ITRM930343A1 - RECOMBINANT PROTEINS, VECTORS FOR THE EXPRESSION AND THEIR USES. - Google Patents
RECOMBINANT PROTEINS, VECTORS FOR THE EXPRESSION AND THEIR USES. Download PDFInfo
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- ITRM930343A1 ITRM930343A1 IT000343A ITRM930343A ITRM930343A1 IT RM930343 A1 ITRM930343 A1 IT RM930343A1 IT 000343 A IT000343 A IT 000343A IT RM930343 A ITRM930343 A IT RM930343A IT RM930343 A1 ITRM930343 A1 IT RM930343A1
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- sequence
- amino acid
- chimeric proteins
- proteins according
- recombinant chimeric
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Description
DESCRIZIONE DESCRIPTION
a corredo di una domanda di brevetto per invenzione dal titolo: "Proteine ricombinanti, vettori per l'espressione e loro usi" accompanying a patent application for an invention entitled: "Recombinant proteins, vectors for expression and their uses"
La presente invenzione concerne proteine ricombinanti, vettori per l'espressione e loro usi. The present invention relates to recombinant proteins, vectors for expression and their uses.
Pi? in particolare l'invenzione riguarda proteine chimeriche ricombinanti, comprendenti sequenze della proteina che si desidera produrre covalentemente legate alla sequenza della ferritina, 0 anche libere da detta sequenza a mezzo di digestione proteolitica specifica della proteina chimerica, ottenibili a seguito di trasformazione di un vettore comprendente le sequenze codificanti per esse, in un ospite. Pi? in particular, the invention relates to recombinant chimeric proteins, comprising sequences of the protein which it is desired to produce covalently linked to the ferritin sequence, or also free from said sequence by means of specific proteolytic digestion of the chimeric protein, obtainable following transformation of a vector comprising the coding sequences for them, in a host.
La ferritina ? una proteina per l'immagazzinamento del ferro, ampiamente distribuita nel mondo vivente (per una recente review sull'argomento vedi Andrews, S.C. et al., 1992, J. Inorg. Biochem., 47, 161 -174). La ferritina ? composta da 24 subunit?, ? termostabile e pu? essere espressa con alte rese dopo trasformazione di vettori ricombinanti opportuni, in cellule di E. coli. (Levi, S. et al. (1987) Gene 51 , 269-274; Levi, S. et al. (1988) J. Biol. Chem. 263, 18086-18092). Ferritin? an iron storage protein, widely distributed in the living world (for a recent review on the subject see Andrews, S.C. et al., 1992, J. Inorg. Biochem., 47, 161-174). Ferritin? composed of 24 subunits,? thermostable and can? be expressed with high yields after transformation of suitable recombinant vectors, in E. coli cells. (Levi, S. et al. (1987) Gene 51, 269-274; Levi, S. et al. (1988) J. Biol. Chem. 263, 18086-18092).
La proteina ottenuta per via ricombinante mantiene la struttura terziaria e quaternaria della proteina naturale ed ? in grado di incorporare il ferro. The recombinant protein obtained maintains the tertiary and quaternary structure of the natural protein and? able to incorporate iron.
La produzione di proteine per via ricombinante su larga scala ? spesso limitata dalle rese non ottimali del procedimento, dalia insolubilit? delle proteine e, ancora, dall'ottenimento di prodotti parzialmente o totalmente denaturati e, comunque, non in grado di esercitare la loro attivit? biologica. Large-scale recombinant protein production? often limited by the non-optimal yields of the procedure, by the insolubility? of proteins and, again, from obtaining partially or totally denatured products and, in any case, not able to exercise their activity? biological.
Nel campo dell'immunologia, ed in particolare dell?allergologia, la produzione di proteine per via ricombinante, che conservino le propriet? antigeniche delle corrispondenti proteine naturali, ? indispensabile per l'allestimento di composizioni farmaceutiche, di vaccini e di saggi immunodiagnostici. Questa esigenza ? ancor pi? sentita in quei casi in cui la proteina antigenica naturale ? ottenibile in quantit? e/o qualit? insufficienti per un suo uso in farmacologia, in diagnostica o nella preparazione di vaccini. In the field of immunology, and in particular of allergology, the production of recombinant proteins, which preserve the properties? antigenic of the corresponding natural proteins,? indispensable for the preparation of pharmaceutical compositions, vaccines and immunodiagnostic assays. This need? even more? felt in those cases where the natural antigenic protein? obtainable in quantity? and / or quality? insufficient for its use in pharmacology, diagnostics or in the preparation of vaccines.
Gli autori della presente invenzione hanno costruito un vettore di espressione in cellule batteriche di sequenze codificanti esogene. (I vettore comprende una sequenza codificante per una porzione della ferritina umana sotto il controllo del promotore P\_ del fago ? e, a valle di detta sequenza, siti di inserzione di un gene esogeno. Pertanto il vettore ? in grado di esprimere una proteina chimerica, dopo trasformazione in un ospite. Sorprendentemente le sequenze della ferritina non alterano le propriet? biologiche e immunoiogiche della proteina da produrre e rendono possibile la purificazione ad alte rese della proteina chimerica ottenuta. Inoltre, inserendo dei siti di attacco per proteasi specifiche tra la porzione ferritina e la porzione codificata dal gene esogeno, ? possibile separare la porzione ferritina da quella che si desidera produrre. The authors of the present invention constructed an expression vector in bacterial cells of exogenous coding sequences. (The vector comprises a coding sequence for a portion of human ferritin under the control of the phage P \ _ promoter and, downstream of said sequence, insertion sites of an exogenous gene. Therefore the vector is capable of expressing a protein chimeric, after transformation into a host. Surprisingly, the ferritin sequences do not alter the biological and immunological properties of the protein to be produced and make it possible to purify the obtained chimeric protein at high yields. Furthermore, by inserting attachment sites for specific proteases between the ferritin portion and the portion encoded by the exogenous gene, it is possible to separate the ferritin portion from the one you want to produce.
Gli autori hanno introdotto diverse sequenze geniche esogene ed hanno ottenuto alte rese delle proteine prodotte ed il mantenimento delle propriet? biologiche di esse. Uno dei geni utilizzati ? quello codificante per l'allergene Lol pii, estratto da polline di Lolium perenne. Il cDNA ? stato isolato e sequenziato dagli autori della presente invenzione. Dopo tentativi fallimentari di espressione della proteina Lol pii utilizzando diversi vettori, la sequenza di cDNA ? stata inserita in un vettore comprendente sequenze codificanti per una porzione della ferritina umana. L'espressione in cellule di E. coli dei vettore ricombinante cos? ottenuto, permette di ottenere con alte rese la proteina chimerica, in forma solubile e con una capacit? antigenica paragonabile a quella della proteina Lol pii naturale. La proteina chimerica ricombinante ? pertanto utilizzata nell'allestimento di saggi aliergenici. Gli autori hanno anche inserito sequenze codificanti per l'allergene Lol pi da L. The authors have introduced several exogenous gene sequences and have obtained high yields of the proteins produced and the maintenance of the properties? biological of them. One of the genes used? the one coding for the Lol pii allergen, extracted from Lolium perennial pollen. The cDNA? been isolated and sequenced by the authors of the present invention. After unsuccessful attempts to express the Lol pii protein using different vectors, the cDNA sequence? was inserted into a vector comprising sequences encoding a portion of human ferritin. The expression in E. coli cells of the recombinant vector cos? obtained, allows to obtain with high yields the chimeric protein, in a soluble form and with a capacity? antigenic comparable to that of the natural Lol pii protein. The recombinant chimeric protein? therefore used in the preparation of alien assays. The authors also inserted coding sequences for the Lol pi allergen from L.
perenne e per il fattore di crescita epidermale umano (hEGF o urogastrone), con risultati soddisfacenti. perennial and for human epidermal growth factor (hEGF or urogastrone), with satisfactory results.
