HRP920714A2 - RECOMBINANT PROTEIN WHICH BINDS TO A COMPLEX VIRAL ANTIGEN OF HIV-a 1 - Google Patents
RECOMBINANT PROTEIN WHICH BINDS TO A COMPLEX VIRAL ANTIGEN OF HIV-a 1 Download PDFInfo
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- HRP920714A2 HRP920714A2 HR920714A HRP920714A HRP920714A2 HR P920714 A2 HRP920714 A2 HR P920714A2 HR 920714 A HR920714 A HR 920714A HR P920714 A HRP920714 A HR P920714A HR P920714 A2 HRP920714 A2 HR P920714A2
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Description
Humana monoklonalna antitijela (mAk) mogu se izraditi, pošto se izdvoje B-limfociti ljudi koji pokazuju imunu reakciju protiv antigena, na primjer uslijed bolesti. Ovi B-limfociti se fuzijom sa pogodnim linijama stanica, naročito sa linijama stanica mieloma, imortaliziraju. Na takav način dobivene linije stanice hibrida, nibridomi, služe kao proizvodni vehikulum za mAk-a. Oni se mogu in vitro upotrijebiti u kulturama stanica i uzgajati se u potrebnom srazmjeru (1). Human monoclonal antibodies (mAk) can be produced by isolating B-lymphocytes from people who show an immune reaction against an antigen, for example due to disease. These B-lymphocytes are immortalized by fusion with suitable cell lines, especially with myeloma cell lines. Hybrid cell lines obtained in this way, nibridomas, serve as a production vehicle for mAk. They can be used in vitro in cell cultures and grown to the required scale (1).
Pri tom proizvedena supstanca po pravilu predstavlja kompletno mAk, koja je okarakterizirana pomoću 2 teška lanca i dva laka lanca, koji su međusobno vezani pomoću disulfidnih mostova i pomoću ne-kovalentnih veza gradeći antitijelo koje specifično vezuje (2). As a rule, the produced substance is a complete mAk, which is characterized by 2 heavy chains and 2 light chains, which are connected to each other by means of disulfide bridges and by means of non-covalent bonds, building an antibody that specifically binds (2).
Struktura takvog antitijela može se podijeliti u konstantan region, koji je odgovoran za takozvane efektorne funkcije, kao npr. aktiviranje komplementa i u promjenjivi region za specifično vezivanje antigena. The structure of such an antibody can be divided into a constant region, which is responsible for so-called effector functions, such as complement activation, and a variable region for specific antigen binding.
Antitijela se mogu cijepati anzimatski pomoću biokemijskih postupaka. Može se na primjer pomoću papaina odn. pomoću pepsina ocijepiti dio konstantnog regiona. Na ovaj način izrađeni Fab' odn. (Fab') fragmenti su na način sličan prvobitnom antitijelu u stanju da vezuju odgovarajući antigen (2). Opisano je također i proteolitičko cijepanje kompletnih konstantnih regiona, koje vodi do takozvanog Fv fragmenta. Ali ono nije izvodljivo da se može reproducirati kako je gore spomenuto cijepanje papainom odn. pepsinom antitijela (3,4). Pomoću postupaka genske tehnike ipak se uspijevaju izraditi Fv fragmenti na način koji se može reproducirati. Za to potrebne pretpostavke kao i primijenjeni postupci opisuju se u sljedećem. Antibodies can be cleaved enzymatically using biochemical procedures. It can be done, for example, using papain or cleave part of the constant region using pepsin. Fab' made in this way or (Fab') fragments are able to bind the corresponding antigen in a manner similar to the original antibody (2). Proteolytic cleavage of complete constant regions, leading to the so-called Fv fragment, has also been described. But it is not feasible that it can be reproduced as the above-mentioned cleavage by papain or. pepsin antibodies (3,4). Using genetic engineering procedures, however, it is possible to produce Fv fragments in a reproducible manner. For this, the necessary assumptions as well as the applied procedures are described in the following.
Pomoću rutinskih postupaka izrađuje se cDNK banka stanice linija hibridoma koji može proizvoditi mAk. Iz hibridoma koji proizvodi mAk izolira se cjelokupna RNK. Ova RNK sadrži pored ribozomalne RNK cjelokupnost transkripata stanice. Prisutni su kako nepotpuno obrađeni, nuklearni transkripti, također i zreli, citoplazmatični transkripti, takozvani mesengeri RNK-a. Ovi se odlikuju poli-adenozinskim repom na 3'-kraju. Ovaj Poli-A region može se primijeniti, da bi se afinitetnom kromatografijom pomoću oligo-dT celuloze izolirale zrele mRNK-e. Pomoću enzima "reversne transkriptaze" može se mRNK preinačiti u takozvanu cDNK. Primjenom pogodnih vektora može se dobivena smjesa cDNK-e klonirati, što vodi do takozvane cDNK banke (5). Imunoglobulin specifične sonde hibridiziranja dozvoljavaju identificiranje i izoliranje klona koji sadrže željene sekvence. Sekvenciranjem DNK ovih klona i uspoređenje sekvence sa poznatim genima imunoglobulina (EMBL Nucleotide Sequence Data Library, Heidelberg, SRN) može se dobiti sigurnost o identitetu klona (5). Na ovaj način mogu se na primjer izolirati kloni, koji nose sekvence lakog odn. teškog lanca mAk-e. Analizom sekvenci na takav način dobivenih imunoglobulin-cDNK-a mogu se uspoređenjem sa poznatim imunglobulinskim sekvencama identificirati pojedinačne domene teškog odn. lakog lanca: moguće je da se identificira promjenjivi i konstantan region, i npr. unutar promjenjivog regiona takozvani "hiperpromjenjivi" ili "complementarity determining regions", koji su zapravo odgovorni za specifično vezivanje antigena (6). Na ovakav način klonirani geni antitijela mogu se dovesti do ekspresije u različitim sustavima. S jedne strane mogu se primijeniti životinjske kulture stanica, kao npr. stanice mieloma, ako se primijene pogodni ekspresioni vektori (7). Primjena kvasca (8) odn. bakterijskih stanica (9) kao ekspresionog vehikuluma za kompletno antitijelo je problematična, pošto takve stanice očigledno nisu u stanju, za njih vrlo velike molekule, kako ih antitijela predstavljaju, korektno sintetizirati. Uspjeh u ovom pravcu se ocrtao tek, kada se pokušalo, da se subfragmenti antitijela dovedu do ekspresije u nižim eukariontima odn. u prokariontima. U sljedećem se opisuju četiri različita postupka, koji dopuštaju ekspresiju Fv. odn. Fab fragmenata u Escherichia coli: Skerra i Pluckthun (1988, (10)) su umetnuli gene za promjenjive regione murinskoganti-fosforilholin - antitijela (McPC603) u svezi sa Lac-Promoter/Operator Region-om, praćena po jednom bakterijelnom Leader-sekvencom, koja služi za izbacivanje proizvoda u periplazmatičan prostor bakterija. Pri tom se radi o Leader-u outer membrane protein A (opmA) kao i alkalnoj fosfatazi (phoA). Poslije transfekcije ovog plazmida u Escherichia coli dokazuje se ekspresija funkcionalnog tj. proteina koji vezuju antigen u periplazmatičnon prostoru bakterija. Pri tom Better i sar. (1988, (11)) proizveli su himerno murinsko/humano antitijelo, koje raspoznaje gangliozid-antigen, kako je otkriveno često na površini humanih stanica karcinoma. Pri tom primijenjena konstrukcija plazmida sastojala se od Salmonella typhimurium araB promotora, kao i pelB leader sekvence uvijek ispred kodirajuće sekvence za svaki lanac. Dobiveni su Fab fragmenti koji stvaraju antigen iz preostatka kulture transformiranih bakterija. Na interesantan način koristili su kako Skerra i Plückthun (1988, (10)) tako i Better i sar. (1988, (11)) takozvane dicistronske konstrukcije, tj. takva, kod kojih na jednu jedinu messenger-RNK molekulu je prisutna informacija za oba lanca na kojima se treba odvojeno eksperimentirati. Autori navode, da je time zagarantirana prostorna blizina nastalih polipeptidnih lanaca, koja stvara pretpostavku za korektno slaganje promjenjivog regiona teškog (VH) sa lakim lancem (VL) lanca. Upravo ovaj problem, naime izradu Fv-peptid-heterodimera (u prirodi nije kovalentno vezan) pokušali su na drugi način riješiti Huston i sar. (1988, (12)) i Bird i sar. (1988, (13)), naime kovalentinim spajanjem lanaca preko aminokiseline-linker sekvence, kako se ona ne nalazi u prirodi. Ova linker sekvenca se odlikuje time, što se ona sastoji od određenog broja i sekvence aminoki-selina, tako da ona može premostiti razmak, koji u prirodnoj konformaciji antitijela postoji između regiona koji se trebaju povezati, a da se ne uvede nepotreban stres u konformaciju: Huston i sar. (1988, (12)) povezali promjenjive regione murinskoh anti-digoksin antitijela preko linkera od 15 aminokiselina sekvence GGGGSGGGGSGGGGS. Odabrani raspored je bio: VH - linker - VL. Using routine procedures, a cDNA bank of hybridoma cell lines capable of producing mAb is created. Total RNA is isolated from the mAk-producing hybridoma. This RNA contains, in addition to ribosomal RNA, the entirety of the cell's transcripts. Both incompletely processed nuclear transcripts and mature cytoplasmic transcripts, the so-called messenger RNAs, are present. These are characterized by a poly-adenosine tail at the 3'-end. This Poly-A region can be used to isolate mature mRNAs by affinity chromatography using oligo-dT cellulose. Using the enzyme "reverse transcriptase", mRNA can be converted into so-called cDNA. By using suitable vectors, the obtained cDNA mixture can be cloned, which leads to the so-called cDNA bank (5). Immunoglobulin specific hybridization probes allow the identification and isolation of clones containing the desired sequences. By sequencing the DNA of these clones and comparing the sequence with known immunoglobulin genes (EMBL Nucleotide Sequence Data Library, Heidelberg, SRN), certainty about the identity of the clone can be obtained (5). In this way, for example, clones can be isolated, which carry sequences of light or of the mAk heavy chain. By analyzing the sequences of immunoglobulin-cDNA obtained in this way, individual domains of difficult or. of the light chain: it is possible to identify a variable and a constant region, and for example within the variable region the so-called "hypervariable" or "complementarity determining regions", which are actually responsible for specific antigen binding (6). In this way, cloned antibody genes can be brought to expression in different systems. On the one hand, animal cell cultures, such as myeloma cells, can be used if suitable expression vectors are used (7). Application of yeast (8) or of bacterial cells (9) as an expression vehicle for a complete antibody is problematic, since such cells are obviously unable to correctly synthesize, for them, very large molecules, as represented by antibodies. Success in this direction was outlined only when an attempt was made to express antibody subfragments in lower eukaryotes or in prokaryotes. In the following, four different procedures are described, which allow the expression of Fv. or Fab fragments in Escherichia coli: Skerra and Pluckthun (1988, (10)) inserted the genes for the variable regions of the murine anti-phosphorylcholine antibody (McPC603) in connection with the Lac-Promoter/Operator Region, followed by one bacterial Leader sequence, which serves to expel the product into the periplasmic space of bacteria. This is about Leader outer membrane protein A (opmA) as well as alkaline phosphatase (phoA). After the transfection of this plasmid in Escherichia coli, the expression of a functional, i.e. antigen-binding, protein in the periplasmic space of the bacteria is demonstrated. At the same time, Better et al. (1988, (11)) produced a chimeric murine/human antibody, which recognizes the ganglioside antigen, as detected frequently on the surface of human carcinoma cells. The plasmid construction used consisted of the Salmonella typhimurium araB promoter, as well as the pelB leader sequence always in front of the coding sequence for each chain. Antigen-producing Fab fragments were obtained from the rest of the culture of transformed bacteria. In an interesting way, both Skerra and Plückthun (1988, (10)) and Better et al. (1988, (11)) so-called dicistronic constructions, i.e., those in which one single messenger-RNA molecule contains information for both chains that should be experimented on separately. The authors state that this guarantees the spatial proximity of the resulting polypeptide chains, which creates an assumption for the correct matching of the heavy variable region (VH) with the light chain (VL) of the chain. Huston et al. tried to solve this problem, namely the production of Fv-peptide-heterodimer (it is not covalently bound in nature) in another way. (1988, (12)) and Bird et al. (1988, (13)), namely by covalent joining of chains via an amino acid-linker sequence, as it is not found in nature. This linker sequence is characterized by the fact that it consists of a certain number and sequence of amino acids, so that it can bridge the gap, which in the natural conformation of the antibody exists between the regions that need to be linked, without introducing unnecessary stress into the conformation: Huston et al. (1988, (12)) connected the variable regions of murine anti-digoxin antibodies via a 15 amino acid linker of the sequence GGGGSGGGGSGGGGS. The chosen arrangement was: VH - linker - VL.
