 60
CAGGATGGGA CTCCTCCTTC TACAACGCTA TCATTG ATG GTT AGT AGA GAT CAG 114 Met Val Ser Arg Asp Gin
000
| ACA AAC GAT | CGC AGC GAT | GAC AAA CCT GCA AGA TCA AAC CCA ACA GAT | 162 | |||||||||||||
| Thr Asn 605 | Asp | Arg | Ser | Asp 090 | Asp | bys | Pro Ala | Arg 09S | Ser | Asn | Pro | Thr | Asp 900 | |||
| TGT | TCC | GTT | CAT | ACG | GAG | CCT | TCT | GAT | GCC | AAC | AAC | CGG | ACC | GGC | GTC | 210 |
| Cys | Ser | Val | His | Thr | Glu | Pro | Ser | Asp | Ala | Asn | Aflft | Arg | Thr | Gly | Val | |
| 905 | 910 | 915 | ||||||||||||||
| CAT | TCC | GGA | CGA | CAC | CCT | GGA | GAA | GCA | CAC | TCT | CAG | GTC | AGA | GAC | CTC | 258 |
| Hle | Ser | Gly | Arg | His | Pro | Gly | Olu | Alo | His | Ser | Gin | Val | Arg | Asp | Leu | |
| 920 | 925 | 930 | ||||||||||||||
| GAC | CTA | CAA | TTT | GAC | TGT | GGG | GGA | CAC | AGG | GTC | AGG | GCT | ΛΛΤ | TGT | CTT | 306 |
| ASp | Leu | Gin | Phe | Asp | Cys | Gly | Gly | His | Arg | Val | Arg | Ala | Asn | Cys | beu | |
| 935 | 940 | 945 | ||||||||||||||
| ITT | ccc | TGG | ATT | CCC | TGG | CTC | ΛΛΤ | TGT | GGG | TGC | TCA | CTA | CAC | ACT | GCA | 354 |
| Phe | Pro | Trp | 31« | Pro | Trp | Leu | Asn | Cys | Gly | Cys | Ser | Leu | Hle | Thr | Ala |
9S0 955 960
-27 CZ 299417 B6
GGO CAA TGG GAA CTA CAA GTT CGA TCA GAT GCT CCT GAC TGC CCA GAA
Gly Gin Trp Glu beu Gin Val Arg Ger Asp Ala Pro Asp Cys Pro Glu
965 970 975 980
CCT ACC GGC CAG TTA CAA CTA CTG CAG GCT AGT GAG TCG GAG TCT CAC
Pro Thr Gly Gin beu Gin lasu bou Gin Ala Ser Glu Ser Glu Ser His
985 990 995
AGT GAG GTC AAG CAC ACT TCC TGG TGG CGT TTA TCC ACT ΛΛΑ CGG CAC
Ser Glu Val bys Kle Thr Ser Trp Trp Arg bou Cya Thr bys Arg Hie
1000 1005 1010
CAT ΑΛΑ CGC CGT GAC CTT CCA AGG AAG CCT GAG TGAACTGACA GATOTTAGCT His bys Arg Arg Asp beu Pro Arg bye Pro Glu
1015 1020
ACAATGGGTT GATGTCTGCA ACAGCCAACA
GGGAAGGGGT CACCGTCCTC AGCTTACCCA
GTGACCCCAT TCCCGCAATA GGGCTTGACC
ACAGGCCCAG AGTCTACACC ATAACTGCAG
AACCAGGTCG GGTAACAATC ACACTGTTCT
GCGTTGGGGG AGAGCTCGTG TTTCAAACAA
TCTACCTCAT AGGCTTTGAT GGGACAACGG
GGCTGACGAC CGGCACCGAC AACCTTATGC 1
TAACCCAGCC AATCACATCC ATCAAACTCG
CAGQGGATCA GATGTCATGG TCGGCAAGAG
ACTATCCAGG GGCCCTCCGT CCCGTCACGC
CCGTCGTTAC GGTCGCTGGG GTGAGCAACT
AGAACCTGGT TACAGAATAC GGCCGATTTG
TACTGAGTGA GAGGGACCGT CTTGGCATCA
ACTTTCGTGA ATACTTCATG GAGGTGGCCO
CATTCGGCTT CAAAGACATA ATCCGGGCCA
CATTGTTCCC ACCTGCCGCT CCCCTAGCCC
TCGGCGATGA GGCACAGGCT GCTTCAGGAA
402
450
498
551
611
671
731
791
851
911
971
1031
1091
1151
1211
1271
1331
1391
1451
1511
1571
1631
TCAACGACAA AATTGGGAAC GTCCTAOTAG
CATCATATGA TCTTGGGTAT GTGAGGCTTG
CAAAAATGGT AGCCACATGT GACAGCAGTG
CCGATCATTA CCAATTCTCA TCACAGTACC
CAGCCAACAT TGATGCCATC ACAAGCCTCA
GCGTCCACGG CCTTGTACTG GGCGCCACCA
TAATCACCAG GGCTGTGGCC GCAAACAATG
CATTCAATCT TGTGATTCCA ACAAACGAGA
AGATAGTCAC CTCCAAAAGT GGTGGTCAGG
GGAGCCTAGC AGTGACGATC CATGGTGGCA
TAGTGGCCTA CGAAAGAGTG GCAACAGGAT
TCGAGCTGAT CCCAAATCCT GAACTAGCAA
ACCCAGGAGC CATGAACTAC ACAAAATTGA
AGACCGTCTG GCCAACAAGG GAGTACACTG
ACCTCAACTC TCCCCTGAAG ATTGCAGGAG
TAAGGAGGAT AGCTGTGCCG GTGGTCTCCA
ATGCAATTGG GGAAGGTGTA GACTACCTGC
CTGCTCGAGC CGCGTCAGGA AAAGCAAGAG
-28CZ 299417 B6
| CTGCCTCAGG | CCGCATAAGG | CAGCTGACTC | TCGCCGCCGA | CAAGGGGTAC | GAGGTAGTCG | 1691 |
| CGAATCTATT | CCAGGTGCCC | CAGAATCCCG | TAGTCGACGG | GATTCTTGCT | TCACCTGGGG | 1751 |
| TACTCCGCGG | TGCACACAAC | OTCGACTGCG | TGTTAAOAGA | GGGTGCCACG | CTATTCCCTG | 1011 |
| TGGTTATTAC | GACAGTGGAA | GACGCCATGA | CACCCAAAGC | ATTGAACAGC | aaaatgtttg | 1871 |
| CTGTCATTGA | AGGCGTGCGA | GAAGACCTCC | AACCTCCATC | TCAAAGAGGA | TCCTTCATAC | 1931 |
| GAACTCTCTC | TGGACACAGA | GTCTATGGAT | ATGCTCCAGA | TGGGGTACTT | CCACTGGAGA | 1991 |
| CTGGGAGAGA | CTACACCGTT | GTCCCAATAG | ATGATGTCTG | OGACGACAGC | ATTATGCTGT | 20S1 |
| CCAAAGATCC | CATACCTCCT | ATTGTGGGAA | ACAGTGGAAA | TCTAGCCATA | GCTTACATGG | 2111 |
| ATGTGTTTCG | ACCCAAAGTC | CCAATCCATG | TGGCTATGAC | GGGAGCCCTC | AATGCTTGTG | 2171 |
| GCGAGATTGA | GAAAGTAAGC | TTTAGAAGCA | CCAAGCTCGC | CAOTGCACAC | CGACTTGGCC | 2231 |
| TTAGGTTGGC | TGGTCCCGGA | GCATTCGATG | TAAACACCGG | GCCCAACTGG | gcaacgttca | 2291 |
| TCAAACGTTT | CCCTCACAAT | CCACGCGACT | GGGACAGGCT | CCCCTACCTC | AACCTACCAT | 2351 |
| ACCnCCACC | CAATGCAGGA | CGCCAGTACC | ACCTTGCCAT | GGCTGCATCA | GAGTTCAAAG | 2411 |
| AOACCCCCGA | ACTCGAGAGT | GCCGTCAGAG | CAATGGAAGC | AGCAGCCAAC | GTGGACCCAC | 2471 |
| TATTCCAATC | TGCACTCAGT | GTGTTCATGT | GGCTGGAAGA | GAATGGGATT | GTGACTGACA | 2531 |
| TGGCCAACTT | CGCACTCAGC | GACCCGAACG | CCCATCGGAT | GCGAAATTTT | CTTGCAAACG | 2591 |
| CACCACAAGC | AGGCAGCAAG | TCGCAAAGGG | CCAAGTACGG | GACAGCAOGC | TACGGAGTGG | 2651 |
| AGGCTCGGGG | CCCCACACCA | GAGGAAGCAC | AGAGGGAAAA | AGACACACGG | ATCTCAAAGA | 2711 |
| AGATGGAGAC | CATGOGCATC | TACTTTGCAA | CACCAGAATG | GGTAGCACTC | AATGGGCACC | 2771 |
| GAGGGCCAAO | CCCCGGCCAG | CTAAAGTACT | GGCAGAACAC | ACGAGAAATA | CCGGACCCAA | 2031 |
| ACGAGGACTA | TCTAGACTAC | GTGCATGCAO | AGAAGAGCCG | GTTGGCATCA | GAAGAACAAA | 2091 |
| TCCTAAGGGC | AGCTACGTCG | ATCTACGGGG | CTCCAGGACA | GGCAGAGCCA | CCCCAAGCTT | 2951 |
| TCATAGACGA | AGTTGCCAAA | GtCTatgaaa | TCAACCATGG | ACGTGGCCCA | AACCAAGAAC | 3011 |
| AGATGAAAGA | TCTGCTCTTG | ACTGCGATGG | AGATGAAGCA | TCGCAATCCC | AGGCGGGCTC | 3071 |
| TACCAAAGCC | CAAGCCAAAA | CCCAATGCTC | CAACACAGAG | ACCCCCTGGT | CGGCTGGGCC | 3131 |
| OCTGGATCAO | GACCGTCTCT | GATGAGGACC | TTGAGTGAOG | CTCCTGGGAG | TCTCCCGACA | 3191 |
| CCACCCGCGC | AGGTGTGGAC | ACCAATTCGG | CCTTACAACA | TCCCAAATTG | GATCCGTTCG | 3251 |
| CGGGTCCCCT | 3261 |
-29CZ 299417 B6
INFORMACE PRO SEQ ID NO:28:
SEKVENČNÍ CHARAKTERISTIKY 5 DÉLKA: 145 AMINOKYSELIN
TYP: AMINOKYSELINA TYPOLOGIE: LINEÁRNÍ
TYP MOLEKULY: protein ío POPIS SEKVENCE: SEQ ID NO:28:
| Met 1 | Val | Ser | Arg | Λβρ 5 | Gin | Thr | Aon | Asp | Arg 10 | Ser | Asp | Asp | Lys | Pro 15 | Ala |
| Arg | Ser | Asn | Pro 20 | Thr | Aep | Cys | Sor | Val 25 | His | Thr | Glu | Pro | Ser 30 | Asp | Ala |
| Asn | Asn | Arg 35 | Thr | Gly | Val | His | Ser 40 | Gly Arg | His | Pro | Gly 45 | Glu | Ala | His | |
| Ser | Gin 50 | val | Arg | Asp | Leu | Asp 55 | Leu | Gin | Phe | Asp | Cys 60 | Gly | Gly | His | Arg |
| Val 65 | Arg | Ala | Asn | Cys | Leu 70 | Phe | Pro | Trp | Ile | Pro 75 | Trp | Leu | Asn | Cys | Gly 00 |
| Cys | Ser | Leu | His | Thr 05 | Ala | Gly | Gin | Trp | Glu 90 | Leu | Gin | Val | Arg | Ser 95 | Asp |
| Ala | Pro | Asp | Cys 100 | Pro | Glu | Pro | Thr | Gly 105 | Gin | Leu | Gin | Leu | Leu 110 | Gin | Ala |
| Ser | Glu | Ser 115 | Glu | Ser | His | Ser | Glu 120 | Val | Lys | His | Thr | Ser 12S | Trp | Trp | Arg |
| l>eu | Cyo 130 | Thr | Lyfl | Arg | His | His 135 | Lys | Arg | Arg | Asp | Leu 140 | Pro | Arg | Lys | Pro |
Glu
145
INFORMACE PRO SEQ ID NO:29:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 3261 PÁRŮ BÁZÍ TYP: NUKLEOVÁ KYSELINA ŘETĚZEC: JEDNODUCHÝ
TYPOLOGIE: KRUHOVÁ
TYP MOLEKULY: cDNA ZNAKY: JMÉNO/KLÍČ: CDS UMÍSTĚNÍ: 131..3166
POPIS SEKVENCE: SEQ ID NO:29:
-30CZ 299417 B6
GGATACGATC GOTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCGTCAA GGCCTTGTTC 60
CAGGATGGGA CTCCTCCTTC TACAACGCTA TCATTGATGG TTAGTAGAGA TCAGACAAAC 120
GATCGCAGCG ATG ACA AAC CTG CAA GAT CAA ACC CAA CAG ATT GTT CCG 169
Met Thr Asn Leu Gin Asp Gin Thr Gin Gin Ile Val Pro
150 155
| TTC Phe | ATA Ilo 160 | CGG AGC | CTT CTG ATG Leu Leu Met 165 | CCA ACA ACC GGA CCG GCG TCC ATT CCG | 217 | |||||||||||
| Arg | Ser | Pro | Thr | Thr | Gly | Pro 170 | Ala | Ser | Ile | Pro | ||||||
| GAC | GAC | ACC | CTG | GAG | AAG | CAC | ACT | CTC | AGG | TCA | GAG | ACC | TCG | ACC | TAC | 265 |
| Asp | Asp | Thr | Leu | Glu | Lys | His | Thr | Leu | Arg | Ser | Glu | Thr | Ser | Thr | Tyr | |
| 175 | 180 | 185 | 190 | |||||||||||||
| AAT | TTG | ACT | GTG | GGG | GAC | ACA | GGG | TCA | GGG | CTA | ATT | GTC | TTT | TTC | CCT | 313 |
| Asn | Leu | Thr | Val | Gly | Asp | Thr | Gly | Ser | Gly | Leu | Ile | Val | Phe | Phe | Pro | |
| 195 | 200 | 205 | ||||||||||||||
| GGA | TTC | CCT | GGC | TCA | ATT | GTG | GGT | GCT | CAC | TAC | ACA | CTG | CAG | GGC | AAT | 361 |
| Gly | Phe | Pro | Gly | Ser | Ile | Val | Gly | Ala | His | Tyr | Thr | Leu | Gin | Gly | Asn | |
| 210 | 215 | 220 | ||||||||||||||