Formano pertanto oggetto delia presente invenzione proteine ricombinanti comprendenti una prima sequenza aminoacidica, compresa nella sequenza della ferritina, covalentemente legata a mezzo di legame peptidico ad una seconda sequenza aminoacidica, e di essere producibili ed estraibili ad alte rese da cellule ospiti, mantenendo almeno una propriet? fisiologica o antigenica di detta seconda sequenza aminoacidica, dette cellule trasformate con un vettore codificante per detta prima e detta seconda sequenza aminoacidica sotto il controllo di un promotore di trascrizione. Therefore, the object of the present invention is recombinant proteins comprising a first amino acid sequence, included in the ferritin sequence, covalently linked by means of peptide binding to a second amino acid sequence, and being producible and extractable at high yields from host cells, maintaining at least one property ? physiological or antigenic of said second amino acid sequence, said cells transformed with a vector coding for said first and said second amino acid sequence under the control of a transcription promoter.
NeH'ambito della presente invenzione per propriet? fisiologica, o biologica, o antigenica si intende almeno una propriet? delia sequenza aminoacidica naturale; per alte rese si intendono rese superiori a 4-5 mg di proteina purificata per litro di coltura batterica, o uguali o superiori al 10% p/p delle proteine batteriche solubili totali. Within the scope of the present invention by property physiological, or biological, or antigenic is meant at least one property? of the natural amino acid sequence; by high yields we mean yields greater than 4-5 mg of purified protein per liter of bacterial culture, or equal to or greater than 10% w / w of total soluble bacterial proteins.
Secondo un aspetto dell'invenzione detta prima sequenza aminoacidica comprende la sequenza dall'aminoacido 1 3 afl'aminoacido 158 della catena H della ferritina umana. According to an aspect of the invention, said first amino acid sequence comprises the sequence from the amino acid 1 3 to the amino acid 158 of the H chain of human ferritin.
Sempre secondo l'invenzione detta prima sequenza aminoacidica pu? essere separata da detta seconda sequenza aminoacidica a mezzo di digestione proteolitica specifica. Still according to the invention, said first amino acid sequence can? be separated from said second amino acid sequence by means of specific proteolytic digestion.
In una forma preferita di attuazione dell'invenzione, detta seconda sequenza aminoacidica comprende una sequenza immunogena, preferibilmente una sequenza allergenica, pi? preferibilmente una sequenza allergenica di un allergene di L. perenne. Preferibilmente detta sequenza allergenica di L. perenne comprende sequenze dell'allergene Lol pii o porzioni di esse, sempre pi? preferibilemente la sequenza descritta nella figura 2, o porzioni di essa ad attivit? allergenica. Alternativamente detta sequenza allergenica di L. perenne comprende sequenze dell'allergene Lol pi o porzioni di esse. In a preferred embodiment of the invention, said second amino acid sequence comprises an immunogenic sequence, preferably an allergenic sequence, more? preferably an allergen sequence of a perennial L. allergen. Preferably, said allergenic sequence of L. perennial comprises sequences of the Lol pii allergen or portions thereof, always more? preferably the sequence described in Figure 2, or portions of it with activity? allergenic. Alternatively, said L. perennial allergen sequence comprises Lol pi allergen sequences or portions thereof.
in una forma alternativa dell' invenzione, detta seconda sequenza aminoacidica comprende una sequenza codificante per un fattore di crescita, preferibilmente il fattore di crescita epidermale o EGF, pi? preferibilmente l'EGF di origine umana. in an alternative form of the invention, said second amino acid sequence comprises a sequence coding for a growth factor, preferably the epidermal growth factor or EGF, plus? preferably the EGF of human origin.
Costituisce ulteriore oggetto dell'invenzione un vettore per l'espressione delie proteine chimeriche ricombinanti dell'invenzione. A further object of the invention is a vector for the expression of the recombinant chimeric proteins of the invention.
Ulteriore oggetto delia presente invenzione ? l'uso delle proteine chimeriche ricombinanti dell'invenzione per la preparazione di composizioni farmacologiche, per vaccini o per fa preparazione di corredi diagnostici per la evidenziazione di anticorpi specifici per detta seconda sequenza aminoacidica da un campione. Further object of the present invention? the use of the recombinant chimeric proteins of the invention for the preparation of pharmacological compositions, for vaccines or for the preparation of diagnostic kits for the detection of specific antibodies for said second amino acid sequence from a sample.
La presente invenzione verr? ora descritta in suoi esempi illustrativi, facendo riferimento alle seguenti figure in cui: The present invention will come now described in its illustrative examples, with reference to the following figures in which:
- la figura 1 rappresenta la sequenza nucleotidica del vettore pVH e quella aminoacidica della catena H della ferritina; Figure 1 represents the nucleotide sequence of the pVH vector and the amino acid sequence of the H chain of ferritin;
- la figura 2 rappresenta la sequenza nucleotidica ed aminoacidica della porzione della proteina allergenica Lol pii, clonata nel vettore pVH-Loipil, ad attivit? allergenica; - figure 2 represents the nucleotide and amino acid sequence of the portion of the allergenic protein Lol pii, cloned in the pVH-Loipil vector, with activity? allergenic;
- la figura 2B rappresenta la sequenza nucleotidica ed aminoacidica della proteina allergenica Lol pii, clonata nel vettore pVH-Lolpil/FX, insieme al sito di attacco della proteasi Xa; - figure 2B represents the nucleotide and amino acid sequence of the allergenic protein Lol pii, cloned in the pVH-Lolpil / FX vector, together with the attachment site of the protease Xa;
- la figura 3 rappresenta una tabella di paragone delle sequenze aminoacidiche di Lol pi, Il e III; - figure 3 represents a comparison table of the amino acid sequences of Lol pi, II and III;
- la figura 4 rappresenta un quadro elettroforetico in condizioni denaturanti (A) e non denaturanti (B) di estratti proteici di cellule di E. coli trasformate con il vettore pVH-Lol pii; - figure 4 represents an electrophoretic picture under denaturing (A) and non-denaturing (B) conditions of protein extracts of E. coli cells transformed with the vector pVH-Lol pii;
- la figura 5 rappresenta un'analisi per "western blot? della proteina ricombinante Ft-Lol pii, in condizioni non denaturanti (A) e denaturanti (B); - figure 5 represents a western blot analysis of the recombinant protein Ft-Lol pii, in non-denaturing (A) and denaturing (B) conditions;
- la figura 6 rappresenta un grafico in cui sono riportati i risultati di un saggio ELISA per la determinazione di IgE anti-Lol pii nei siero (A) e (B); - figure 6 represents a graph showing the results of an ELISA assay for the determination of IgE anti-Lol pii in serum (A) and (B);
- la figura 7 rappresenta un grafico in cui sono riportati i risultati di un saggio di rilascio di istamina in soggetti allergici a Lol pll; - figure 7 represents a graph showing the results of a histamine release assay in subjects allergic to Lol pll;
- la figura 8 rappresenta una foto di un gel di proteine SDS-PAGE, in cui 1 ) Ceppo E. coli GC76 non indotto; 2) Ceppo E. coli GC76 indotto; 3) Ceppo E. coli GC76 con plasmide ricombinante Ft-Lol p I, indotto; 4) Ceppo E. coli GC76 con plasmide ricombinante, non indotto; - figure 8 represents a photo of an SDS-PAGE protein gel, in which 1) strain E. coli GC76 not induced; 2) E. coli GC76 induced strain; 3) E. coli GC76 strain with induced recombinant plasmid Ft-Lol p I; 4) Strain E. coli GC76 with recombinant plasmid, not induced;
- la figura 9 rappresenta la sequenza del gene sintetico per hEGF, in cui le sequenze specifiche per gli enzimi Xbal, Kpnl e per la protessi fattore Xa sono indicate e il sito di taglio specifico per la protessi ? indicato da una freccia; - Figure 9 represents the sequence of the synthetic gene for hEGF, in which the specific sequences for enzymes Xbal, Kpnl and for the protected factor Xa are indicated and the specific cleavage site for the protected? indicated by an arrow;
- la figura 10 rappresenta una foto di un gel di proteine SDS-PAGE, in cui : 1 ) Ceppo E. coli GC76; 2) Ceppo E. coli GC76, sonicato e scaldato a 65 ?C; 3) Ceppo GC76 con plasmide ricombinante Ft-hEGF; 4) Ceppo GC76 con plasmide ricombinante Ft-hEGF, sonicato e scaldato a 65 ?C. Gli stessi campioni sono trasferiti (western blotting) e incubati con un anticorpo monocfonale specifico anti-ferritina unama H: 5) Ceppo GC76 con plasmide ricombinante Ft-hEGF, sonicato e scaldato a 65?C; 6} Ceppo GC76 con plasmide ricombinante Ft-hEGF; 7) Ceppo E. coli GC76, sonicato e scaldato a 65?C; 8) Ceppo E. coli GC76; 9) Ferritina umana H ricombinante. - figure 10 represents a photo of an SDS-PAGE protein gel, in which: 1) strain E. coli GC76; 2) Strain E. coli GC76, sonicated and heated to 65 ° C; 3) GC76 strain with recombinant Ft-hEGF plasmid; 4) Strain GC76 with recombinant Ft-hEGF plasmid, sonicated and heated to 65 ° C. The same samples are transferred (western blotting) and incubated with a specific mono-phonal antibody anti-ferritin unama H: 5) Strain GC76 with recombinant Ft-hEGF plasmid, sonicated and heated to 65 ° C; 6} GC76 strain with recombinant Ft-hEGF plasmid; 7) Strain E. coli GC76, sonicated and heated to 65 ° C; 8) Strain E. coli GC76; 9) Recombinant human ferritin H.
