WO2006106331A2 - Amorces de souris - Google Patents

Amorces de souris Download PDF

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Publication number
WO2006106331A2
WO2006106331A2 PCT/GB2006/001251 GB2006001251W WO2006106331A2 WO 2006106331 A2 WO2006106331 A2 WO 2006106331A2 GB 2006001251 W GB2006001251 W GB 2006001251W WO 2006106331 A2 WO2006106331 A2 WO 2006106331A2
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WIPO (PCT)
Prior art keywords
seq
primer
nos
primer set
group
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PCT/GB2006/001251
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WO2006106331A3 (fr
Inventor
Ralph Adams
Andrew George Popplewell
Patrick Marcel Slocombe
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UCB SA
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UCB SA
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Publication of WO2006106331A3 publication Critical patent/WO2006106331A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • telomere sequences coding for the variable regions exist, e.g. cloning rearranged genomic DNA, or mRNA via cDNA.
  • Methods to obtain full length cDNAs or to extend short cDNAs are also known in the art, for example RACE (Rapid amplification of cDNA ends; e.g. Frohman et al., 1988, Proc. Natl. Acad. Sci USA 85:8998-9002) and for example, using MarathonTM technology (Clontech Laboratories, Inc.).
  • RACE Rapid amplification of cDNA ends
  • MarathonTM technology Clontech Laboratories, Inc.
  • a primer set as used herein includes a primer set comprising at least one primer.
  • the primer sets described herein are useful for the isolation of mouse antibodies but may be adapted for use with other species, for example, other rodents.
  • oligonucleotide primer set comprising or consisting of:
  • Primers of SEQ ID NOS.-152 to 156 are reverse primers; SEQ ID NOS:157-203 are forward primers.
  • any one of the primer sets defined in ( ⁇ )-(r), above, in a PCR enables the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody lambda light chain.
  • These primer sets have the following restriction sites incorporated: 5'-Hind III (bold) and 3'-Bsi WI (bold and underlined). It will be understood by one skilled in the art that any restriction site desired may be incorporated into the primers, for example by replacing the restriction site encoded with coding for the desired restriction site.
  • the oligonucleotide primer set comprises SEQ ID NOS:205-212 (Set K).
  • Primers of SEQ ID NO:204 is a reverse primer; SEQ ID NOS:205-212 are forward primers.
  • primer sets Preferably, one primer set is used to isolate a nucleic acid comprising the variable heavy chain region sequence, and one primer set is used to isolate a nucleic acid comprising the variable light chain region sequence of a mouse antibody. Alternatively, two or more primer sets may be used.
  • primer set B or primer set H is used in conjunction with primer set D or J (or primer set F or L), above, and even more preferably, primer set B is used in conjunction with primer set D (or primer set F) and primer set H is used in conjunction with primer set J (or primer set L) to enable the isolation of nucleic acids comprising the variable heavy and light chain region sequences of a mouse antibody.
  • nucleic acid sequences are of use in cloning full length antibody heavy and light chain sequences.
  • nucleic acids are of also use in designing a humanised antibody or fragment thereof.
  • (V) at least one primer selected from the group consisting of SEQ ID NOS : 103 to 107; or
  • (VI) at least one primer selected from the group consisting of SEQ ID NOS : 108 to 151.
  • PCRs may be used, for example, but not limited to three, four, five or more PCRs.
  • fewer numbers of cycles are used then one or more additional PCRs may be performed to produce the amplification required.
  • the invention described herein also provides methods for the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody kappa light chain sequence comprising use of an oligonucleotide primer set in a PCR wherein the primer set comprises or consists of:
  • (VIII) at least one primer selected from the group consisting of SEQ ID NOS :48 to 93; or
  • any of the primer set defined in, (VII)-(DC), above is used in a PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • the method comprises use of the oligonucleotide primer Set D, defined above, in a PCR, preferably a first PCR enabling the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody kappa light chain.
  • any of the primer sets defined in (VII)-(IX), above may be used in a second PCR in conjunction with template derived from a first PCR.
  • any one of the primer sets defined in (VII)-(IX), above can be used in a first and a second PCR.
  • (X) at least one primer selected from the group consisting of SEQ ID NOS : 152 to 156, and at least one primer selected from the group consisting of SEQ ID NOS:157 to 203;
  • (XI) at least one primer selected from the group consisting of SEQ ID NOS : 157 to 203; or
  • (XII) at least one primer selected from the group consisting of SEQ ID NOS: 152 to 156.
  • any one of the primer sets defined in (X)-(XII), above is used in a PCR.
  • the method comprises use of the oligonucleotide primer Set J, defined above, in a PCR, preferably a second PCR, and enables the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody kappa light chain.
  • any of the primer sets defined in (X)- (XII), above is used in a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • any one of said primer sets can be used in a first and a second PCR.
  • the method comprises two PCRs, a first PCR and a second PCR using as template, DNA from the first PCR. Accordingly, provided is a method for the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody kappa light chain comprising use of at least one primer set in a first PCR and at least one primer set in a second PCR, said first primer set comrpsing or consisting of any one of the primer sets defined in (VII)-(IX), above, and said second primer set comprising or consisting of any one of the primer sets defined in (X)-(XII), above.
  • the invention described herein also provides a method for the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody lambda light chain comprising use of an oligonucleotide primer set in a PCR, wherein the primer set comprises or consists of:
  • any one of the primer sets defined in, (XHI)-(XV), above is used as a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • the method comprises use of the oligonucleotide primer set F, defined above, in a PCR, preferably a first PCR enabling the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody.
  • any one of the primer sets defined in (XIII)-(XV), above may be used in a second PCR in conjunction with template derived from a first PCR.
  • any one of said primer sets can be used in a first and a second PCR.