| GGG | AAC | TAC | AAG | TTC | GAT | CAG | ATG | CTC | CTG | ACT | GCC | CAG | AAC | CTA | CCG | 409 |
| Oly | Aon | Tyr | Lys | Phe | Asp | Gin | Met | Leu | Leu | Thr | Ala | Gin | Asn | Leu | Pro | |
| 225 | 230 | 235 | ||||||||||||||
| GCC | AGT | TAC | AAC | TAC | TGC | AGG | CTA | GTG | AGT | CGG | AGT | CTC | ACA | GTG | AGG | 457 |
| Ala | Ser | Tyr | Asn | Tyr | Cye | Arg | Leu | Val | Ser | Arg | Ser | Leu | Thr | Val | Arg | |
| 240 | 245 | 250 | ||||||||||||||
| TCA | AGC | ACA | CTT | CCT | GGT | GGC | GTT | TAT | GCA | CTA | AAC | GGC | ACC | ATA | AAC | 505 |
| Sor | Ser | Thr | Leu | pro | Gly | Gly | Val | Tyr | Ala | LeU | Asn | Gly | Thr | Ile | Asn | |
| 255 | 260 | 2GS | 270 | |||||||||||||
| GCC | GTG | ACC | TTC | CAA | GGA | AGC | CTG | AGT | GAA | CTG | ACA | GAT | GTT | AGC | TAC | 553 |
-31 CZ 299417 B6
| Ala | Val | Thr Phe Gin Gly 275 | Ser Leu Ser | Glu Leu Thr 280 | Asp | Val | Ser 285 | Tyr | ||||||||
| ΑΛΤ | GGG | TTG | ATG | tct | GCA | ACA | GCC | AAC | ATC | AAC | GAC | ΛΑΑ | ATT | GGG | AAC | 601 |
| Asn | Gly | Leu | Met | Ser | Ala | Thr | Ala | Asn | Ile | Asn | Asp | Lys | Ile | Gly | Asn | |
| 290 | 295 | 300 | ||||||||||||||
| GTC | CTA | GTA | GGG | GAA | GGG | GTC | ACC | GTC | CTC | AGC | ΤΤΛ | CCC | ACA | TCA | TAT | 649 |
| Val | Leu | Val | Gly | Glu | Gly | Val | Thr | Val | Leu | Ser | Leu | Pro | Thr | Ser | Tyr | |
| 305 | 310 | 315 |
| GAT CTT GGG | TAT GTG AGG | CTT GGT GAC CCC ATT CCC GCA ATA GGG CTT | 697 | ||||||||||
| Asp Leu 320 | Gly | Tyr val | Arg | Lau 325 | Gly Asp | Pro | Ile | Pro 330 | Ala | Ile | Gly | Leu | |
| GAC CCA | ΛΑΛ | ATG GTA | GCC | ACA | TGT GAC | AGC | AGT | GAC | AGG | CCC | AGA | GTC | 74S |
| Asp Pro 335 | Lys | Met Val | Ala 340 | Thr | Cys Asp | Ser | Ser 345 | Asp | Arg | Pro | Arg | Val 350 | |
| TAC ACC | ATA | ACT GCA | GCC | GAT | GAT TAC | CAA | TTC | TCA | TCA | CAG | TAC | CAA | 793 |
| Tyr Thr | Ile | Thr Ala 3SS | Ala | Asp | Asp Tyr | Gin 360 | Phe | Ser | Ser | Gin | Tyr 365 | Gin | |
| CCA GGT | GGG | GTA ACA | ATC | ACA | CTG TTC | TCA | GCC | AAC | ATT | GAT | GCC | ATC | 841 |
| Pro Gly | Gly | Val Thr 370 | Ile | Thr | Leu Phe 375 | Ser | Ala | Asn | Ile | Λ»Ρ 380 | Ala | Ile | |
| ACA AGC | CTC | AGC GTT | GGG | CGA | GAG CTC | GTG | TTT | CAA | ACA | AGC | GTC | CAC | 889 |
| Thr ser | Iieu 385 | Ser Val | Gly | Gly | Glu Lftu 390 | Val | Phe | Gin | Thr 395 | Ser | Val | lilo | |
| GGC CTT | GTA | CTG GGC | GCC | ACC | ATC TAC | CTC | ATA | GGC | TTT | GAT | GGG | ACA | 937 |
| Gly Leu 400 | Vnl | Lau Gly | Ala | Thr 405 | Ile Tyr | Leu | Ile | Gly 410 | Phe | Asp | Gly | Thr | |
| ACG GTA | ATC | ACC AGG | GCT | GTG | GCC GCA | AAC | ΛΛΤ | GGG | CTG | ACG | ACC | GGC | 985 |
| Thr Val | Ile | Thr Arg | Ala | Val | Ala Ala | Asn | Asn | Gly | Leu | Thr | Thr | Gly |
415 420 425 430
ACC GAC AAC CTT ATG CCA TTC ΛΛΤ CTT GTG ATT CCA ACA AAC GAG ΛΤΛ 1033
Thr Aep Asn Leu Mot Pro Phe Asn Leu Val Ile Pro Thr Asn Glu Ile
435 440 445
| ACC Thr | CAG CCA ATC ACA | TCC ATC ΑΛΛ CTG GAG | ATA GTG ACC | TCC ΛΑΛ ser Lys 460 | AGT ser | 1081 | ||||||||||
| Gin | Pro | Ile 450 | Thr | Ser | Ile | bys | Leu Glu 455 | Ile Val | Thr | |||||||
| GGT | GGT | CAG | GCA | GGG | GAT | CAG | ATG | TCA | TGG | TCG | GCA | AGA | GGG | AGC | CTA | 1129 |
| Gly | Gly | Gin | Ala | Gly | Asp | Gin | Met | Ser | Trp | Ser | Ala | Arg | Gly | Ser | Leu | |
| 465 | 470 | 47S |
-32CZ 299417 B6
| GCA GTG ACG ATC | CAT GGT | GGC AAC TAT CCA GGG GCC CTC CGT CCC GTC | 1177 | |||||||||||||
| Ala Val Thr 480 | Ile | His | Gly | Gly 485 | Asn Tyr | Pro | Gly | Ala 490 | Leu | Arg | Pro | Val | ||||
| ACG | CTA | GTG | GCC | TAC | GAA | AGA | GTG | GCA | ACA | GGA | TCC | GTC | GTT | ACG | GTC | 1225 |
| Thr | Leu | Val | Ala | Tyr | Glu | Arg | Val | Ala | Thr | Gly | Ser | Val | Val | Thr | Val | |
| 49S | 500 | 505 | 510 | |||||||||||||
| GCT | GGG | GTG | AGC | AAC | TTC | GAG | CTG | ATC | CCA | AAT | CCT | GAA | CTA | GCA | AAG | 1273 |
| Ala | Gly | Val | ser | Aan | Phe | Glu | Leu | ile | Pro | Asn | Pro | Glu | Leu | Ala | Lya | |
| 515 | 520 | 525 | ||||||||||||||
| AAC | CTG | GTT | ACA | GAA | TAC | GGC | CGA | τττ | GAC | CCA | GGA | GCC | ATG | AAC | TAC | 1321 |
| Asn | Leu | Val | Thr | Glu | Tyr | Gly | Arg | Phe | Asp | Pro | Gly | Ala | Met | Asn | Tyr | |
| 530 | 535 | 540 | ||||||||||||||
| ACA | ΛΑΛ | TTG | ΛΤΑ | CTG | AGT | GAG | AGG | GAC | CGT | CTT | GGC | ATC | AAG | ACC | GTC | 1369 |
| Thr | Lys | Leu | Ila | Leu | Ser | Glu | Arg | Asp | Arg | Leu | Gly | Ile | Lys | Thr | Val | |
| 545 | 550 | 555 | ||||||||||||||
| TGG | Cca | ACA | AGG | GAG | TAC | ACT | GAC | TTT | CGT | GAA | TAC | TTC | ATG | GAG | GTG | 1417 |
| Trp | Pro | Thr | Arg | Glu | Tyr | Thr | Asp | Phe | Arg | Glu | Tyr | Phe | Met | Glu | Val | |
| 560 | 565 | 570 | ||||||||||||||
| GCC | GLAC | CTC | AAC | TCT | CCC | CTG | AAG | ATT | GCA | GGA | GCA | TTC | GGC | TTC | ΛΛΛ | 1465 |
| Ala | Asp | Leu | Asn | Ser | Pro | Lou | Lys | Ile | Ala | Gly | Ala | Phe | Gly | Phe | Lys | |
| 575 | 580 | 585 | 590 | |||||||||||||
| GAC | ΛΤΛ | ATC | CGG | GCC | ΛΤΛ | AGG | AGG | ΑΤΛ | GCT | GTG | CCG | GTG | GTC | TCC | ACA | 1513 |
| Αβρ | Ile | Ile | Arg | Ala | Ile | Arg | Arg | Ile | Ala | Val | Pro | Val | Val | Ser | Thr | |
| 595 | 600 | 605 | ||||||||||||||
| TTG | TTC | CCA | CCT | GCC | GCT | CCC | CTA | GCC | CAT | GCA | ATT | GGG | GAA | GGT | GTA | 1561 |
| Lou | Phe | Pro | Pro | Ala | Ala | Pro | Leu | Ala | His | Ala | Ile | Gly | Glu | Gly | Val | |
| 610 | 615 | 620 | ||||||||||||||
| GAC | TAC | CTG | CTG | GGC | GAT | GAG | GCA | CAG | GCT | GCT | TCA | GGA | ACT | GCT | CGA | 1609 |
| Asp | Tyr | Leu | Leu | Gly | Asp | Glu | Ala | Gin | Ala | Ala | Ser | Gly | Thr | Ala | Arg | |
| 625 | 630 | 635 | ||||||||||||||
| GCC | GCG | TCA | GGA | ΛΛΛ | GCA | AGA | GCT | GCC | TCA | GGC | CGC | ΛΤΛ | AGG | CAG | CTG | 1657 |
| Ala | Ala | Ser | Gly | Lys | Ala | Arg | Ala | Ala | Ser | Gly | Arg | Ile | Arg | Gin | Leu |
640 645 650
| ACT Thr 655 | CTC GCC GCC GAC | AAG GGG | TAC GAG GTA GTC | GCG AAT CTA | TTC CAG Phe Gin 670 | ||||||||||
| Leu Ala Ala | Asp | Lys 660 | Gly | Tyr | Glu | Val | Val 665 | Ala | Asn | Leu | |||||
| GTG | CCC | CAG | AAT | CCC | GTA | GTC | GAC | GGG | ATT | CTT | GCT | TCA | CCT | GGG | GTA |
| Val | Pro | Gin | Asn | Pro | Val | Val | Aap | Gly | Ile | Leu | Ala | Ser | Pro | Gly | Val |
| 675 | 680 | 685 |
-33CZ 299417 B6
| CTC CGC GGT GCA | CAC AAC | CTC GAC TGC GTG ΤΤΛ AGA GAG GGT GCC ACG | 1801 | |||||||||||||
| Leu | Arg | Oly | Ala 690 | His | Aan | Lou Asp | Cys Val 695 | Leu | Arg | Glu | Gly 700 | Ala Thr | ||||
| CTA | TTC | CCT | GTG | GTT | ATT | ACG | ACA | GTG | GAA | GAC | GCC | ATG | ACA | CCC | AAA | 1849 |
| Leu | Phe | Pro | val | Val | Ile | Thr | Thr | Val | Glu | Asp | Ala | Met | Thr | Pro | Lys | |
| 705 | 710 | 715 | ||||||||||||||
| GCA | TTG | AAC | AGC | ΛΑΛ | ATG | TTT | GCT | GTC | ATT | GAA | GGC | GTG | CGA | GAA | GAC | 1097 |
| Ala | Leu | Asn | Ser | Lys | Met | Phe | Ala | Val | Ile | Glu | Gly | Val | Arg | Glu | Asp | |
| 720 | 72S | 730 | ||||||||||||||
| CTC | CAA | CCT | CCA | TCT | CAA | AGA | GGA | TCC | TTC | ATA | CGA | ACT | CTC | TCT | GGA | 194S |
| Leu | Gin | Pro | Pro | Ser | Gin | Arg | Gly | Ser | Phe | Tle | Arg | Thr | Leu | Ser | Gly | |
| 735 | 740 | 745 | 750 | |||||||||||||
| CAC | AGA | GTC | TAT | GGA | TAT | GCT | CCA | GAT | GGG | GTA | CTT | CCA | CTG | GAG | ACT | 1993 |
| His | Arg | Val | Tyr | Gly | Tyr | Ala | Pro | Asp | Gly | Val | Leu | Pro | Leu | Glu | Thr | |
| 755 | 760 | 765 | ||||||||||||||
| GGG | AGA | GAC | TAC | ACC | GTT | GTC | CCA | ATA | GAT | GAT | GTC | TGG | GAC | GAC | AGC | 2041 |
| Gly | Arg | Asp | Tyr | Thr | Val | Val | Pro | Ile | Asp | Asp | Val | Trp | Asp | Aep | Ser | |
| 770 | 77S | 780 | ||||||||||||||
| ATT | ATG | CTG | TCC | AAA | GAT | CCC | ATA | CCT | CCT | ATT | GTG | GGA | AAC | AGT | GGA | 2089 |
| Ile | Met | Leu | Ser | Lys | ASp | Pro | Ile | Pro | Pro | Ile | Val | Gly | Asn | Ser | Gly | |
| 705 | 790 | 795 | ||||||||||||||
| AAT | CTA | GCC | ATA | GCT | TAC | ATG | GAT | GTG | TTT | CGA | CCC | AAA | GTC | CCA | ATC | 2137 |