ESEMPIO 1 Costruzione del vettore di espressione DVH EXAMPLE 1 Construction of the DVH expression vector
Il vettore plasmidico pVH ? derivato dal vettore pEMBL8 (ATCC N. 37396), secondo metodiche note agli esperti del settore e descritte nei riferimenti bibliografici seguenti. Il plasmide pEMBL8 ? sostituito nel poivlinker come descritto in Sollazzo et al. Gene 37, 1 99-206, originando il vettore pEMBLex2; in questo vettore ? inserita la sequenza codificante per la catena H della ferritina, originando il vettore pEMBLex2-HFt (Levi et al., 1987 Gene 51 , 269-274), modificato per mutagenesi sito-diretta secondo metodiche convenzionali. Questo vettore codifica per un mutante delia catena H della ferritina umana denominato 222, che porta le sostituzioni K86Q, per ottenere la cristallizzazione della proteina; E62K e H65G che aboliscono l?attivit? ferrossidasica ed aumentano la stabilit? della ferritina, come dimostrato in Lawson, M.D. et al. (1991 ) Nature 349, 541-544 e in Santambrogio, P. et al. (1992) J. Biol. Chem. 267, 14077-14083. Gli autori dell'Invenzione hanno inserito sequenze di riconoscimento per gli enzimi Xba I e Kpn I, per poter clonare, nella stessa cornice di lettura della catena della ferritina, geni esogeni codificanti per le proteine da produrre, i siti di restrizione sono ottenuti per mutagenesi sito diretta, come descritto in Levi, S. et al. (1988) J. Biol. Chem. 263, 18086-18092. L'innesco per la mutagenesi sito diretta attuato sul vettore pEMBLexHFT/222 ? il seguente: The pVH plasmid vector? derived from the vector pEMBL8 (ATCC N. 37396), according to methods known to those skilled in the art and described in the following bibliographical references. The pEMBL8 plasmid? substituted in the poivlinker as described in Sollazzo et al. Gene 37, 1 99-206, giving rise to the vector pEMBLex2; in this vector? inserted the coding sequence for the ferritin H chain, giving rise to the pEMBLex2-HFt vector (Levi et al., 1987 Gene 51, 269-274), modified for site-directed mutagenesis according to conventional methods. This vector encodes a human ferritin H-chain mutant named 222, carrying the K86Q substitutions, to achieve protein crystallization; E62K and H65G that abolish the activity? ferroxidase and increase the stability? of ferritin, as demonstrated in Lawson, M.D. et al. (1991) Nature 349, 541-544 and in Santambrogio, P. et al. (1992) J. Biol. Chem. 267, 14077-14083. The authors of the invention have inserted recognition sequences for the enzymes Xba I and Kpn I, in order to clone, in the same reading frame of the ferritin chain, exogenous genes coding for the proteins to be produced, the restriction sites are obtained by mutagenesis direct site, as described in Levi, S. et al. (1988) J. Biol. Chem. 263, 18086-18092. Is the primer for site-directed mutagenesis implemented on the pEMBLexHFT / 222 vector? the following:
innesco SH10: 5'-GAC-AAG-CAC-ACT-CTA-GAA-GAC-AGT-GGT-ACC-GAA-AGC-TAG-3? SH10 primer: 5'-GAC-AAG-CAC-ACT-CTA-GAA-GAC-AGT-GGT-ACC-GAA-AGC-TAG-3?
L'inserzione dei due siti di restrizione nella regione del vettore induce le sostituzioni G176 a E, D179 a G e N180 a T alla estremit? C-terminale della catena H della ferritina. The insertion of the two restriction sites in the vector region induces the substitutions G176 to E, D179 to G and N180 to T at the extremity. C-terminal of the H chain of ferritin.
La sequenza nucleotidica dei vettore pVH, insieme a quella aminoacidica della ferritina ? illustrata in figura 1 . The nucleotide sequence of the pVH vector, together with the amino acid sequence of ferritin? illustrated in figure 1.
ESEMPIO 2 Isolamento del cDNA codificante oer la oroteina LOLPH EXAMPLE 2 Isolation of the cDNA encoding for the LOLPH orotein
Tutti i reagenti chimici sono della BDH Biochemical Reagents (Dorset, U.K.), a meno che indicato diversamente. La catena H della ferritina umana ricombinante ? ottenuta per purificazione come descritto in Levi, S. et al. (1987) Gene 51 , 269-274. L'estratto crudo da polline di L. perenne (Allergon AB, Engelholm, S.) ? preparato seguendo le procedure descritte in Ansari, A. A., Sthenbagamurthi, P., and Marsh, D.G. (1 989) J. Biol. Chem. 264, 1 1 181 -1 1185. All chemical reagents are from BDH Biochemical Reagents (Dorset, U.K.), unless otherwise indicated. The H-chain of recombinant human ferritin? obtained by purification as described in Levi, S. et al. (1987) Gene 51, 269-274. The raw extract from pollen of L. perennial (Allergon AB, Engelholm, S.)? prepared following the procedures described in Ansari, A. A., Sthenbagamurthi, P., and Marsh, D.G. (1 989) J. Biol. Chem. 264, 1 1 181 -1 1185.
Preparazione del RNA Preparation of the RNA
Si macerano in un mortaio 5 g di polline da L. perenne sotto azoto liquido per 15 minuti. Si aggiungono 20 mi di soluzione di lisi di guanidina isotiocianato (GTC, 4M guanidina ?sotiocianato, 5mM EDTA, 25mM citrato trisodico, pH 7.0, 0.5% sodio N-laurilsarcosilato, 0.1 M 2-mercaptoetanolo) e si agita gentilmente la preparazione. Si filtra la soluzione attraverso una garza, e si centrifuga a 5000 rpm per 10 minuti. Si stratifica il sovranatante sopra una soluzione di 5.7 M cloruro di cesio in 25 mM acetato di sodio pH 5.4, 100 mM EDTA. Si centrifugano i campioni a 31.000 rpm per 20 ore a 20?C in un rotore SW40 (Beckman, Fullerton, CA). Si lava il centrifugato con 70% etanolo e si risospende in tampone TE {10 mM Tris, 1 mM EDTA, pH 7.0). Dopo una estrazione con c!oroformio:isoamilalcol (24:1 ), si precipita a freddo l'RNA con etanolo e si risospende in tampone TE. 5 g of L. perennial pollen are macerated in a mortar under liquid nitrogen for 15 minutes. 20 ml of guanidine isothiocyanate lysis solution (GTC, 4M guanidine? Sothiocyanate, 5mM EDTA, 25mM trisodium citrate, pH 7.0, 0.5% sodium N-laurylsarcosylate, 0.1 M 2-mercaptoethanol) are added and the preparation is stirred gently. The solution is filtered through gauze and centrifuged at 5000 rpm for 10 minutes. The supernatant is layered on top of a solution of 5.7 M cesium chloride in 25 mM sodium acetate pH 5.4, 100 mM EDTA. Samples are centrifuged at 31,000 rpm for 20 hours at 20 ° C in a SW40 rotor (Beckman, Fullerton, CA). The centrifugate is washed with 70% ethanol and resuspended in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.0). After an extraction with c! Oroform: isoamyl alcohol (24: 1), the RNA is cold precipitated with ethanol and resuspended in TE buffer.