Ovaj takozvani single-chain Fv fragment se eksprimirao u vezi sa MLE leader sekvencom pod kontrolom sintetskog trp-promoter/operatora u obliku netopljivih Inclusion bodies. Poslije njihovog otapanja u 6M gvanidin-HCl i odstranjivanju leadera kiselom hidrolizom između aminokiselina Asp i Pro, kao i poslije nekoliko stupnjeva kromatografiranja dobiva se single-chain Fv fragment koji stvara antigen. This so-called single-chain Fv fragment was expressed in connection with the MLE leader sequence under the control of the synthetic trp-promoter/operator in the form of insoluble inclusion bodies. After their dissolution in 6M guanidine-HCl and removal of the leader by acid hydrolysis between the amino acids Asp and Pro, as well as after several stages of chromatography, a single-chain Fv fragment is obtained that creates an antigen.
U principu sličan način postupanja odabrao je Bird sa sasr. (1988, (13)) za konstrukciju proteina koji stvara murinski antigen, koji specifično vezuje fluorescein. Ova grupa je ipak primijenila linker od 18 aminokiselina sa sekvencom KESGSVSSEQLAQFRSLD. Ovaj linker je dio sekvence humane "karbonske anhidraze", i odabrana je iz Brookhaven Proteinstruktur-Datenbank-a kao Loopstruktura, koja se točno slaže prostorno na položaj aminokiselina Fv fragmenta koje se trebaju međusobno spojiti. Raspored pojedinačnih regiona je ovdje bio drugačiji nego kod Husron i sar. (1988, (12)), naime VL- linker - Vh. Gore opisane konstrukcije gena za proizvodnju fragmenata antitijela u Eshcerichia coli odnose na murinske sekvence odn. u jednom slučaju na murinsko/humane himeru. Još nisu objavljeni odgovarajući ogledi sa humanim sekvencama. In principle, a similar way of proceeding was chosen by Bird with sasr. (1988, (13)) for the construction of a murine antigen-forming protein that specifically binds fluorescein. However, this group used a linker of 18 amino acids with the sequence KESGSVSSEQLAQFRSLD. This linker is part of the human "carbonic anhydrase" sequence, and was selected from the Brookhaven Proteinstruktur-Datenbank as a Loop structure, which exactly fits spatially to the position of the amino acids of the Fv fragment to be linked together. The distribution of individual regions was different here than in Husron et al. (1988, (12)), namely VL-linker - Vh. The above-described gene constructs for the production of antibody fragments in Eshcerichia coli relate to murine sequences or. in one case to a murine/human chimera. Corresponding trials with human sequences have not yet been published.
Feb', (Fab')2 i Fv fragmenti pružaju različite prednosti u odnosu na kompletna antitijela. Na osnovu njihove male veličine u usporedbi prema kompletnim antitijelima oni mogu lakše i brže difundirati, kako in vitro tako i također kod eventualnih in vivo primjena. Iz istog razloga sa njima se može općenito lakše rukovati, i treba u većini slučajeva, kod kojih funkcije konstantnih regiona (npr. efektorne funkcije, vezivanje na receptorne stanice, vezivanje na druge molekule) nisu potrebne odn. čak donose nedostatke, kompletna antitijela jednako vrijedno odn. u danom slučaju čak pretpostaviti. Na primjer kod tumor-imaginga pri primjeni kompletnog antitijela često nastaju problemi uslijed nespecifičnih pozadinskih signala, koji su uvjetovani nespecifičnim vezivanjem antitijela na receptorske stanice, potpomognuti konstantnim regionima antitijela. Poznato je da pri primjeni Fab-fragmenata takvi se problemi mogu smanjiti. Feb', (Fab')2 and Fv fragments provide distinct advantages over complete antibodies. Based on their small size compared to complete antibodies, they can diffuse more easily and quickly, both in vitro and also in possible in vivo applications. For the same reason, they can generally be handled more easily, and should be in most cases, where the functions of constant regions (eg effector functions, binding to receptor cells, binding to other molecules) are not required or they even bring deficiencies, complete antibodies as valuable or in a given case even assume. For example, in tumor-imaging when using a complete antibody, problems often arise due to non-specific background signals, which are conditioned by non-specific binding of antibodies to receptor cells, supported by constant regions of the antibody. It is known that when using Fab-fragments, such problems can be reduced.
Prema tome treba očekivati da će upotreba Fv-fragmenata odn. single-chain Fv-fragmenata donijeti daljnje poboljšanje u ovom pogledu (13, 12). Therefore, it should be expected that the use of Fv-fragments or single-chain Fv-fragments bring further improvement in this regard (13, 12).
Do sada se radilo sa antitijelima murinskog porijekla, koja se vezuju na male, dobro opisane antigene, kao fluorescein odn. digoksin. Cjelokupna konstrukcija gena je sazdana na tome, da se kao antigen vezuje niskomolekularna supstanca (Mm manja od 1000). Antigene supstance koje se u prirodni u velikoj mjeri najčešće nalaze su peptidi, peptidoglikani, proteini i polisaharidi, i kao takvi visokomolekularni. So far, we have worked with antibodies of murine origin, which bind to small, well-described antigens, such as fluorescein or digoxin. The entire construction of the gene is based on the binding of a low-molecular substance (Mm less than 1000) as an antigen. Antigenic substances that are found in large quantities in nature are peptides, peptidoglycans, proteins and polysaccharides, and as such high molecular weight.
Prema izumu sadrži protein u uvode navedene vrste regione koji vezuju antigen antitijela koje potječe od linije stanica 3D6 (Accession Nr. 87110301, PHLS, Porotn Down, UK (1, 14, 15, 16). Time će se prvi put dobiti protein humanog porijekla, koji ima željena svojstva vezivanja i koji se također može eksprimirati u jednostanične mikroorganizme, kao što su kvasac ili bakterije. Osim toga opisat će se prema ovom izumu izrade single-chain konstrukcije, koja je izvedena iz humanog antitijela. Ova single-chain konstrukcija se vezuje na visokomolekularan, kompleksan, virusni antigen, nasuprot malim, dobro definiranim antigenima. According to the invention, it contains a protein in the introduction of the specified type of antigen-binding region of antibodies derived from the cell line 3D6 (Accession Nr. 87110301, PHLS, Porotn Down, UK (1, 14, 15, 16). This will be the first time to obtain a protein of human origin , which has the desired binding properties and which can also be expressed in single-celled microorganisms, such as yeast or bacteria. In addition, the invention will describe the production of a single-chain construct, which is derived from a human antibody. This single-chain construct is binds to high-molecular, complex, viral antigen, as opposed to small, well-defined antigens.
Nije se moglo pretpostaviti, da bi odgovarajući postupci za konstrukciju single-chain fragmenata mogli voditi također i kod drugih osim objavljenih antitijela, naročito kod humanih antitijela, do funkcionalnih, tj. molekula koji vezuju antigen. Isto tako nije očevidno, da kompleksni antigeni kao npr. antigeni na površini virusa, kod kojih prema iskustvu sudjeluje veći broj aminokiselina na vezivanje antigen-antitijelo nego kod malih antigena, toleriraju na isti način manipulacije u području promjenjivih regiona odgovarajućih antitijela koja se vezuju. It could not be assumed that appropriate procedures for the construction of single-chain fragments could also lead to functional, i.e. antigen-binding, molecules other than published antibodies, especially human antibodies. It is also not obvious that complex antigens, such as antigens on the surface of viruses, in which, according to experience, a greater number of amino acids participate in antigen-antibody binding than in the case of small antigens, tolerate in the same way manipulations in the area of variable regions of the corresponding antibodies that bind.