| Asn | Leu | Ala | Ile | Ala | Tyr | Met | Asp | Val | Phe | Arg | Pro | tys | Val | Pro | Ile | |
| 800 | 805 | 810 | ||||||||||||||
| CAT | GTG | GCT | ATG | ACG | GtíA | GCC | CTC | AAT | GCT | TGT | GGC | GAG | ATT | GAG | AAA | 2185 |
| lila | Val | Ala | Met | Thr | Gly | Ala | Leu | Asn | Ala | Cys | Gly | Glu | Ile | Glu | Lys | |
| 81S | 820 | 825 | 830 | |||||||||||||
| OTA | AGC | TTT | AGA | AGC | ACC | AAG | CTC | GCC | ACT | GCA | CAC | CGA | CTT | GGC | CTT | 2233 |
| Val | Ser | Phe | Arg | Ser | Thr | Lys | Leu | Ala | Thr | Ala | His | Arg | Leu | Gly | Leu | |
| 035 | 840 | 845 | ||||||||||||||
| AGG | TTG | GCT | GGT | CCC | GGA | GCA | TTC | GAT | GTA | AAC | ACC | GGG | CCC | AAC | TGG | 2281 |
| Arg | Leu | Ala | Gly | Pro | Gly | Ala | Phe | Aap | Val | Asn | Thr | Gly | Pro | Asn | Trp | |
| 850 | 855 | 860 | ||||||||||||||
| GCA | ACG | TTC | ATC | AAA | CGT | TTC | CCT | CAC | AAT | CCA | CGC | GAC | TGG | GAC | AGG | 2329 |
| Ala | Thr | Phe | Ile | Lye | Arg | Phe | Pro | His | Asn | Pro | Arg | Asp | Trp | Asp | Arg | |
| oeS | 870 | 875 | ||||||||||||||
| CTC | CCC | TAC | CTC | AAC | CTA | CCA | TAC | CTT | CCA | CCC | AAT | GCA | GGA | CGC | CAG | 2377 |
| Leu | Pro | Tyr | Leu | Aon | Leu | Pro | Tyr | Leu | Pro | Pro | Asn | Ala | Gly | Arg | Gin | |
| 080 | 885 | 890 |
-34CZ 299417 B6
| TAC CAC CTT GCC ATG | GCT GCA TCA GAG TTC AAA GAG ACC CCC GAA | CTC Leu 910 | 2425 | ||||||||||||
| Tyr 895 | His Leu | Ala | Met | Ala 900 | Ala | Ser | Glu | Phe | Lya 905 | Glu | Thr | Pro | Glu | ||
| GAG | AGT GCC | GTC | AGA | GCA | ATG | GAA | GCA | GCA | GCC | AAC | GTG | GAC | CCA | CTA | 2473 |
| Glu | Ser Ala | Val | Arg 915 | Ala | Met | Glu | Ala | Ala 920 | Ala | Asn | Val | Asp | Pro 925 | Leu | |
| TTC | CAA TCT | GCA | CTC | AGT | GTG | TTC | ATG | TGG | CTG | GAA | GAG | AAT | GGG | ATT | 2521 |
| Phe | Oln Sar | Ala 930 | Leu | Ser | Val | Phe | Met 935 | Trp | Leu | Glu | Glu | Asn 940 | Gly | Ile | |
| GTG | ACT GAC | ATG | GCC | AAC | TTC | GCA | CTC | AGC | GAC | CCG | AAC | GCC | CAT | CGG | 2569 |
| Val | Thr Asp 945 | Met | Ala | Asn | Phe | Ala 950 | Leu | Ser | Asp | Pro | Asn 955 | Ala | His | Arg | |
| ATG | CGA AAT | TTT | CTT | GCA | AAC | GCA | CCA | CAA | GCA | GGC | AGC | AAG | TCG | CAA | 2617 |
| Mat | Arg Asn 960 | Phe | Leu | Ala | Asn 965 | Ala | Pro | Gin | Ala | Gly 970 | Ser | Lys | Ser | Gin | |
| AGG | GCC AAG | TAC | GGG | ACA | GCA | GGC | TAC | GGA | GTG | GAG | GCT | CGG | GGC | CCC | 2665 |
| Arg 975 | Ala Lys | Tyr | Gly | Thr 980 | Ala | Gly | Tyr | Gly | Val 985 | Glu | Ala | Arg | Gly | Pro 990 | |
| ACA | CCA GAG | GAA | GCA | CAG | AGG | GAA | AAA | GAC | ACA | CGG | ATC | TCA | AAG | AAG | 2713 |
| Thr | Pro Glu | Glu | Ala 995 | Gin | Arg | Glu | Lys | Asp Thr 1000 | Arg | Ile | Ser | Lys Lys 1005 | |||
| ATG | GAG ACC | ATG | GGC | ATC | TAC | TTT | GCA | ACA | CCA | GAA | TGG | GTA | GCA | CTC | 2761 |
| Met | Glu Thr | Met Gly 1010 | Ile | Tyr | Phe | Ala Thr 1015 | Pro | Glu | Trp | Val Ala 1020 | Leu | ||||
| AAT | GGG CAC | CGA | GGG | CCA | AGC | CCC | GGC | CAG | CTA | AAG | TAC | TGG | CAG | AAC | 2809 |
| Asn | Gly His Arg 1025 | Gly | Pro | Ser | Pro Gly 1030 | Gin | Leu | Lys | Tyr Trp 1035 | Gin | Asn | ||||
| ACA | CGA GAA | ATA | CCG | GAC | CCA | AAC | GAG | GAC | TAT | CTA | GAC | TAC | GTG | CAT | 2857 |
| Thr | Arg Glu 1040 | Ile | Pro | Asp | Pro Asn 1045 | Glu | Asp | Tyr | Leu Asp 1050 | Tyr | Val | His | |||
| GCA | GAG AAG | AGC | CGG | TTG | GCA | TCA | GAA | GAA | CAA | ATC | CTA | AGG | GCA | GCT | 2905 |
| Ala Glu Lyc 1055 | Ser | Arg | Leu Ala 1060 | Ger | Glu | Glu | Gin Ile. 1065 | Leu | Arg | Ala | Ala 107O | ||||
| ACG | TCG ATC | TAC | GGG | GCT | CCA | GGA | CAG | GCA | GAG | CCA | CCC | CAA | GCT | TTC | 2953 |
| Thr | Ser Ile | Tyr | Gly Ala 1075 | Pro | Gly | Gin | Ala Glu 1080 | Pro | pro | Gin | Ala Phe 1085 | ||||
| ΛΤΛ | GAC GAA | GTT | GCC | AAA | GTC | TAT | GAA | ATC | AAC | CAT | GGA | CGT | GGC | CCA | 3001 |
| Ile | Aop GlU | Val Ala 1090 | Lys | Val | Tyr | Glu Ile 1095 | Asn | Hle | Gly | Arg Gly 1100 | Pro |
-35CZ 299417 B6
| AAC CAA GAA CAG | ATG AAA GAT CTG CTC TTG ACT GCG ATG GAG ATG AAG | 3049 | |||||||
| Aen | Gin Glu Gin 1105 | Met | Lys | Aap | Leu Leu 1110 | Leu | Thr Ala Met Glu Met 1115 | Lye | |
| CAT | CGC AAT CCC | AGG | CGG | GCT | CTA CCA | AAG | CCC AAG CCA AAA CCC | AAT | 3097 |
| Mis | Arg Aen Pro | Arg | Arg | Ala | Leu Pro | Lys | Pro Lys Pro Lya Pro | Asn | |
| 1120 | 1125 | 1130 | |||||||
| GCT | CCA ACA CAG | AGA | CCC | CCT | GGT CGG | CTG | GGC CGC TGG ATC AGG | ACC | 3145 |
| Ala | Pro Thr Gin | Arg | Pro | Pro | Gly Arg | Leu | Gly Arg Trp lle Arg | Thr | |
| 1135 | 1140 | 114 5 | 1150 |
QTC TCT GAT GAG GAC CTT GAG TGAGGCTCCT GGGAGTCTCC CGACACCACC 3196
Val Ser Asp Glu Asp Leu Olu
1155
COCGCAGGTG TGGACACCAA TTCGGCCTTA CAACATCCCA AATTGGATCC GTTCGCGGGT 3256
CCCCT 3261
INFORMACE PRO SEQ ID NO:30:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 1012 AMINOKYSELIN TYP: AMINOKYSELINA TYPOLOGIE: LINEÁRNÍ
TYP MOLEKULY: protein POPIS SEKVENCE: SEQ ID NO:30:
| Mot 1 | Thr | Asn | Leu | Gin 5 | Asp | Gin | Thr | Gin | Gin 10 | Ile | Val | Pro | Phe | ile 15 | Arg |
| Ser | Leu | Leu | Met 20 | Pro | Thr | Thr | Gly | Pro 25 | Ala | Ser | Ile | Pro | Asp 30 | Asp | Thr |
| Leu | Glu | Lys 35 | His | Thr | Leu | Arg | Ser 40 | Glu | Thr | Ser | Thr | Tyr 45 | Asn | Leu | Thr |
| Val | Gly 50 | Asp | Thr | Gly | Ser | Gly 55 | Leu | Ile | Val | Phe | Phe 60 | Pro | Gly | Phe | Pro |
| Gly 65 | Sar | Ile | Val | Gly | Ala 70 | Mls | Tyr | Thr | Leu | Gin 75 | Gly | Asn | Gly | Asn | Tyr 80 |
| Lye | Pho | Asp | Gin | Met 85 | Leu | Leu | Thr | Ala | Gin 90 | Asn | Leu | Pro | Ala | Ser 95 | Tyr |
| λβη | Tyr | Cys | Arg | Leu | Val | Ser | Arg | Ser | Leu | Thr | Val | Arg | Ser | Ser | Thr |
100 los 110
-36CZ 299417 B6
| Leu | Pro | Gly 115 | Gly | Val | Tyr | Ala | heu 120 | Asn | Gly | Thr | Ile | Asn 125 | Ala | Val | Thr |
| Phe | Gin 130 | Oly | Ser | Leu | Ser | Glu 135 | Leu | Thr | Asp | Val | Ser 140 | Tyr | Asn | Gly | Leu |
| Met 145 | Ser | Ala | Thr | Ala | Aan 150 | Ile | Asn | Asp | Lys | Ile 155 | Gly | Asn | Val | Leu | Val 160 |
| Gly | Glu | Gly | Val | Thr 16S | Val | Leu | Ser | Leu | Pro 170 | Thr | Ser | Tyr | Asp | Leu 175 | Gly |
| Tyr | Val | Arg | Leu 180 | Oly Asp | Pro | Ile | Pro 185 | Ala | Ile | Gly | Leu | Asp 190 | Pro | Lys | |
| Met | Val | Ala 195 | Thr | cys | Asp | Ser | Ser 200 | Asp | Arg | Pro | Arg | Val 205 | Tyr | Thr | Ile |
| Thr | Ala 210 | Ala | Asp | Asp | Tyr | Gin 215 | Phe | Ser | Ser | Gin | Tyr 220 | Gin | Pro | Gly | Gly |
| Val 225 | Thr | Ile | Thr | Leu | Phe 230 | Ser | Ala | Asn | Ile | Asp 235 | Ala | Ile | Thr | Ser | Leu 240 |
| Ser | Val | Gly | Gly | Glu 245 | Leu | Val | Phe | Gin | Thr 2S0 | Ser | Val | His | Gly | Leu 255 | Val |
| Lou | Gly | Ala | Thr 260 | Ile | Tyr | Leu | Ile | Gly 265 | Phe | Asp | Gly | Thr | Thr 270 | Val | Ile |
| Thr | Arg | Ala 275 | Val | Ala | Ala | Asn | Asn 200 | Gly | Leu | Thr | Thr | Gly 285 | Thr | Asp | Asn |
| Leu | Met 290 | Pro | Phe | Asn | Leu | Val 295 | Ile | Pro | Thr | Asn | Glu 300 | Ile | Thr | Gin | Pro |
| Ile 305 | Thr | Ser | Ile | Lys | Leu 310 | Glu | Ile | Val | Thr | Ser 315 | Lys | Ser | Gly | Gly | Gin 320 |
| Ala | Gly | Asp | Gin | Met 325 | Ser | Trp | Ser | Ala | Arg 330 | Gly | Ser | Leu | Ala | Val 335 | Thr |
| Ile | His | Gly | Gly 340 | Asn | Tyr | Pro | Gly | Ala 345 | Leu | Arg | Pro | val | Thr 350 | Leu | Val |
| Ala | •tyt | Glu 355 | Axg | Val | Ala | Thr | Gly 360 | Ser | Val | Val | Thr | Val 365 | Ala | Gly | Val |
| ser | Aan 370 | Pbe | Glu | Leu | Ile | Pro 375 | Asn | Pro | Glu | Leu | Ala 380 | Lys | Asn | Leu | Val |
| Thr | GlU | Tyr | Gly | Arg | Phe | Asp | Pro | Gly | Ala | Met | Asn | Tyr | Thr | Lys | Leu |
-37CZ 299417 B6
385 390
Ile Leu Ser Glu Arg Asp Arg 405
Arg Glu Tyr Thr Asp Phe Arg 420
Asn Ser Pro Leu Lys Ile Ala 435
Arg Ala Ile Arg Arg Ile Ala 4S0 455
Pro Ala Ala Pro Leu Ala His 465 470
Leu Gly Asp Glu Ala Gin Ala 485
Gly Lys Ala Arg Ala Ala Ser 500
