SK173099A3 - Vaccine compositions comprising the helicobacter pylori flge polypeptide - Google Patents
Vaccine compositions comprising the helicobacter pylori flge polypeptide Download PDFInfo
- Publication number
- SK173099A3 SK173099A3 SK1730-99A SK173099A SK173099A3 SK 173099 A3 SK173099 A3 SK 173099A3 SK 173099 A SK173099 A SK 173099A SK 173099 A3 SK173099 A3 SK 173099A3
- Authority
- SK
- Slovakia
- Prior art keywords
- polypeptide
- gly
- ala
- ser
- flge
- Prior art date
Links
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 59
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
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Abstract
Description
Kompozície očkovacej látky obsahujúce FlgE polypeptid Helicobacter pyloríVaccine compositions comprising a FlgE polypeptide of Helicobacter pylori
Oblasť technikyTechnical field
Predložený vynález sa týka polypeptidov a kompozícií očkovacej látky na vyvolanie ochrannej imunitnej odozvy voči infekcii Helicobacter pylón. Vynález sa ďalej týka použitia polypeptidov Helicobacter pylorí na prípravu kompozícií na liečenie alebo profýlaxiu infekcie Helicobacter pylorí.The present invention relates to polypeptides and vaccine compositions for inducing a protective immune response against Helicobacter pylon infection. The invention further relates to the use of Helicobacter pylori polypeptides for the preparation of compositions for the treatment or prophylaxis of Helicobacter pylori infection.
Doterajší stav technikyBACKGROUND OF THE INVENTION
Helicobacter pyloríHelicobacter pylori
Gram-negatívna baktéria Helicobacter pylorí (H. pylorí) je významným ľudským patogénom, ktorý je príčinou niektorých gastroduodenálnych ochorení. Kolonizácia epitelu žalúdka baktériami vedie k aktívnemu zápalu a progresívnej chronickej gastritíde, so značne zvýšeným rizikom progresie kpeptickému vredovému ochoreniu. Dlhodobý zápal sliznice žalúdka je vo veľmi úzkom vzájomnom vzťahu so signifikantne zvýšeným rizikom rakoviny žalúdka.The Gram-negative bacterium Helicobacter pylori (H. pylori) is an important human pathogen that is responsible for some gastroduodenal diseases. Colonization of the stomach epithelium by bacteria leads to active inflammation and progressive chronic gastritis, with a significantly increased risk of progression to peptic ulcer disease. Long-term gastric mucosal inflammation is in a very close correlation with a significantly increased risk of gastric cancer.
Pri kolonizácii sliznice žalúdka H. pylorí sa uplatňuje celý rad virulentných faktorov. Takéto virulentné faktory zahrňujú adhezíny (zrasty), ktorými sa baktéria spája so sliznicou a/alebo sa viaže k epiteliálnym bunkám; močoviny, ktoré pomáhajú neutralizovať kyslé prostredie; a proteolytické enzýmy, ktoré robia sliznicu tekutejšou. Okrem toho H. pylorí je veľmi pohyblivá, steká v sliznici a dolu do vnútra. Ukázalo sa, že pohyblivosť je základným virulentným faktorom, pretože H. pylorí nedokázala infikovať sliznicu pri experimentálnych modeloch, ktoré uskutočnili Eaton a kol. (Infection & Immunity 64(7), 2455-2448 1996).A number of virulent factors are involved in the colonization of the gastric mucosa of H. pylori. Such virulent factors include adhesins (adhesions) by which the bacterium attaches to the mucosa and / or binds to epithelial cells; ureas that help neutralize the acidic environment; and proteolytic enzymes that make the mucosa more fluid. In addition, H. pylori is very mobile, running down the mucosa and down into the interior. Mobility has been shown to be an essential virulence factor because H. pylori has failed to infect the mucosa in experimental models performed by Eaton et al. (Infection & Immunity 64 (7), 2455-2448 1996).
Pre toto jestvuje veľa možných príčin, najpochopiteľnejšou z nich je neschopnosť stekať dolu a dosiahnuť bunky sliznice a neschopnosť vyhnúť sa škodlivým činidlám v žalúdku.There are many possible causes for this, the most understandable of which is the inability to flow down and reach the mucosal cells and the inability to avoid harmful agents in the stomach.
Napriek silnej zdanlivej imunitnej odozve hostiteľa na H. pylorí, pri produkcii ako lokálnych (mukozálnych) tak i systémových protilátok, patogén zotrváva v sliznici žalúdka bežne počas života pacienta. Dôvodom tohto je pravdepodobne to, že spontánne vyvolané imunitné odozvy sú neadekvátne alebo zamerané vočiDespite a strong apparent host immune response to H. pylori, producing both local (mucosal) and systemic antibodies, the pathogen persists in the gastric mucosa normally during the life of the patient. The reason for this is probably that spontaneously induced immune responses are inadequate or directed against
-2nesprávnym epitopom antigénov. Alternatívne môže byť imunitná odozva nesprávneho druhu, pretože imunitný systém môže pokladať H. pylori za symbiotickú (ako je znázornené zo vzájomného pomeru životnosť hostiteľa/baktérie).- incorrect epitope of antigens. Alternatively, the immune response may be of the wrong species since the immune system may consider H. pylori to be symbiotic (as shown by the host / bacterial life ratio).
Aby sme pochopili patogenézu a imunológiu infekcií H. pylori, má veľký význam definovať antigénnu štruktúru tejto baktérie. Predovšetkým, jestvuje tu potreba charakterizácie vystaveného povrchu, spojeného povrchu ako aj vylúčených proteínov, ktoré, ako sa preukázalo, predstavujú pri mnohých bakteriálnych patogénoch hlavný virulentné faktory, a ktoré môžu byť užitočné pri diagnóze H. pylori a pri príprave očkovacích kompozícií. Ak sú takéto proteíny okrem toho, že sú povrchovo pripojené, tiež základnými pre prežitie a/alebo kolonizáciu, ich využiteľnosť ako cieľ pre očkovacou látkou sprostredkované imunoterapeutické ciele sa zvyšuje.In order to understand the pathogenesis and immunology of H. pylori infections, it is of great importance to define the antigenic structure of this bacterium. In particular, there is a need for characterization of the exposed surface, the bonded surface as well as the secreted proteins, which have been shown to be major virulent factors in many bacterial pathogens and which may be useful in diagnosing H. pylori and in preparing vaccine compositions. When such proteins, in addition to being surface-linked, are also essential for survival and / or colonization, their utility as a target for vaccine-mediated immunotherapeutic targets increases.
Pri strese alebo v ohrození sa bunka H. pylori transformuje z bacilárnej formy na kokoidnú formu. V kokoidnej forme je bunka H. pylori menej citlivá voči antibiotikám a ďalším antibakteriálnym činidlám. Podrobné výskumy naznačujú, že H. pylori sa môže prenášať medzi jednotlivcami v tejto forme, pravdepodobne vodou alebo priamym kontaktom (orálno-orálne, fekálno-orálne). Účinná kompozícia očkovacej látky by teda mala vyvolať imunitnú odozvu voči obom formám H. pylori, kokoidnej a bacilárnej. Pretože systémová imunita hrá pravdepodobne iba obmedzenú úlohu pri ochrane pred infekciami sliznice, je tiež dôležité, aby kompozícia očkovacej látky zvyšovala ochranný imunitný mechanizmus lokálne v žalúdku.In stress or at risk, a H. pylori cell is transformed from a bacillary form to a cocoid form. In a cocoid form, the H. pylori cell is less sensitive to antibiotics and other antibacterial agents. Detailed research suggests that H. pylori may be transmitted between individuals in this form, probably by water or direct contact (oral-oral, faecal-oral). Thus, an effective vaccine composition should elicit an immune response against both forms of H. pylori, cocoid and bacillary. Since systemic immunity is likely to play only a limited role in protecting against mucosal infections, it is also important that the vaccine composition enhances the protective immune mechanism locally in the stomach.
Flagelámy hook proteínFlagellam hook protein
Ukázalo sa, že Flagelárne hook z H. pylori pozostávajú z FlgE podjednotiek 78kDa (O'Toole a kol., Molecular Microbiology, 14(4), 691 - 703, 1994). Úlohou Flagelámeho hook je spojiť Flagela so submembránovým Flagelárnym hook pohybom. Časť hook vytláčaná mimo membránu je krátka, približne 60 nanometrov (v porovnaní ku približne 10 mikrometrom pre Flagela). Podobne ako Flagela H. pylori, hook je pravdepodobne pokrytý poťahom (Geis a kol., (1993), J. Med. Microbiol. 38(5), 371 - 377).H. pylori flagellar hooks have been shown to consist of the 78kDa FlgE subunits (O'Toole et al., Molecular Microbiology, 14 (4), 691-703, 1994). The task of Flagelame hook is to connect Flagela with submembrane Flagellar hook movement. The portion of the extruded hook is short, about 60 nanometers (compared to about 10 micrometers for Flagela). Like Flagela H. pylori, the hook is probably covered with a coating (Geis et al., (1993), J. Med. Microbiol. 38 (5), 371-377).
-3Aminokyselinová sekvencia FlgE polypeptidu má signifikantnú podobnosť s ďalšími známymi hook proteínmi, vrátane limitovanej homológie k ďalším druhom Helicobacter, ako je mustelae (O'Toole a kol., uvedené vyššie). Polyklonálne protilátky vyvolané proti FlgE polypetidu vykázali skríženú reaktivitu voči Flagelámym proteínom A a B, čo pravdepodobne indikuje existenciu spoločných epitopov. Produkcia FlgE paralyzuje H. pylón, výsledkom čoho je aflageláma nepohyblivá baktéria, kde sa FlgE polypetid produkoval, ale mohol sa znova zachytiť v cytoplazme.The -3 amino acid sequence of the FlgE polypeptide has significant similarity to other known hook proteins, including limited homology to other Helicobacter species such as mustelae (O'Toole et al., Supra). Polyclonal antibodies raised against FlgE polypetide showed cross-reactivity to Flagela proteins A and B, probably indicating the existence of common epitopes. FlgE production paralyzes H. pylon, resulting in an aflagelama immobilized bacterium where FlgE polypeptide was produced but could be recaptured in the cytoplasm.