Polazeći od linije stanica 3D6, koja proizvodi humano monoklonalno antitijelo tipa IgGl/kappa, koje specifično reagira sa HIV-1-gp41, i pokazuje slabo reakciju ukrštanja sa HIV-1 gp120 (3D6; (1, 14, 16, 16)), izolirana je cjelokupna RNK. Pri tom je primijenjen postupak gvanidin-izocijanatne ekstrakcije i ultracentrifugiranje preko jastuka 5,7M CsCl (5). Iz ukupne RNK se adsorbiranjem na oligo-dt, celulozi izolirala poli A+ - frakcija, dakle mRNK, (mRNK kit za pročišćavanje, Fa. Pharmacia, Švedska). Starting from the 3D6 cell line, which produces a human IgGl/kappa-type monoclonal antibody that specifically reacts with HIV-1-gp41, and shows weak cross-reactivity with HIV-1 gp120 (3D6; (1, 14, 16, 16)), total RNA was isolated. The procedure of guanidine-isocyanate extraction and ultracentrifugation over a 5.7M CsCl cushion was used (5). The poly A+ fraction, i.e. mRNA, was isolated from the total RNA by adsorbing on oligo-dt, cellulose (mRNA purification kit, Pharmacia, Sweden).
mRNK je služila kao podloga za sintezu cDNK (cDNK kit za sintezu, Fa. Pharmacia, Švedska). mRNA served as a substrate for cDNA synthesis (cDNA synthesis kit, Fa. Pharmacia, Sweden).
Kloniranje cDNK banke je obavljeno u plazmidnom vektoru pUC19. Rekombinantni plazmidi se transformiraju u Escherichia coli, spoj HB101, i uzgajaju na LB podlozi (5). Pozitivni kloni se identificiraju pomoću hibridiziranja pomoću specifičnih oligonukleotidnih sondi. Sekvence za sonde se uzimaju iz EMBL DNA-Sewuenz Datenbank-e iz konstantnih regiona humanih IgGl. teških odn. Kappa-lakih lanaca. Kloni identificirani pomoću pozitivnih signala hibridizacije se dalje karakteriziraju restrikcionom analizom, i oni kloni identificiraju, koji nose plazmide sa najdužim umecima. Pomoću analize sekvence ovih klona identificira se po klon sa kompletnim kodirajućim regionom za težak odn. za lak lanac antitijela. Ovi kloni nose oznaku pUC3D6HC (Seq ID NO: 1) odn. pUC3D6LC (SEQ ID NO: 2). The cDNA bank was cloned in the pUC19 plasmid vector. Recombinant plasmids are transformed into Escherichia coli, accession HB101, and grown on LB medium (5). Positive clones are identified by hybridization with specific oligonucleotide probes. The sequences for the probes are taken from the EMBL DNA-Sewuenz Databank from the constant regions of human IgG1. heavy or Kappa-light chains. Clones identified by positive hybridization signals are further characterized by restriction analysis, and those clones are identified as carrying plasmids with the longest inserts. By means of sequence analysis of these clones, each clone with the complete coding region for heavy or for the light chain of antibodies. These clones bear the designation pUC3D6HC (Seq ID NO: 1) or pUC3D6LC (SEQ ID NO: 2).
Primjer 1 Example 1
U sekvenci inseta klona pUC3D6HC (SEQ ID NO: 1) odn. pUC3D6LC (SEQ ID NO: 2) identificiraju se prelazna mjesta između regiona leaderpeptida i promjenjivog regiona, kao i između promjenjivog regiona konstantnog regiona. Pomoću oligonukleotidno usmjerene mutageneze (in vitro mutagenesis system, Amersham, UK) izvedene su na ovim prelaznim mjestima sljedeće mutacije (vidi također i sheme 1 - 4): In the inset sequence of clone pUC3D6HC (SEQ ID NO: 1) or pUC3D6LC (SEQ ID NO: 2) the transition sites between the leader peptide region and the variable region, as well as between the variable region of the constant region, are identified. Using oligonucleotide-directed mutagenesis (in vitro mutagenesis system, Amersham, UK), the following mutations were performed at these transition sites (see also Schemes 1 - 4):
1) Sekvence raspoznavanja za određene restrikcione enzime se umutiraju. Pomoću ovih restrikcionih mjesta iz odnosnih plazmida isijecaju promjenjivi regioni teškog odn. lakog lanca antitijela 3D6. 1) Recognition sequences for certain restriction enzymes are mutated. Using these restriction sites, variable regions of heavy or of light chain antibody 3D6.
2) Za kasniju ekspresiju potrebni startni i zaustavni kodoni se umutiraju. 2) The start and stop codons required for later expression are mutated.
Da bi se promjenjivi regioni antitijela 3D6 mogli vezati sa linkerom, izrađuju se dva sintetska oligonukleotida, koji grade oba DNK-konca linkera. Oba oligonukleotida, se tako odabiru, da, kada se međusobno hibridiziraju, nastaje duplog konca DNK, na čijim se krajevima nalaze pojedinačno končasti DNK regioni koji izlaze, koji odgovaraju točno onim krajevima koji izlaze, koji pri sječenju pomoću odgovarajućih restrikcionih enzima nastaju na gore spomenutim umutiranim restrikcionim mjestima. Ovo omogućuje vezivanje pomoću ovih restrikcionih enzima izoliranih promjenjivih regiona sa sintetskim oligonukleotidima linkera. Vezivanjem međusobno prethodno tretiranih fragmenata (VH, linker, VL) koji odgovara 3 dobiva se gen, koji je na prijelaznim mjestima između promjenjivih regiona i linkera još nosio umutirana restrikciona mjesta, koja sadrže nukleotide, koji odgovaraju nukleotidima koji se prirodno ne nalaze na ovim mjestima. Time se dobila također i izmijenjena sekvenca amino kiseline (sheme 5 i 6). In order for the variable regions of the 3D6 antibody to be attached to the linker, two synthetic oligonucleotides are made, which build both DNA strands of the linker. Both oligonucleotides are selected in such a way that, when they hybridize with each other, a double-stranded DNA is formed, at the ends of which there are single-stranded DNA regions that come out, which correspond exactly to those ends that come out, which, when cut with the appropriate restriction enzymes, are created at the above-mentioned mutated restriction sites. This allows binding of isolated variable regions with synthetic linker oligonucleotides by these restriction enzymes. By binding to each other the previously treated fragments (VH, linker, VL) corresponding to 3, a gene is obtained, which at the transition points between the variable regions and the linker still carried scrambled restriction sites, which contain nucleotides, which correspond to nucleotides that are not naturally found in these places . This also resulted in an altered amino acid sequence (Schemes 5 and 6).
Da bi se prvobitna sekvenca aminokiselina ponovo uspostavila, izrađuje se na spomenutim prelaznim mjestima pomoću ponovnog mutacionog procesa željena DNK sekvenca (sheme 6 i 6). Pomoću ovih postupaka je konstruiran također gen, što ima strukturu VH = linker - VL. Ovaj konstrukt se označava kao cs3D6, i umeće se u vektor kloniranja pUC19 (SEQ ID NO: 3). Vektor koji nastaje nosi oznaku pUCsc3D6. In order to re-establish the original sequence of amino acids, the desired DNA sequence is created at the mentioned transition sites by means of a re-mutation process (schemes 6 and 6). Using these procedures, a gene was also constructed, which has the structure VH = linker - VL. This construct is designated as cs3D6, and inserted into the cloning vector pUC19 (SEQ ID NO: 3). The resulting vector is labeled pUCsc3D6.
sc3D6-gen se isijeca iz plazmida pUCsc3D6 pomoću restrikcionih enzima, i umeće u bakterijelni ekspresioni vektor pKK223-3 (Pharmacia), koji sadrži tacpromoter koji se može inducirati pomoću izopropil-β tio-glaktozidom (IPTG). Vektor koji nastaje nosi oznaku pKKsc3D6 i transformira se u E.coli spoj JM105. The sc3D6 gene was excised from plasmid pUCsc3D6 using restriction enzymes, and inserted into the bacterial expression vector pKK223-3 (Pharmacia), which contains a tacpromoter inducible with isopropyl-β thio-galactoside (IPTG). The resulting vector is labeled pKKsc3D6 and is transformed into E.coli compound JM105.
Uzgoj bakterija Cultivation of bacteria
Transformirane bakterije se uzgajaju u laboratorijskom fermentoru čak do OD600 od 2,0 u LB hranjivoj podlozi (5). Poslije toga se obavila indukcija ekspresije dodavanjem izopropiltioga-laktozida (IPTG). Bakterije se uzgajaju 3 sata u prisustvu IPGT dalje, potom pomoću echtrifugiranja sakupe i skladište na -80°C. Poslije toga se protein ekstrahira i pročišćuje. Transformed bacteria are grown in a laboratory fermenter up to an OD600 of 2.0 in LB nutrient medium (5). After that, expression was induced by adding isopropylthioga-lactoside (IPTG). The bacteria are grown for 3 hours in the presence of IPGT further, then collected by echtrifugation and stored at -80°C. After that, the protein is extracted and purified.
Ekstrakcija i pročišćavanje Extraction and purification
Za svaku oglednu šaržu stavlja se 10g biomase (vlažna masa). Stanice se pomoću lizozima otapaju. Zamrznuta pasta E.coli se izlomi u male komade i priprema pomoću STE-pufera (10mM Tris, 100 mM Na Cl, 1 mM EDTA, pH 8.0) 10%-tna suspenzija. Ovoj suspenziji se dodaje koktel otapanja, koji sadrži nukleaze, lizozim i inhibitore (vidi Tabelu 1).Ova E.coli suspenzija se inkubira 15 minuta na 42°C. Dodavanjem Triton-X-100 (krajnja koncentracija 0,5%) i ponovnom petominutnom inkubacijom na 42°C liziraju se stanice. 10g of biomass (moist mass) is added to each sample batch. Cells are dissolved using lysozyme. Frozen E.coli paste is broken into small pieces and prepared using STE-buffer (10mM Tris, 100mM NaCl, 1mM EDTA, pH 8.0) 10% suspension. To this suspension is added a dissolution cocktail, which contains nucleases, lysozyme and inhibitors (see Table 1). This E.coli suspension is incubated for 15 minutes at 42°C. Cells are lysed by adding Triton-X-100 (final concentration 0.5%) and re-incubating for five minutes at 42°C.