Ala Asp Lys Gly Tyr Glu Val 515
Asn Pro Val Val Asp Gly Ile 530 535
Ala His Asn Leu Asp Cys Val 545 550
Val Val ile Thr Thr Val Glu 565
Ser Lya Met Phe Ala Val Ile 580
Pro Ser Gin Arg Gly Ser Phe 595
Tyr Gly Tyr Ala Pro Asp Gly 610 615
Tyr Thr Val Val Pro Ile Asp 625 630
Ser Lys Asp pro Ile Pro Pro 645
Ile Ala Tyr Met Asp Val Phe 660
395 400
| Leu | Gly | Ile 410 | Lys | Thr | Val | Trp | Pro 415 | Thr |
| Glu | Tyr 425 | Phe | Met | Glu | Val | Ala 430 | Asp | Leu |
| Gly 440 | Ala | Phe | Gly | Phe | Lys 445 | Asp | Ile | Ile |
| Val | Pro | val | Val | Ser 460 | Thr | Leu | Phe | Pro |
| Ala | Ile | Gly | Glu 475 | Gly | Val | Asp | Tyr | Leu 480 |
| Ala | Ser | Gly 490 | Thr | Ala | Arg | Ala | Ala 495 | Ser |
| Gly | Arg 505 | Ile | Arg | Gin | Leu | Thr 510 | Leu | Ala |
| Val 520 | Ala | Asn | Leu | Phe | Gin 525 | Val | Pro | Gin |
| Leu | Ala | Sex | Pro | Gly 540 | Val | Leu | Arg | Gly |
| Leu | Arg | Glu | Gly 555 | Ala | Thr | Leu | Phe | Pro 560 |
| Asp | Ala | Met 570 | Thr | Pro | Lys | Ala | Leu 575 | Asn |
| Glu | Gly 585 | Val | Arg | Glu | Asp | Leu 590 | Gin | Pro |
| Ile 600 | Arg | Thr | Leu | Ser | Gly 605 | His | Arg | Val |
| Val | Leu | Pro | Leu | Glu 620 | Thr | Gly | Arg | Asp |
| Asp | Val | Trp | Asp 635 | Asp | Ser | ile | Met | Leu 640 |
| Ile | Val | oiy 650 | Asn | Ser | Gly | Asn | Leu 655 | Ala |
| Arg | Pro | bys | Val | Pro | Ile | His | Val | Ala |
665 670
-38CZ 299417 B6
| Met | Thr | Gly G75 | Ala | Leu | Asn | Ala | cys 680 | Gly | GlU | Ile | Glu | Lys 685 | Val | Ser | Phe |
| Arg | Ser 690 | Thr | Lye | Leu | Ala | Thr 695 | Ala | His | Arg | Leu | Gly 700 | Leu | Arg | Leu | Ala |
| Gly 705 | Pro | Gly | Ala | Phe | Asp 710 | Val | Asn | Thr | Gly | Pro 715 | Asn | Trp | Ala | Thr | Phe 720 |
| Ile | hye | Arg | Phe | Pro 725 | His | Asn | Pro | Arg | Asp 730 | Trp | Asp | Arg | Leu | Pro 735 | Tyr |
| beu | Aen | Leu | Pro 740 | Tyr | Leu | Pro | Pro | Asn 745 | Ala | Gly | Arg | Gin | Tyr 750 | His | Leu |
| Ala | Met | Ala 755 | Ala | ser | Glu | Phe | Lya 760 | Glu | Thr | Pro | Glu | Leu 765 | Glu | Ser | Ala |
| Val | Arg 770 | Ala | Met | Glu | Ala | Ala 775 | Ala | Aen | Val | Aep | Pro 780 | Leu | Phe | Gin | Ser |
| Ale 785 | Leu | Ser | Val | Phe | Met 790 | Trp | Leu | Glu | Glu | Asn 795 | Oly | Ile | Val | Thr | Asp 800 |
| Met | Ala | Aon | Phe | Ala 005 | Leu | Ser | Asp | Pro | Asn 810 | Ala | Hle | Arg | Met | Arg BIS | Asn |
| Phe | Leu | Ala | Asn 820 | Ala | Pro | Gin | Ala | Gly 825 | Ser | Lya | Ser | Gin | Arg 830 | Ala | Lys |
| Tyr | Gly | Thr 83S | Ala | Gly | Tyr | Gly | Val 840 | Glu | Ala | Arg | Gly | Pro 845 | Thr | Pro | Glu |
| Glu | Ala 850 | Gin | Ary | Glu | Lys | Asp 855 | Thr | Arg | Ile | Ser | Lys 860 | Lys | Met | Glu | Thr |
| Met 865 | Gly | Ile | Tyr | Phe | Ala 870 | Thr | Pro | Glu | Trp | val 875 | Ala | Leu | Aen | Gly | Hle 880 |
| Arg | Gly | Pro | Ser | Pro 085 | Gly | Gin | Leu | Lys | Tyr 890 | Trp | Gin | Asn | Thr | Arg 895 | Glu |
| Ile | Pro | Asp | Pro 900 | Asn | Glu | Asp | Tyr | Leu 90S | Asp | Tyr | val | His | Ala 910 | Glu | Lys |
| Ser | Arg | beu 915 | Ala | Ser | Glu | Glu | Gin 920 | Ile | Leu | Arg | Ala | Ala 925 | Thr | Ser | Ile |
| Tyr | Gly 930 | Ala | Pro | Gly | Gin | Ala 935 | Glu | Pro | Pro | Gin | Ala 940 | Phe | Ile | Asp | Glu |
| Val | Ala | Lys | Val | Tyr | Glu | Ile | Asn | His | Gly | Arg | Gly | Pro | Aen | om | Glu |
-39CZ 299417 B6
945 950 955 960
| Gin | Met | Lys | Asp | Leu 965 | Leu | Leu | Thr | Ala | Met 970 | Glu | Met | Lys | His | Arg 975 | Asn |
| Pro | Arg | Arg | Ala 980 | Leu | Pro | Lys | Pro | Lys 905 | Pro | Lys | Pro | Asn | Ala 990 | Pro | Thr |
| Gin | Arg | Pro | Pro | Gly | Arg | Leu | Gly | Arg | Trp | Ile | Arg | Thr | Val | Ser | Asp |
995 1000 1005
Glu Aep Leu Glu 1010
INFORMACE PRO SEQ ID NO:31:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 3264 PÁRŮ BÁZÍ TYP: NUKLEOVÁ KYSELINA ŘETĚZEC: JEDNODUCHÝ ío TYPOLOGIE: KRUHOVÁ
TYP MOLEKULY: cDNA ZNAKY: JMÉNO/KLÍČ: CDS UMÍSTĚNÍ: 97..531
POPIS SEKVENCE: SEQ IDNO:31:
GGATACGATC GGTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCATCAC TGCCTTGTTC 60
CTGGTTGGAA CTCCTCTTTC TGCTGTACTA TCGTTG ATG GTG AGT AGA GAT CAG 114
Met Val Ser Arg Aep Gin
1015
ACA AAC GAT CGC AGC GAT GAC AAA CCT GAT GGA TCA CAC CCA ACA GAT 162
Thr Asn Asp Arg Ser Asp AGp Lys Pro Asp Gly Ser His Pro Thr Asp
1020 1025 1030
TGT TCC GTT CAT J\C<3 GAG CCT TCT GAT GCC AAC GAC CGG ACC GGC GTC 210
Cys Ser Val His Thr Glu Pro Ser Asp Ala Asn Asp Arg Thr Gly Val
1035 1040 1045 1050
CAT TCC GGA CGA CAC CCT GGA GAA GCA CAC ACT CAG GTC CGA AAC CTC 250
Hio Ser Gly Arg Hie Pro Gly Glu Ala His Thr Gin Val Arg Asn Leu
1055 1060 1065
GAC TTA CAA CTT GAC TGT AGG GGA TAC AGO GTC AGG ACT AAT TGT CTT 306
-40CZ 299417 B6
| Asp Leu | Gin | Leu Aap Cys Arg Gly Tyr Arg | Val | Arg Thr | Asn Cys 1080 | Leu | |
| 1070 | 1075 | ||||||
| TTT CCC | TGG | ATT CCC | TGG TTC AGT TGT AGG | TGC | TCA CTA | CAC ACT | GCA 354 |
| Phe Pro | Trp | Ile Pro | Trp Phe Ser Cys Arg | Cys | Ser Leu | His Thr | Ala |
| 1085 | 1090 | 1095 | |||||
| GAG CAG | TGG | GAA CTA | CCA ATT CGA CCA GAT | GCT | CCT GAC | AGC GCA | GAA 402 |
| Glu Gin | Trp | Glu Leu | Pro Ile Arg Pro Asp | Ala | Pro Asp | Ser Ala | Glu |
| 1100 | 110S | 1110 | |||||
| CCT GCC | TGC | CAG CTA | CAA CTA CTG CAG GCT | AGT | GAG CAG | GAG TCT | AAC 450 |
| Pro Ala | Cye | Gin Leu | Gin Leu Leu Gin Ala | Ser | Olu Gin | Glu Ser | Asn |
| 1115 | 1120 | 1125 | 1130 | ||||
| CGT ACG | GTC | AAG CAC | ACT CCC TGG TGG CGT | TTA | TGC ACT | AAA CGG | AAC 490 |
| Arg Thr | Val | Lys His | Thr Pro Trp Trp Arg | Leu | cys Thr | Lya Arg | Asn |
| 1135 1140 | 1145 | ||||||
| CAT AAA | CGC | AGT GAC | CTT CCA CGG AAG CCT | GAG | TGAGTTGACT GACTACAGCT 551 | ||
| His Lys | Arg | Ser Asp | Leu Pro Arg Lys Pro | GlU |
1150 1155
ACAACGGGCT GATGTCAGCC ACTGCGAACA TCAACGACAA GATCGGGAAC GTTCTAGTTO Sil
GAGAAGGGGT GACTGTTCTC AGTCTACCGA CTTCATATGA CCTTAGTTAT GTGAGACTCG 671
GTGACCCCAT CCCCGCAGCA GGACTCGACC CGAAGTTGAT GGCCACGTOC GACAGTAGTG 731
ACAGACCCAG AGTCTACACC ATAACAGCTG CAGATGAATA CCAATTCTCG TCACAACTCA 791
TCCCGAGTGG CGTGAAGACC ACACTGTTCT CCGCCAACAT CGATGCTCTC ACCAGCTTCA 851
GCGTTGGTGG TGAGCTTGTC TTCAGCCAAG TAACGATCCA AAGCATTGAA GTGGACGTCA 911
CCATTCACTT CATTGGGTTT GACGGGACAG ACGTAGCAGT CAAGGCAGTT GCAACAGACT 971
TTGGGCTGAC AACTGGGACA AACAACCTTG TGCCATTCAA CCTGCTGGTC CCAACAAATG 1031
AGATCACCCA GCCCATCACT TCCATGAAAC TAGAGGTTGT GACCTACAAG ATTGGCGGCA 1091
CCGCTGGTGA CCCAATATCA TGGACAGTQA GTGGTACACT AGCTOTGACG GTGCACGGAG 1151
GCAACTACCC TGGGGCTCTC CGTCCTGTCA CCCTGGTGGC CTATGAACGA GTGGCTGCAG 1211
GATCTGTTGT CACAGTTGCA GGQGTGAGCA ACTTCGAGCT AATCCCCAAC CCTGAGCTTG 1271
CAAAGAACCT AGTTACAGAG TATGGCCGCT TTGACCCCGG AGCAATGAAC TACACCAAAC 1331
TAATACTGAG TGAGAGAGAT CGTCTAGGCA TCAAGACAGT CTGGCCCACC AGGGAGTACA 1391 .CCGATTTCAG GGAGTACTTC ATGGAGGTTG CAGATCTCAA CTCACCCCTA AAGATTGCAG 1451
-41 CZ 299417 B6
| GAGCATTTGG | CTTTAAGGAC | ATAATCCGAG | CCATTCGGAA | GATTGCGGTG | CCAGTGGTAT | 1511 |
| CCACACTCTT | CCCTCCAGCT | gcacccctag | CACATGCAAT | CGGAGAAGGT | GTAGACTACC | 1571 |
| TCCTGGGCGA | CGAGGCCCAA | GCAGCCTCAG | GGACAGCTCG | AGCCGCGTCA | GGAAAAGCTA | 1631 |
| GAGCTGCCTC | AGGACGAATA | AGGCAGCTAA | CTCTCGCAGC | TGACAAGGGG | TGCGAGGTAG | 1691 |
| TCGCCAACAT | GTTCCAGGTG | CCCCAGAATC | CCATTGTTGA | TGGCATTCTG | GCATCCCCAG | 1751 |
| GAATCCTGCG | TGGCGCACAC | AACCTCGACT | GCGTGCTATG | GGAGGGAGCC | actcttttcc | 1811 |
| CTGTTGTCAT | TACGACACTC | GAGGATGAGC | TGACCCCCAA | GGCACTGAAC | AGCAAAATGT | 1B71 |
| TTGCTGTCAT | TGAAGGTGTG | CGAGAGGACC | TCCAGCCTCC | ATCCCAACGG | GGATCCTTCA | 1931 |
| TTCGAACTCT | CTCTGGCCAT | AGAGTCTATG | GCTATGCCCC | AGACGOAGTA | CTGCCTCTGG | 1991 |
| AGACCGGGAG | AGACTACACC | GTTGTCCCAA | TTGATGATGT | GTGGGACGAT | AGCATAATGC | 2051 |
| TGTCGCAGGA | CCCCATACCT | CCAATCATAG | GGAACAGCGG | CAACCTAGCC | ATAGCATACA | 2111 |
| TGGATGTCTT | CAGGCCCAAG | GTCCCCATCC | ACGTGGCTAT | GACAGGGGCC | CTCAATGCCC | 2171 |
| GCGGTGAGAT | CGAGAGTGTT | ACGTTCCGCA | GCACCAAACT | CGCCACAGCC | CACCGACTTG | 2231 |
| GCATGAAGTT | AGCTGGTCCT | GGAGCCTATG | ACATTAATAC | AGGACCTAAC | TGGGCAACGT | 2291 |