Prehľad obrázkov na výkresochBRIEF DESCRIPTION OF THE DRAWINGS
Obrázok 1Figure 1
Vplyv terapeutickej imunizácie myší infikovaných H. pylorí (n = 9-10/skupinu) s FlgE polypeptidom. Výsledky sú uvedené ako stredná ±SEM počtu H. pylorí spojených s antrum (=A), telieskom (=B) alebo celkovo (A+B) (=C). Skratky: CFU, jednotky tvoriace kolóniu (počet baktérií); nešrafované stĺpce = DOC+CT, fosfátom pufrovaný fyziologický roztok s 0,5 % deoxycholátu sa podával spolu s toxínom cholery 10 pg/myš; šrafované stĺpce = FlgE+CT, myši dostávali 100 pg FlgE a 10pg toxínu cholery. Pokles CFU bol signifikantný vantrume a vypočítal sa pre celý žalúdok.Effect of therapeutic immunization of H. pylori-infected mice (n = 9-10 / group) with FlgE polypeptide. Results are presented as mean ± SEM of the number of H. pylori associated with anthra (= A), body (= B) or total (A + B) (= C). Abbreviations: CFU, colony forming units (number of bacteria); unhatched bars = DOC + CT, phosphate buffered saline with 0.5% deoxycholate was co-administered with cholera toxin 10 µg / mouse; shaded bars = FlgE + CT, mice received 100 µg FlgE and 10 µg cholera toxin. The decrease in CFU was a significant vantrume and was calculated for the entire stomach.
**p<0,01; *p<0,05 (Wilcoxon-Mann-Whittneyov klasifikačný test znakov)** p <0.01; * p <0.05 (Wilcoxon-Mann-Whittney character classification test)
Obrázok 2Figure 2
Sérum IgG z myší merané pomocou ELISA metodiky: odozvy na infekciu a na imunizáciu s FlgE. Hodnoty boli vyjadrené ako priemerné titre ±SEM, n = 9 až 10/skupinu. ELISA pokrytá s kmeňom H. pylorí 244; ako príznak infekcie H. pylorí sa v sére zvierat ošetrených s DOC + CT (=A, kontrola/244) dali zistiť špecifické protilátky. Následná imunizácia s FlgE + toxínom cholery (=B, FlgE/244) túto reaktivitu zvyšovala štvornásobne (**p<0,01; (Wicoxon-Mann-Whittneyov klasifikačný test znakov). C = FlgE špecifické. Špecifické FlgE sa zvyšovalo u zvierat, ktoré dostávali FlgE + CT, avšak nedalo sa stanoviť pre kontrolné zvieratá.IgG serum from mice measured by ELISA methodology: response to infection and immunization with FlgE. Values were expressed as mean titers ± SEM, n = 9-10 / group. ELISA coated with H. pylori 244; specific antibodies could be detected as a symptom of H. pylori infection in the sera of animals treated with DOC + CT (= A, control / 244). Subsequent immunization with FlgE + cholera toxin (= B, FlgE / 244) increased this reactivity fourfold (** p <0.01; (Wicoxon-Mann-Whittney character test). C = FlgE specific. Specific FlgE increased in animals that received FlgE + CT but could not be determined for control animals.
-4Podstata vynálezu4. Summary of the Invention
Cieľom tohto vynálezu je poskytnúť antigénny polypetid H. pylori, ktorý sa môže použiť na vyvolanie ochrannej imunitnej odozvy voči H. pylori a na diagnostikovanie infekcie H. pylori. Tento cieľ sa dosiahol pomocou rekombinantného klonovania génu H. pylori, ktorý kóduje dobre uchovaný základný polypeptid. Sekvencia nukleových kyselín tohto génu je podobná sekvencií génu FlgE, ako publikovali O'Toole a kol., Molecular Microbiology, 14(4), 691 - 703, 1994. Ako základný proteín motility je gén FlgE exprimovaný všetkými kmeňmi H. pylori.It is an object of the present invention to provide an antigenic H. pylori polypeptide that can be used to elicit a protective immune response against H. pylori and to diagnose H. pylori infection. This goal was achieved by recombinant cloning of the H. pylori gene, which encodes a well conserved parent polypeptide. The nucleic acid sequence of this gene is similar to that of the FlgE gene as reported by O'Toole et al., Molecular Microbiology, 14 (4), 691-703, 1994. As a basic motility protein, the FlgE gene is expressed by all H. pylori strains.
Prekvapujúco sa zistilo, že FlgE polypetid H. pylori, napriek skutočnosti, že len malá časť hook proteínu jestvuje mimo baktérie a že je pravdepodobne pokrytý poťahom, môže slúžiť ako terapeutický antigén pri modele myší infikovaných H. pylori, ak sa podáva spolu s pomocnou látkou toxínom cholery. Nižšie uvedené experimentálne údaje teda indikujú, že FlgE polypetid H. pylori, ak sa použije ako orálny imunogén, pôsobí ako stimulátor imunitnej odozvy, ktorý vedie k signifikantnému zníženiu kolonizácie H. pylori u myší, ktoré boli infikované s H. pylori jeden mesiac pred imunizáciou.Surprisingly, it has been found that FlgE H. pylori polypeptide, despite the fact that only a small portion of the hook protein exists outside the bacteria and that it is likely to be coated, may serve as a therapeutic antigen in a model of H. pylori-infected mice when co-administered with an adjuvant. cholera toxin. Thus, the experimental data below indicates that FlgE polypeptide of H. pylori, when used as an oral immunogen, acts as an immune response stimulator leading to a significant reduction in H. pylori colonization in mice infected with H. pylori one month prior to immunization. .
Tieto výsledky silne podporujú použitie FlgE polypeptidu H. pylori v orálnych prípravkoch očkovacej látky na použitie pri liečení alebo prevencii infekcií H. pylori u človeka. Samotný FlgE proteín bude využiteľný ako pri stanovení infekcií H. pylori tak aj pri výrobe kompozícií očkovacej látky, ktoré, ak sa podajú vo vhodných farmaceutických prípravkoch, budú vyvolávať ochrannú alebo terapeutickú imunitnú odozvu voči takýmto infekciám.These results strongly support the use of the H. pylori FlgE polypeptide in oral vaccine formulations for use in treating or preventing H. pylori infections in humans. FlgE protein itself will be useful in both H. pylori infections as well as in the manufacture of vaccine compositions which, when administered in suitable pharmaceutical formulations, will produce a protective or therapeutic immune response to such infections.
Preto vynález ďalej poskytuje FlgE polypetid Helicobacter pylori na použitie na vyvolanie ochrannej imunitnej odozvy voči infekciám. Termín “FlgE polypetid Helicobacter pylori je mienený ako polypetid, ktorý opísali O'Toole a kol. v Molecular Microbiology, 14(4), 691 - 703, 1994, a ktorý je kódovaný pomocou génu, ktorého nukleotidová sekvencia je uvedená ako SEQ ID NO. 1, alebo sa môže získať z National Center for Biotechnology Information (Accession number (prírastkové číslo) U09549) alebo v podstate podobná modifikovaná forma uvedeného polypeptidu, ktorá si zachováva funkčnú ekvivalentnú antigenicitu.Therefore, the invention further provides a Helgobacter pylori FlgE polypeptide for use in eliciting a protective immune response against infections. The term "FlgE polypeptide Helicobacter pylori" is intended to be the polypeptide described by O'Toole et al. in Molecular Microbiology, 14 (4), 691-703, 1994, and which is encoded by a gene whose nucleotide sequence is shown as SEQ ID NO. 1, or may be obtained from the National Center for Biotechnology Information (Accession Number U09549) or a substantially similar modified form of said polypeptide that retains functional equivalent antigenicity.
-5Pod termínom ochranná imunitná odozvy” je potrebné rozumieť imunitnú odozvu, ktorá robí kompozíciu vhodnou na terapeutické a/alebo profylaktické účely.-5 "Protective immune response" means an immune response that makes the composition suitable for therapeutic and / or prophylactic purposes.
Pod termínom “funkčná ekvivalentná antigenicita” treba chápať ako schopnosť vyvolať systémovú a mukozálnu imunitnú odozvu, pričom sa znižuje počet buniek H. pylori spojených so sliznicou žalúdka. Odborníci skúsení v odbore budú schopní identifikovať modifikované formy FlgE polypeptidu, ktorý si zachováva funkčnú ekvivalentnú antigenicitu, s použitím známych postupov, ako je zobrazenie epitopov s protilátkami vyvolanými in vivo.By "functional equivalent antigenicity" is meant the ability to elicit a systemic and mucosal immune response, reducing the number of H. pylori cells associated with the gastric mucosa. Those of skill in the art will be able to identify modified forms of FlgE polypeptide that retain functional equivalent antigenicity, using known procedures, such as imaging epitopes with antibodies elicited in vivo.
Vo výhodnom spôsobe uskutočnenia vynálezu FlgE polypeptid Heiicobacter pylori na použitie na vyvolanie ochrannej imunitnej odozvy voči infekcii Heiicobacter pylori, má v podstate aminokyselinovú sekvenciu uvedenú ako SEQ ID NO. 2 v zozname sekvencií, alebo jej modifikovanú formu, ktorá sa zachováva funkčnú ekvivalentnú antigenicitu.In a preferred embodiment of the invention, the Heiicobacter pylori FlgE polypeptide for use in eliciting a protective immune response against Heiicobacter pylori infection, has substantially the amino acid sequence shown as SEQ ID NO. 2 of the Sequence Listing, or a modified form thereof that retains functional equivalent antigenicity.