Sintesi della prima elica e reazione PCR Synthesis of the first helix and PCR reaction
Si ottiene una sintesi della prima elica di DNA da RNA totale con un innesco di oligo dT (Amersham cDNA Synthesis System Plus, Amersham U.K.). Una miscela di reazione tipica di 60 ?? contiene: trascrittasi inversa dal virus della leucemia murina di Moloney (MMLV) (0.2?/??), inibitori della ribonucleasi da placenta umana (HPRI) (0.45?/??), 1 x tampone per la trascrittasi inversa MMLV (50 mM TRIS-HCI pH 8.2, 70 mM KCI, 5 mM ditiotreitolo, 10 mM MgCl2), innesco oligo dT12-18 (8.33 ng/??), miscela di dNTPs (dATP, dCTP, dGTP, dTTP, 1 mM), RNA totale 10 ?g. Si incuba la reazione a 42?C per 40 minuti. Si utilizza il cDNA cosi ottenuto come stampo per la amplificazione. Gli inneschi per la amplificazione da PCR sono i seguenti: A synthesis of the first DNA helix from total RNA is obtained with a primer of oligo dT (Amersham cDNA Synthesis System Plus, Amersham U.K.). A typical reaction mixture of 60 ?? contains: Moloney murine leukemia virus (MMLV) reverse transcriptase (0.2? / ??), human placenta ribonuclease inhibitors (HPRI) (0.45? / ??), 1 x MMLV reverse transcriptase buffer (50 mM TRIS-HCI pH 8.2, 70 mM KCI, 5 mM dithiothreitol, 10 mM MgCl2), primer oligo dT12-18 (8.33 ng / ??), mixture of dNTPs (dATP, dCTP, dGTP, dTTP, 1 mM), total RNA 10? G. The reaction is incubated at 42 ° C for 40 minutes. The cDNA thus obtained is used as a template for amplification. The primers for PCR amplification are as follows:
LH/SP: innesco senso 5'-AAT-CTA-GA(A/G)-TT(T/C)-AC(T/G)-GT(T/G)-GA(A/G)-AA-3' LH / SP: 5'-AAT-CTA-GA (A / G) -TT (T / C) -AC (T / G) -GT (T / G) -GA (A / G) -AA- trigger 3 '
LII/AP: innesco antisenso LII / AP: antisense trigger
5?-TTG-GTA-CC{A/G/C/T)-AC(T/C)-TT{A/G)-AA(A/G)-TC-3' I due inneschi, corrispondenti ai terminali 5' e 3' sono progettati per amplificare la maggior parte della regione codificante e per inserire i siti di restrizione Xbai e Kpnl per il clonaggio in vettori opportuni, come il plasmide pVH. Questi oligonucleotidi contengono solo 2 degenerazioni per gli aminoacidi glieina, treonina e vaiina, considerando i legami ad alta energia tra le basi guanina e timina. 5? -TTG-GTA-CC {A / G / C / T) -AC (T / C) -TT {A / G) -AA (A / G) -TC-3 'The two primers, corresponding to the terminals 5 'and 3' are designed to amplify most of the coding region and to insert the restriction sites Xbai and Kpnl for cloning into suitable vectors, such as the pVH plasmid. These oligonucleotides contain only 2 degenerations for the amino acids gliein, threonine and goin, considering the high energy bonds between the guanine and thymine bases.
Si attua la reazione PCR in 50 ?? di una soluzione contenente: 2 ?? della prima elica dj cDNA; 1 x tampone di reazione (50 mM KCI, 10 mM TRIS-HCI pH 8.3, 1.5 mM MgCl2); miscela di dNTPs 1 mM; DNA polimerasi Taq (Perkin Elmer-Cetus, 50 mU/??) come descritto in Scharf, S.J., Horn, G.T., and Erlich, H.A. (1988) Science 233, 1076-1079 e in Saiki, R.K. et al. (1 988) Science 239, 487-492. Ciascuno stampo ? aggiunto ad una concentrazione finale di 8 ng/?? e la miscela ? sottoposta a 40 cicli di amplificazione PCR, utilizzando un apparecchio automatico (Perkin Elmer, USA). Le temperature per questa reazione sono le seguenti: 93?C, 45 sec.; 48?C, 45 sec.; 72?C, 90 sec. Si utilizzano gel di agarosio 1 ,2 %, colorati con bromuro di etidio, per analizzare e separare i frammenti di PCR. The PCR reaction is carried out in 50 ?? of a solution containing: 2 ?? of the first helix dj cDNA; 1 x reaction buffer (50 mM KCl, 10 mM TRIS-HCI pH 8.3, 1.5 mM MgCl2); mixture of 1 mM dNTPs; DNA polymerase Taq (Perkin Elmer-Cetus, 50 mU / ??) as described in Scharf, S.J., Horn, G.T., and Erlich, H.A. (1988) Science 233, 1076-1079 and in Saiki, R.K. et al. (1 988) Science 239, 487-492. Each mold? added to a final concentration of 8 ng / ?? and the mixture? subjected to 40 PCR amplification cycles, using an automatic device (Perkin Elmer, USA). The temperatures for this reaction are as follows: 93 ° C, 45 sec .; 48? C, 45 sec .; 72? C, 90 sec. 1.2% agarose gels stained with ethidium bromide are used to analyze and separate the PCR fragments.
Si produce dopo amplificazione un singolo frammento di DNA, di circa 300 bp, in accordo con la dimensione attesa dalla sequenza di 97 aminoacidi della proteina Lol pii, descritta in Ansari, A. A., Shenbagamurthi, P., and Marsh, D.G. (1989) J. Biol. Chem. 264, 11 181 -11 185. A single DNA fragment of about 300 bp is produced after amplification, in accordance with the size expected from the 97 amino acid sequence of the Lol pii protein, described in Ansari, A. A., Shenbagamurthi, P., and Marsh, D.G. (1989) J. Biol. Chem. 264, 11 181 -11 185.
Clonaaaio e sequenza del DNA Cloning and DNA sequence
Si digeriscono i prodotti da PCR purificati su gei con gli enzimi di restrizione Xbal e Kpnl. Si ligano i frammenti nei vettori di sequenza M13 mp18 e mp19 come descritto in Messing, J. et al. (1981 ) ISIucl. Acids Res. 9, 309-321 e in Norrander, J., Kempe, T., and Messing, J. (1983) Gene 26, 101-106. Le reazioni di restrizione e di ligazione seguono protocolli abituali descritti in Sambrook, J., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY. Gli enzimi di restrizione e di modificazione sono forniti da Boehringer (Mannheim, Germany). La sequenza del cDNA ? ottenuta utilizzando il metodo "di-deossi? ed un sequenziatore Applied Biosystems 370A (Foster City, CA, USA). La sequenza nucleotidica e aminoacidica derivata ? mostrata in figura 2. A causa dei procedimenti di cfonaggio, la sequenza codifica per una proteina deleta per i primi quattro e per gli ultimi cinque aminoacidi, in riferimento alla sequenza gi? riportata in Ansari, A. A., Shenbagamurthi, P., and Marsh, D.G. (1989) J. Biol. Chem. 264, 1 1181-1 1185. La sequenza aminoacidica derivata dal cDNA ? in accordo completo con quella pubblicata da Ansari et al. (citato), eccetto che un residuo di triptofano al posto di uno di arginina ? in posizione 77, come mostrato in fig. 3. The PCR products purified on gei are digested with the restriction enzymes Xbal and Kpnl. The fragments are ligated in the M13 sequence vectors mp18 and mp19 as described in Messing, J. et al. (1981) ISIucl. Acids Res. 9, 309-321 and in Norrander, J., Kempe, T., and Messing, J. (1983) Gene 26, 101-106. Restriction and ligation reactions follow customary protocols described in Sambrook, J., Fritsch, E.F., and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor NY. Restriction and modification enzymes are provided by Boehringer (Mannheim, Germany). The cDNA sequence? obtained using the "di-deoxy" method and an Applied Biosystems 370A sequencer (Foster City, CA, USA). The derived nucleotide and amino acid sequence is shown in Figure 2. Due to the binding procedures, the sequence codes for a deleted protein for the first four and for the last five amino acids, with reference to the sequence already reported in Ansari, A. A., Shenbagamurthi, P., and Marsh, D.G. (1989) J. Biol. Chem. 264, 1 1181-1 1185. La cDNA-derived amino acid sequence? in complete agreement with that published by Ansari et al. (quoted), except that a tryptophan residue instead of an arginine residue? at position 77, as shown in Fig. 3.