Izdvajanje inkluzionih tijela Separation of inclusion bodies
Talog se ponovo suspendira u STE-puferu i miješa 8 sati na 4°C. Inkluziona tijela se obogaćuju centrifugiranjem. Za ovo se stavi glicerinski jastuk (50% glicerina/u fosfatno puferiranoj fiziološkoj otopini soli (PBS)) u epruvetama za centrifugiranje, preša istom zapreminom suspenzije i centrifugira. (30 minuta, 6000 o/m, 4°C, JA-20 Rotor, J2-21 Zentrifuge, Fa. Beckman). The precipitate is resuspended in STE-buffer and stirred for 8 hours at 4°C. Inclusion bodies are enriched by centrifugation. For this, a glycerin cushion (50% glycerin/in phosphate-buffered saline (PBS)) is placed in centrifuge tubes, pressed with the same volume of suspension and centrifuged. (30 minutes, 6000 rpm, 4°C, JA-20 Rotor, J2-21 Centrifuge, Fa. Beckman).
Otapanje inkluzionih tijela Dissolution of inclusion bodies
Obogaćena inkluziona tijela se otapaju 6M GuHCl (gvandin hidro-klorid) u PBS, pH 8,3 uz miješanje na 4°C (12 h). Zatim se određuje sadržaj proteina fotometrijski. Enriched inclusion bodies are dissolved with 6M GuHCl (guandine hydrochloride) in PBS, pH 8.3 with stirring at 4°C (12 h). The protein content is then determined photometrically.
Refolding Refolding
U GuHCl otopljeni protein se ovija u prisustvu stranih proteina. Najprije je obavljeno određivanje proteina. Otopljena inkluziona tijela se tako razblaže pomoću refolding šufera (GuHCl IM, glutation reducirani 30 mM, glutation oksidiran 3mM, EDTA 100 μM, u PBS, pH 8,3) da krajnja koncentracija iznosi 80 mg proteina/1. Razblaživanje otopljenih inkluzionih tijela se obavlja u kaboratorijskom srazmjeru uz primjenu birete laganim dokapavanjem otopine proteina u refolding pufer. Najbolje se radi na 37°C. Preklapanje je praćeno pomoću reversne faze HPLC. Za to su uzimani uzorci, pH vrijednost podešena na 5,5, da bi se spriječio daljnji refolding, uzorci su centrifugirani (Millipore stona centrifuga. 4700 o/m, sobna temperatura), sterilno filtrirani (veličina pora 0,22 μm, nisko vezivanje proteina), u slučaju potrebe koncentrirani (Millipore, stona centrifuga, 4700 o/m, 20°C) i svakad 250 μl analizirani pomoću reversne faze HPLC (Nucleosil 300, 5 μm. 4x125 mm., Fa. Macerey i Vogel, SRN. Na kolonu je postavljen linearni gradijent 0,1 % TFA / acetonitril 10 - 60% u toku 40 minuta. Protein dissolved in GuHCl is wrapped in the presence of foreign proteins. First, protein determination was performed. Dissolved inclusion bodies are so diluted using refolding buffer (GuHCl IM, reduced glutathione 30 mM, oxidized glutathione 3 mM, EDTA 100 μM, in PBS, pH 8.3) that the final concentration is 80 mg protein/1. Dilution of the dissolved inclusion bodies is done in a carbonator ratio with the use of a burette by gently adding the protein solution to the refolding buffer. It works best at 37°C. Overlap was monitored using reverse phase HPLC. For this, samples were taken, the pH value was adjusted to 5.5, to prevent further refolding, the samples were centrifuged (Millipore table centrifuge. 4700 rpm, room temperature), sterile filtered (pore size 0.22 μm, low binding protein), if necessary concentrated (Millipore, table centrifuge, 4700 rpm, 20°C) and each time 250 μl analyzed using reverse phase HPLC (Nucleosil 300, 5 μm. 4x125 mm., Fa. Macerey and Vogel, SRN. A linear gradient of 0.1% TFA / acetonitrile 10 - 60% was placed on the column in the course of 40 minutes.
Preklopljeni sc3D6 se ultradiafiltrira. Primjenjuje se 10000 Daltona granice propuštanja polisulfon membrana. Diafiltrirana otopina proteina se nanese na anionski izmjenjivač i potom pomoću 100 mM NaCl eluira iz kolone. sc3D6 se pomoću Sephadex G-25 (Fa. Pharmacia, Švedska) gelfiltriranjem oslobodi soli i konjugira po postupku Nakane i sar. (17) pomoću alkalne fosfataze. Pročišćeni sc3D6 protein se kontrolira pomoću SDS-PAGE (Slika 7). Za dokaz funkcionalnosti sc3D6 proteina izveden je Western Blot Test HIV-1-trake za ogled (Fa.RioRad, SAD). Kao pozitivna kontrola izvodi se sličan ogled sa prirodnim antitijelom izoliranim iz životinjskih stanica. Kao negativna kontrola služio je preparat cjelokupnog proteina iz E.coli. Rezultat ovog ogleda je bio pozitivan i prikazan je na slici 8. Folded sc3D6 is ultradiafiltered. The 10,000 Dalton permeation limit of polysulfone membranes is applied. The diafiltered protein solution is applied to the anion exchanger and then eluted from the column using 100 mM NaCl. sc3D6 is freed from salts by gel filtration using Sephadex G-25 (Fa. Pharmacia, Sweden) and conjugated according to the procedure of Nakana et al. (17) using alkaline phosphatase. Purified sc3D6 protein is controlled by SDS-PAGE (Figure 7). To prove the functionality of the sc3D6 protein, a Western Blot Test of the HIV-1 test strip was performed (Fa.RioRad, USA). As a positive control, a similar experiment is performed with a natural antibody isolated from animal cells. A preparation of the entire protein from E.coli served as a negative control. The result of this experiment was positive and is shown in Figure 8.
Pročišćavanje sc3D6 proteina, pomoću afinitetne kromatografije Sa odgovarajući pročišćenim sc3D6 proteinom izrađen je serum kunića pod standardnim uvjetima pomoću kompletnog Feund-ovog adjuvansa. Pomoću CM-Sepharoze Fast Flow Chromatografije (fa. Pharmacie, Švedska) dobivena je IgG frakcija iz seruma kunića. Specifičnost je utvrđena pomoću ELISA. Pomoću peptidnog sintesajzera izrađen je 15 amino kiselina dug linker peptid (Sekvenca: GGGGSGGGGSGGGGS) i zatim konjugiran pomoću karbodiimidne kondenzacije sa albuminom goveđeg seruma (BSA) u molarnom odnosu od 6:1. Sa ovim konjugatom se nanosi sloj na ploče za mikrotitriranje. Serumski uzorak se inkubira u pločama za mikrotitriranje na kojima se nalazi sloj i vezano antitijelo se dokazuje pomoću peroksidaze-markiranog koza-anti-zamorci IgG. Purification of sc3D6 protein, using affinity chromatography Rabbit serum was prepared with appropriately purified sc3D6 protein under standard conditions using Feund's complete adjuvant. IgG fraction was obtained from rabbit serum using CM-Sepharose Fast Flow Chromatography (Pharmacie, Sweden). Specificity was determined using ELISA. Using a peptide synthesizer, a 15 amino acid long linker peptide (Sequence: GGGGSGGGGSGGGGS) was made and then conjugated using carbodiimide condensation with bovine serum albumin (BSA) in a molar ratio of 6:1. This conjugate is coated on microtiter plates. The serum sample is incubated in coated microtiter plates and the bound antibody is detected using peroxidase-labeled goat-anti-guinea pig IgG.
Na taj način izrađen i ispitan anti-sc3D6 IgG vezan na BrCN aktiviranu Sepharose 4B (Fa.Pharmacia, Švedska). Materijal koji nije vezan ispere se. Prethodno pročišćeni ekstrakt sc3D6 proteina, koji je kako što je gore opisano preklopljen i oslobođen soli pomoću Sephadex G-25 (Fa.Pharmacia, Švedska), nanese se na anti-sc3D6 kolonu. Materijal koji nije vezan se ispire i specifično vezani sc3D6 protein se eluira eluira pomoću 0,1M glicin HCl pufera pH 2,5. Potom se eluat neutralizira pomoću 1M Tris pufera i sc3D6 karakterizira kako je opisano pomoću SDS elektroforeze i ispita pomoću Western Blot-a na funkcionalnost. Drugi postupak za imunafinitethromatografsko pročišćavanje sc3D6 proteina je kao što slijedi: In this way, anti-sc3D6 IgG bound to BrCN-activated Sepharose 4B (Fa.Pharmacia, Sweden) was produced and tested. The unbound material is washed away. The pre-purified sc3D6 protein extract, which was folded and desalted as described above using Sephadex G-25 (Fa.Pharmacia, Sweden), was applied to an anti-sc3D6 column. Unbound material is washed away and specifically bound sc3D6 protein is eluted using 0.1M glycine HCl buffer pH 2.5. The eluate was then neutralized with 1M Tris buffer and sc3D6 was characterized as described by SDS electrophoresis and assayed by Western Blot for functionality. Another procedure for immunoaffinity chromatographic purification of the sc3D6 protein is as follows:
Sa gore opisanim linker peptidom kupliranim na BSA izrađuje se serum kunića pomoću kompletnog Frend-ovog adjuvansa. IgG frakcija se dobiva CM-Sepharose Fast Flow kromatografijom (fa. Pharmacia, Švedska) i dalje pročišćuje preko BSA-Sepharose 4B kolone (Fa. Pharmacia, Švedska), da bi se odstranilo anti-BSA antitijelo. Tako dobiveni anti-linker IgG kuplira se na BrCN aktiviranoj Sepharose 4B (Pa.Pharmacia, Švedska). Prethodno pročišćeni ekstrakt sc3D6 proteina, koji je preklopljen kako je opisano gore i oslobođen soli sa Sephadex G-25 (Fa. Pharmacia, Švedska), nanese na antilinker kolonu. Materijal koji nije vezan se ispire i specifično vezani sc3D6 protein se eluira pomoću 0,1M glicin HCl pufera pH 3,0. Zatim se eluat neutralizira pomoću 1M Tris pufera i sc3D6 protein kako je opisano karakterizira pomoću SDS elektroforeze i funkcionalnost ispita pomoću Western Blot-a. With the linker peptide described above coupled to BSA, rabbit serum is prepared using complete Frend's adjuvant. The IgG fraction is obtained by CM-Sepharose Fast Flow chromatography (Pharmacia, Sweden) and further purified over a BSA-Sepharose 4B column (Pharmacia, Sweden) to remove the anti-BSA antibody. The thus obtained anti-linker IgG is coupled to BrCN activated Sepharose 4B (Pa.Pharmacia, Sweden). The previously purified sc3D6 protein extract, which was folded as described above and desalted with Sephadex G-25 (Fa. Pharmacia, Sweden), was applied to an antilinker column. Unbound material is washed away and specifically bound sc3D6 protein is eluted with 0.1 M glycine HCl buffer pH 3.0. The eluate is then neutralized with 1M Tris buffer and the sc3D6 protein is characterized as described by SDS electrophoresis and functionality assayed by Western Blot.