Preto je treba chápať, že definícia FlgE polypeptidu Heiicobacter pylori nie je prísne obmedzená na polypeptid, s aminokyselinovou sekvenclou, ktorý je identický s SEQ ID č. 2 v zozname sekvencií. Vynález skôr zahrňuje polypeptidy, ktoré nesú modifikácie, ako sú substitúcie, malé vyradenia, vloženia alebo inverzie, pričom tieto polypeptidy napriek tomu majú v podstate biologickú aktivitu FlgE polypeptidu Heiicobacter pylori a zachováva sa funkčná ekvivalentná antigenicita. Do definície FlgE polypetidu Heiicobacter pylori sú preto zahrnuté polypeptidy, aminokyselinová sekvencia ktorých je najmenej na 90 % homologických, výhodne najmenej na 95 % homologických, s aminokyselinovou sekvenciou uvedenou ako SEQ ID No 2, v zozname sekvencií.Therefore, it is to be understood that the definition of the FlgE polypeptide of Heiicobacter pylori is not strictly limited to a polypeptide having an amino acid sequence that is identical to SEQ ID NO. 2 in the sequence list. Rather, the invention encompasses polypeptides that carry modifications, such as substitutions, small knockouts, insertions, or inversions, but which nevertheless have substantially the biological activity of the Heiicobacter pylori FlgE polypeptide and retain functional equivalent antigenicity. Therefore, polypeptides having an amino acid sequence of at least 90% homology, preferably at least 95% homology, with the amino acid sequence shown as SEQ ID No 2 in the Sequence Listing are included in the definition of Hegicobacter pylori FlgE polypeptide.
Vynález sa ďalej poskytuje kompozíciu očkovacej látky na vyvolanie ochrannej imunitnej odozvy voči infekcii Heiicobacter pylori, ktorá obsahuje imunogénne účinné množstvo FlgE polypetidu Heiicobacter pylori, ako je definovaný vyššie, prípadne spolu s farmaceutický prijateľným nosičom alebo riedidlom.The invention further provides a vaccine composition for eliciting a protective immune response against a Heiicobacter pylori infection comprising an immunogenically effective amount of a Heiicobacter pylori FlgE polypeptide as defined above, optionally together with a pharmaceutically acceptable carrier or diluent.
V uvedenom kontexte termín “imunologický účinné množstvo treba chápať ako množstvo, ktoré vyvolá signiflkantnú ochrannú odozvu Heiicobacter pylori, ktorá vykorení infekciu H. pylori u infikovaných cicavcov alebo zabráni infekcii u náchylných cicavcov. Typicky bude imunologický účinné množstvo obsahovaťIn the present context, the term "immunologically effective amount" is understood to be an amount that elicits a significant protective response of Heiicobacter pylori that eradicates H. pylori infection in infected mammals or prevents infection in susceptible mammals. Typically, the immunologically effective amount will comprise
-6približne 1 gg až 1000 mg, výhodne približne 10 pg až 100 mg, antigénu H. pylori na orálne podávanie alebo približne menej ako 100 pg na parenterálne podávanie.About 1 gg to 1000 mg, preferably about 10 µg to 100 mg, of H. pylori antigen for oral administration or about less than 100 µg for parenteral administration.
Kompozícia očkovacej látky obsahuje prípadne okrem farmaceutický prijateľného nosiča alebo riedidla jeden alebo viac ďalších farmaceutický účinných antigénov na profylaktické alebo terapeutické použitie. Fyziologicky prijateľné nosiče a riedidlá sú pre odborníkov v odbore dobre známe a zahrňujú napríklad fosfátom pufrovaný fyziologický roztok (PBS) alebo, v prípade orálnych očkovacích látok, prípravky na báze HCO3- alebo entericky poťahované práškové prípravky.The vaccine composition optionally comprises, in addition to a pharmaceutically acceptable carrier or diluent, one or more other pharmaceutically effective antigens for prophylactic or therapeutic use. Physiologically acceptable carriers and diluents are well known to those skilled in the art and include, for example, phosphate buffered saline (PBS) or, in the case of oral vaccines, HCO3- or enteric-coated powder formulations.
Kompozícia očkovacej látky môže prípadne obsahovať alebo sa môže podávať spolu s inhibítormi sekrécie, výhodne inhibítormi protónovej pumpy (PPI), napríklad omeprazolom. Očkovacia látka sa potom môže formulovať do známych systémov dodávania, ako sú lipozómy, ISCOM, kochley, a podobne (pozri napríklad Rabinovich a kol., (1994), Science 265, 1401 až 1404) alebo sa pripojiť alebo začleniť do mikrosfér polyméru odbúrateľnej alebo neodbúrateľnej povahy. Antigény môžu byť spojené so živými oslabenými baktériami, vírusmi alebo fágmi alebo s usmrtenými vektormi rovnakého druhu. Antigény môžu byť chemicky alebo geneticky pripojené k proteínom nosiča inertného alebo pomocného typu (napríklad podjednotka cholery B). Ďalej sa vynález týka kompozície očkovacej látky podľa vyššie uvedeného, ktorá okrem toho obsahuje pomocnú látku, ako je toxín cholery. Takéto farmaceutický prijateľné formy toxínu cholery sú známe z doterajšieho stavu techniky, napríklad z Rappuoli a kol., (1995), Int. Árch. Allergy & Immunol. 108(4), 327 až 333; a Dickinson a kol., (1995), Infection and Immunity 63(5), 1617 až 1623.The vaccine composition may optionally contain or be administered together with secretion inhibitors, preferably proton pump inhibitors (PPIs), for example omeprazole. The vaccine may then be formulated in known delivery systems such as liposomes, ISCOM, cochley, and the like (see, for example, Rabinovich et al., (1994), Science 265, 1401-1404) or attached or incorporated into the microspheres of a degradable or polymer. non-degradable nature. Antigens may be associated with live attenuated bacteria, viruses or phages, or with killed vectors of the same species. The antigens may be chemically or genetically linked to carrier or inert type carrier proteins (e.g., cholera B subunit). Furthermore, the invention relates to a vaccine composition according to the above, which additionally comprises an adjuvant such as cholera toxin. Such pharmaceutically acceptable forms of cholera toxin are known in the art, for example, from Rappuoli et al., (1995) Int. Arch. Allergy & Immunol. 108 (4), 327-333; and Dickinson et al., (1995) Infection and Immunity 63 (5), 1617-1623.
Kompozícia očkovacej látky podľa vynálezu sa môže použiť ako na terapeutické tak i na profylaktické účely. Vynález teda zahrňuje kompozíciu očkovacej látky, ako je definovaná vyššie, na použitie ako liečivo alebo profylaktickú očkovaciu látku pre cicavca, vrátane človeka, ktorý je infikovaný Helicobacter pylori. V tomto kontexte termín “profylaktický účel” znamená vyvolanie imunitnej odozvy, ktorá zabráni budúcej infekcii s Helicobacter pylori, zatiaľ čo termín “terapeutický účel” znamená vyvolanie imunitnej odozvy, ktorá môže vykoreniť jestvujúce infekcie Helicobacter pylori.The vaccine composition of the invention can be used for both therapeutic and prophylactic purposes. Thus, the invention encompasses a vaccine composition as defined above for use as a medicament or prophylactic vaccine for a mammal, including a human, who is infected with Helicobacter pylori. In this context, the term "prophylactic purpose" means inducing an immune response that will prevent future infection with Helicobacter pylori, while the term "therapeutic purpose" means inducing an immune response that may eradicate existing Helicobacter pylori infections.
-7Kompozícia očkovacej látky podľa vynálezu sa výhodne podáva cez sliznicu cicavca, napríklad cez bukáinu, nasálnu, tonzilárnu, žalúdočnú alebo intestinálnu (tenké a hrubé črevo), rektálnu a vaginálnu sliznicu. Mukozálna očkovacia látka sa môže podávať spolu s pomocnými látkami vhodnými na tento účel. Očkovacia látka sa môže podávať tiež orálne alebo parenterálne, subkutánnou, intrakutánnou alebo intramuskulárnou cestou, prípadne spolu s vhodnými pomocnými látkami. Kompozícia očkovacej látky sa môže prípadne podávať spolu s vhodnou pomocnou látkou. Kompozícia účinnej látky sa môže prípadne podávať spolu s antimikrobiálnym terapeutickým činidlom.The vaccine composition of the invention is preferably administered through the mammalian mucosa, for example, through the buccal, nasal, tonsillar, stomach or intestinal (small and large intestine), rectal and vaginal mucosa. The mucosal vaccine may be administered together with excipients suitable for this purpose. The vaccine can also be administered orally or parenterally, subcutaneously, intracutaneously or intramuscularly, optionally together with suitable excipients. The vaccine composition may optionally be administered together with a suitable excipient. Optionally, the active ingredient composition may be co-administered with an antimicrobial therapeutic agent.
Vynález sa ďalej týka použitia FlgE polypeptidu Helicobacter pylorí, ako je definovaný vyššie, na prípravu (i) kompozície na liečenie, profylaxiu alebo diagnostikovanie infekcie Helicobacter pylón;The invention further relates to the use of a Helgobacter pylori FlgE polypeptide as defined above for the preparation of (i) a composition for the treatment, prophylaxis or diagnosis of a Helicobacter pylon infection;
(ii) očkovacej látky na použitie pri vyvolaní ochrannej imunitnej odozvy proti Helicobacter pylón; a (iii) diagnostického kitu na diagnostikovanie infekcie Helicobacter pylorí.(ii) a vaccine for use in eliciting a protective immune response against Helicobacter pylon; and (iii) a diagnostic kit for diagnosing Helicobacter pylori infection.
Okrem toho vynález ešte poskytuje spôsob in vitro diagnostikovania infekcie Helicobacter pylorí, ktorý zahrňuje najmenej jeden krok, pričom FlgE polypetid Helicobacter pylorí, definovaný vyššie, sa prípadne použije značený alebo spojený s pevným podkladom. Uvedený spôsob by mohol napríklad zahrňovať kroky (a) uvedenie do kontaktu uvedeného FlgE polypeptidu Helicobacter pylorí , prípadne viazaného k pevnému podkladu, s telesnou kvapalinou odobranou cicavcovi; a (b) detegovaním protilátok z uvedenej telesnej kvapaliny naviazaním na uvedený FlgE polypeptid. Výhodnými spôsobmi detekcie protilátok sú spôsoby ELISA (Enzýme linked imunoabsorbed assay, enzýmom viazané imunoabsorbčné skúšky), ktoré sú dobre známe z doterajšieho stavu techniky.In addition, the invention still provides a method for in vitro diagnosing Helicobacter pylori infection, which comprises at least one step, wherein the Helicobacter pylori FlgE polypeptide as defined above is optionally used labeled or associated with a solid support. For example, said method could comprise the steps of (a) contacting said Helicobacter pylori FlgE polypeptide, optionally bound to a solid support, with a body fluid removed from the mammal; and (b) detecting antibodies from said body fluid by binding to said FlgE polypeptide. Preferred methods for detecting antibodies are ELISA (Enzyme-linked immunoabsorbed assay) methods, which are well known in the art.