ESEMPIO 3 Costruzione del sistema di espressione ricombinante dell'allergene Lol pii EXAMPLE 3 Construction of the recombinant expression system of the Lol pii allergen
Tentativi di esprimere la proteina matura Lol p II in L? coli a mezzo di altri vettori di espressione sono deludenti, probabilmente a causa della piccola quantit? di materiale espresso. Per ottenere grandi quantit? di proteina Lol p II, la sequenza ? stata fusa al C-terminale della catena H della ferritina umana, che ? una proteina espressa ad alte rese in coli e facile da purificare. Il frammento di DNA codificante per la proteina Lol pii (dall1 aminoacido 5 all'aminoacido 92) ? clonato nei siti di restrizione Xba I e Kpn I del plasmide pVH; il gene ottenuto codifica per una proteina di 266 aminoacidi, dei quali i primi 176 residui appartengono alla ferritina H, dal residuo 177 al residuo 264 alla proteina Lol pii, e gii ultimi 2 aminoacidi alla sequenza della ferritina. Il vettore ? denominato pVH-Lolpll. Attempts to express the mature protein Lol p II in L? coli by means of other expression vectors are disappointing, probably due to the small amount? of express material. To obtain large quantities? of protein Lol p II, the sequence? been fused to the C-terminus of the H chain of human ferritin, which? a protein expressed at high yields in coli and easy to purify. The DNA fragment encoding the Lol pii protein (from amino acid 5 to amino acid 92)? cloned in the restriction sites Xba I and Kpn I of the pVH plasmid; the gene obtained encodes a protein of 266 amino acids, of which the first 176 residues belong to ferritin H, from residue 177 to residue 264 to the Lol pii protein, and the last 2 amino acids to the ferritin sequence. The vector ? called pVH-Lolpll.
Gli autori hanno anche a mezzo di amplificazione con PCR usando i seguenti oligonucleotidi originato un vettore comprendente anche la sequenza per i primi 4 e gli ultimi 5 aminoacidi dell?allergene Lol pii, nonch? per il sito di taglio del fattore proteolitico Xa, tra la sequenza della ferritina e quella dell'allergene Lol pii. The authors have also by means of amplification with PCR using the following oligonucleotides originated a vector including the sequence for the first 4 and the last 5 amino acids of the allergen Lol pii, as well as? for the cleavage site of the proteolytic factor Xa, between the sequence of ferritin and that of the allergen Lol pii.
5'innesco: 5'-TTCACTCTAGACATTGAAGGTAGAGCTGCT CCGGTTGAGTTTACGGTGGAGAAG-3' 5 'trigger: 5'-TTCACTCTAGACATTGAAGGTAGAGCTGCT CCGGTTGAGTTTACGGTGGAGAAG-3'
3' innesco: 5?-TGCAAGGTACCTTATTACTCCGGGTTATAA GTTGTGCCGACTTTGAAGTC-3' 3 'trigger: 5? -TGCAAGGTACCTTATTACTCCGGGTTATAA GTTGTGCCGACTTTGAAGTC-3'
Il DNA amplificato con tali inneschi, utilizzando il DNA di pVH-Lol pii, come stampo ? digerito con gli enzimi Xba I e Kpa I ed ? poi clonato nel frammento vettoriale Xba ?-??? I del piasmide pVH. il frammento clonato ha la sequenza mostrata in figura 2B. Il piasmide ? denominato pVH-Loipii Bis. DNA amplified with such primers, using pVH-Lol pii DNA as a template? digested with the enzymes Xba I and Kpa I and? then cloned into Xba vector fragment? - ??? I of the piasmide pVH. the cloned fragment has the sequence shown in figure 2B. The piasmid? called pVH-Loipii Bis.
ESEMPIO 4 Espressione e purificazione della proteina chimerica ricombinante Lo! oli (Ft/Loll) EXAMPLE 4 Expression and purification of the recombinant chimeric protein Lo! oils (Ft / Loll)
L'espressione dell'allergene ferritina-Lol pii ? sotto il controllo del promotore di X pj_, ed ? indotta aumentando la temperatura da 30?C a 42?C ed incubando per ulteriori 3 ore come descritto da Levi et al. (1988) J. Biol. Chem. 263, 18086-18092 e da Levi, S. (1989) Biochem. J. 264, 381-388. Il ceppo batterico utilizzato per l'espressione ? E. coli GC76 (ara strr ? lac-oro F'Iac IQ lacZ ? M15 ??? ). La fermentazione del ceppo ricombinante ? attuata in un fermentatore da 7 litri, seguendo condizioni abituali. Le cellule sono raccolte per centrifugazione e si ottengono da 4 litri di cultura circa 20 grammi di pasta cellulare (peso umido). The expression of the ferritin-Lol pii allergen? under the control of the promoter of X pj_, and? induced by increasing the temperature from 30 ° C to 42 ° C and incubating for a further 3 hours as described by Levi et al. (1988) J. Biol. Chem. 263, 18086-18092 and by Levi, S. (1989) Biochem. J. 264, 381-388. The bacterial strain used for expression? E. coli GC76 (ara strr? Lac-gold F'Iac IQ lacZ? M15 ???). Fermentation of the recombinant strain? carried out in a 7 liter fermenter, following usual conditions. The cells are collected by centrifugation and approximately 20 grams of cellular paste (wet weight) are obtained from 4 liters of culture.
Si purificano le proteine ricombinanti con un procedimento utilizzato per le ferritine ricombinanti descritto in Levi, S. et al. (1987) Gene 51 , 269-274, Levi et al. (1988) J. Biol. Chem. 263, 18086-18092 e Levi, S. et al. (1989) Biochem. J. 264, 381 -388. Si distrugge la pasta cellulare per sonicazione e si evidenzia la presenza di proteine ricombinanti per elettroforesi su gel della frazione solubile dall'omogenato cellulare. Si analizzano i campioni per elettroforesi in SDS o in condizioni native su gel di poliacriiammide come descritto in Arosio, P., Adelman, T.G., and Drysdale, J.W. (1978) J. Biol. Chem. 253, 4451 -4458. Recombinant proteins are purified with a procedure used for recombinant ferritins described in Levi, S. et al. (1987) Gene 51, 269-274, Levi et al. (1988) J. Biol. Chem. 263, 18086-18092 and Levi, S. et al. (1989) Biochem. J. 264, 381 -388. The cellular pulp is destroyed by sonication and the presence of recombinant proteins is highlighted by gel electrophoresis of the soluble fraction from the cellular homogenate. Samples are analyzed by electrophoresis in SDS or in native conditions on polyacryamide gels as described in Arosio, P., Adelman, T.G., and Drysdale, J.W. (1978) J. Biol. Chem. 253, 4451 -4458.
Si riscalda l'omogenato cellulare solubile a 65?C per 10 minuti, si chiarifica e si tratta con DNAsi e RNAsi. Si precipita poi il campione con solfato di ammonio (520 g/l), si risospende e si dializza contro 20 mM Tris HCI pH 7.4. Si carica poi il campione su una colonna di sefarosio 6B, si frazionano i picchi, si concentrano e si conservano a 4?C con aggiunta di 1 mM EDTA, 0.1 % sodio azide, 1 mM PMSF, 1?? pepstatina A e 10 ?? leupeptina. La proteina ricombinante ha una purit? circa dell'80%, come evidenziabile da elettroforesi. Almeno 4 -5 mg della proteina modificata si ottengono da 1 litro di coltura cellulare. The soluble cell homogenate is heated to 65 ° C for 10 minutes, clarified and treated with DNAse and RNase. The sample is then precipitated with ammonium sulphate (520 g / l), resuspended and dialyzed against 20 mM Tris HCI pH 7.4. The sample is then loaded onto a 6B sepharose column, the peaks are fractionated, concentrated and stored at 4 ° C with the addition of 1 mM EDTA, 0.1% sodium azide, 1 mM PMSF, 1 ?? pepstatin A and 10 ?? leupeptin. Does the recombinant protein have a purity? about 80%, as evidenced by electrophoresis. At least 4-5 mg of the modified protein is obtained from 1 liter of cell culture.