| TCGTCAAACG | TTTCCCTCAC | AATCCCCGAG | ACTGGGACAG | GTTGCCCTAC | CTCAACCTTC | 2351 |
| CTTATCTCCC | ACCAACAGCA | GGACGTCAGT | TCCATCTAGC | CCTGGCTGCC | TCCGAQTTCA | 2411 |
| AAGAGACCCC | AGAACTCGAA | úacgctgtgc | GCGCAATGGA | TGCCGCTGCA | AATGCCGACC | 2471 |
| CATTGTTCCG | CTCAGCTCTC | CAGGTCTTCA | TGTGGTTGGA | AGAAAACGGG | ATTGTGACCG | 2531 |
| ACATGGCTAA | CTTCGCCCTC | AGCGACCCAA | ACGCGCATAG | GATGAAAAAC | 'ITCCTAGCAA | 2591 |
| ACGCACCCCA | GGCTGGAAGC | AAGTCGCAGA | GGGCCAAGTA | TGGCACGGCA | GGCTACGGAG | 2651 |
| TGGAGGCTCG | AGGCCCCACA | CCAGAAGAGG | CACAGAGGGA | AAAAGACACA | CGGATCTCCA | 2711 |
| AGAAGATGGA | AACAATGGGC | ATCTACTTCG | CGACACCGGA | ATGGGTGGCT | CTCAACGGGC | 2771 |
| ACCGAGGCCC | AAGCCCCGGC | CAACTCAAGT | ACTGGCAAAA | CACAAGAGAA | ATACCAGAGC | 2831 |
| CCAATOAGGA | CTACCCAGAC | TATGTGCACG | CGGAGAAGAG | CCGGTTGGCG | TCAGAAGAAC | 2891 |
| AGATCCTACQ | i GGCAGCCACG | TCGATCTACG | ; GGGCTCCAGG | i ACAGGCTGAA | CCACCCCAGG | 2951 |
| CCTTCATAGA CGAGGTCGCC | ; AGGGTCTATG | ! AAATCAACCA TGGGCGTGGT | ' CCAAACCAGG | 3011 |
-42CZ 299417 B6
AGCAGaTGAA GGACCTOCTC CTGACTGCGA TGGAGATGAA GCATCGCAAT CCCAGGCGGG 3071 CTCCACCAAA GCCAAAGCCA AAACCCAATG CTCCATCACA GAGACCCCCT GGACGGCTGG 3131 GCCGCTGGAT CAGGACGGTG TCCGACGAGQ ACTTGGAGTG AGGCTCCTGG GAGTCTCCCQ 31S1 ACACTACCCG CGCAGGTGTG GACACCAATT CGGCCTTCTA CCATCCCAAA TTGGATCCGT 3251
TCGCGGGTCC CCT
INFORMACE PRO SEQ ID NO:32:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 145 AMINOKYSELIN TYP: AMINOKYSELINA TYPOLOGIE: LINEÁRNÍ
TYP MOLEKULY: protein POPIS SEKVENCE: SEQ IDNO:32:
| Met 1 | Val | Ser | Arg | Asp 5 | Gin | Thr | ASn | Asp | Arg 10 | Ser | ASp | Aep | bys | Pro 15 | Asp |
| Gly | Ser | His | Pro 20 | Thr | Asp | Cys | Ser | Val 25 | His | Thr | Glu | Pro | Ser 30 | Asp | Ala |
| Asn | Asp | Arg 35 | Thr | Gly | Val | Hic | Ser 40 | Gly | Arg | His | Pro | Gly 45 | Glu | Ala | His |
| Thr | Gin 50 | Val | Arg | Asn | beu | Asp 55 | beu | Gin | I^eu | Asp | Cys 60 | Arg | Gly | Tyr | Arg |
| Vol 65 | Arg | Thr | Asn | Cys | beu 70 | Phe | Pro | Trp | Ile | Pro 75 | Tip | Phe | Ser | Cys | Aťg 80 |
| Cye | Ser | Leu | Hie | Thr 85 | Ala | Glu | Gin | Trp | Glu 90 | b«!U | Pro | Ile | Arg | Pro 95 | Asp |
| Ala | Pro | Asp | Ser 100 | Ala | Glu | Pro | Ala | Cys 10S | Gin | beu | Gin | beu | beu 110 | Gin | Ala |
| Ser | Glu | Gin 115 | Glu | Ser | Asn | Arg | Thr 120 | Val | bys | His | Thr | Pro 125 | Trp | Trp | Arg |
| beu | Cys 130 | Thr | bys | Arg | Asn | His 135 | bys | Arg | Ser | Aep | beu 140 | Pro | Arg | bys | Pro |
Glu
145
INFORMACE PRO SEQ ID NO:33:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 3264 PÁRŮ BÁZÍ
TYP: NUKLEOVÁ KYSELINA
-43 CZ 299417 B6
ŘETĚZEC: JEDNODUCHÝ TYPOLOGIE: KRUHOVÁ
TYP MOLEKULY: cDNA 5 ZNAKY: JMÉNO/KLÍČ: CDS
UMÍSTĚNÍ: 131..3169
POPIS SEKVENCE: SEQ ID NO:33:
GGATACGATG GGTCTGACCC CGGGGGAGTC ACCCGGGGAC AGGCCATCAC TGCCTTGTTC 60
CTGGTTGGAA CTCCTCTTTC TGCTGTACTA TCGTTGATGO TGAGTAGAGA TCAGACAAAC 120
GATCGCAGCG ATG ACA AAC CTG ATG GAT CAC ACC CAA CAG ATT GTT CCG 169
Met Thr Asn Leu Met Asp His Thr Gin Gin Ile Val Pro
ISO 155
| TTC Phe | ΛΤΛ 1l e 160 | CGG AGC CTT | CTG ATG CCA ACG ACC GGA CCG GCG TCC ATT | CCG Pro | 217 | |||||||||||
| Arg Ser | Leu | Leu Met 165 | Pro Thr Thr | Gly Pro Ala 170 | Ser | ile | ||||||||||
| GAC | GAC | ACC | CTG | GAG | AAG | CAC | ACA | CTC | AGG | TCC | GAA | ACC | TCG | ACT | TAC | 265 |
| Asp | Asp | Thr | Leu | Glu | Lys | His | Thr | Leu | Arg | Ser | Glu | Thr | Ser | Thr | Tyr | |
| 175 | 180 | ies | 190 | |||||||||||||
| AAC | TTG | ACT | GTA | GGG | GAT | ACA | GGG | TCA | GGA | CTA | ATT | GTC | TTT | TTC | CCT | 313 |
| Asn | Leu | Thr | Val | Gly | Asp | Thr | Gly | Ser | Gly | Leu | Ile | Val | Phe | Phe | Pro | |
| 195 | 200 | 205 | ||||||||||||||
| GGA | TTC | CCT | GGT | TCA | GTT | GTA | GGT | GCT | CAC | TAC | ACA | CTG | CAG | AGC | AGT | 361 |
| Gly | Pho | Pro | Gly | Ser | Val | Val | Gly | Ala | His | Tyr | Thr | Leu | Gin | Ser | Ser | |
| 210 | 215 | 220 | ||||||||||||||
| GGG | AAC | TAC | CAA | TTC | GAC | CAG | ATG | CTC | CTG | ACA | GCG | CAG | AAC | CTG | CCT | 409 |
| Gly | Asn | Tyr | Gin | Phe | Asp | Gin | Met | Leu | Leu | Thr | Ala | Gin | Asn | Leu | Pro | |
| 225 | 230 | 235 | ||||||||||||||
| GCC | AGC | TAC | AAC | TAC | TGC | AGG | CTA | GTG | AGC | AGG | AGT | CTA | ACC | GTA | CGG | 4S7 |
| Ala | Ser | Tyr | Asn | Tyr | Cyo | Arg | Leu | Val | Ser | Arg | Ser | Leu | Thr | Val | Arg | |
| 240 | 245 | 250 |
-44CZ 299417 B6
| TCA AGC ACA CTC CCT | GGT GGC GTT TAT GCA CTA AAC GGA ACC ATA | AAC Aon 270 | 505 | |||||||||||||
| 6er 255 | ser Thr | Leu | Pro | Gly 260 | Gly | Val | Tyr | Ala | Leu 265 | Aan | Gly | Thr | Ile | |||
| GCA | GTG | ACC | TTC | CAC | GGA | AGC | CTG | AGT | GAG | TTG | ACT | GAC | TAC | AGC | TAC | 553 |
| Ala | Val | Thr | Phe | His | Gly | Ser | Leu | Ser | Glu | Leu | Thr | Asp | Tyr | Ser | Tyr | |
| 275 | 280 | 285 | ||||||||||||||
| AAC | GGG | CTG | ATG | TCA | GCC | ACT | GCG | AAC | ATC | AAC | GAC | AAG | ATC | GGG | AAC | 601 |
| Aan | Gly | Leu | Met | Ser | Ala | Thr | Ala | Asn | Ile | Asn | Λβρ | Lys | Ile | Gly | Asn | |
| 290 | 235 | 300 | ||||||||||||||
| GTT | CTA | GTT | GGA | GAA | GGG | GTG | ACT | GTT | CTC | AGT | CTA | CCG | ACT | TCA | TAT | 643 |
| Val | Leu | Val | Gly | GlU | Gly | Val | Thr | Val | Leu | Ser | Leu | Pro | Thr | Ser | Tyr | |
| 305 | 310 | 315 | ||||||||||||||
| GAC | CTT | AGT | TAT | GTG | AGA | CTC | GGT | GAC | CCC | ATC | CCC | GCA | GCA | GGA | CTC | 697 |
| Asp | Leu | Ser | Tyr | Val | Leu | Gly Asp | Pro | Ile | Pro | Ala | Ala | Gly | Leu | |||
| 320 | 325 | 330 | ||||||||||||||
| GAC | CCG | AAG | TTG | ATG | GCC | ACG | TGC | GAC | AGT | AGT | GAC | AGA | CCC | AGA | GTC | 745 |
| ASp | Pro | Lys | Leu | Mot | Ala | Thr | Cys | Aop | Ber | Ser | Asp | Arg | Pro | Arg | Val | |
| 335 | 340 | 345 | 350 | |||||||||||||
| TAC | ACC | ATA | ACA | GCT | GCA | GAT | GAA | TAC | CAA | TTC | TCG | TCA | CAA | CTC | ATC | 793 |
| Tyr | Thr | Ile | Thr | Ala | Ala | Asp | Glu | Tyr | Gin | Phe | ser | Ser | Gin | Leu | Ile | |
| 355 | 360 | 365 | ||||||||||||||
| CCG | AGT | GGC | GTG | AAG | ACC | ACA | CTG | TTC | TCC | GCC | AAC | ATC | GAT | GCT | CTC | 841 |
| Pro | Ser | Gly | Val | Lye | Thr | Thr | Leu | Phe | Ser | Ala | Asn | Ile | Asp | Ala | Leu | |
| 370 | 375 | 380 | ||||||||||||||
| ACC | AGC | TTC | AGC | GTT | GGT | GGT | GAG | CTT | GTC | rrc | AGC | CAA | GTA | ACG | ATC | 809 |
| Thr | Ser | Phe | Ser | Val | Gly Gly | Glu | Leu | Val | Phe | Ser | Gin | Val | Thr | Ile | ||
| 385 | 390 | 395 | ||||||||||||||
| CAA | AGC | ATT | GAA | GTG | GAC | GTC | ACC | ATT | CAC | TTC | ATT | GGG | TTT | GAC | GGG | 937 |
| Gin | Ser | Ile | Glu | Val | Asp | Val | Thr | Ile | His | Phe | Ile | Gly | Phe | Asp | Gly | |
| 400 | 405 | 410 | ||||||||||||||
| ACA | GAC | GTA | GCA | GTC | AAG | GCA | GTT | GCA | ACA | GAC | TTT | GGG | CTG | ACA | ACT | 985 |
| Thr | Asp | Val | Ala | Val | Lys | Ala | Val | Ala | Thr | Asp | Phe | Gly | Leu | Thr | Thr | |
| 415 | 420 | 425 | 430 | |||||||||||||
| GGG | ACA | AAC | AAC | CTT | GTG | CCA | TTC | AAC | CTG | GTG | GTC | CCA | ACA | AAT | GAG | 1033 |
| Gly | Thr | Aen | Aon | Leu | Val | Pro | Phe | Asn | Leu | Val | Val | Pro | Thr | Aen | Glu | |
| 435 | 440 | 445 | ||||||||||||||
| ATC | ACC | CAG | CCC | ATC | ACT | TCC | ATG | AAA | CTA | GAG | GTT | GTG | ACC | TAC | AAG | 1001 |
| I le | Thr | Gin | Pro | Xle | Thr | Ser | Met | Lys | Len | Glu | Val | Val | Thr | Tyr | Lys | |
| 450 | 455 | 460 |
-45CZ 299417 B6
| ΑΪΤ GGC GGC | ACC Thr | GCT GGT GAC | CCA ATA TCA TGG ACA GTG AGT GGT ACA | 1129 | ||||||||||
| Ile | Gly Gly 465 | Ala | Gly Asp | Pro 470 | Ile | Ser | Trp | Thr | Val 475 | Ser | Gly | Thr | ||
| CTA | GCT GTG | ACG | GTG | CAC GGA | GGC | AAC | TAC | CCT | GGG | GCT | CTC | CGT | CCT | 1177 |
| Leu | Ala Val 400 | Thr | val | Hle Gly Gly 485 | Aen | Tyr | Pro | Gly 490 | Ala | Leu | Arg | Pro | ||
| GTC | ACC CTG | GTG | GCC | TAT GAA | CGA | GTG | GCT | GCA | GGA | TCT | GTT | GTC | ACA | 1225 |
| Val 495 | Thr Leu | Val | Ala | Tyr Glu 500 | Arg | Val | Ala | Ala 505 | Gly | Ser | Val | Val | Thr 510 | |