Predložený vynález v ďalšom poskytuje diagnostický kit na detegovanie infekcie Helicobacter pylorí u cicavca, vrátane človeka, obsahujúci zložky, ktoré umožňujú uskutočnenie spôsobu diagnostikovania in vitro, ako je opísané vyššie. Uvedený diagnostický kit môže obsahovať napríklad: (a) FlgE polypeptid Helicobacter pylorí;The present invention further provides a diagnostic kit for detecting a Helicobacter pylori infection in a mammal, including a human, comprising components that enable the method of in vitro diagnosis as described above to be performed. Said diagnostic kit may comprise, for example: (a) a Helicobacter pylori FlgE polypeptide;
-8a (b) reagenty na detegovanie protilátok viažúcich sa k uvedenému FlgE polypeptidu. Uvedenými reagentami na detegovanie protilátok by mohol byť napríklad enzýmom značený anti-imunoglobulín a chromogénny substrát pre uvedený enzým.(B) reagents for detecting antibodies binding to said FlgE polypeptide. Said antibody detection reagents could be, for example, an enzyme-labeled anti-immunoglobulin and a chromogenic substrate for said enzyme.
Ešte ďalej sa vynález týka poskytnutia spôsobu vyvolania u cicavca, vrátane človeka, ochrannej Imunitnej odozvy proti infekcii Helicobacter pylón, pričom uvedený spôsob zahrňuje krok podávania uvedenému cicavcovi imunologický účinné množstvo FlgE polypeptidu Helicobacter pylori ako je definovaný vyššie, alebo alternatívne podávanie uvedenému cicavcovi imunologický účinné množstvo kompozície očkovacej látky, definovanej vyššie.Still further, the invention provides a method of inducing in a mammal, including a human, a protective immune response against Helicobacter pylon infection, said method comprising the step of administering to said mammal an immunologically effective amount of a Helicobacter pylori FlgE polypeptide as defined above or alternatively administering to said mammal an immunologically effective amount. the vaccine composition as defined above.
Experimentálne metódyExperimental methods
V celom tomto opise termín “štandardný protokol” a “štandardný postup” použité v kontexte metód génového inžinierstva, treba chápať ako protokoly a postupy nájdené v štandardnom laboratórnom manuále, ako je: Current Protocols in Molecular Biology, Ed. F. Ausubel a kol., John Woley and Sons, Inc. 1994, alebo Sambrook, J., Fritsch, E. F. a Maniatis, T., Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989. Príprava rekombinantného FlgE polypeptidu Helicobacter pyloriThroughout this specification, the terms "standard protocol" and "standard procedure" used in the context of genetic engineering methods are to be understood as protocols and procedures found in a standard laboratory manual such as: Current Protocols in Molecular Biology, Ed. F. Ausubel et al., John Woley and Sons, Inc. 1994, or Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A laboratory manual, 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 1989. Preparation of Recombinant Helicobacter pylori FlgE Polypeptide
DNA sekvenčná informáciaDNA sequence information
Sekvečná informácia pre gén kódovaný pre FlgE polypeptid sa získala od National Center for Biotechnology Information (Accession number (prírastkové číslo) U09549; SEQ ID No: 1).Sequence information for the gene encoded by the FlgE polypeptide was obtained from the National Center for Biotechnology Information (Accession number U09549; SEQ ID No: 1).
PCR amplifikácia a klonovanie DNA sekvencií obsahujúcich ORF pre membrány a vylúčené proteíny z J99 kmeňa Helicobacter pylori.PCR amplification and cloning of DNA sequences containing ORFs for membranes and secreted proteins from the J99 strain Helicobacter pylori.
Sekvencie sa klonovali zJ99 kmeňa H. pylori amplifikačným klonovaním s použitím polymerázovej reťazovej reakcie (PCR). Navrhli a získali sa syntetické oligonukleotidové priméry (pozri nižšie) špecifické pre 5'a 3'-konce otvorených čítacích rámcov (GibcoBRL Life Technologies, Gaithersburg, MD, USA). Pôvodné priméry (špecifické pre 5'-konce sekvencie) pre FlgE sa navrhli tak, aby zahrňovaliThe sequences were cloned from the J99 strain of H. pylori by amplification cloning using a polymerase chain reaction (PCR). Synthetic oligonucleotide primers (see below) specific for the 5 'and 3' ends of the open reading frames (GibcoBRL Life Technologies, Gaithersburg, MD) were designed and obtained. The original primers (specific for the 5'-ends of the sequence) for FlgE were designed to include
-9Ncol klonované miesto na extrémnom 5'-konci, pričom reverzné priméry zahrňovali EcoRI miesto na extrémnom 5'-konci, aby sa umožnilo klonovanie každej sekvencie H. pylori do čítacieho rámca vektora pET-28b. Inzerty klonované do NcolEcoR\ miest vektora pET-28b sa fúzovali na vektor sekvencie DNA kódujúci prídavných 20 karboxy-terminálnych amino, vrátane šesť histidínových zvyškov (na extrémnom C-konci).The 99 NcoI cloned site at the extreme 5'-end, wherein the reverse primers included an EcoRI site at the extreme 5'-end to allow cloning of each H. pylori sequence into the reading frame of the pET-28b vector. The inserts cloned into the NcoEcoR1 sites of the pET-28b vector were fused to a DNA sequence vector encoding additional 20 carboxy-terminal amino acids, including six histidine residues (at the extreme C-terminus).
Pôvodný primér (SEQ ID NO: 3)Original primer (SEQ ID NO: 3)
5'-TAT ACC ATG GTG CTT AGG TCT TTA T-3'5 'TAT ACC ATG GTG CTT AGG TCT TTA T-3'
Reverzný primér (SEQ ID NO: 4)Reverse primer (SEQ ID NO: 4)
5'-GCG AAT TCA ATT GCT TAA GAT TCA A-3'5 '-GCG AAT TCA ATT GCT TAA GAT TCA A-3'
Genómová DNA pripravená z kmeňa J99 Helicobacter pylori sa použila ako zdroj matrice DNA na PCR amplifikačné reakcie (Current Protocols in Molecular Biology, Ed. F. Ausubel a kol., John Wiley and Sons, Inc. 1994). Na amplifikovanie DNA sekvencie obsahujúcej ORF H. pylori, sa genómová DNA (50 ng) zaviedla do reakčnej fľaštičky, obsahujúcej 2 mM MgCI2, 1 μΜ syntetických oligonukleotidových primérov (pôvodné a reverzné priméry) komplementárna k a lemujúca definovanú ORF H. pylori, 0,2 mM každého deoxynukleotidového trifosfátu dATP, dGTP, dCTP, dTTP a 2,5 jednotiek tepelne stabilizovanej DNA polymerázy (Amplitaq, Roche Molecular Systems, Inc. Branchburg, NJ, USA) v konečnom objeme 100 μΙ. Použili sa nasledovné cyklické podmienky na získanie amplifikovaných DNA produktov pre každý použitý ORF, s použitím Perkin Elmer Cetus/GeneAmp PCR systému 9600 termálneho cyklu:Genomic DNA prepared from Helicobacter pylori J99 strain was used as a source of DNA matrix for PCR amplification reactions (Current Protocols in Molecular Biology, Ed. F. Ausubel et al., John Wiley and Sons, Inc. 1994). To amplify the DNA sequence containing the H. pylori ORF, genomic DNA (50 ng) was introduced into a reaction vial containing 2 mM MgCl2, 1 μΜ of synthetic oligonucleotide primers (parent and reverse primers) complementary to the defined H. pylori ORF, 0.2 mM of each deoxynucleotide triphosphate dATP, dGTP, dCTP, dTTP and 2.5 units of heat stabilized DNA polymerase (Amplitaq, Roche Molecular Systems, Inc., Branchburg, NJ, USA) in a final volume of 100 μΙ. The following cyclic conditions were used to obtain amplified DNA products for each ORF used, using a Perkin Elmer Cetus / GeneAmp 9600 thermal cycle PCR system:
Denaturácia pri +94 °C počas 2 min;Denaturation at +94 ° C for 2 min;
cykly pri +94 “C počas 15 sek., +30 °C počas 15 sek. a +72 eC počas 1,5 min.;cycles at +94 ° C for 15 sec., +30 ° C for 15 sec. and +72 e C for 1.5 min;
cyklov pri +94 °C počas 15 sek., +58 °C počas 15 sek. a +72 °C počas 1,5 min.; Reakcie sa ukončili pri +72 eC počas 6 minút.cycles at +94 ° C for 15 sec., +58 ° C for 15 sec. and + 72 ° C for 1.5 min; Reactions were concluded at + 72 e C for 6 minutes.
Po ukončení termálnych cyklických reakcií sa každá vzorka amplifikovanej DNA sa premyla a prečistila s použitím Qiaquick Spin PCR kitu na prečistenie (Qiagen, Gaithersburg, MD, USA). Amplifikované DNA vzorky sa podrobili digesciiAfter thermal cycling reactions were completed, each amplified DNA sample was washed and purified using a Qiaquick Spin PCR purification kit (Qiagen, Gaithersburg, MD, USA). The amplified DNA samples were digested
-10s reštrikčnými endonukleázami Nde\ a EcoRI s použitím štandardných postupov. Vzorky DNA sa potom podrobili elektroforéze na 1,0 % NuSeive (FMC BioProducts, Rockland, ME, USA) agarózovom géle. DNA sa vizualizovala expozíciou etídiumbromidu a ožiareniu UV dlhými vlnami. DNA obsiahnutá v podieloch izolovaných zagarózového gélu sa prečistila s použitím Bio 101 GeneClean Kit protokolu (Bio 101 Vista, CA, USA).-10 with Nde I and Eco RI restriction endonucleases using standard procedures. DNA samples were then subjected to agarose gel electrophoresis on a 1.0% NuSeive (FMC BioProducts, Rockland, ME, USA). DNA was visualized by exposure to ethidium bromide and UV long wave irradiation. DNA contained in aliquots of isolated zagarose gel was purified using the Bio 101 GeneClean Kit protocol (Bio 101 Vista, CA, USA).