Imunafinitetkromatografsko pročišćavanje HIV-1 gp160 Immunoaffinity chromatography purification of HIV-1 gp160
Za izradu sceD6-imunafinitetne kolone pročišćeni sc3D6 protein se vezuje ns 1m1 NHS kolonu (Fa. Pharmacia, Švedska) (prema propisu Fa.Pharmacia). To create a sceD6 immunoaffinity column, the purified sc3D6 protein is bound to a ns 1m1 NHS column (Fa. Pharmacia, Sweden) (according to Fa.Pharmacia's instructions).
Prethodno pročišćavanje gp160 (coat proteina HIVa-1 koji specifično vezuje antitijelo 3D6 kao i sc3D6) izvodi se po Barrett i sar. (18). Pre-purification of gp160 (HIVa-1 coat protein that specifically binds antibody 3D6 as well as sc3D6) is performed according to Barrett et al. (18).
Prethodno pročišćeni materijal, koji sadrži rekombinantni gp160, koncentrira se preko ultra/diafiltriranja i kondicionira za sc3D6-imunafinitetnu kromatografiju. Ovaj kondicionirani materijal se nanosi na sc3D6-imunafinitetnu kolonu. Kao ekvilibracioni pufer primjenjuje se 100mM Tris pufer pH 7,4 sa 0,1% Tween-a 20. Rekombinantni antigen se eluira pomoću 3Mrodanida. The pre-purified material, containing recombinant gp160, is concentrated via ultra/diafiltration and conditioned for sc3D6-immunaffinity chromatography. This conditioned material is applied to a sc3D6-immunaffinity column. 100mM Tris buffer pH 7.4 with 0.1% Tween 20 is used as equilibration buffer. Recombinant antigen is eluted using 3Mrodanide.
Prinosi pojedinačnih stupnjeva složeni su u Tabeli 2. The yields of individual grades are compiled in Table 2.
Primjer 2 Example 2
Drugo kloniranje sc3D6, kod koga se sc3D6 gen fuzionira sa genom izoliranim iz Escherichia coli sa alkalnu fosfatazu, izvodi se kako slijedi: sc3D6 gen se restrikcionim enzimima isiječe iz plazmida pUCsc3D6, i umetne u vektor pEcphoAMut3 (19). Vektor koji nastaje nosi oznaku pAPsc3D6. Vektor pEcphoAMut3 sadrži gen za alkalnu fosfatazu (20) koji je izoliran iz Esherichia coli u koju se pomoću oligonukleotid usmjerene mutageneze umutira na 3' kraju kodirajućeg regiona restrikciono mjesto, koje dozvoljava fuziju EcphoA gena sa drugim genima. Na ovaj način mogu ekspresijom fuzionog gena izraditi fuzioni proteini, tj. proteini, kod kojih su uvijek kodirajući regioni međusobno vezani pomoću peptidnih veza preko aminokiselina. The second cloning of sc3D6, in which the sc3D6 gene is fused with the gene isolated from Escherichia coli with alkaline phosphatase, is performed as follows: the sc3D6 gene is cut with restriction enzymes from the pUCsc3D6 plasmid, and inserted into the pEcphoAMut3 vector (19). The resulting vector is labeled pAPsc3D6. The vector pEcphoAMut3 contains the gene for alkaline phosphatase (20) that was isolated from Esherichia coli in which, by means of oligonucleotide directed mutagenesis, a restriction site was inserted at the 3' end of the coding region, which allows the fusion of the EcphoA gene with other genes. In this way, by expressing the fusion gene, fusion proteins can be created, i.e. proteins in which the coding regions are always connected to each other by means of peptide bonds via amino acids.
EcphoA - sc3D6 fuzioni gen se isijeca iz pAPsc3D6 pomoću restrikcionih enzima i umetnu u bakterijski ekspresioni vektor pKK223-3 (Fa.Pharmacia, Švedska). Plazmid koji se dobiva nosi oznaku pKKAPsc3D6. The EcphoA - sc3D6 fusion gene was cut from pAPsc3D6 using restriction enzymes and inserted into the bacterial expression vector pKK223-3 (Fa.Pharmacia, Sweden). The resulting plasmid is labeled pKKAPsc3D6.
Plazmid pKKPsc3D6 se transformira u Escherichia coli soj JM 105 i transformirane bakterije uzgajaju u LB hranjivoj sredini (5). Poslije induciranja pomoću IPTG aktivni EcPhoA - sc3D6 fuzioni protein iz periplazmatičnog prostora bakterija pročišćuje se kako slijedi: Bakterije se sakupe centrifugiranjem i isperu sa 10mM Tris puferom pH 7,5, koji je pomiješan 30mM NaCl. Isprane bakterije se ponovo suspendiraju u 33Mm Tris pufera pH 7,5 i pomiješaju sa istom zapreminom 40% rastvora saharoze u (33mM Tris pufera) i dodaje EDTA na krajnju koncentraciju od 0,1mM. Poslije 10 minutne inkubacije na sobnoj temperaturi bakterije se odstrane centrifugiranjem i prihvate u 0,5mM MgCl2 otopini. Poslije 10 minutne inkubacije na 0°C dodaje se koktel inhibicije proteaze, koji se sastoji iz PMSF i EGTA i bakterije odstrane centrifugiranjem. Tekućina iznad se pomoću 1M Tris otopine pH 7,5 dovede na krajnju koncentraciju od 25mM Tris-a. Ovim postupkom se oslobađa periplazmatičan prostor E.coli stanica. Plasmid pKKPsc3D6 is transformed into Escherichia coli strain JM 105 and the transformed bacteria are grown in LB medium (5). After induction by IPTG, the active EcPhoA - sc3D6 fusion protein from the periplasmic space of bacteria is purified as follows: Bacteria are collected by centrifugation and washed with 10mM Tris buffer pH 7.5, which is mixed with 30mM NaCl. The washed bacteria are resuspended in 33 mM Tris buffer pH 7.5 and mixed with the same volume of 40% sucrose solution in (33 mM Tris buffer) and EDTA is added to a final concentration of 0.1 mM. After a 10-minute incubation at room temperature, the bacteria are removed by centrifugation and accepted in a 0.5mM MgCl2 solution. After a 10-minute incubation at 0°C, a protease inhibition cocktail is added, which consists of PMSF and EGTA and bacteria removed by centrifugation. The liquid above is brought to a final concentration of 25mM Tris using 1M Tris solution pH 7.5. This procedure frees the periplasmic space of E.coli cells.
Centrifugiranjem pri 12000 g bistri se proteinska otopina i zatim koncentrira preko ultrafiltriranja. EcPhOA - sc3D6 protein se dalje pročišćuje pomoću hidrofobne interakcione kromatografije. Phenylsepharose Fast Flow (Fa.Pharmacia, Švedska) se ekvilibrira sa 60% zasićenim amonij sulfatnom otopinom u 25mM Tris puferu pH 7,5. Proteinska otopina se naizmjenično sa ekvilibracionim puferom nanosi na kolonu. sc3D6 se pomoću linearnog gradijenta eluira od 60% amonij sulfata na 0% amonij sulfata. Frakcije koje sadrže EcPhOA - sc3D6 protein oslobađaju se soli gelfiltraciju. Za dokaz funkcionalnosti EcPhoA - sc3D6 proteina izvodi se Western Blot ogled sa HIV-1 oglednim trakama (Fa.BioRad, SAD). Za kontrolu izveden je sličan ogled sa prirodnim antitijelom izoliranim iz životinjskih stanica. Kao negativna kontrola služio je preparat ukupnog proteina iz E.coli. Rezultat ovog ogleda je bio pozitivan i pokazan je na Slici 8. The protein solution is clarified by centrifugation at 12,000 g and then concentrated via ultrafiltration. The EcPhOA - sc3D6 protein is further purified using hydrophobic interaction chromatography. Phenylsepharose Fast Flow (Fa.Pharmacia, Sweden) is equilibrated with 60% saturated ammonium sulfate solution in 25mM Tris buffer pH 7.5. The protein solution is applied to the column alternately with the equilibration buffer. sc3D6 is eluted using a linear gradient from 60% ammonium sulfate to 0% ammonium sulfate. Fractions containing EcPhOA - sc3D6 protein are freed from salt by gel filtration. To prove the functionality of the EcPhoA - sc3D6 protein, a Western Blot test is performed with HIV-1 test strips (Fa.BioRad, USA). As a control, a similar experiment was performed with a natural antibody isolated from animal cells. A preparation of total protein from E.coli served as a negative control. The result of this experiment was positive and is shown in Figure 8.
Direktan dokaz HIV-1 antigena pomoću ELISA Direct detection of HIV-1 antigen by ELISA
gp120-specifično monoklonalno antitijelo (Klon 25 C2, Accession Nr. 89120601, PHLS, Porton Down, UK) nanosi se u obliku sloja na ploče za mikrotitar (Frade I, Fa. Nunc, Danska). Tekućina iznad kulture (16) koja sadrži HIV-1 nanosi se na slojem obložene ploče za mikrotitraciju. Kao standard se primjenjuje rekombinantni gp160 (18). A gp120-specific monoclonal antibody (Clone 25 C2, Accession Nr. 89120601, PHLS, Porton Down, UK) is coated on microtiter plates (Frade I, Fa. Nunc, Denmark). The culture supernatant (16) containing HIV-1 is applied to a coated microtiter plate. Recombinant gp160 (18) is used as a standard.