| GTT | GCA GGG | GTG | AGC | AAC TTC | GAG | CTA | ATC | CCC | AAC | CCT | GAG | CTT | GCA | 1273 |
| val | Ala Gly | Val | Ser 515 | Aen Phe | GlU | Leu | Ile 520 | Pro | Asn | Pro | Glu | Leu 525 | Ala | |
| AAG | AAC CTA | GTT | ACA | GAG TAT | GGC | CGC | TTT | GAC | CCC | GGA | GCA | ATG | AAC | 1321 |
| Lye | Asn Leu | val 530 | Thr | Glu Tyr | Gly | Arg 535 | Phe | Asp | Pro | Gly | Ala 540 | Met | Asn | |
| TAC | ACC AAA | CTA | ATA | CTG AGT | GAG | AGA | GAT | CGT | CTA | GGC | ATC | AAG | ACA | 1369 |
| Tyr | Thr Lys 545 | Leu | Ile | Leu Ser | Glu SSO | Arg | Aep | Arg | Leu | Gly 555 | Ile | Lys | Thr | |
| GTC | TGG CCC | ACC | AGG | GAG TAC | ACC | GAT | TTC | AGG | GAG | TAC | TTC | ATG | GAG | 1417 |
| Val | Trp Pro 560 | Thr | Arg | Glu Tyr 565 | Thr | Asp | Phe | Arg | GlU 570 | Tyr | Phe | Met | Glu | |
| GTT | GCA GAT | CTC | AAC | TCA CCC | CTA | AAG | ATT | GCA | GGA | GCA | TTT | GGC | TTT | 1465 |
| val 575 | Ala Aep | Leu | Aen | Ser Pro 500 | Leu | Lys | Ile | Ala 505 | Gly | Ala | Phe | Gly | Phe 590 | |
| Aag | GAC ATA | ATC | CGA | GCC ATT | CGG | AAG | ATT | GCG | GTG | CCA | GTG | GTA | TCC | 1513 |
| Lye | Λερ Ile | Ile | Arg 595 | Ala Ile | Arg | Lye | Ile 600 | Ala | Val | Pro | Val | Val 605 | Ser | |
| ACA | CTC TTC | CCT | CCA | GCT GCA | CCC | CTA | GCA | CAT | GCA | ATC | GGA | GAA | GGT | 1561 |
| Thr | Leu Phe | Pro 610 | Pro | Ala Ala | Pro | Leu 615 | Ala | ills | Ala | Ile | Gly 620 | Glu | Gly | |
| GTA | GAC TAC | CTC | CTG | GGC GAC | GAG | GCC | CAA | GCA | GCC | TCA | GGG | ACA | GCT | 1609 |
| Val | Asp Tyr 62S | Leu | Leu | Gly Aep | Glu 630 | Ala | Gin | Ala | Ala | Ser 635 | Gly | Thr | Ala | |
| CGA | GCC GCG | TCA | GGA | AAA GCT | AGA | GCT | GCC | TCA | GGA | CGA | ATA | AGG | CAG | 1657 |
| Arg | Ala Ala 640 | Ser | Gly | Lys Ala €45 | Arg | Ala | Ala | Ser | Gly 650 | Arg | Ile | Arg | Gin | |
| CTA | ACT CTC | GCA | GCT | GAC AAG | GGG | TGC | GAG | GTA | GTC | GCC | AAC | ATG | TTC | 1705 |
| Leu 655 | Thr Leu | Ala | Ala | Asp Lye 660 | Gly | Cys | Glu | Val 665 | Val | Ala | Asn | Met | Phe 670 |
-46CZ 299417 B6
| CAS GTG | CCC CAG AAT CCC | ATT GTT GAT GGC ATT CTG GCA TCC CCA GGA | 1753 | |||||||||||||
| Gin | Val | Pro | Gin Aan Pro 67S | Ile Val | Asp | Gly 680 | Ile | Leu | Ala | Ser | Pro 685 | Gly | ||||
| ATC | CTG | CGT | GGC | GCA | CAC | AAC | CTC | GAC | TGC | GTG | CTA | TGG | GAG | GGA | GCC | 1801 |
| Ile | Leu | Arg | Gly | Ala | His | Asn | Leu | Asp | Cys | Val | Leu | Trp | Glu | Gly | Ala | |
| 630 | 695 | 700 | ||||||||||||||
| ACT | CTT | TTC | CCT | GTT | GTC | ATT | ACG | ACA | CTC | GAG | GAT | GAG | CTG | ACC | CCC | 1849 |
| Thr | Leu | Phe | Pro | Val | Val | Ile | Thr | Thr | Leu | Glu | Asp | Glu | Leu | Thr | Pro | |
| 705 | 710 | 715 | ||||||||||||||
| AAG | GCA | CTG | AAC | AGC | AAA | ATG | TTT | GCT | GTC | ATT | GAA | GGT | GTG | CGA | GAG | 1897 |
| Lye | Ala | I<eu | Aen | Ser | Lye | Met | Phe | Ala | Val | Ile | Glu | Gly | Val | Arg | Glu | |
| 720 | 725 | 730 | ||||||||||||||
| GAC | ctc | CAG | CCT | CCA | TCC | CAA | CGG | GGA | TCC | TTC | ATT | CGA | ACT | CTC | TCT | 1945 |
| Asp | Leu | Gin | Pro | Pro | Ser | Gin | Arg | Gly | Ser | Phe | Ile | Arg | Thr | Leu | Ser | |
| 735 | 740 | 745 | 750 | |||||||||||||
| GGC | CAT | AGA | GTC | TAT | GGC | TAT | GCC | CCA | GAC | GGA | GTA | CTG | CCT | CTG | GAG | 1993 |
| Gly | Hie | Arg | Val | Tyr | Gly | Tyr | Ala | Pro | Asp | Gly | Val | Leu | Pro | Leu | Glu | |
| 755 | 760 | 765 | ||||||||||||||
| ACC | GGG | AGA | GAC | TAC | ACC | GTT | GTC | CCA | ATT | GAT | GAT | GTG | TGG | GAC | GAT | 2041 |
| Thr | Gly | Arg | Asp | Tyr | Thr | Val | Val | Pro | Ile | Asp | Asp | Val | Trp | Asp | Asp | |
| 770 | 775 | 780 | ||||||||||||||
| AGC | ATA | ATG | CTG | TCG | CAG | GAC | CCC | ATA | CCT | CCA | ATC | ΛΤΛ | GGG | AAC | AGC | 20B9 |
| Ser | Ilo | Mot | Leu | Ser | Gin | Λερ | Pro | Ilo | Pro | Pro | Ile | Ile | Gly | Aen | Ser | |
| 785 | 790 | 795 | ||||||||||||||
| GGC | AAC | CTA | GCC | ATA | GCA | TAC | ATG | GAT | GTC | TTC | AGG | CCC | AAG | GTC | CCC | 2137 |
| Gly | Asn | Leu | Ala | Ile | Ala | Tyr | Met | Aap Val | Phe | Arg | Pro | Lya | Val | Pro | ||
| 800 | 805 | 810 | ||||||||||||||
| ATC | CAC | GTG | GCT | ATG | ACA | GGG | GCC | CTC | AAT | GCC | CGC | GGT | GAG | ATC | GAG | 2185 |
| Ile | Hie | Val | Ala | Met | Thr | Gly | Ala | Leu | Asn | Ala | Arg | Gly | Glu | Ile | Glu | |
| 815 | 820 | 825 | 830 | |||||||||||||
| AGT | GTT | ACG | TTC | CGC | AGC | ACC | AAA | CTC | GCC | ACA | GCC | CAC | CGA | CTT | GGC | 2233 |
| ser | Val | The | Phe | Arg | Ser | Thr | Lys | Leu | Ala | Thr | Ala | His | Arg | Leu | Gly | |
| 835 | 840 | 845 | ||||||||||||||
| ATG | AAG | TTA | GCT | GGT | CCT | GGA | GCC | TAT | GAC | ATT | AAT | ACA | GGA | CCT | AAC | 2281 |
| Mec | Lys | Leu | Ala | Gly | pro | Gly | Ala | Tyr | Λερ | Ile | Aen | Thr | Gly | Pro | Asn | |
| 850 | 855 | 860 | ||||||||||||||
| TGG | GCA | ACG | TTC | GTC | AAA | CGT | TTC | CCT | CAC | AAT | CCC | CGA | GAC | TGG | GAC | 2329 |
| Trp | Ala | Thr | Phe | Val | Lys | Arg | Phe | Pro | His | Asn | Pro | Arg | Αερ | Trp | Asp | |
| 865 | 070 | 875 |
-47CZ 299417 B6
| AGG TTG CCC | TAC CTC AAC | CTT CCT TAT CTC CCA CCA ACA Leu Pro Tyr Leu Pro Pro Thr | GCA GGA CGT | 2377 | |||||
| Arg Leu 880 | Pro | Tyr | Leu Asn | Ala | Gly | Arg | |||
| 885 | 890 | ||||||||
| CAG TTC | CAT | CTA | GCC CTG | GCT GCC TCC | GAG TTC AAA GAG | ACC | CCA | GAA | 2425 |
| Gin Phe 895 | His | Leu | Ala Leu 900 | Ala Ala Ser | Glu Phe Lys Glu 905 | Thr | Pro | Glu 910 | |
| CTC GAA | GAC | GCT | GTG CGC | GCA ATG GAT | GCC GCT GCA AAT | GCC | GAC | CCA | 2473 |
| Lau Glu | Asp | Ala | Val Arg 915 | Ala Met Asp | Ala Ala Ala Asn 920 | Ala | Asp 925 | Pro | |
| TTG TTC | CGC | TCA | GCT CTC | CAG GTC TTC | ATG TGG TTG GAA | GAA | AAC | GGG | 2S21 |
| Leu Phe | Arg | Ser 930 | Ala Leu | Gin Val Phe 935 | Met Trp Leu Glu | Glu 940 | Aen | Oly | |
| ATT GTG | ACC | GAC | ATG GCT | AAC TTC GCC | CTC AGC GAC CCA | AAC | GCG | CAT | 2569 |
| Ile Val | Thr 945 | Asp | Met Ala | Asn Phe Ala 950 | Leu Ser Asp Pro 955 | Asn | Ala | His | |
| AGG ATG | AAA | AAC | TTC CTA | GCA AAC GCA | CCC CAG GCT GGA | AGC | AAG | TCG | 2617 |
| Arg Met 960 | Lya | Asn | Phe Leu | Ala Asn Ala 965 | Pro Gin Ala Gly 970 | Ser | Lys | Ser | |
| CAG AGG | GCC | AAG | TAT GGC | ACG GCA GGC | TAC GGA GTG GAG | GCT | CGA | GGC | 2665 |
| Gin Arg 975 | Ala | Lys | Tyr Gly 980 | Thr Ala Gly | Tyr Gly val Glu 98S | Ala | Arg | Gly 990 | |
| CCC ACA | CCA | GAA | GAG GCA | CAG AGG GAA | AAA GAC ACA CGG | ATC | TCC | AAG | 2713 |
| Pro Thr | Pro | Glu | Glu Ala 995 | Gin Arg Glu | Lys Asp Thr Arg 1000 | Ile | Ser Lye 1005 | ||
| AAG ATG | GAA | ACA | ATG GGC | ATC TAC TTC | GCG ACA CCG GAA | TGG | GTG | GCT | 2761 |
| Lye Met | Glu | Thr Met Gly 1010 | Ile Tyr Phe Ala Thr Pro Glu 1015 | Trp Val 1020 | Ala | ||||
| CTC AAC | GGG | CAC | CGA GGC | CCA AGC CCC | GGC CAA CTC AAG | TAC | TGG | CAA | 2809 |
| Lau Asn | Gly His 1025 | Arg Gly | Pro Ser Pro 1030 | Gly Gin Leu Lys Tyr 1035 | Trp | Gin | |||
| AAC ACA | AGA | GAA | ATA CCA | GAG CCC ΛΛΤ | GAG GAC TAC CCA | GAC | TAT | GTG | 2857 |
| Asn Thr Arg 1040 | Glu | Ile Pro | Glu Pro Asn 1045 | Glu Asp Tyr Pro 1050 | Asp | Tyr | val | ||
| CAC GCG | GAG | AAG | AGC CGG | TTG GCG TCA | GAA GAA CAG ATC | CTA | CGG | GCA | 2905 |
| His Ala 1055 | Glu | Lys | Ser Arg Leu Ala Ser 1060 | Glu Glu Gin Ile 1065 | Leu | Arg | Ala 1070 | ||
| GCC ACG | TCG | ATC | TAC GGG | GCT CCA GGA | CAG GCT GAA CCA | CCC | CAG | GCC | 2953 |
| Ala Thr | Ser | Ile | Tyr Gly Ala Pro Gly 1075 | Gin Ala Glu Pro 1080 | Pro | Gin Ala 1085 |
-48CZ 299417 B6
TTC ATA GAC GAG GTC GCC AGG GTC ΤΛΪ GAA ATC AAC CAT GGG CGT GGT 3001
Phe Ile Aep Glu Val Ala Arg Val Tyr Glu Ile Asn Mis Gly Arg Gly
1090 1095 1100
CCA AAC CAG GAG CAG ATG AAG GAC CTG CTC CTG ACT GCG ATG GAG ATG 3049
Pro Asn Gin Glu Gin Met Lye Aep Leu Leu Leu Thr Ala Met Glu Met
1105 1110 1115
AAG CAT CGC AAT CCC AGG CGG GCT CCA CCA AAG CCA AAG CCA AAA CCC 3097
Lye His Arg Aen Pro Arg Arg Ala Pro Pro Lys Pro Lye Pro Lye Pro