Klonovanie DNA sekvencií H. pylori do pET-28b prokaryotického expresívneho vektora pET-28b vektor sa pripravil na klonovanie digesciou s Λ/col a EcoRI podľa štandardných postupov. Po digescii sa DNA inzerty klonovali podľa štandardných postupov do pôvodne digerovaného pET-28b expresívneho vektora. Produkty ligačnej reakcie sa potom použili na transformovanie BL21 kmeňa E. coli, ako je opísané ďalej.Cloning of H. pylori DNA sequences into pET-28b prokaryotic expression vector pET-28b vector was prepared for cloning by digestion with s / col and EcoRI according to standard procedures. After digestion, the DNA inserts were cloned according to standard procedures into the originally digested pET-28b expression vector. The ligation reaction products were then used to transform the BL21 strain of E. coli as described below.
Transformácia vhodnej baktérie s rekombinantnými plazmidmiTransformation of a suitable bacterium with recombinant plasmids
Vhodné baktérie E. coli kmeňa BL21 alebo E. coli kmeňa BL21(DE3) sa transformovali s rekombinantnými pET expresívnymi plazmidmi, ktoré nesú klonované sekvencie H. pylori podľa štandardných postupov. Stručne 1 μΙ ligačnej reakčnej zmesi sa zmiešalo s 50 μΙ elektrokompetentných buniek a podrobilo sa impulzu s vysokým napätím, po ktorom sa vzorky inkubovali v 0,45 ml SOC média (0,5 % extrakt kvasiniek, 2,0 % tryptónu, 10 mM NaCI, 2,5 mM KCI, 10 mM MgCI2, 10 mM MgSO4 a 20 mM glukózy) pri teplote +37 °C za trepania počas jednej hodiny. Vzorky sa potom naniesli na LB agarové platne obsahujúce 25 μg/ml kanamynsulfátu na a nechali sa rásť cez noc. Transformované kolónie BL21 sa potom zozbierali a analyzovali sa na vyhodnotenie kolovaných inzertov opísaných nižšie.Appropriate E. coli strain BL21 or E. coli strain BL21 (DE3) was transformed with recombinant pET expression plasmids carrying cloned H. pylori sequences according to standard procedures. Briefly, 1 μΙ of the ligation reaction mixture was mixed with 50 μΙ of electrocompetent cells and subjected to a high voltage pulse, after which the samples were incubated in 0.45 ml SOC medium (0.5% yeast extract, 2.0% tryptone, 10 mM NaCl). , 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4, and 20 mM glucose) at + 37 ° C with shaking for one hour. Samples were then plated on LB agar plates containing 25 µg / ml kanamynsulfate on and allowed to grow overnight. Transformed BL21 colonies were then harvested and analyzed for evaluation of the rounded inserts described below.
Identifikácia rekombinantných pET expresívnych plazmidov nesúcich sekvencie H. pyloriIdentification of recombinant pET expression plasmids carrying H. pylori sequences
Jednotlivé BL21 klony transformované s rekombinantnými pET-28b génmi H. pylori sa analyzovali pomocou PCR amplifikácie klonovaných inzertov s použitím pôvodných a reverzných primérov, špecificky pre každú sekvenciu H. pylori , ktoréIndividual BL21 clones transformed with recombinant H. pylori pET-28b genes were analyzed by PCR amplification of the cloned inserts using the original and reverse primers, specifically for each H. pylori sequence that
-11sa použili v pôvodných PCR amplifikačných klonovacích reakciách. Následná amplifikácia verifikovala integráciu sekvencií H. pylón v expresívnom vektore podľa štandardných postupov.Were used in the original PCR amplification cloning reactions. Subsequent amplification verified the integration of H. pylon sequences in the expression vector according to standard procedures.
Izolácia a príprava plazmidu DNA z BL21 transformantovIsolation and preparation of plasmid DNA from BL21 transformants
Jednotlivé klony rekombinantných pET-28b vektorov, ktoré nesú vhodne klonované ORF H. pylorí sa zozbierali a inkubovali sa cez noc v 5 ml LB bujónu plus 25 pg/ml kanamycínsulfátu. Nasledujúci deň sa plazmid DNA izoloval a prečistil s použitím Qiagen plazmidového čistiaceho protokolu (Qiagen Inc., Chatsworth, CA, USA).Individual clones of recombinant pET-28b vectors carrying appropriately cloned H. pylori ORFs were harvested and incubated overnight in 5 ml LB broth plus 25 µg / ml kanamycin sulfate. The following day, plasmid DNA was isolated and purified using a Qiagen plasmid purification protocol (Qiagen Inc., Chatsworth, CA, USA).
Expresia rekombinantnej sekvencie H. pylón v E. coliExpression of the recombinant H. pylon sequence in E. coli
Vektor pET sa môže množiť v ktoromkoľvek K-12 kmeni E. coli, napríklad HMS174, HB101, JM109, DH5a, a podobne, pre účely klonovania alebo prípravy plazmidu. Hostitelia pre expresiu zahrňujú kmene E. coli obsahujúce chromozomálnu kópiu génu pre T7 RNA polymerázu. Týmito hostiteľmi sú lyzogény bakteriofágu DE3, lambda derivát, ktorý nesie lacl gén, lacUV5 promótor a gén pre T7 RNA polymerázu. T7 RNA polymeráza sa vyvolá pridaním izopropyl-p-Dtiogalaktozidu (IPTG) a T7 RNA polymeráza transkribuje akýkoľvek cieľový plazmid, ako je pET-28b, nesúci svoj významný gén. Kmene použité v našom laboratóriu zahrňujú: BL21(DE3), (Studier, F. W., Rosenberg, A.H., Dunn, J. J. a Dubendorff, J. W. (1990), Methods Enzymol. 185, 60 -89).The pET vector can be propagated in any K-12 strain of E. coli, for example HMS174, HB101, JM109, DH5α, and the like, for cloning or plasmid preparation purposes. Expression hosts include E. coli strains containing a chromosomal copy of the T7 RNA polymerase gene. These hosts are the bacteriophage DE3 lysogens, a lambda derivative that carries the lacI gene, the lacUV5 promoter, and the T7 RNA polymerase gene. T7 RNA polymerase is induced by the addition of isopropyl-β-Dtiogalactoside (IPTG) and T7 RNA polymerase transcribes any target plasmid, such as pET-28b, carrying its significant gene. Strains used in our laboratory include: BL21 (DE3), (Studier, F.W., Rosenberg, A.H., Dunn, J.J., and Dubendorff, J.W. (1990), Methods Enzymol. 185, 60-89).
Na expresiu rekombinantných sekvencií H. pylorí sa použilo 50 ng plazmidu DNA, izolovaného ako je opísané vyššie, na transformovanie príslušnej BL21(DE3) baktérie, ako je opísané vyššie (ktorú poskytol Novagen ako súčasť kitu pET expresívneho systému). Transformované bunky sa kultivovali v SOC médiu počas jednej hodiny a kultúry sa potom naniesli na LB platne obsahujúce 25 pg/ml kanamycínsulfátu. Nasledujúci deň sa bakteriálne kolónie spojili a nechali sa rásť v LB médiu obsahujúcom kanamycínsulfát (25 pg/ml) do optickej hustoty pri 600 nm 0,5 až 1,0 O.D. jednotiek, pričom v tomto bode sa ku kultúre pridal počas 3 hodín 1mM IPTG na vyvolanie expresie H. pylorí rekombinantných DNA konštrukcií.For the expression of recombinant H. pylori sequences, 50 ng of plasmid DNA, isolated as described above, was used to transform the respective BL21 (DE3) bacterium as described above (provided by Novagen as part of the pET expression system kit). The transformed cells were cultured in SOC medium for one hour and the cultures were then plated on LB plates containing 25 µg / ml kanamycin sulfate. The following day, the bacterial colonies were pooled and grown in LB medium containing kanamycin sulfate (25 µg / ml) to an optical density at 600 nm of 0.5 to 1.0 O.D. units, at which point 1 mM IPTG was added to the culture for 3 hours to induce the expression of H. pylori recombinant DNA constructs.
-12Po vyvolaní expresie génu sIPTG sa baktérie granulovali centrifúgovaním v Sorval RC-3B centrifúge pri 3500 x g počas 15 minút pri teplote 4 “C. Granule sa resuspendovali v 50 ml chladného 10 mM Tris-HCI, pH 8,0, 0,1 M NaCI a 0,1 mM EDTA (STE pufer). Bunky sa potom centrifúgovali pri 2000 x g počas 20 minút pri teplote +4 °C. Vlhké granule sa odvážili a zmrazili pri teplote -80 °C, až kým boli pripravené na prečistenie proteínu.After inducing expression of the sIPTG gene, the bacteria were granulated by centrifugation in a Sorval RC-3B centrifuge at 3500 x g for 15 minutes at 4 ° C. The granules were resuspended in 50 ml cold 10 mM Tris-HCl, pH 8.0, 0.1 M NaCl and 0.1 mM EDTA (STE buffer). The cells were then centrifuged at 2000 x g for 20 minutes at + 4 ° C. The wet granules were weighed and frozen at -80 ° C until ready for protein purification.