La fig. 4A mostra che cellule di E. coli trasformate con il plasmide pVH-Lol p II esprimono ad alte rese una proteina di circa 30 Kdal (colonne 4-6), che non ? presente in cellule di E. coli non trasformate (colonne 2-3), e che ? pi? pesante della ferritina selvatica di circa 20 Kdal (colonna 1 ). Elettroforesi su gel in condizioni non denaturanti degli estratti cellulari solubili mostrano, come ? evidente in fig. 4B che i peptidi chimerici si assemblano in una proteina (colonne 4-6), con una mobilit? leggermente minore della ferritina naturale (colonna 1 ). La resa di proteina assemblata e solubile sembra comparabile a quella delia ferritina H selvatica. Fig. 4A shows that E. coli cells transformed with the pVH-Lol p II plasmid express at high yields a protein of about 30 Kdal (columns 4-6), which does not? present in untransformed E. coli cells (columns 2-3), and what? pi? weight of wild ferritin of about 20 Kdal (column 1). Gel electrophoresis under non-denaturing conditions of the soluble cell extracts show, how? evident in fig. 4B that the chimeric peptides assemble into a protein (columns 4-6), with a mobility? slightly lower than natural ferritin (column 1). The yield of assembled and soluble protein appears to be comparable to that of wild ferritin H.
La proteina di fusione ? assoggettata a saggi preliminari di termostabilit?, e mostra di essere resistente fino a riscaldamento a 65 ?C, mentre la ferritina selvatica resiste a 80 ?C. Si purifica la proteina chimerica con il procedimento utilizzato per le ferritine ricombinanti, coinvolgendo un trattamento a caldo a 65?C ed una filtrazione su gel di Sefarosio 6B. Le preparazioni cos? ottenute sono considerate sufficientemente pure (pi? dell'80% della proteina ricombinante), per una ulteriore caratterizzazione. Le rese sono di circa 10 mg/L di cultura, dando circa 5 mg/L di proteina purificata. The fusion protein? subjected to preliminary tests of thermostability, and shown to be resistant up to heating to 65 ° C, while wild ferritin withstands 80 ° C. The chimeric protein is purified with the procedure used for recombinant ferritins, involving a heat treatment at 65 ° C and filtration on Sepharose 6B gel. The preparations cos? obtained are considered sufficiently pure (more than 80% of the recombinant protein), for further characterization. Yields are approximately 10 mg / L of culture, giving approximately 5 mg / L of purified protein.
ESEMPIO 5 Caratterizzazione biochimica e immunoloqica della proteina ricombinante Lol oli (Ft/Loll) EXAMPLE 5 Biochemical and immunological characterization of the recombinant protein Lol oli (Ft / Loll)
Si attua una gel filtrazione su una colonna FPLC superosio 6 (Pharmacia) in 20 mM Tris HCI pH 7.4, 0.15 M NaCI, calibrata con standards adeguati. La proteina chimerica eluisce con un peso molecolare apparente di circa 700 Kdal ed ? resistente all'incubazione in 0.1 % SOS a temperatura ambiente. Si pu? pertanto concludere che la proteina chimerica si assembla in una proteina a 24 monomeri, analogamente alla ferritina. Una incubazione con enzimi proteolitici induce una riduzione del peso molecolare dei peptidi nella proteina di fusione mentre non influenza la dimensione del peptide selvatico ferritina H. Questo indica che l'estensione C-terminale con la porzione Lol pii ? esposta al solvente. Gel filtration is carried out on a superose 6 FPLC column (Pharmacia) in 20 mM Tris HCI pH 7.4, 0.15 M NaCI, calibrated with suitable standards. The chimeric protein elutes with an apparent molecular weight of about 700 Kdal and? resistant to incubation in 0.1% SOS at room temperature. Can you? therefore conclude that the chimeric protein assembles into a 24 monomer protein, similar to ferritin. An incubation with proteolytic enzymes induces a reduction in the molecular weight of the peptides in the fusion protein while not affecting the size of the wild ferritin H peptide. This indicates that the C-terminal extension with the Lol pii? exposed to the solvent.
Le proteine di fusione ricombinanti, separate su gel in condizioni non denaturanti (Fig. 5A) o con SDS {Fig. 5B), vengono trasferite su membrane di nitrocellulosa come descritto in Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Nati. Acad. Sci. U.S.A. 76, 4350-4358 e in Burnette, W.N. (1981 ) Anal. Biochem. 112, 195-203. Dopo trasferimento, i siti di legame non specifici sono bloccati con 5% di latte in polvere in tampone TBS (50 mM TRIS-HCI pH 7.5; 0.9% NaCI). Si incubano le membrane di nitrocellulosa per 2 ore a temperatura ambiente con diversi sieri umani gi? saggiati a mezzo di RAST (Radio Adergo Sorbent Test, Pharmacia). Dopo il lavaggio, si incubano le membrane per 2 ore a temperatura ambiente con immunogiobuline di coniglio anti-lgE umane, coniugate con perossidasi (diluizione 1 :250, Sigma). Lo sviluppo del colore si ottiene dopo aggiunta di 4-cloro-1 -naftolo (BioRad), seguendo le istruzioni della casa fornitrice. Recombinant fusion proteins, separated on gel under non-denaturing conditions (Fig. 5A) or with SDS {Fig. 5B), are transferred onto nitrocellulose membranes as described in Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Nati. Acad. Sci. U.S.A. 76, 4350-4358 and in Burnette, W.N. (1981) Anal. Biochem. 112, 195-203. After transfer, the non-specific binding sites are blocked with 5% milk powder in TBS buffer (50 mM TRIS-HCI pH 7.5; 0.9% NaCl). Nitrocellulose membranes are incubated for 2 hours at room temperature with different human sera. tested by means of RAST (Radio Adergo Sorbent Test, Pharmacia). After washing, the membranes are incubated for 2 hours at room temperature with peroxidase conjugated rabbit anti-IgE immunoglobins (dilution 1: 250, Sigma). Color development is obtained after adding 4-chloro-1-naphthol (BioRad), following the instructions of the supplier.
Studi immunologie! mostrano che la proteina chimerica ? riconosciuta da anticorpi monoclonali anti ferritina H per doppia diffusione e in esperimenti di trasferimento come mostrato in fig. 5A, colonna 5. Immunology studies! show that the chimeric protein? recognized by anti-ferritin H monoclonal antibodies by double diffusion and in transfer experiments as shown in fig. 5A, column 5.
i filtri sono incubati con sieri da pazienti allergici atopici e con sieri di controllo negativi, seguiti da incubazione con anticorpi marcati anti IgE. I risultati mostrano che la ferritina selvatica non ? riconosciuta dalle IgE dei pazienti {fig. 5A e B, colonna 4} e dai soggetti di controllo negativo (fig. 5A e B, colonna 8), mentre la proteina chimerica ? riconosciuta dalle IgE contenute nei sieri di pazienti allergici (fig. 5A e B, colonna 3) ma non dai soggetti di controllo negativo (fig. 3A e B, colonna 7). the filters are incubated with sera from atopic allergic patients and with negative control sera, followed by incubation with anti-IgE labeled antibodies. The results show that wild ferritin is not? recognized by patients' IgE {fig. 5A and B, column 4} and from the negative control subjects (fig. 5A and B, column 8), while the chimeric protein? recognized by the IgE contained in the sera of allergic patients (fig. 5A and B, column 3) but not by the negative control subjects (fig. 3A and B, column 7).
Per valutare se la proteina chimerica sia utilizzabile per lo sviluppo di saggi immunologie! pi? semplici ? sviluppato un saggio ELISA. To evaluate whether the chimeric protein is usable for the development of immunoassays! pi? simple? developed an ELISA assay.