Poslije ispiranja nevezanog materijala nanosi se EcPhoA-sc3D6 protein i inkubira. Nevezani materijal se ponovo ispire i pomoću p-nitrofenilfosfata se dokazuje fotometrijski na 602 nm vezani EcPhoA-sc3D6 protein. Na slici 9 je predstavljena standardna krivulja i različiti uzroci HIV-1 pozitivnih tekućina iznad kulture. After washing off the unbound material, the EcPhoA-sc3D6 protein is applied and incubated. The unbound material is washed again and the bound EcPhoA-sc3D6 protein is detected photometrically at 602 nm using p-nitrophenylphosphate. Figure 9 presents the standard curve and various causes of HIV-1 positive supernatants.
Kompetitivni anti-HIV-1 ELISA Competitive anti-HIV-1 ELISA
Mikrotitracione ploče (Grade I, Fa.Nunc, Danska) se oblože sa otopinom 10 mg/ml rekombinantnog gp160 (18). Potom se ploče ispiru sa PBS + 0,1% Tween-a 20 + BSA. Otopina 5 μg/ml EcPhoA - sc3D6 fuzionog proteina se pomiješa u odnosu 1:1 sa HIV-1 pozitivnim odn. HIV-1 negativnim serumima i nanese na obložene ploče. Kao kontrola nanese se pomoću pufera za razblaživanje pomiješani EcPhoA - sc3D6 -fuzioni protein i inkubira na 37°C 60 min. Poslije toga se nevezani materijal ispire. Microtiter plates (Grade I, Fa.Nunc, Denmark) are coated with a solution of 10 mg/ml recombinant gp160 (18). Then the plates are washed with PBS + 0.1% Tween 20 + BSA. A solution of 5 μg/ml EcPhoA - sc3D6 fusion protein is mixed in a 1:1 ratio with HIV-1 positive or. HIV-1 negative sera and applied to coated plates. As a control, the mixed EcPhoA - sc3D6 -fusion protein was applied using the dilution buffer and incubated at 37°C for 60 min. After that, the unbound material is washed away.
Dodavanjem p-nitrofenilfosfata dokazuje se udio vezanog EcPhoA -sc3D6 protein. Boja se kvantificira fotometrijski na 602 nm. Inhibicija seruma je određena u procentima ekstrinkcije EcPhoA -sc3D6 proteina bez seruma. Kako je predstavljeno na Slici 10, svi HIV-1 pozitivni serumi inhibiraju vezivanje EcPhoA - sc3D6 fuzionog proteina na gp160. Svi HIV-1 negativni serumi su pokazivali manje inhibicije od HIV-1 pozitivnih seruma. Adding p-nitrophenylphosphate proves the proportion of bound EcPhoA-sc3D6 protein. Color is quantified photometrically at 602 nm. Serum inhibition was determined as percentage of EcPhoA-sc3D6 protein extrinsic without serum. As presented in Figure 10, all HIV-1 positive sera inhibit the binding of the EcPhoA - sc3D6 fusion protein to gp160. All HIV-1 negative sera showed less inhibition than HIV-1 positive sera.
Primjer 3 Example 3
Druga vrsta ekspresije za sc3D6 protein, kod koje se kao stanice domaćina koriste stanice mieloma miša, izvedena je kako slijedi: Another type of expression for the sc3D6 protein, using mouse myeloma cells as host cells, was performed as follows:
3' dio sc3D6 gena izolira se iz plazmida pUCsc3D6 (SEQ ID NO:3) parcijalnim EcoRV Verdau kao i kompletnim HindIII Verdau (dužina fragmenta: 401 bp). Iz plazmida pUC3D6HC (SEQ ID NO: 1) se odstranjuje 3' dio gena za teški lanac sječenjem pomoću EcoRV i HindIII. U vektor koji zaostaje umeće se preko agarozne-gel elektroforeze izdvojeni i pročišćeni 401 bp -fragment sc3D6 gena. The 3' part of sc3D6 gene was isolated from plasmid pUCsc3D6 (SEQ ID NO:3) by partial EcoRV Verdau as well as complete HindIII Verdau (fragment length: 401 bp). From the plasmid pUC3D6HC (SEQ ID NO: 1), the 3' part of the heavy chain gene is removed by cutting with EcoRV and HindIII. The separated and purified 401 bp fragment of the sc3D6 gene is inserted into the lagging vector via agarose-gel electrophoresis.
Tako rekombinirani gen se sastoji također i iz sekvence za leader peptid teškog lanca antitijela 3D6, iza koga slijedi sekvenca sc3D6 gena. Plazmid nosi oznaku pLsc3D6. Ova konstrukcija dozvoljava oslobađanje sc3D6 proteina iz životinjskih stanica. Kodirajući gen se izolira iz pLsc3D6 pomoću enzima NcoI i HindIII, popunjava ispuštene krajeve Klenow-polimerazom i klonira u SmaI mjesto ekspresionog vektora pRcRSV (Invitrogen, SAD) koji pogodan za životinjske stanice. SmaI mjesto ovog ekspresionog vektora leži između Long Terminal Repeat BSV-a, dakle jako virusnog promotera, i transkipcionih-završnih sekvenci, koje prvobitno potječu od goveđeg hormona rasta. Umetanjem u ovo restrikciono mjesto je moguće da se dovedu bilo koji strukturni geni u molekularnu okolinu, koja dozvoljava ekspresiju gena u životinjskim stanicama. Osim toga vektor pRcRSV posjeduje još sekcioni obilježivač "neomicinske rezistencije", koji dozvoljava selekciju uspješno transformiranih životinjskih stanica u kulturi. The thus recombined gene also consists of the sequence for the leader peptide of the heavy chain of antibody 3D6, followed by the sequence of the sc3D6 gene. The plasmid is labeled pLsc3D6. This construct allows release of the sc3D6 protein from animal cells. The coding gene was isolated from pLsc3D6 using NcoI and HindIII enzymes, filling in the missing ends with Klenow polymerase and cloned into the SmaI site of the expression vector pRcRSV (Invitrogen, USA) suitable for animal cells. The SmaI site of this expression vector lies between the Long Terminal Repeat BSV, thus a highly viral promoter, and the transcription-termination sequences, originally derived from bovine growth hormone. By inserting into this restriction site, it is possible to bring any structural genes into a molecular environment that allows gene expression in animal cells. In addition, the pRcRSV vector has a "neomycin resistance" section marker, which allows the selection of successfully transformed animal cells in culture.
Na taj način konstruirani plazmid nosi oznaku pRcRSVLsc3D6. Poslije selekcije transformiranih stanica pomoću antibiotika neomicina odabiru se u 2 osnove kloniranja i osnove screening-a ukupno 5 klona, koji eksprimiraju sc3D6 gen. Ekspresioni nivoi pojedinačnih klona se testiraju pomoću ELISA, specifičnog antigena i nalaze se između 0,5 i 1 μg/ml. Tekućina iznad kulture transficiranih stanica mieloma miša koja sadrži sc3D6 protein izbistri se centrifugiranjem pri 5000 g u centrifugi sa posudom. Izbistrena tekućina iznad kulture se ultrafiltriranjem (Minitan, PTGC, odsječak 100000 Daltona, Fa. Millipore) 10-struko koncentrira i pomoću 50 mM Tris pufera pH 7,2 diafiltrira sa 5-strukom zapreminom. Diafiltrirana otopina proteina se dalje pročišćuje pomoću Q-Se-Diafiltrirana otopina proteina se dalje pročišćuje pomoću Q-Sepharose Fast Flow (Fa. Pharmacia, Švedska) (ekvilibracioni pufer 50 mM Tris pufer pH 7,2). Eluiranje sc3D6 proteina se izvodilo pomoću 150 mM NaCl. Pročišćeni protein je testiran pomoću ELISA specifičnog antigena. The plasmid constructed in this way is labeled pRcRSVLsc3D6. After the selection of transformed cells using the antibiotic neomycin, a total of 5 clones expressing the sc3D6 gene are selected in 2 cloning bases and screening bases. Expression levels of individual clones are tested by ELISA, specific antigen and are between 0.5 and 1 μg/ml. The culture supernatant of transfected mouse myeloma cells containing the sc3D6 protein was clarified by centrifugation at 5000 g in a bowl centrifuge. The clarified liquid above the culture is concentrated 10-fold by ultrafiltration (Minitan, PTGC, cut-off 100,000 Daltons, Millipore) and diafiltered with a 5-fold volume using 50 mM Tris buffer pH 7.2. The diafiltered protein solution is further purified using Q-Se-The diafiltered protein solution is further purified using Q-Sepharose Fast Flow (Fa. Pharmacia, Sweden) (equilibration buffer 50 mM Tris buffer pH 7.2). Elution of sc3D6 protein was performed using 150 mM NaCl. The purified protein was tested using a specific antigen ELISA.
Prinosi pojedinačnih stupnjeva pročišćavanja su predstavljeni na Tabeli 3. The yields of individual stages of purification are presented in Table 3.
Primjer 4 Example 4
Plazmid pRcRSVLsc3D6 je transficiran u kineskog hrčka ovarijske (CHO) stanice. Na sličan način kako je opisano u Primjeru 3, odabiru se transformirane stanice i skriniraju i sc3D6 protein pročišćuje iz tekućine iznad kulture. Testiranje ekspresionih nivoa pomoću ELISA specifičnog antigena donijelo je vrijednosti između 1 i 5 μg/ml antitijela. Plasmid pRcRSVLsc3D6 was transfected into Chinese hamster ovary (CHO) cells. In a similar manner as described in Example 3, transformed cells are selected and screened and sc3D6 protein is purified from the culture supernatant. Testing of expression levels using ELISA specific antigen yielded values between 1 and 5 μg/ml of antibody.
Primjer 5 Example 5
sc3D6 gen se isijeca iz plazmida pUCsc3D6 pomoću restrikcionih enzima i umeće u ekspresioni vektor pGI (Clontech Laboratories Inc., Palo Alto, Sad) kvasca. U ovoj konstrukciji se sc3D6 gen stavlja uz reguliranje GALI-promotera koji mogu inducirati galaktozom. Konstrukt se transficira u Saccharomyses carevisiae soj SHY2 (trpl-) i odabira u podlozi bez triptofana na komplementiranje triptofan-auksotrofije. Pozitivni transformanti se izoliraju i upotrebljavaju za proizvodnju sc3D6 proteina. Uvjeti za kultiviranje proizvodnog soja, kao i za izoliranje, obradu i pročišćavanje proizvoda obavljali su se prema standardnim propisima (22). The sc3D6 gene was excised from the plasmid pUCsc3D6 using restriction enzymes and inserted into the yeast expression vector pGI (Clontech Laboratories Inc., Palo Alto, CA). In this construction, the sc3D6 gene is placed under the regulation of the galactose-inducible GALI-promoter. The construct is transfected into Saccharomyses carevisiae strain SHY2 (trpl-) and selected in tryptophan-free medium for complementation of tryptophan-auxotrophy. Positive transformants are isolated and used to produce sc3D6 protein. The conditions for cultivation of the production strain, as well as for the isolation, processing and purification of the product were carried out according to standard regulations (22).