Analytické metódyAnalytical methods
Koncentrácie prečistených proteínových prípravkov sa kvantifikovali spektrofotometricky s použitím koeficientov absorbancie vypočítaných z obsahu aminokyseliny (Perkins, S. J., 1986 Eur. J. Biochem, 157,169 - 180). Koncentrácie proteínu sa tiež merali pomocou metódy, ktorú opísal Bradford, M. M. (1976) Anál. Biochem. 72, 248 - 254 a Lowry, O. H., Rosebrough, N., Farr, A. L. & Randall, R. J. (1951), s použitím albumínu hovädzieho séra ako štandardu.Concentrations of purified protein preparations were quantified spectrophotometrically using absorbance coefficients calculated from the amino acid content (Perkins, S.J., 1986 Eur. J. Biochem, 157, 169-180). Protein concentrations were also measured using the method described by Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 and Lowry, O.H., Rosebrough, N., Farr, A.L. & Randall, R.J. (1951), using bovine serum albumin as a standard.
Gély dodecylsulfát sodný-polyakrylamid (SDS-PAGE) (gradient akrylamidu 12 % alebo 4 až 25 %) sa získali od BioRad (Hercules, CA, USA) a zafarbili sa s použitím Coomasie Brilliant Blue. Markéry molekulovej hmotnosti obsahujúce skeletálny svalový myozín králika (200 kDa), E. coli β-galaktozidázu (116 kDa), svalovú fosforylázu B králika (97,4 kDa), albumín hovädzieho séra (66,2 kDa), ovalbumín (45 kDa), hovädziu uhličitú anhydrázu (31 kDa), inhibítor trypsínu sóje (21,5 kDa), lyzozým vaječného bielka (14,4 kDa) a hovädzí aprotinín (6,5 kDa).Sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gels (acrylamide gradient of 12% or 4 to 25%) were obtained from BioRad (Hercules, CA, USA) and stained using Coomasie Brilliant Blue. Molecular weight markers containing rabbit skeletal muscle myosin (200 kDa), E. coli β-galactosidase (116 kDa), rabbit muscle phosphorylase B (97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa) , bovine carbon anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa), egg white lysozyme (14.4 kDa) and bovine aprotinin (6.5 kDa).
Prečistenie FlgE z implikovaných teliesokPurification of FlgE from implied bodies
Nasledujúce kroky sa uskutočnili pri teplote +4 °C. Granule buniek sa resuspendovali v lýznom pufre s 10 % glycerolu, 200 pg/ml lyzozómu, 5 mM EDTA, mM PMSF a 0,1 % β-merkaptoetanolu. Po pasáži cez rozrušovač buniek sa výsledný homogenizát spracoval s 0,2 % DOC, miešal sa počas 10 minút, potom sa centrifúgoval (10,000 x g 30 min). Granule sa najskôr premyli s lýznym pufrom obsahujúcim 10 % glycerolu, 10 mM EDTA, 1 % Triton X-100,1 mM PMSF a 0,1 % β-merkaptoetanolu, potom s lýznym pufrom obsahujúcim 1 M močoviny, 1 mM PMSF a 0,1 % β-merkaptoetanolu. Výsledné biele granule primárne pozostávaliThe following steps were performed at +4 ° C. The cell granules were resuspended in lysis buffer with 10% glycerol, 200 µg / ml lysosome, 5 mM EDTA, mM PMSF and 0.1% β-mercaptoethanol. After passage through the cell disrupter, the resulting homogenate was treated with 0.2% DOC, stirred for 10 minutes, then centrifuged (10,000 x g for 30 minutes). The granules were first washed with lysis buffer containing 10% glycerol, 10 mM EDTA, 1% Triton X-100.1 mM PMSF and 0.1% β-mercaptoethanol, then with lysis buffer containing 1 M urea, 1 mM PMSF and 0, 1% β-mercaptoethanol. The resulting white granules consisted primarily
-13z implikovaných teliesok, ktoré neobsahovali rozrušené bunky a membranózne materiály.-13 from implied bodies that did not contain disrupted cells and membranous materials.
Nasledujúce kroky sa uskutočnili pri laboratórnej teplote, implikované telieska sa rozpustili v 20 ml 8 M močoviny v lýznom roztoku s 1 mM PMSF a 0,1 % βmerkaptoetanolu a inkubovali sa pri laboratórnej teplote počas jednej hodiny. Látky, ktoré sa nerozpustili sa odstránili centrifúgovaním (100,00 x g počas 30 min). Číry supernatant sa odfiltroval a naniesol na Ni2+-NTA agarózovú kolónu, ktorá sa ekvilibrovala s 8 M močoviny v lýznom pufre. Kolóna sa premyla s 250 ml (50 vrstvových objemov) lýzneho pufra obsahujúceho 8 M močoviny, 1 mM PMSF a 0,1 % β-merkaptoetanolu a vyvíjala sa s postupnými krokmi lýzneho roztoku obsahujúceho 8 mM močoviny, 1 mM PMSF, 0,1 % β-merkaptoetanolu a 20, 100, 200 a 500 mM imidazolu. Frakcie sa monitorovali pomocou absorbancie pri OD280 nm a piky frakcií sa analyzovali s SDS-PAGE. Dva pásy sa vizualizovali vyfarbením s použitím Coomassie Brilliant Blue, hlavný pás Mr=78 kDa a minoritný pás Mr=60 kDa. Čistota rekombinantnej FlgE (78 kDa) sa vyhodnotila vyššia ako 90 %. Tak ako pri prečistení rozpustných proteínov, frakcie obsahujúce rekombinantný proteín sa eluovali pri 100 mM midazolu.The following steps were performed at room temperature, the implied bodies were dissolved in 20 ml of 8 M urea in lysis solution with 1 mM PMSF and 0.1% β-mercaptoethanol and incubated at room temperature for one hour. Substances that did not dissolve were removed by centrifugation (100.00 xg for 30 min). The clear supernatant was filtered off and loaded onto a Ni 2+ -NTA agarose column which was equilibrated with 8 M urea in lysis buffer. The column was washed with 250 ml (50 bed volumes) lysis buffer containing 8 M urea, 1 mM PMSF and 0.1% β-mercaptoethanol and developed with stepwise lysis solution containing 8 mM urea, 1 mM PMSF, 0.1% β-mercaptoethanol and 20, 100, 200 and 500 mM imidazole. Fractions were monitored by absorbance at OD 280 nm and fractions peaks were analyzed by SDS-PAGE. Two bands were visualized by staining using Coomassie Brilliant Blue, the main band Mr = 78 kDa and the minor band Mr = 60 kDa. The purity of recombinant FlgE (78 kDa) was evaluated to be greater than 90%. As with the purification of soluble proteins, the fractions containing the recombinant protein were eluted at 100 mM midazole.
Močovina sa pomaly odstránila z FlgE polypeptidu dialýzou oproti TBS obsahujúcom 0,5 % DOC s nasledovným postupným znížením močoviny: 6 M, 4 M, 3 M, 2 M, 1 M, 0,5 M a potom 0 M. Každý krok dialýzy sa uskutočňoval počas najmenej 4 hodín pri laboratórnej teplote.Urea was slowly removed from FlgE polypeptide by dialysis versus TBS containing 0.5% DOC with the following gradual decrease in urea: 6 M, 4 M, 3 M, 2 M, 1 M, 0.5 M and then 0 M. Each dialysis step was performed for at least 4 hours at room temperature.
Po dialýze sa vzorky zahustili tlakovou filtráciou s použitím Amicon miešaných buniek. Koncentrácie proteínu sa potom stanovili pomocou postupov Perkinsa, Bradforda a Lowryho.After dialysis, samples were concentrated by pressure filtration using Amicon stirred cells. Protein concentrations were then determined using Perkins, Bradford and Lowry procedures.
Príklady uskutočnenia vynálezuDETAILED DESCRIPTION OF THE INVENTION
Príklad 1Example 1
Terapeutická imunizáciaTherapeutic immunization
1. Materiál a metódy1. Material and methods
- ΜΙ. 1. Zvieratá- ΜΙ. 1. Animals
Samice SPF BALB/c myší sa získali od Bomholt Breeding centre (Dánsko).Female SPF BALB / c mice were obtained from the Bomholt Breeding Center (Denmark).
Udržiavali sa v bežných makrokolónových klietkach s voľným podávaním vody a potravy. Pri dodaní boli zvieratá vo veku 4 až 6 týždňov.They were kept in conventional macrocolumn cages with free water and food. At delivery, the animals were 4-6 weeks of age.
1.2. Infikovanie1.2. infection
Po minimálne jednom týždni aklimatizácie sa zvieratá nainfikovali s typom 2 kmeňa H. pylori (kmeň 244, pôvodne izolovaný od ulceratívneho pacienta). Tento kmeň sa už predtým preukázal ako dobrý kolonizátor v žalúdku myší. Baktérie z kmeňa udržiavané pri teplote -70 °C sa nechali rásť cez noc v Brucela bujóne doplnenom s 10 % fetálneho teľacieho séra, pri teplote +37 °C v mikroaerofilnej atmosfére (10 % CO2, 5 % O2). Zvieratám sa podala orálna dávka omeprazolu (400 pmol/kg) a po 3 až 5 hodinách orálna inokulácia H. pylori (približne 10'7 až 10 8 CFU/zviera). Infekcia sa skúmala na kontrolných zvieratách 2 až 3 týždne po inokulácii.After at least one week of acclimatization, the animals were infected with type 2 strain H. pylori (strain 244, originally isolated from an ulcerative patient). This strain has previously been shown to be a good colonizer in the stomach of mice. Bacteria from the strain maintained at -70 ° C were grown overnight in Brucel broth supplemented with 10% fetal calf serum, at + 37 ° C in a microaerophilic atmosphere (10% CO 2, 5% O 2). The animals were given an oral dose of omeprazole (400 pmol / kg) and after 3-5 hours an oral inoculation of H. pylori (approximately 10 7 to 10 8 CFU / animal). Infection was examined in control animals 2 to 3 weeks after inoculation.