Piastre da microtitolazione (Greiner) sono saturate per incubazione per 18 ore a 4?C in 50 mM sodio carbonato pH 9.6 con 1 ?g di proteina ricombinante purificata per pozzetto (100 ??). Si lavano le piastre in tampone fosfato salino pH 7.4 (PBS) e con 0.5% Tween 20. Si incubano poi le piastre per 2 ore a 37?C con diluizioni seriali di sieri da pazienti in 2% (atte magro in PBS, 0.5% Tween 20. Si incuba per 2 ore a 37?C con immunoglobuiine anti IgE coniugate con perossidasi diluite 1 :500 in 2% latte magro in PBS pH 7.4, 0.5% Tween 20. Si attua la reazione colorimetrica per aggiunta di O-fenilendiammina (OPD, Sigma) e si misura l'assorbanza a 492 nm utilizzando un lettore ELISA. Microtiter plates (Greiner) are saturated by incubation for 18 hours at 4 ° C in 50 mM sodium carbonate pH 9.6 with 1? G of purified recombinant protein per well (100 ??). The plates are washed in phosphate buffer pH 7.4 (PBS) and with 0.5% Tween 20. The plates are then incubated for 2 hours at 37 ° C with serial dilutions of patient sera in 2% (lean in PBS, 0.5% Tween 20. It is incubated for 2 hours at 37 ° C with anti IgE immunoglobin conjugated with peroxidase diluted 1: 500 in 2% skim milk in PBS pH 7.4, 0.5% Tween 20. The colorimetric reaction is carried out by adding O-phenylenediamine ( OPD, Sigma) and absorbance is measured at 492 nm using an ELISA reader.
La figura 6A mostra che due sieri positivi ai RAST danno risposte dipendenti dalla diluizione, significativamente maggiori dei sieri di controllo normali e di un siero positivo preadsorbito con la proteina Lol pii ricombinante. Il segnale specifico sparisce a diluizioni di 1 su 16 e maggiori, e la diluizione 1 su 4 ? indicativa come quella a cui effettuare un'analisi su alcuni campioni clinici. I saggi hanno una buona riproducibilit?, i risultati dei saggio ELISA su sieri di 26 individui atopici classificati sulla base del saggio RAST e su 9 soggetti di controllo non allergici sono mostrati in fig. 6B. Tutti i sieri di controllo negativi danno risposte sotto 0.2 A, mentre la maggior parte dei sieri allergici produce dei segnali sopra 0.2 A. I valori medi maggiori sono osservati nei pazienti di classe 5 e 6. Utilizzando 0.2 A a 492 nm come limite di normalit?, solo 3 pazienti (12%) risultano negativi con il saggio ELISA. Figure 6A shows that two RAST positive sera give dilution dependent responses, significantly greater than normal control sera and one positive serum preadsorbed with the recombinant Lol pii protein. The specific signal disappears at dilutions of 1 in 16 and higher, and the dilution 1 in 4? indicative as the one to be analyzed on some clinical samples. The assays have good reproducibility, the results of the ELISA assay on sera from 26 atopic individuals classified on the basis of the RAST assay and on 9 non-allergic control subjects are shown in fig. 6B. All negative control sera give responses below 0.2 A, while most allergic sera produce signals above 0.2 A. The highest mean values are observed in class 5 and 6 patients. Using 0.2 A at 492 nm as the limit of normal ?, only 3 patients (12%) were negative with the ELISA assay.
ESEMPIO 6 Saggio di rilascio di istamina EXAMPLE 6 Histamine release assay
Si attua un saggio per il rilascio di istamina su sangue intero con un corredo commerciale radioimmunologico (Immunotech International, Francia), seguendo le istruzioni della casa produttrice. Il saggio immunologico si basa sulla competizione tra la istamina modificata nel campione e l'istamina tracciante iodinata per il legame all'anticorpo legato su PB come descritto in More!, A.M., and Deiaage, M.A. (1988) J. Allergy Clin. Immunol. 82, 646-654. Si raccoglie il sangue in tubi eparinizzati e si attuano i saggi entro poche ore dalla raccolta. Le cellule vengono esposte a diverse diluizioni dell'estratto da polline di L. perenne e con l'allergene ricombinante purificato o come controllo con la ferritina H ricombinante selvatica. Dopo incubazione, si determina la quantit? di istamina rilasciata. An assay for the release of histamine on whole blood is carried out with a commercial radioimmunoassay kit (Immunotech International, France), following the manufacturer's instructions. The immunoassay is based on competition between the modified histamine in the sample and the iodinated tracer histamine for binding to the PB bound antibody as described in More !, A.M., and Deiaage, M.A. (1988) J. Allergy Clin. Immunol. 82, 646-654. The blood is collected in heparinized tubes and the tests are carried out within a few hours of collection. The cells are exposed to different dilutions of the perennial L. pollen extract and with the purified recombinant allergen or as a control with the wild recombinant ferritin H. After incubation, the quantity is determined. of histamine released.
I risultati dei saggi di rilascio di istamina da basofili di un soggetto allergico sono mostrati in fig. 7. La proteina Lol pii ricombinante induce una significativa risposta allergica paragonata a quella indotta dall'estratto di polline, mentre la proteina ricombinante ferritina selvatica non produce alcun rilascio evidenziabile di istamina. Un paragone tra le potenze dei due campioni allergenici ? difficile, a causa della composizione eterogenea del campione naturale che contiene almeno un altro allergene maggiore (Lol pi). Inoltre, solo 1 /3 della proteina FT-Lol pii ricombinante ? rappresentato dalia proteina Lol pii. The results of histamine release assays from basophils of an allergic subject are shown in fig. 7. Recombinant Lol pii protein induces a significant allergic response compared to that induced by pollen extract, whereas recombinant wild ferritin protein produces no detectable histamine release. A comparison between the potencies of the two allergenic samples? difficult, due to the heterogeneous composition of the natural sample which contains at least one other major allergen (Lol pi). Also, only 1/3 of the recombinant FT-Lol pii protein? represented by the protein Lol pii.
ESEMPIO 7 Reazione allergica da puntura cutanea EXAMPLE 7 Allergic reaction from skin prick
L'attivit? biologica della proteina ricombinante ? anche analizzata su reazione allergica alla cute su volontari non allergici e sani, come mostrato in tabella 1 . The activity? biological recombinant protein? also analyzed on allergic skin reaction on non-allergic and healthy volunteers, as shown in table 1.
Tabella 1 Table 1
Reazione allergica a Allergic reaction to
Paziente NaCI Istamina Estratto rFerritina Ft-Lolpll 10 mg/ml Crudo Patient NaCI Histamine Extract rFerritin Ft-Lolpll 10 mg / ml Raw
A.S. 0 64 0/0/0 0/0/0 0/0/0 E.T. 0 81 144/25/16 0/0/0 49/9/0 a La superficie della reazione ? indicata in mm^. Sono riportati i risultati con concentrazioni di proteine di 1 600, 16 e 1.6 Mg/ml. A.S. 0 64 0/0/0 0/0/0 0/0/0 E.T. 0 81 144/25/16 0/0/0 49/9/0 a The surface of the reaction? indicated in mm ^. Results with protein concentrations of 1 600, 16 and 1.6 Mg / mL are reported.
Il soggetto sano non reagisce con nessuna delle preparazioni proteiche, mentre il soggetto allergico d? dei risultati positivi con l'estratto di polline ad una concentrazione fino a 1.6 Mg/ml e con l'allergene ricombinante ad una concentrazione di 16 M9/ml. La ferritina H ricombinante come controllo non induce alcuna risposta allergica. The healthy subject does not react with any of the protein preparations, while the allergic subject d? positive results with the pollen extract at a concentration of up to 1.6 Mg / ml and with the recombinant allergen at a concentration of 16 M9 / ml. Recombinant ferritin H as a control does not induce any allergic response.