1120 1125 1130
AAT GCT CCA TCA CAG AGA CCC CCT GGA CGG CTG GGC CGC TGG ATC AGG 3145
Asn Ala Pro aer Gin Arg Pro Pro Gly Arg Leu Gly Arg Trp ile Arg
1135 1140 1145 1150
ACG GTC TCC GAC GAG GAC TTG GAG TGAGGCTCCT GGGAGTCTCC CGACACTACC 3199 Thr Val ser Aep Olu Aep Leu Glu
1155
CGCGCAGGTG TGGACACCAA TTCGGCCTTC TACCATCCCA AATTGGATCC GTTCGCGGGT 3259 CCCCT 3264
INFORMACE PRO SEQ ID NO:34:
SEKVENČNÍ CHARAKTERISTIKY DÉLKA: 1013 AMINOKYSELIN TYP: AMINOKYSELINA TYPOLOGIE: LINEÁRNÍ
TYP MOLEKULY: protein POPIS SEKVENCE: SEQ ID N0:34:
| Mot 1 | Thr | Asn | Leu | Met 5 | Asp | His | Thr | Gin | Gin 10 | Ile | Val | Pro | Phe | Ile 15 | Arg |
| Ser | Leu | Leu | Met 20 | Pro | Thr | Thr | Gly | Pro 25 | Ala | Ser | Ile | Pro | Asp 30 | Asp | Thr |
| Leu | Glu | Lys 35 | His | Thr | Leu | Arg | Ser 40 | Glu | Thr | Ser | Thr | Tyr 45 | Asn | Leu | Thr |
| Val | Gly 50 | Aap | Thr | Gly | Ser | Gly 55 | Leu | Ile | Val | Phe | phe 60 | Pro | Gly | Phe | Pro |
| cly 65 | Ser | Val | Val | Gly | Ala 70 | His | Tyr | Thr | Leu | Gin 75 | Ser | Ser | Gly | Asn | Tyr 80 |
| Gin | Pho | Asp | Gin | Met | Leu | Leu | Thr | Ala | Gin | Asn | Leu | Pro | Ala | Ser | Tyr |
-49CZ 299417 B6
90 95
| Asn | Tyr | Cys | Arg 100 | Leu | Val | Sor | Arg | Ser 105 | Leu | Thr | Val | Arg | ser 110 | Ser | Thr |
| Len | Pro | Gly 115 | cly | Val | Tyr | Ala | Leu 120 | Asn | Gly | Thr | ile | Asn 125 | Ala | Val | Thr |
| Phe | His 130 | Gly | Ser | Leu | Ber | Glu 135 | Leu | Thr | Aep | Tyr | Ser 140 | Tyr | Asn | Gly | Leu |
| Met 145 | Ser | Ala | Thr | Ala | Asn 150 | Ile | Asn | Asp | Lys | Ile 155 | Gly | Asn | Val | Leu | Val 160 |
| Gly | Glu | Gly | Val | Thr 165 | Val | Leu | Ser | Leu | Pro 170 | Thr | Ser | Tyr | Asp | Leu 175 | Ser |
| Tyr | Val | Arg | Leu 180 | Gly | Asp | Pro | Ile | Pro 185 | Ala | Ala | Gly | Leu | Asp 190 | Pro | Lys |
| Leu | Met | Ala 195 | Thr | Cys | Aap | Ser | Ser 200 | Asp | Arg | Pro | Arg | Val 205 | Tyr | Thr | Ile |
| Thr | Ala 210 | Ala | Asp | Olu | Tyr | Gin 215 | Phe | Ser | Ser | Gin | Leu 220 | Ile | Pro | Ser | Gly |
| Val 225 | Lys | Thr | Thr | Leu | Phe 230 | Ser | Ala | Asn | Ile | Aep 235 | Ala | Leu | Thr | Ser | Phe 240 |
| Ser | Val | Gly Gly | Glu 245 | Leu | Val | Phe | Ser | Gin 250 | val | Thr | Ile | Gin | Ser 255 | Ile | |
| Glu | Val | Asp | Val 260 | Thr | Ile | His | Phe | Ile 265 | Gly | Phe | Aep | Gly | Thr 270 | Asp | Val |
| Ala | Val | Lya 275 | Ala | Val | Ala | Thr | Asp 280 | Phe | Gly | Leu | Thr | Thr 285 | Gly | Thr | Asn |
| Asn | Leu 290 | Val | Pro | Phe | Asn | Leu 295 | Val | Val | Pro | Thr | Asn 300 | Glu | Ile | Thr | Gin |
| Pro 305 | Ilo | Thr | Ser | Met | Lys 310 | Leu | Glu | Val | Val | Thr 315 | Tyr | Lye | Ile | Gly | Gly 320 |
| Thr | Ala | Gly | Asp | Pro 325 | Ile | Ser | Trp | Thr | Val 330 | Ser | Gly | Thr | Leu | Ala 335 | Val |
| Thr | Val | His | Gly 340 | Gly | Asn | Tyr | Pro | Gly 345 | Ala | Leu | Arg | Pro | Val 350 | Thr | Leu |
| Val | Ala | Tyr 355 | Glu | Arg | Val | Ala | Ala 360 | Gly | Ser | Val | Val | Thr 365 | Val | Ala | Gly |
-50CZ 299417 B6
| Vol | Ser 370 | Asn | Phe | GlU Leu | Ile 375 | Pro | Asn | Pro | Glu | Leu 380 | Ala | Lys | Asn | |
| Val 305 | Thr | Glu | Tyr | Gly Arg 390 | Phe | ASp | pro | oiy | Ala 395 | Met | Λεη | Tyr | Thr | |
| Leu | Xle | Leu | Ser | GlU 405 | Arg | Asp | Arg | Leu | Gly 410 | Ile | Lys | Thr | Val | Trp 415 |
| Thr | Arg | Glu | Tyr 420 | Thr | Aep | Phe | Arg | Glu 425 | Tyr | Phe | Met | Glu | Val 430 | Ala |
| Leu | Aon | Ser 435 | Pro | Leu | Lye | Ile | Ala 440 | Gly | Ala | Phe | Gly | Phe 445 | Lys | Aep |
| Ile | Arg 450 | Ala | Uc | Arg | Lye | Ile 455 | Ala | Val | Pro | Val | Val 460 | Ser | Thr | Leu |
| Pro 465 | Pro | Ala | Ala | Pro | Leu 470 | Ala | His | Ala | Xle | Gly 47S | Glu | Gly | val | Asp |
| Leu | Leu | Gly | Asp | Glu 405 | Ala | Gin | Ala | Ala | Ser 490 | Gly | Thr | Ala | Arg | Ala 495 |
| Ser | Gly | Lys | Ala 500 | Arg | Ala | Ala | Ser | Gly 505 | Arg | Ile | Arg | Gin | Leu 510 | Thr |
| Ala | Ala | Asp 515 | Lys | Gly | Cye | Glu | Val 520 | Val | Ala | Asn | Met | Phe 525 | Gin | Val |
| Gin | Asn 530 | Pro | Ile | Val | Asp | Gly 535 | Ile | Leu | Ala | Ser | Pro 540 | Gly | Ile | Leu |
| Gly 545 | Ala | Mis | Asn | Leu | Asp 550 | cys | Val | Leu | Trp | Glu 555 | Gly | Ala | Tht | Leu |
| Pro | Val | Val | Ile | Thr 565 | Thr | Letí | Glu | Asp | Glu S70 | Leu | Thr | Pro | Lys | Ala 575 |
| Aen | Ser | Lyo | Met 500 | Phe | Ala | val | Ile | Glu 585 | Gly | Val | Arg | Glu | Asp 590 | Leu |
| Pro | Pro | Ser 595 | Gin | Arg | Gly | Ser | Phe 600 | Ile | Arg | Thr | Leu | Sesr 605 | Gly | His |
| Val | Tyr 610 | Gly | Tyr | Ala | Pro | Asp 615 | Gly | Val | Leu | Pro | Leu 620 | Glu | Thr | Gly |
| Asp | Tyr | Thr | Val | Val | Pro | Ile | ASp | Asp | Val | Trp | Asp | Asp | Ser | Ile |
625 630 635
Leu
Lys
400
Pro
Λερ
Ile
Phe
Tyr
400
Ala
Leu
Pro
Arg
Phe
560
Leu
Gin
Arg
Arg
Met
640
-51 CZ 299417 B6
| Leu | Ser | Gin | Asp | Pro 645 | Ile | Pro | Pro | Ile | Ile 650 | Gly | Asn | Ser | Gly | Asn 655 | Leu |
| Ala | Ile | Tyr 660 | Met | Asp | Val | Phe | Arg 665 | Pro | Lys | Val | Pro | Ile 670 | His | Val | |
| Ala | Met | Thr 675 | Oly | Ala | Leu | Asn | Ala 680 | Arg | Gly | Glu | Ile | Glu 685 | Ser | Val | Thr |
| Phe | Arg 690 | Ser | Thr | Lys | Leu | Ala 695 | Thr | Ala | Hle | Arg | Leu 700 | cly | Met | bys | Leu |
| Ala 705 | Gly | Pro | Gly | Ala | Tyr 710 | Asp | Ile | Asn | Thr | Gly 715 | Pro | Asn | Trp | Ala | Thr 720 |
| Phe | Val | Lya | Arg | Phe 725 | Pro | His | Asn | Pro | Arg 730 | Asp | Trp | Asp | Arg | Leu 735 | Pro |
| Tyr | Leu | Asn | Leu 740 | Pro | Tyr | Leu | Pro | Pro 745 | Thr | Ala | Gly | Arg | Gin 750 | Phe | Hle |
| Leu | Ala | Leu 7S5 | Ala | Ala | Ser | Glu | Phe 760 | Lys | Glu | Thr | Pro | Glu 765 | Leu | Glu | Asp |
| Ala | Val 770 | Arg | Ala | Met | Asp | Ala 775 | Ala | Ala | Aen | Ala | Asp 780 | Pro | Leu | Phe | Arg |
| Sor 785 | Ala | Leu | Gin | Val | Phe 790 | Met | Trp | Leu | Glu | Glu 795 | Asn | Gly | Ile | Val | Thr 800 |
| Asp | Met | Ala | Aon | Phe 005 | Ale | Leu | Ser | Asp | Pro 010 | Asn | Ala | His | Arg | Met 815 | Lys |
| Aon | Phe | Leu | Ala 820 | Asn | Ala | Pro | Gin | Ala 825 | Gly | Ser | Lys | Ser | Gin 830 | Arg | Ala |
| Lys | 'JTyr | Gly 83S | Thr | Ala | Gly | Tyr Gly 840 | Val | Glu | Ala | Arg | Gly 845 | Pro | Thr | Pro | |
| Glu | Glu 850 | Ala | Gin | Arg | Glu | bys 855 | Asp | Thr | Arg | Ile | Ser 060 | Lys | Lys | Met | Glu |
| Thr 865 | Met | Gly | Ile | Tyr | Phe 870 | Ala | Thr | Pro | Glu | Trp 875 | Val | Ala | Leu | Asn | Gly 880 |
| His | Arg | Gly | Pro | Ser 805 | Pro | Gly | Gin | Leu | Lys 090 | Tyr | Trp | Gin | Asn | Thr 89S | Arg |
| Glu | Ile | Pro | Glu | pro | Asn | Glu | Asp | Tyr | Pro | Asp | Tyr | Val | His | Ala | Glu |
900 905 910
| Lya | Ser | Arg 915 | Leu | Ala | Ser | Glu | Glu 920 | Gin | ile | Leu Arg | Ala 925 | Ala | Thr | Ser |
| Ile | Tyr 93Q | Gly | Ala | Pro | Gly | Gin 935 | Ala | Glu | Pro | Pro Gin 940 | Ala | Phe | Ile | Asp |
| Olu 945 | Val | Ala | Arg | Val | Tyr 950 | Glu | Ile | Aen | Mls | Gly Arg 955 | Oly | Pro | Asn | Gin 960 |
| Olu | Gin | Met | Lys | Asp 965 | Leu | Leu | Leu | Thr | Ala 970 | Met GlU | Met | Lys | Mls 975 | Arg |
| Asn | Pro | Arg | Arg 900 | Ala | Pro | Pro | Lya | Pro 985 | Lys | Pro Lys | Pro | Asn 990 | Ala | Pro |
| Ser | Gin | Arg 995 | Pro | Pro | Gly | Arg | Leu Gly 1000 | Arg | Trp Ile | Arg Thr 1005 | Val | Ser |
Ληρ Glu Asp Leu Glu
Claims (10)
- PATENTOVÉ NÁROKY10 1. Způsob přípravy živého Bimaviru, kterým je virus infekční choroby bursy, vyznačující se t í m , že zahrnuje následující kroky:přípravu jedné nebo více cDNA genomových segmentů A a B viru infekční choroby bursy, transkripci této jedné nebo těchto více cDNA za vzniku syntetických RNA transkriptů, kde tyto RNA transkripty jsou pozitivními RNA transkripty uvedených segmentů A a B,15 transfekci hostitelských buněk těmito syntetickými RNA transkripty, inkubaci těchto hostitelských buněk v kultivačním médiu a izolaci živého infekčního viru infekční choroby bursy z uvedeného kultivačního média.