1.3. Imúnizácia1.3 Immunizations
Jeden mesiac po infekcii sa dve skupiny myší (10 myší/skupina) imunizovali štyrikrát v priebehu 34-dňového obdobia (deň 1, 15, 25 a 35). Prečistený rekombinantný FlgE rozpustený v PBS plus 0,5 % deoxycholátu (DOC) sa podával v dávke 100 mikrogramov/myš.One month after infection, two groups of mice (10 mice / group) were immunized four times over a 34-day period (days 1, 15, 25 and 35). Purified recombinant FlgE dissolved in PBS plus 0.5% deoxycholate (DOC) was administered at a dose of 100 micrograms / mouse.
Ako pomocná látka sa zvieratám v oboch kontrolných skupinách ako aj FlgE skupine podávalo 10 pg/myš toxínu cholery (CT) s každou imunizáciou. Omeprazol (400 μηιοΙ/kg) sa podával orálne všetkým zvieratám 3 až 5 hodín pred imunizáciou ako spôsob ochrany antigénov pred kyslou degradáciou. Zvieratá sa utratili 1 až 2 týždne po poslednej imunízácii.As an adjuvant, animals in both control groups and FlgE group were given 10 µg / mouse cholera toxin (CT) with each immunization. Omeprazole (400 μηιοΙ / kg) was administered orally to all animals 3 to 5 hours prior to immunization as a way to protect antigens from acid degradation. Animals were sacrificed 1-2 weeks after the last immunization.
Skupina 1: 300 μΙ PBS s 0,5 % DOC obsahujúceho 10 gg CTGroup 1: 300 μΙ PBS with 0.5% DOC containing 10 gg CT
Skupina 2: 300 μΙ PBS s 0,5 % DOC obsahujúceho 100 μg FlgE a 10 μg CT.Group 2: 300 μΙ PBS with 0.5% DOC containing 100 μg FlgE and 10 μg CT.
1.4. Analýza infekcie1.4. Analysis of infection
-15Myši sa utratili pomocou CO2 a zlomenia šije. Brucho a hrudná dutina sa otvorili a krv sa odobrala punkciou zo srdca. Následne sa žalúdok odstránil. Po rozrezaní žalúdka pozdĺž najväčšieho zakrivenia, sa žalúdok premyl fyziologickým roztokom a potom sa rozrezal na dve rovnaké časti. Oblasť 25 mm2 sliznice zantrumu a z korpusu sa zoškrabali oddelene s použitím chirurgického skalpela. Zoškrabaná sliznica sa suspendovala vBrucela bujóne, zriedila sa a naniesla na Blood Skirrowove platničky. Platničky sa inkubovali pri mikroaerofilných podmienkach počas 3 až 5 dní a počítal sa počet kolónií. Identita H. pylón sa vyhodnotila pomocou močoviny a testu s katalázou a priamou mikroskopiou alebo Gram vyfarbením.-15Mice were sacrificed using CO 2 and neck breakage. The abdomen and thoracic cavity were opened and blood was collected by puncture from the heart. Subsequently, the stomach was removed. After cutting the stomach along the greatest curvature, the stomach was washed with saline and then cut into two equal parts. The 25 mm 2 area of the mucosa of the zantrum and the corpus were scraped separately using a surgical scalpel. The scraped mucosa was suspended in Brucel broth, diluted and plated on Blood Skirrow plates. The plates were incubated under microaerophilic conditions for 3-5 days and the number of colonies counted. The identity of H. pylon was evaluated by urea and a catalase and direct microscopy or Gram stain assay.
1.5. Meranie protilátok1.5. Measurement of antibodies
Protilátky v sére sa odobrali z krvi. Pred centifúgovaním sa krv zriedila rovnakým množstvom PBS. Sérum sa udržiavalo pri teplote -20 °C až do analýzy. Protilátky v sére sa merali s použitím ELISA, pričom platničky boli potiahnuté buď rozdrobenými frakciami kmeňa 244 H. pylón alebo s FlgE a následne sa pridali rozličné zriedenia séra. ELISA sa vyvíjala s alkalickou fosfatázou značenými myšími anti-lg-protilátkami. Anti-lg protilátky boli typu reťazca anti-ťažký/anti-ľahký, ktorý by mal detegovať všetky typy protilátok.Serum antibodies were collected from blood. Prior to centrifugation, blood was diluted with an equal amount of PBS. The serum was maintained at -20 ° C until analysis. Serum antibodies were measured using ELISA, wherein the plates were coated with either fragmented H. pylon strain or FlgE, and various serum dilutions were then added. ELISA was developed with alkaline phosphatase labeled mouse anti-Ig antibodies. Anti-Ig antibodies were of the anti-heavy / anti-light chain type, which should detect all types of antibodies.
2. Výsledky2. Results
2.1. Terapeutická imunizácia: vplyv na CFU2.1. Therapeutic immunization: effect on CFU
Zvieratá v tejto štúdii boli infikované kmeňom 244 H. pylori jeden mesiac pred imunizáciou. Myši v skupinách po desať sa potom imunizovali buď s toxínom cholery (CT) alebo CT spolu s rekombinantným FlgE polypeptídom. Štyri týždne po poslednej imunizácii sa zvieratá utratili a stanovila sa CFU (Obrázok 1). Zvieratá ošetrené so samotným CT boli vysoko infikované, ako v korpuse tak i v antrume. Zvieratá aktívne imunizované s rekombinantným FlgE polypeptidom a CT vykazovali signifikantné zníženie CFU hodnôt v antrume a v žalúdku, v porovnaní so zvieratami imunizovanými s CT (p<0,01 a p<0,05; Wilcoxon-Mann-Whittneyov klasifikačný test znakov).Animals in this study were infected with strain H. H. pylori one month before immunization. Mice in groups of ten were then immunized with either cholera toxin (CT) or CT together with the recombinant FlgE polypeptide. Four weeks after the last immunization, animals were sacrificed and CFU was determined (Figure 1). Animals treated with CT alone were highly infected in both the corpus and the antrum. The animals actively immunized with the recombinant FlgE polypeptide and CT showed a significant decrease in CFU values in the antrum and stomach compared to animals immunized with CT (p <0.01 and p <0.05; Wilcoxon-Mann-Whittney character classification test).
2.2. Terapeutická imunizácia: vplyv na tvorbu protilátok a sekréciu2.2 Therapeutic immunization: effect on antibody production and secretion
-16Ako príznak infekcie H. pylorí sa môžu v sére nájsť špecifické protilátky (kontrola/244). U zvierat, ktoré dostávali FlgE + CT sa titer voči kmeňu 244 (ako membránové proteíny) zvýšil štvornásobne (p<0.01). Len u zvierat, ktoré dostávaliAs a symptom of H. pylori infection, specific antibodies can be found in the serum (control / 244). In animals receiving FlgE + CT, titer to strain 244 (as membrane proteins) increased fourfold (p <0.01). Only in the animals they received
FlgE + CT sa dal namerať špecifický IgG titer voči FlgE (Obrázok 2).FlgE + CT could be measured for specific IgG titer to FlgE (Figure 2).
FlgE špecifická IgG sa zvýšila u zvierat, ktoré dostávali FlgE + CT, avšak nedala sa detegovať pri kontrolných zvieratách.FlgE specific IgG was increased in animals receiving FlgE + CT but could not be detected in control animals.
Uvedené výsledky ukazujú, že rekombinantný FlgE polypeptid H. pylón je vysoko imunogénny, ak sa podáva orálne, spolu s toxínom cholery ako pomocnou látkou, merané ako zvýšenie systémových FlgE špecifických Ig protilátok. Imunizácia s FlgE mala tiež za následok signifikantné zvýšenie ig titrov voči rozdrobenej frakcii H. pylón. Okrem toho sa zistilo dramatické zníženie počtu H. pylorí kolonizujúcich sliznicu žalúdka infikovaných myší po imunizácii s FlgE spolu s toxínom cholery.The results show that the recombinant H. pylon FlgE polypeptide is highly immunogenic when administered orally, together with cholera toxin as an adjuvant, measured as an increase in systemic FlgE specific Ig antibodies. Immunization with FlgE also resulted in a significant increase in ig titers relative to the fragmented H. pylon fraction. In addition, a dramatic decrease in the number of H. pylori colonizing the mucosa of the stomach of infected mice was found after immunization with FlgE together with cholera toxin.
-17Zoznam sekvencií (1) Všeobecné informácie (i) Prihlasovateľ (A) Meno: Astra AB (B) Ulica: Västra Mälarehamnen 9 (C) Mesto: Sôdertälje (E) Štát: Švédsko (F) Kód pošty (ZIP): S-151 85 (G) Telefón: +46 8 553 260 00 (H) Telefax: +46 8 553 288 20 (i) Názov vynálezu: Kompozície očkovacej látky obsahujúce FlgE polypeptid Helicobacter pylori (ii) Počet sekvencií: 4 (iii) Počítačom čitateľná forma:-17 Sequence Listing (1) General (i) Applicant (A) Name: Astra AB (B) Street: Västra Mälarehamnen 9 (C) City: Södertälje (E) Country: Sweden (F) Post Code (ZIP): S- 151 85 (G) Telephone: +46 8 553 260 00 (H) Fax: +46 8 553 288 20 (i) Title of the invention: Vaccine compositions containing the Helicobacter pylori FlgE polypeptide (ii) Number of sequences: 4 (iii) Computer readable form:
(A) Typ média: Floppy disk (B) Počítač: IBM PC kompatibilný (C) Operačný systém: PC-DOS/MS-DOS (D) Software: Patentln Reease #1.0, verzia #1,30 (EPO) (2) Informácie pre SEQ ID NO: 1:(A) Media Type: Floppy Disk (B) Computer: IBM PC Compatible (C) Operating System: PC-DOS / MS-DOS (D) Software: Patentln Reease # 1.0, Version # 1.30 (EPO) (2) Information for SEQ ID NO: 1:
(i) Charakteristiky sekvencie:(i) Sequence characteristics:
(A) Dĺžka: 2550 základných párov (B) Typ: nukleová kyselina (C) Reťazec: dvojitý (D) Topológia: lineárna (ii) Typ molekuly: cDNA (vi) Pôvodný zdroj:(A) Length: 2550 base pairs (B) Type: nucleic acid (C) String: double (D) Topology: linear (ii) Molecule type: cDNA (vi) Original source:
(A) Organizmus: Helicobacter pylori (ix) Charakteristika:(A) Organism: Helicobacter pylori (ix) Characteristics:
(A) Názov/kľúč: CDS (B) Lokalizácia: 321..2477 (D) Ďalšie informácie: /produkt = “FlgE Flagelámy hook proteín (x) Publikačné informácie:(A) Name / Key: CDS (B) Localization: 321..2477 (D) Other Information: / Product = “FlgE Flagellam Hook Protein (x) Editorial Information:
-18(A) Autori: O'Toole, Paul W., Kostrzynska, Magdaléna, Trust, Trevor J.-18 (A) Authors: O'Toole, Paul W., Kostrzynska, Magdalena, Trust, Trevor J.