Esempio 8 Proteina chimerica Ferritina- Lol D I Example 8 Chimeric protein Ferritin - Lol D I
Il gene (cDNA) codificante per ? allergene di L. perenne Lol p I ? clonato seguendo le stesse procedure sperimentali utilizzate per il clonaggio dell'allergene Lol pii. in particolare, gii inneschi oiigonucieotidici utilizzati per la reazione di amplificazione (PCR) dall' RNA estratto dal polline, sono, sulla base delle sequenze descritte in Perez M. et al. 1990 Journ. of Biol. Chem. 265, 16210-16215; Griffith I.J. et al. 1991 FEBS 279, 210-215; Singh M.B. et al. 1991 Proc. Nati. Acad. Sci. USA 88, 1384-1388: The gene (cDNA) encoding for? allergen of L. perennial Lol p I? cloned following the same experimental procedures used for the cloning of the Lol pii allergen. in particular, on the basis of the sequences described in Perez M. et al. 1990 Journ. of Biol. Chem. 265, 16210-16215; Griffith I.J. et al. 1991 FEBS 279, 210-215; Singh M.B. et al. 1991 Proc. Nati. Acad. Sci. USA 88, 1384-1388:
innesco senso pl/S , 25-mer priming sense pl / S, 25-Wed
5'- ATGCACTGCAGAGTACGGGGACAAA -3' 5 '- ATGCACTGCAGAGTACGGGGACAAA -3'
Pst I Pst I
innesco anti-senso pl/A, 24-mer anti-sense trigger pl / A, 24-Wed
5'- CGTAGGATCCGTGTCGGCTTTCCA -3' 5 '- CGTAGGATCCGTGTCGGCTTTCCA -3'
Barn HI Barn HI
La sequenza dei frammento amplificato, ottenuta dopo clonaggio in un vettore di sequenza, ? differente alle sequenze gi? note nelle posizioni indicate nella seguente Tabella 2. The sequence of the amplified fragment, obtained after cloning into a sequence vector,? different to the sequences already? notes in the positions indicated in the following Table 2.
Tabella 2 Table 2
Posizione Isoforma isolata Isoforme descr?tte Position Isolated isoform Described isoforms
45 Asp Asn (JBC, clone 5A) 45 Asp Asn (JBC, clone 5A)
144 Gly Asp (FEBS) 144 Gly Asp (FEBS)
154 Gly Ala (JBC,cione 5A) 154 Gly Ala (JBC, cione 5A)
187 Ile Thr (JBC, clone 5A) 187 Ile Thr (JBC, clone 5A)
209 Ser Phe (FEBS e JBC, 5A e IA) 223 Val Phe (JBC, 5A) 209 Ser Phe (FEBS and JBC, 5A and IA) 223 Val Phe (JBC, 5A)
Il clone ? poi riamplificato per il clonaggio nel vettore pVH per la fusione con la ferritina H. Gli inneschi utilizzati sono: The clone? then re-amplified for cloning in the pVH vector for fusion with ferritin H. The primers used are:
innesco senso Lpi, 31 mer priming sense Lpi, 31 wed
5?- ACCCGGTCTAGAAACTGCAGAGTACGGGGAC- 3? 5? - ACCCGGTCTAGAAACTGCAGAGTACGGGGAC- 3?
Xba I Xba I
innesco anti-senso Lpl, 30 mer anti-sense trigger Lpl, 30 wed
5?- GCATGCGGTACCCGTGTCGGCTTTCCAGCC- 3? 5? - GCATGCGGTACCCGTGTCGGCTTTCCAGCC- 3?
Kpn I Kpn I
il gene ottenuto codifica per una proteina monomerica di 405 aminoacidi, di cui i primi 1 76 appartengono alla ferritina H umana, gli aminoacidi 177-401 codificano per i' allergene Lol pi (corrispondenti agli aminoacidi 11 -235 delie sequenze pubblicate), e gli ultimi 4 residui corrispondono a due aminoacidi derivati dai clonaggio e a due residui della ferritina, prima dei codone di terminazione della traduzione. the gene obtained encodes a monomeric protein of 405 amino acids, of which the first 1 76 belong to human ferritin H, the amino acids 177-401 encode the allergens Lol pi (corresponding to the amino acids 11-235 of the published sequences), and the the last 4 residues correspond to two amino acids derived from cloning and to two ferritin residues, before the translation termination codon.
La proteina chimerica ? espressa e purificata con le procedure applicate alla proteina ferritina-Lol p II. Le rese di espressione e di proteina purificata sono analoghe a quelle della ferritina non modificata. In figura 8 sono presentati i risultati dell' espressione in E. coli. The chimeric protein? expressed and purified with the procedures applied to the ferritin-Lol p II protein. The yields of expression and purified protein are similar to those of unmodified ferritin. Figure 8 shows the results of expression in E. coli.
Analisi biochimiche indicano che la proteina chimerica ? riconosciuta da anticorpi per la ferritina, ? stabile e forma un multimero di 24 subunit?, analogamente alia ferritina naturale. Esempio 9 Proteina chimerica Ferritina- hEGF Biochemical analyzes indicate that the chimeric protein? recognized by antibodies to ferritin,? stable and forms a multimer of 24 subunits, similar to natural ferritin. Example 9 Chimeric protein Ferritin-hEGF
Il frammento di DNA codificante per la sequenza dell' Epidermal Growth Factor umano (hEGF o urogastrone) (Gregory H. 1975 Nature , 257, 325-327; Scott J. et al. 1983 Science 221 , 236-240; Heath W.F. and Merrifield R.B., 1986, Proc. Nati. Acad. Sci. USA 83, 6367-6371 ;Smith J. et al. The DNA fragment encoding the human Epidermal Growth Factor sequence (hEGF or urogastrone) (Gregory H. 1975 Nature, 257, 325-327; Scott J. et al. 1983 Science 221, 236-240; Heath W.F. and Merrifield R.B., 1986, Proc. Nati. Acad. Sci. USA 83, 6367-6371; Smith J. et al.
1 982 Nucl. Acid Res. 10, 4467) ? prodotto per sintesi chimica ed ha la sequenza indicata in figura 9. Esso contiene siti di restrizione per gli enzimi Xbal e Kpnl per il clonaggio in opportuni vettori, ed un codone di terminazione di traduzione. 1 982 Nucl. Acid Res. 10, 4467)? produced by chemical synthesis and has the sequence indicated in figure 9. It contains restriction sites for the enzymes Xbal and Kpnl for cloning into suitable vectors, and a translation termination codon.
Si costruisce il plasmide per la superespressione della proteina chimerica ferritina- hEGF inserendo tale sequenza codificante nel vettore pVH. Il gene ottenuto codifica per una proteina monomerica di 236 aminoacidi: i primi 176 appartengono alla ferritina H, gli aminoacidi 177-182 fungono da collegamento e gii aminoacidi 183-236 codificano per la forma matura dell' EGF umano. E' possibile ottenere la forma matura del hEGF mediante trattamento della proteina con fattore Xa bovino, una protessi specifica per la sequenza aminoacidica IEGR, posta a valle della sequenza della ferritina. Tale proteina chimerica ? espressa e purificata con le procedure applicate alia proteina ferritina-Lol p II. Le rese di espressione e di proteina purificata sono analoghe a quelle della ferritina non modificata. Analisi biochimiche indicano che la proteina chimerica ? riconosciuta da anticorpi per la ferritina, come mostrato nella figura 10, ed ? stabile e forma un multimero di 24 subunit?, analogamente alla ferritina naturale. La proteina chimerica prodotta ? anche riconosciuta da anticorpi commerciali specifici anti-EGF. The plasmid for the super-expression of the chimeric ferritin-hEGF protein is constructed by inserting this coding sequence in the pVH vector. The gene obtained encodes a monomeric protein of 236 amino acids: the first 176 belong to ferritin H, the amino acids 177-182 act as a link and the amino acids 183-236 encode the mature form of human EGF. It is possible to obtain the mature form of hEGF by treating the protein with bovine factor Xa, a specific protecte for the amino acid sequence IEGR, located downstream of the ferritin sequence. Such a chimeric protein? expressed and purified with the procedures applied to the ferritin-Lol p II protein. The yields of expression and purified protein are similar to those of unmodified ferritin. Biochemical analyzes indicate that the chimeric protein? recognized by antibodies to ferritin, as shown in Figure 10, and? stable and forms a multimer of 24 subunits, similar to natural ferritin. The chimeric protein produced? also recognized by specific commercial anti-EGF antibodies.
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