Primjer 6 Example 6
sc3D6 se isijeca iz plazmida pUCsc3D6 pomoću restrikcionih enzima i umeće u vektor pAc373 (23). Ovaj rekombinantni plazmid se zajedno sa DNK Baculovirus Autographa californica nuclear polyhedrosis virusa (AcMNPV) transficira liniju stanica Sf: koja potječe od Spodoptera frugiperda. Kultiviranje Sf9 stanica se izvodilo po standardnom postupku, kako je opisano u katalogu American Culture Collection. 3 do 5 dana poslije transfekcije se obložene ploče rekombinantnih virusa mikroskopski identificiraju i izoliraju. Da bi se išlo sigurno, da izolirani rekombinantni virusi nisu zagađeni virusima divljeg tipa, nadovezana su tri daljnja procesa pročišćavanja obloženih ploča. Infekcija Sf9 stanica s rekombinantnim virusom poslije 3 do 5 dana vodilo je do liziranja inficiranih stanica, i, sa tim u vezi, do proizvodnje sc3D6 proteina u tekućini iznad lizata stanica. sc3D6 protein se iz ove tekućine iznad lizata pročišćuje i analizira na sličan način kako je opisano u Primjeru 3. Na ovaj način se moglo dokazati funkcionalnost ovog rekombinantnog proteina. sc3D6 is excised from plasmid pUCsc3D6 using restriction enzymes and inserted into vector pAc373 (23). This recombinant plasmid is transfected together with Baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA into the Sf: cell line derived from Spodoptera frugiperda. Cultivation of Sf9 cells was performed according to a standard procedure, as described in the American Culture Collection catalog. 3 to 5 days after transfection, coated plates of recombinant viruses are microscopically identified and isolated. In order to make sure that the isolated recombinant viruses were not contaminated with wild-type viruses, three further purification processes of the coated plates were followed. Infection of Sf9 cells with recombinant virus after 3 to 5 days led to the lysis of infected cells, and, in this connection, to the production of sc3D6 protein in the supernatant of cell lysates. The sc3D6 protein is purified from this liquid above the lysate and analyzed in a similar way as described in Example 3. In this way, the functionality of this recombinant protein could be proven.
Primjer 7 Example 7
Za protein Avidin kodirajuća sekvenca (24) se izrađuje pomoću sintetskog oligonukleotida kao sintetskog gena, i to tako, što se dodatno na 5'-kraju gena leader peptida nalazi za E.coli al-kalna fosfataza (20) i na 3'-kraju polilinker region za umetanje drugih gena. Geni umetnuti u ovaj polilinker region eksprimiraju se pod pogodnim uvjetima kao fuzioni proteini sa Avidinom kao fuzionim partnerom. Pomoću leadera koji se nalazi na 5' kraju izbacuju se ovi fuzioni proteini u aktivnom obliku u plazmatični prostor Escherichia coli. Ovaj konstrukt umeće u pogodnom restrikcionom mjestu bakterijelnog ekspresionog vektora pET-3a (25), koji za ekspresiju kloniranih gena sadrži bakteriofag T7-∅10 promotor kao i ∅ terminator. Nastali vektor nosi oznaku pET-3a-Av. For the Avidin protein, the coding sequence (24) is made using a synthetic oligonucleotide as a synthetic gene, which is additionally located at the 5'-end of the leader peptide gene for E.coli alkaline phosphatase (20) and at the 3'-end polylinker region for insertion of other genes. Genes inserted into this polylinker region are expressed under suitable conditions as fusion proteins with Avidin as a fusion partner. With the help of the leader located at the 5' end, these fusion proteins are released in an active form into the plasmatic space of Escherichia coli. This construct is inserted into a suitable restriction site of the bacterial expression vector pET-3a (25), which contains the bacteriophage T7-∅10 promoter as well as the ∅ terminator for the expression of cloned genes. The resulting vector is labeled pET-3a-Av.
Bakteriofag T7-∅10 promotor ima svojstvo, da se ne transkribira u odsustvu bakteriofaga T7 RNK polimeraze u E.coli stanice. Ako se pak na primjer gusto narasla E.coli kultura inficira sa vektorom faga, koji nosi genetsku informaciju za T7 polimerazu, tada vodi time proizvedena T7 polimeraza do ekspresije gena, koji se na primjer nalaze klonirani u vektorima kao gore opisanom. Ovo svojstvo je za ekspresiju Avidina i Avidin-fuzionih proteina u B.coli vrlo važno, pošto je Avidin toksičan za kulture E.coli koje rastu. Bacteriophage T7-∅10 promoter has the property that it is not transcribed in the absence of bacteriophage T7 RNA polymerase in E.coli cells. If, for example, a densely grown E.coli culture is infected with a phage vector, which carries the genetic information for T7 polymerase, then the T7 polymerase produced thereby leads to the expression of genes, which are, for example, cloned in the vectors as described above. This property is very important for the expression of Avidin and Avidin-fusion proteins in B.coli, since Avidin is toxic to growing E.coli cultures.
sc3D6 gen se isijeca iz vektora pUGsc3D6 restrikcionim enzimima i umeće u polilinker region vektora pET-3a-Av. Vektor koji nastaje nosi oznaku pET-3a-Av-sc3D6. Pogodne E.coli stanice domaćin (npr. HMS174) se transformiraju sa ovim vektorom i uzgajaju. Čim je kultura dostigla OD600 od 0,6, inficira se sa bakteriofagom CE6 (Lambda cIts857Sam7) (25) koji nosi bakteriofag T7 Genl. Time stvorena T7 polimeraza vodila je do ekspresije Avidin-sc3D6 fuzionog proteina u periplazmatičnom prostoru E.coli. Čim je ekspresija dostigla svoj maksimum (u zavisnosti od uvjeta kultiviranja između 3 i 12 sati poslije infekcije sa fagima), rekombinantni protein se oslobađa na sličan način kako je opisano u Primjeru 2 i koncentrira ultrafiltriranjem. Koncentrirana otopina proteina se dalje pročišćuje preko Sephacryl S 200 (Fa.Pharmacia) i drugi put koncentrira pomoću ultrafiltriranja. Ova otopina se nanese na biotinsku kolonu. Odgovarajući fuzioni protein Avidin-sc3D6 ostaje specifično vezan. Nečistoće se isperu. Tako izrađena afinitetna kolona se upotrebljava za pročišćavanje rekombinantnog gp160 slično Primjeru 1, to jest, prethodno pročišćena otopina proteina prema Barrett i sar. (18) nanosi se na afinitetnu kolonu i poslije ispiranja nevezanog materijala eluira se rekombinantni gp160 sa 3M rodanidom. Pri tom postignuti prinosi se ponašaju slično rezultatima prikazanim u Tabeli 2. The sc3D6 gene is cut from the pUGsc3D6 vector with restriction enzymes and inserted into the polylinker region of the pET-3a-Av vector. The resulting vector is designated pET-3a-Av-sc3D6. Suitable E.coli host cells (eg HMS174) are transformed with this vector and cultured. As soon as the culture reached an OD600 of 0.6, it was infected with bacteriophage CE6 (Lambda cIts857Sam7) (25) carrying bacteriophage T7 Genl. The resulting T7 polymerase led to the expression of the Avidin-sc3D6 fusion protein in the periplasmic space of E.coli. As soon as the expression has reached its maximum (depending on the cultivation conditions between 3 and 12 hours after phage infection), the recombinant protein is released in a similar way as described in Example 2 and concentrated by ultrafiltration. The concentrated protein solution is further purified over Sephacryl S 200 (Fa.Pharmacia) and concentrated a second time using ultrafiltration. This solution is applied to the biotin column. The corresponding Avidin-sc3D6 fusion protein remains specifically bound. The impurities are washed away. The affinity column made in this way is used to purify recombinant gp160 similar to Example 1, that is, a previously purified protein solution according to Barrett et al. (18) is applied to the affinity column and after washing the unbound material, the recombinant gp160 is eluted with 3M rhodanide. At the same time, the achieved yields behave similarly to the results shown in Table 2.
SEQ ID NO: 1 SEQ ID NO: 1
VRSTA SEKVENCE: Nukleotidi sa odgovarajućim proteinom SEQUENCE TYPE: Nucleotides with corresponding protein
DUŽINA SEKVENCE: 1548 baznih parova SEQUENCE LENGTH: 1548 base pairs
OBLIK NITI: Jedinična nit THREAD FORM: Single thread
TOPLOGIJA: kružna TOPLOGY: circular
VRSTA MOLEKULA: Plazmid-DNK sa umetkom humane sCNK TYPE OF MOLECULE: Plasmid-DNA with human sCNK insert
PRVOBITNO PORIJEKLO ORGANIZMA: čovjek ORIGINAL ORIGIN OF THE ORGANISM: man
NEPOSREDNO EKSPERIMENTALNO PORIJEKLO: IMMEDIATE EXPERIMENTAL ORIGIN:
NAZIV LINIJE STANICE: 3D6 STATION LINE NAME: 3D6
OZNAKE: MARKS:
od 1 do 36 bp Plazmid pUC19 polilinker from 1 to 36 bp Plasmid pUC19 polylinker
od 37 do 1527 bp Umetak teški lanac antitijela 3D6 from 37 to 1527 bp Insert heavy chain of antibody 3D6
od 37 do 98 bp 5' ne premješten region from 37 to 98 bp 5' not moved region
od 99 do 1526 bp kodirajući reagion from 99 to 1526 bp coding reaction
od 99 do 155 bp signalni peptid from 99 to 155 bp signal peptide
od 156 do 1526 bp zreli peptid from 156 to 1526 bp mature peptide
od 156 do 533 bp promjenjivi region from 156 to 533 bp variable region
od 156 do 245 bp Framework 1 from 156 to 245 bp Framework 1
od 246 do 260 bp comolementarity determining region 1 from 246 to 260 bp co-elementarity determining region 1
od 261 do 302 bp Framework 2 from 261 to 302 bp Framework 2
od 303 do 353 bp complementarity determining region 2 from 303 to 353 bp complementarity determining region 2
od 354 do 449 bp Framework 3 from 354 to 449 bp Framework 3
od 450 do 500 bp complementarity determining region 3 from 450 to 500 bp complementarity determining region 3
od 501 do 533 bp Framework 4 from 501 to 533 bp Framework 4
od 534 do 1526 bp konstantan region from 534 to 1526 bp constant region
od 1527 do 1547 bp Plazmid pUc 19 polilinker from 1527 to 1547 bp Plasmid pUc 19 polylinker
SVOJSTVA: cDNK klon teškog lanca antitijela 3D6 koji je umetnut u plazmid PUC19. PROPERTIES: A cDNA clone of the antibody 3D6 heavy chain that was inserted into plasmid PUC19.