(B) Názov: Non-motile mutants of Helicobacter pylón and Helicobacter mustelae defective in flagellar hook production (C) Časopis: Mol. Microbiology (D) Ročník: 14 (E) Číslo: 4 (F) Strany: 691 - 703 (G) Dátum: 1994 (xi) Popis sekvencie: SEQ ID NO:1:(B) Title: Non-motile mutants of Helicobacter pylon and Helicobacter mustelae defective in flagellar hook production (C) Magazine: Mol. Microbiology (D) Volume: 14 (E) Number: 4 (F) Pages: 691 - 703 (G) Date: 1994 (xi) Description of Sequence: SEQ ID NO: 1:
AACAAAGCGA TAACTCCTTT GTCTTATTAG CGACACAATT TAACCCATTG ACTTTAAATC GCGCTTCAGC cgaagagatt caagatcatg aatgcgcgat tttgcactaa AGCGAGTTÄG attcttaäat ttgagcgata acctttaaaa AGCGTAATTA AGGGGTGGTG ttacaaaaccAACAAAGCGA TAACTCCTTT GTCTTATTAG CGACACAATT TAACCCATTG ACTTTAAATC GCGCTTCAGC cgaagagatt caagatcatg aatgcgcgat tttgcactaa AGCGAGTTÄG attcttaäat ttgagcgata
19CCCTATCCCC TTATGAATTT19CCCTATCCCC TTATGAATTT
CÄATCATTCT AAAAAGCTATCÄATCATTCT AAAAAGCTAT
CTTTAAATTA AAGGATAACCCTTTAAATTA AAGGATAACC
GACCGATCTT TTTGATTAAC AAAACTTTAA AATCCGCAATGACCGATCTT TTTGATTAAC AAAACTTTAA AATCCGCAAT
TTAGGAACAA CTTTTGCTTT ATTTTGCATA GATTGAATTTTTAGGAACAA CTTTTGCTTT ATTTTGCATA GATTGAATTT
ATG CTT AGG TCT TTA TGG TCT GGT GTC AAT Met Leu Arg Ser Leu Tro Ser Gly Val AsnATG CTT AGG TCT TTA TGG TCT GGT GTC AAT Met Leu Arg Ser Leu Tro Ser Gly Val Asn
1010
220 225 230 'CT TTA TAC AAT GAA GAT GGC GAC GCT CTT TTG TTG AAT GAA AAT CAA220 225 230 'CT TTA TAC AAT GAA GAT GG GAT GCT CTT TTG TTG AAT GAA AAT CAA
495 500 505495 500 505
-21GGA GCG GAT TGG AAT TTT AGA GTG ATC GTG CCT GAG CCT GGG GAA TTA Gly Ala Asp Trp Asn Phe Arg Val íle Val Pro Glu Pro Gly Glu Leu-21GGA GAT GAT TGG AAT TTT AGA GTG ATC GTG CCT GAG CCT GGG GAA TTA Gly Ala Asp
510 515 520(+420) 510 515 520
GTA GGG GGG TCA GCG GCT AGG CCT AAT GTG TTT GAA GGA GGC CGT TTG Val Gly Gly Ser Ala Ala Arg Pro Asn Val Phe Glu Gly Gly Arg LeuGTA GGG GGG TCA GCG GCT AGG CCT AAT GTG TTT GAA GGA GGC CGT TTG Val Gly Gly Ser Ala Ala
525 530 535525 530 535
AAT CTT AAG CAA TAA ACTAAAGGAT TACTCTAATA CAATATAATA GGGGCTAATT Asn Leu Lys Gin *AAT CTT AAG CAA TAA ACTAAAGGAT TACTCTAATA CAATATAATA GGGGCTAATT Asn Leu Lys Gin
715715
TAAAGATTAA GGTTTAGTAT GCATGAATAC TCGTAAAGATTAA GGTTTAGTAT GCATGAATAC TCG
-22(2) Informácie pre SEQ ID NO: 2:(2) Information for SEQ ID NO: 2:
(i) Charakteristiky sekvencie:(i) Sequence characteristics:
(A) Dĺžka: 719 aminokyselín (B) Typ: aminokyselina (C) Topológia: lineárna (ii) Typ molekuly: proteín (xi) Popis sekvencie: SEQ ID NO:2:(A) Length: 719 amino acids (B) Type: amino acid (C) Topology: linear (ii) Molecule type: protein (xi) Sequence description: SEQ ID NO: 2:
325 330 335(+420) 325 330 335
Ser Lys Leu Glu Ser Ser Asn Val Asp Leu Ser Arg Ser Leu Thr AsnSer Lys Leu Glu Ser Ser Asn Val Asp Ser Le Ser Ser Ser Le Thr Asn
-25675 680 685-25675 680 685
Leu íle Val Val Gin Arg Gly ?he Gin Ala Asn Ser Lys Ala Val Th’· 690 695 700Leu il Val Val Gin Arg Gly? He Gin Ala Asn Ser Lys Ala Val Th '· 690 695 700
Thr Ser Asp Gin íle Leu Asn Thr Leu Leu Asn Leu Lys Gin *Thr Ser Asp Gin White Leu Asn Thr Leu Leu Asn Leu Lys Gin *
705 710 715 (2) Informácie pre SEQ ID NO: 3:705 710 715 (2) Information for SEQ ID NO: 3:
(i) Charakteristiky sekvencie:(i) Sequence characteristics:
(A) Dĺžka: 25 základných párov (B) Typ: nukleová kyselina (C) Reťazec: jednoduchý (D) Topológia: lineárna (ii) Typ molekuly: Ďalšie nukleové kyseliny (A) Popis: /dese = PRC primér” (xi) Popis sekvencie: SEQ ID NO:3:(A) Length: 25 base pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: Other nucleic acids (A) Description: / dese = PRC primer ”(xi) Sequence description: SEQ ID NO: 3:
TATACCATGG TGCTTAGGTC TTTAT (2) Informácie pre SEQ ID NO: 4:TATACCATGG TGCTTAGGTC TTTAT (2) Information for SEQ ID NO: 4:
(i) Charakteristiky sekvencie:(i) Sequence characteristics:
(A) Dĺžka: 25 základných párov (B) Typ: nukleová kyselina (C) Reťazec: jednoduchý (D) Topológia: lineárna (ii) Typ molekuly: Ďalšie nukleové kyseliny (A) Popis: /dese = “PCR primér” (xi) Popis sekvencie: SEQ ID NO:4:(A) Length: 25 base pairs (B) Type: nucleic acid (C) String: single (D) Topology: linear (ii) Molecule type: Other nucleic acids (A) Description: / dese = “PCR primer” (xi ) Sequence Description: SEQ ID NO: 4:
GCGAATTCAA TTGCTTAAGA TTCAAGCGAATTCAA TTGCTTAAGA TTCAA
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- 1998-06-08 EP EP98928772A patent/EP1009764A1/en not_active Withdrawn
- 1998-06-08 CA CA002293293A patent/CA2293293A1/en not_active Abandoned
- 1998-06-08 PL PL98337503A patent/PL337503A1/en unknown
- 1998-06-08 IL IL13314498A patent/IL133144A0/en unknown
- 1998-06-08 CN CN98806101A patent/CN1259960A/en active Pending
- 1998-06-08 NZ NZ501427A patent/NZ501427A/en unknown
- 1998-06-08 AU AU80487/98A patent/AU8048798A/en not_active Abandoned
- 1998-06-08 KR KR1019997011714A patent/KR20010013699A/en not_active Application Discontinuation
- 1998-06-08 ID IDW991542A patent/ID23052A/en unknown
- 1998-06-08 TR TR1999/03060T patent/TR199903060T2/en unknown
- 1998-06-08 HU HU0003164A patent/HUP0003164A3/en unknown
- 1998-06-08 JP JP50228399A patent/JP2002507118A/en active Pending
- 1998-06-08 WO PCT/SE1998/001093 patent/WO1998056816A1/en not_active Application Discontinuation
- 1998-06-08 BR BR9810026-2A patent/BR9810026A/en not_active IP Right Cessation
-
1999
- 1999-12-08 IS IS5288A patent/IS5288A/en unknown
- 1999-12-10 NO NO996132A patent/NO996132L/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
NO996132L (en) | 2000-01-28 |
BR9810026A (en) | 2000-09-19 |
JP2002507118A (en) | 2002-03-05 |
NO996132D0 (en) | 1999-12-10 |
WO1998056816A1 (en) | 1998-12-17 |
EP1009764A1 (en) | 2000-06-21 |
AU8048798A (en) | 1998-12-30 |
PL337503A1 (en) | 2000-08-28 |
CA2293293A1 (en) | 1998-12-17 |
IS5288A (en) | 1999-12-08 |
CN1259960A (en) | 2000-07-12 |
ID23052A (en) | 2000-01-20 |
HUP0003164A2 (en) | 2000-12-28 |
ZA984696B (en) | 1999-01-04 |
HUP0003164A3 (en) | 2001-10-29 |
TR199903060T2 (en) | 2000-09-21 |
EE9900566A (en) | 2000-06-15 |
SE9702242D0 (en) | 1997-06-12 |
AR012896A1 (en) | 2000-11-22 |
KR20010013699A (en) | 2001-02-26 |
NZ501427A (en) | 2000-09-29 |
IL133144A0 (en) | 2001-03-19 |
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