- 2. Způsob podle nároku 1,vyznačuj ící se tím, že uvedenými hostitelskými buňkami 20 jsou buňky Věro z kočkodana.
- 3. Způsob podle nároku 1,vyznačuj ící se tím, že uvedené genomové segmenty A a B viru infekční choroby bursy jsou nezávisle na sobě připraveny z různých kmenů viru infekční choroby bursy.
- 4. Způsob podle nároku 3,vyznačující se tím, že uvedený segment A je přítomný v plasmidů pUC19FLAD78 mající SEQ ID NO: 27 a 29 nebo pUC18FLA23 mající SEQ ID NO: 31 a33.30 5. Způsob podle nároku3, vyznač u j í cí se t í m , že uvedený segment B je přítomný v plasmidů pUCI18FLBP2 mající SEQ ID NO: 25.6. Způsob přípravy živého chimérického viru infekční choroby bursy, vyznačující se t í m , že zahrnuje kroky35 přípravy jedné nebo více cDNA genomových segmentů A a B viru infekční choroby bursy,-53CZ 299417 B6 kde jsou tyto cDNA odvozeny z více než jednoho kmene viru infekční choroby bursy, transkripci této jedné nebo těchto více cDNA za vzniku syntetických RNA transkriptů, kde tyto RNA transkripty jsou pozitivními RNA transkripty uvedených segmentů A a B, transfekci hostitelské buňky těmito syntetickými RNA transkripty,
- 5 inkubaci této hostitelské buňky v kultivačním médiu a izolaci živého chimérického viru infekční choroby bursy z uvedeného kultivačního média.
- 7. Způsob podle nároku 6, vyznačující se tím, že uvedené genomové segmenty A a B viru infekční choroby bursy jsou nezávisle na sobě připraveny z různých kmenů viru infekční ío choroby bursy.
- 8. Plasmid vybraný ze skupiny sestávající z pUCI
- 9FLAD78 mající SEQ ID NO: 27 a 29, pUC18FLA23 mající SEQ ID NO: 31 a 33 a pUC19FLBP2 mající SEQ ID NO: 25.15 9. Způsob přípravy vakcíny, vy zn ač u j í cí se tí m , že zahrnuje přípravu živého chimérického viru infekční choroby bursy podle nároku 6 a inaktívaci tohoto živého chimérického viru infekční choroby bursy před podáváním.
- 10. Způsob podle nároku 1,vyznačující se tím, že uvedenými hostitelskými buňkami20 jsou drůbeží buňky.
- 11. Způsob podle nároku 10, vyznačující se tím, že uvedenými drůbežími buňkami jsou kuřecí nebo krocaní buňky.25 12. Způsob podle nároku 11, vyznačující se tím, že uvedenými drůbežími buňkami jsou kuřecí embryonální fibroblastové buňky nebo kuřecí embryonální ledvinové buňky.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US08/708,541 US5871744A (en) | 1996-09-05 | 1996-09-05 | Method for generating birnavirus from synthetic RNA transcripts |
| PCT/US1997/012955 WO1998009646A1 (en) | 1996-09-05 | 1997-07-31 | A method for generating birnavirus from synthetic rna transcripts |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CZ74299A3 CZ74299A3 (cs) | 1999-08-11 |
| CZ299417B6 true CZ299417B6 (cs) | 2008-07-16 |
Family
ID=24846203
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CZ0074299A CZ299417B6 (cs) | 1996-09-05 | 1997-07-31 | Zpusob prípravy živého Birnaviru, kterým je virusinfekcní choroby bursy, plasmid a zpusob prípravyvakcíny |
Country Status (18)
| Country | Link |
|---|---|
| US (1) | US5871744A (cs) |
| EP (2) | EP0959901A4 (cs) |
| JP (1) | JP4670025B2 (cs) |
| KR (1) | KR100475427B1 (cs) |
| CN (1) | CN1168500C (cs) |
| AT (1) | ATE460178T1 (cs) |
| AU (1) | AU741055B2 (cs) |
| BR (1) | BR9712012B1 (cs) |
| CA (1) | CA2264488C (cs) |
| CZ (1) | CZ299417B6 (cs) |
| DE (1) | DE69739805D1 (cs) |
| ES (1) | ES2342325T3 (cs) |
| HU (1) | HUP9904365A3 (cs) |
| IL (2) | IL128804A0 (cs) |
| NO (1) | NO991074L (cs) |
| NZ (1) | NZ334667A (cs) |
| PL (1) | PL190220B1 (cs) |
| WO (1) | WO1998009646A1 (cs) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6596280B1 (en) * | 1997-07-31 | 2003-07-22 | University Of Maryland Biotechnology Institute | Method for generating birnavirus from synthetic RNA transcripts |
| CA2324478C (en) * | 1998-03-31 | 2011-08-23 | University Of Maryland Biotechnology Institute | A method for generating nonpathogenic, infectious pancreatic necrosis virus (ipnv) from synthetic rna transcripts |
| WO2000012677A2 (en) * | 1998-09-01 | 2000-03-09 | The University Of Hong Kong | Generation of recombinant infectious bursal disease viruses by reverse genetics technology and the use of the recombinant viruses as attenuated vaccines |
| HUP9802974A1 (hu) * | 1998-12-19 | 2000-09-28 | Mezőgazdasági Biotechnológiai Kutatóközpont | Fertőző bursitis elleni DNS-vakcina |
| US6492148B1 (en) * | 1999-03-05 | 2002-12-10 | Akzo Nobel Nv | Genetically engineered cell culture adapted infectious bursal disease virus (IBDV) mutants |
| US6468984B1 (en) * | 1999-06-08 | 2002-10-22 | Innovo Biotechnologies Ltd. | DNA vaccine for protecting an avian against infectious bursal disease virus |
| EP1069187A1 (en) * | 1999-07-14 | 2001-01-17 | Stichting Dienst Landbouwkundig Onderzoek | Mosaic Infectious Bursal Disease Virus vaccines |
| US7431930B2 (en) * | 2002-03-01 | 2008-10-07 | Intervet International B.V. | Infectious bursal disease virus (IBDV) mutant expressing virus neutralising epitopes specific for classic-and variant IBDV strains |
| ES2217967B1 (es) * | 2003-03-31 | 2006-01-01 | Consejo Sup. Investig. Cientificas | Procedimiento de produccion de particulas virales vacias (vlps) del virus inductor de la bursitis infecciosa (ibdv), composiciones necesarias para su puesta a punto y su uso en la elaboracion de vacunas frente al ibdv. |
| ES2307345B1 (es) * | 2004-01-21 | 2009-11-13 | Consejo Sup. Investig. Cientificas | Capsidas vacias quimericas del virus causante de la enfermedad de la bursitis infecciosa (ibdv), su procedimiento de obtencion y aplicaciones. |
| ES2307346B1 (es) * | 2004-01-21 | 2009-11-13 | Consejo Sup. Investig. Cientificas | Capsidas vacias (vlps(-vp4)) del virus causante de la enfermedad de la bursitis infecciosa (ibdv), su procedimiento de obtencion y aplicaciones. |
| US7244432B2 (en) * | 2004-12-08 | 2007-07-17 | University Of Maryland Biotechnology Institute | Infectious bursal disease virus (IBDV) variant from Georgia |
| ES2310062B1 (es) * | 2005-07-15 | 2009-11-13 | Bionostra, S.L. | Particulas pseudovirales vacias quimericas derivadas del virus causante de la enfermedad de la bursitis infecciosa (ibdv), procedimiento de obtencion y aplicaciones. |
| KR100757541B1 (ko) * | 2005-11-08 | 2007-09-10 | 엘지전자 주식회사 | 플라즈마 디스플레이 장치 및 그의 화상 처리방법 |
| EP2101797B1 (en) * | 2006-12-01 | 2015-08-26 | HepC Terápia Kereskedelmi és Szolgáltató Zártkörüen Müködö Részvénytársaság | Compositions and methods for the treatment of viral hepatitis |
| CN101016547B (zh) * | 2007-01-25 | 2010-07-28 | 浙江大学 | 海洋双rna病毒mabv重组蛋白的制备方法与应用 |
| EP2407534A1 (en) | 2010-07-14 | 2012-01-18 | Neo Virnatech, S.L. | Methods and reagents for obtaining transcriptionally active virus-like particles and recombinant virions |
| CN109321583B (zh) * | 2018-09-29 | 2023-08-15 | 福建省农业科学院畜牧兽医研究所 | 一种构建番鸭呼肠孤病毒反向遗传系统的方法 |
| CA3149497A1 (en) | 2019-08-09 | 2021-02-18 | Nutcracker Therapeutics, Inc. | Methods and apparatuses for manufacturing for removing material from a therapeutic composition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US4530831A (en) * | 1982-11-02 | 1985-07-23 | Akzo N.V. | Infectious Bursal Disease vaccine |
| US5192539A (en) * | 1988-07-21 | 1993-03-09 | Akzo N.V. | Infectious bursal disease virus production in continuous cell lines |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1334941C (en) * | 1985-05-30 | 1995-03-28 | Ahmed Azad Azad | Cloning and expression of a host-protective immunogens of ibdv |
| AU602875B2 (en) * | 1985-12-18 | 1990-11-01 | British Technology Group Limited | Newcastle disease virus gene clones |
| US5518724A (en) * | 1988-08-02 | 1996-05-21 | University Of Maryland | Infectious bursal disease virus |
| NZ235474A (en) * | 1989-10-18 | 1992-12-23 | Univ Maryland | Virus capable of inducing infectious bursal disease; assay and vaccine |
| AU7955591A (en) * | 1990-05-04 | 1991-11-27 | University Of Maryland At College Park, The | Specific dna sequences related to an ibdv protein including vectors, hosts and vaccines |
| JPH06509235A (ja) * | 1991-07-26 | 1994-10-20 | ヴァイロジェネティクス コーポレイション | 伝染性ファブリキウス嚢病ウイルス組換えポックスウイルスワクチン |
| US5788970A (en) * | 1994-03-29 | 1998-08-04 | The University Of Maryland College Park | Chimeric infectious bursal disease virus CDNA clones, expression products and vaccines based thereon |
-
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- 1996-09-05 US US08/708,541 patent/US5871744A/en not_active Expired - Lifetime
-
1997
- 1997-07-31 KR KR10-1999-7001899A patent/KR100475427B1/ko not_active IP Right Cessation
- 1997-07-31 PL PL97332184A patent/PL190220B1/pl not_active IP Right Cessation
- 1997-07-31 AT AT07012332T patent/ATE460178T1/de not_active IP Right Cessation
- 1997-07-31 CN CNB971976880A patent/CN1168500C/zh not_active Expired - Fee Related
- 1997-07-31 AU AU38918/97A patent/AU741055B2/en not_active Ceased
- 1997-07-31 CZ CZ0074299A patent/CZ299417B6/cs not_active IP Right Cessation
- 1997-07-31 DE DE69739805T patent/DE69739805D1/de not_active Expired - Lifetime
- 1997-07-31 HU HU9904365A patent/HUP9904365A3/hu unknown
- 1997-07-31 CA CA002264488A patent/CA2264488C/en not_active Expired - Fee Related
- 1997-07-31 JP JP51264398A patent/JP4670025B2/ja not_active Expired - Fee Related
- 1997-07-31 IL IL12880497A patent/IL128804A0/xx active IP Right Grant
- 1997-07-31 NZ NZ334667A patent/NZ334667A/xx not_active IP Right Cessation
- 1997-07-31 ES ES07012332T patent/ES2342325T3/es not_active Expired - Lifetime
- 1997-07-31 BR BRPI9712012-0A patent/BR9712012B1/pt not_active IP Right Cessation
- 1997-07-31 WO PCT/US1997/012955 patent/WO1998009646A1/en not_active Application Discontinuation
- 1997-07-31 EP EP19970936185 patent/EP0959901A4/en not_active Withdrawn
- 1997-07-31 EP EP07012332A patent/EP1930026B1/en not_active Expired - Lifetime
-
1999
- 1999-03-03 IL IL128804A patent/IL128804A/en not_active IP Right Cessation
- 1999-03-04 NO NO19991074A patent/NO991074L/no not_active Application Discontinuation
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Also Published As
| Publication number | Publication date |
|---|---|
| CZ74299A3 (cs) | 1999-08-11 |
| DE69739805D1 (de) | 2010-04-22 |
| KR100475427B1 (ko) | 2005-03-10 |
| EP0959901A4 (en) | 2002-11-04 |
| ATE460178T1 (de) | 2010-03-15 |
| AU741055B2 (en) | 2001-11-22 |
| JP4670025B2 (ja) | 2011-04-13 |
| EP0959901A1 (en) | 1999-12-01 |
| CA2264488C (en) | 2009-04-07 |
| EP1930026A1 (en) | 2008-06-11 |
| CN1168500C (zh) | 2004-09-29 |
| NZ334667A (en) | 2000-09-29 |
| NO991074L (no) | 1999-04-29 |
| KR20010029487A (ko) | 2001-04-06 |
| CN1229358A (zh) | 1999-09-22 |
| BR9712012A (pt) | 2000-01-18 |
| EP1930026B1 (en) | 2010-03-10 |
| HUP9904365A3 (en) | 2001-06-28 |
| BR9712012B1 (pt) | 2009-08-11 |
| US5871744A (en) | 1999-02-16 |
| HUP9904365A2 (hu) | 2000-04-28 |
| PL190220B1 (pl) | 2005-11-30 |
| JP2001501082A (ja) | 2001-01-30 |
| PL332184A1 (en) | 1999-08-30 |
| WO1998009646A1 (en) | 1998-03-12 |
| IL128804A (en) | 2006-08-01 |
| IL128804A0 (en) | 2000-01-31 |
| CA2264488A1 (en) | 1998-03-12 |
| NO991074D0 (no) | 1999-03-04 |
| AU3891897A (en) | 1998-03-26 |
| ES2342325T3 (es) | 2010-07-05 |
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