[image] [image]
SEQ ID NO: 2 SEQ ID NO: 2
VRSTA SEKVENCE: Nukleotidi sa odgovarajućim proteinom SEQUENCE TYPE: Nucleotides with corresponding protein
DUŽINA SEKVENCE: 945 baznih parova SEQUENCE LENGTH: 945 base pairs
OBLIK NITI: Jedinična nit THREAD FORM: Single thread
TOPOLOGIJA: kružna TOPOLOGY: circular
VRSTA MOLEKULA: Plazmid-DNK sa umetkom humane cDNK TYPE OF MOLECULE: Plasmid-DNA with human cDNA insert
PRVOBITNO PORIJEKLO ORGANIZMA: čovjek ORIGINAL ORIGIN OF THE ORGANISM: man
NEPOSREDNO EKSPERIMENTALNO PORIJEKLO: IMMEDIATE EXPERIMENTAL ORIGIN:
NAZIV LINIJE STANICE: 3D6 STATION LINE NAME: 3D6
OZNAKE: MARKS:
od 1 do 21 bp Plazmid pUCl9 polilinker from 1 to 21 bp Plasmid pUCl9 polylinker
od 22 do 732 bp umetak laki lanac antitijela 3D6 from 22 to 732 bp insert light chain of antibody 3D6
od 22 do 27 bp 5'ne premješten region from 22 to 27 bp 5'ne moved region
od 28 do 732 bp kodirajući region from 28 to 732 bp coding region
od 28 do 93 bp signalni peptid from 28 to 93 bp signal peptide
od 94 do 732 bp zreli peptid from 94 to 732 bp mature peptide
od 94 do 408 bp promjenjivi region from 94 to 408 bp variable region
od 94 do 162 bp Framework 1 from 94 to 162 bp Framework 1
od 163 do 195 bp complementarity determining region 1 from 163 to 195 bp complementarity determining region 1
od 196 do 240 bp Framework 2 from 196 to 240 bp Framework 2
od 241 do 261 bp complementarity determining region from 241 to 261 bp complementarity determining region
od 262 do 357 bp, Framework 3 from 262 to 357 bp, Framework 3
od 358 do 378 bp complementarity determining region from 358 to 378 bp complementarity determining region
od 379 do 408 bp Framework 4 from 379 to 408 bp Framework 4
od 409 do 732 bp konstantan region from 409 to 732 bp constant region
od 733 do 905 bp 3'ne premješten region from 733 to 905 bp 3'ne moved region
od 906 do 945 bp plazmid pUC 19 polilinker from 906 to 945 bp plasmid pUC 19 polylinker
SVOJSTVA: cDNK klon lakog lanca antitijela 3D6 koje je umetnuto u plazmid PUC19. PROPERTIES: A cDNA clone of the light chain of antibody 3D6 that was inserted into plasmid PUC19.
[image] [image]
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SHEME SCHEMES
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Shema 1: Scheme 1:
Mutacija na prijelazu između leader regiona i promjenjivog regiona teškog lanca antitijela 3D6 (SEQ ID NO: 1). Mutirane baze su označene sa "x". Navedene su kodirane aminokiseline u divljem tipu DNK, osim toga restrikciona mjesta EcoRI i Ncol koja nastaju mutacijom kao i startni kodon ATG Mutation at the transition between the leader region and the variable region of the heavy chain of antibody 3D6 (SEQ ID NO: 1). Mutated bases are marked with "x". The coded amino acids in the wild-type DNA are listed, in addition to the restriction sites EcoRI and NcoI, which are created by mutation, as well as the start codon ATG
[image] [image]
Shema 2: Scheme 2:
Mutacija na prijelazu između promjenjivog regiona i konstantnog regiona teškog lanca antitijela 3D6 (SEQ ID NO: 1). Mutirane baze su označene sa "x". Kodirane aminokiseline u divljem tipu DNK su navedene, osim toga restrikciono mjesto BamHI koje nastaje mutacijom. Mutation at the transition between the variable region and the constant region of the heavy chain of antibody 3D6 (SEQ ID NO: 1). Mutated bases are marked with "x". The encoded amino acids in the wild-type DNA are indicated, in addition to the BamHI restriction site created by the mutation.
[image] [image]
Shema 3: Scheme 3:
Mutacija na prijelazu između leader regiona i promjenjivog regiona lakog lanca antitijela 3D6 (SEQ ID NO: 2). Mutirane baze su označene sa "x". Navedene su kodirane aminokiseline u divljem tipu DNK, osim toga restrikciono mjesto Sall koje nastaje mutacijom Mutation at the transition between the leader region and the variable region of the light chain of antibody 3D6 (SEQ ID NO: 2). Mutated bases are marked with "x". The coded amino acids in wild-type DNA are indicated, in addition to the SalI restriction site that is created by mutation
[image] [image]
Shema 4: Scheme 4:
Mutacija na prijelazu između promjenjivog regiona i konstantnog regiona lakog lanca antitijela 3D6 (SEQ ID NO: 2). Mutirane baze su označene sa "x". Navedene su kodirane aminokiseline u divljem tipu DNK, osim toga restrikciono mjesto HindIII koje nastaje mutacijom kao i stop kodon TAA. Mutation at the transition between the variable region and the constant region of the light chain of antibody 3D6 (SEQ ID NO: 2). Mutated bases are marked with "x". The coded amino acids in the wild-type DNA are listed, in addition to the HindIII restriction site that is created by mutation as well as the TAA stop codon.
[image] [image]
Shema 5: Scheme 5:
dnk sekvenca mjesta vezivanja VH - linker prije i poslije ponovne mutacije za ponovnu izradu prirodne sekvence aminokiselina u području VH regiona (SEQ ID NO: 3). Mutirane baze su označene sa "x". Navedena je konačna sekvenca aminokiselina. dnk sequence of the VH binding site - linker before and after re-mutation to recreate the natural amino acid sequence in the VH region (SEQ ID NO: 3). Mutated bases are marked with "x". The final amino acid sequence is indicated.
[image] [image]
Shema 6: Scheme 6:
DNK sekvenca mjesta vezivanja linker - VI prije i poslije ponovne mutacije za ponovnu izradu prirodne sekvence aminokiselina u području VL regiona (SEQ I2D NO: 3). Mutirane baze su označene sa "x". Navedena je konačna sekvenca aminokiselina. DNA sequence of the linker - VI binding site before and after re-mutation to recreate the natural sequence of amino acids in the VL region (SEQ I2D NO: 3). Mutated bases are marked with "x". The final amino acid sequence is indicated.
DIREKTAN DOKAZ HIV-1 ANTIGENA DIRECT EVIDENCE OF HIV-1 ANTIGEN
Standardna krivulja Standard curve
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Slika 9: Figure 9:
Standardna krivulja ELISA za direktan dokaz HIV-1 antigena pomoću sc3D6 proteina ELISA standard curve for direct detection of HIV-1 antigen using sc3D6 protein
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Slika 7: Figure 7:
SDS gel pročišćenog sc3D6. Molekularne mase primijenjenog standarda su navedene. SDS gel of purified sc3D6. The molecular weights of the applied standard are listed.
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Slika 8: Figure 8:
HI-1 Westernblot trake. Protein koji vezuje antigen: HI-1 Westernblot strips. Antigen binding protein:
1. sc3D6 protein 1. sc3D6 protein
2. APsc3D6 fuzioni protein 2. APsc3D6 fusion protein
3. Antitijelo 3d6 3. Antibody 3d6
4. ukupan protein iz E.coli 4. total protein from E.coli
Tabele Tables
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Tabela 1: Table 1:
Krajnja koncentracija kemikalija za razlaganje u suspenziji stanica. Final concentration of degradation chemicals in cell suspension.
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Tabela 2: Table 2:
Prinose pojedinačnih stupnjeva imunafinitetno kromatografskog pročišćavanja rekombinantnog gp160 pomoću sc3D6 kao afinitetnog liganda. Yields of individual steps of immunoaffinity chromatographic purification of recombinant gp160 using sc3D6 as an affinity ligand.
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Tabela 3: Table 3:
Pronose pojedinačnih stupnjeva pročišćavanja sa3D6 proteina iz tekućine iznad kulture transformiranih stanica mieloma miša. Passage of individual steps of purification of the sa3D6 protein from the culture supernatant of transformed mouse myeloma cells.
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Slika 10: Figure 10:
Kompetitivna ELISA sa APsc3D6 proteinom. Kod uzora-lijevo od strelice radi se o HIV-1 negativnim serumima. Competitive ELISA with APsc3D6 protein. The samples to the left of the arrow are HIV-1 negative sera.
Claims (12)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT117890A AT396939B (en) | 1990-05-29 | 1990-05-29 | COMPLEX VIRAL ANTIQUE OF HIV-1 BINDING RECOMBINANT PROTEIN |
| YU114691A YU114691A (en) | 1990-05-29 | 1991-05-28 | COMPLEX VIRAL ANTIGEN HIV-1 WHICH BINDS RECOMBINANT PROTEIN |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HRP920714A2 true HRP920714A2 (en) | 1995-06-30 |
Family
ID=25595095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HR920714A HRP920714A2 (en) | 1990-05-29 | 1992-10-01 | RECOMBINANT PROTEIN WHICH BINDS TO A COMPLEX VIRAL ANTIGEN OF HIV-a 1 |
Country Status (1)
| Country | Link |
|---|---|
| HR (1) | HRP920714A2 (en) |
-
1992
- 1992-10-01 HR HR920714A patent/HRP920714A2/en not_active Application Discontinuation
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