  • any one of the primer sets defined in (XVI)-(XVIII), above is used in a PCR.
  • the method comprises use of the oligonucleotide primer Set L, defined above, in a PCR, most preferably a second PCR, using as template nucleic acid derived from a first PCR, said method enabling the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody lambda light chain.
  • any one of the primer sets defined in (XVI)-(XVIII), above is used in a first PCR in conjunction with template derived from at least one antibody producing cell of interest, for example by reverse transcription of mRNA into cDNA.
  • a method for the isolation of a nucleic acid comprising the variable region sequence of a mouse antibody lambda light chain comprising use of at least one primer set in a first PCR and at least one primer set in a second PCR, said first primer set comprising or consisting of any one of the primer sets defined in (XII)-(XV), above, and said second primer set comprising or consisting of any one of the primer sets defined in (XVI)-(XVIII), above.
  • the method comprises use of primer Set F, defined above, in a first PCR and primer Set L, defined above, in a second PCR, using as template, DNA from the first PCR.
  • Said method enables the isolation of the variable region of a mouse antibody lambda light chain.
  • said PCRs are performed individually.
  • said PCRs are performed jointly.
  • Such antibody regions isolated are useful for the cloning of the full sequence of a mouse antibody lambda light chain.
  • nucleic acids are of also use in designing a humanised antibody or fragment thereof.
  • more than two PCRs may be used, for example, but not limited to three, four, five or more PCRs. Thus, one skilled in the art will appreciate that if, for example, fewer numbers of cycles are used then one or more additional PCRs may be performed to produce the amplification required.
  • the sequence of at least the variable regions of both the heavy and light chains of a mouse antibody can be determined by use of the primer sets and methods described above.
  • All methods described herein for the isolation of at least the variable region of a mouse antibody sequence comprise the performance of at least one PCR and provision of: i. at least one primer set as provided herein; ii. optionally one or more additional primer sets; iii. template DNA from an antibody producing cell of interest; and iv. PCR performance reagents.
  • the additional primer set as required can be a forward or a reverse primer set depending upon whether the primer set provided herein is a forward or a reverse primer set.
  • Such additional primer sets may be designed to anneal to any desired region of an antibody, particularly a mouse antibody. It will also be understood that suitable use of primer sets requires that forward and reverse primers are used.
  • Reverse transcription and PCR performance reagents include reagents as known in the art, such as without limitation, DNA polymerase, dNTPs, buffers such as but not limited toTris buffers, cations such as Mg2+, detergents, for example but not limited, to Nonidet NP-40, reducing agents such as dithiothreitol or mercaptoethanol and RNAasin.
  • any one of the primer Sets B, D, F, H, J and L provided herein are utilised in a single PCR.
  • suitable use means that one or more additional primers will be required.
  • one or more PCRs may be performed utilising a portion of the primers present within a primer set, for example but without limitation, one or two or more primers from any one of Sets A to L 5 plus additional primers where required, may be utilised in a single PCR.
  • one or more of the primer sets A, C, E, G 5 1 or K (which are designed to complement the variable regions of an antibody, in particular a mouse antibody), or one or more primers comprising any of the sets, may be used in combination with primers designed to anneal to downstream regions of a nucleic acid encoding a mouse antibody of interest.
  • one or more of the primer sets A, C, E, G, I or K 5 or one or more primers comprising any of the sets may be used for RACE, where the source cDNA can be homopolynucleotide tailed at the 3 '-end using terminal transferase enzyme, providing a location for strand synthesis using a complementary primer.
  • primers described herein can be made as desired.
  • sequence of one or more primers may be shortened or lengthened.
  • restriction sites may be altered or removed altogether.
  • Suitable annealing temperatures for using such primers may be selected as known in the art, for example but without limitation, lowering the annealing temperature may allow less stringent priming and result in a PCR product where previously, at higher temperatures, no product was isolated.
  • Antibody sequences provided using the methods of the invention described herein include sequences of functionally active fragments, derivatives or analogues and may be, but are not limited to, polyclonal, monoclonal, bi-, tri- or tetra-valent antibodies, humanised or chimeric antibodies, single chain antibodies, Fab fragments, Fab' and Fab' 2 fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.
  • Humanised antibodies are antibody molecules from non-human species having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (see, e.g.
  • an antibody can be used therapeutically alone or in combination with a cytotoxic factor(s) and/or cytokine(s).
  • antibodies provided using the primer sets and methods of the invention can be conjugated to a therapeutic agent, such as a cytotoxic agent, a radionuclide or drug moiety to modify a given biological response.
  • a therapeutic agent such as a cytotoxic agent, a radionuclide or drug moiety to modify a given biological response.
  • the therapeutic agent is not to be construed as limited to classical chemical therapeutic agents.
  • the therapeutic agent may be a drug moiety which may be a protein or polypeptide possessing a desired biological activity.
  • Such moieties may include, for example and without limitation, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin, maytansinoid (DMl), a protein such as tumour necrosis factor, ⁇ -interferon, ⁇ - interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumour necrosis factor, ⁇ -interferon, ⁇ - interferon, nerve growth factor, platelet derived growth factor or tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g.
  • angiostatin or endostatin angiostatin or endostatin; angiogenin, gelonin, dolstatins, minor groove-binders, bis-iodo-phenol mustard, or, a biological response modifier such as a lymphokine, interleukin-1 (IL-I), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM- CSF), granulocyte colony stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor.
  • IL-I interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • GM- CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • NGF nerve growth factor
  • Therapeutic agents also include cytotoxins or cytotoxic agents including any agent that is detrimental to (e.g. kills) cells.
  • cytotoxins or cytotoxic agents including any agent that is detrimental to (e.g. kills) cells.
  • examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vinca alkaloids, e.g.
  • Therapeutic agents also include, but are not limited to, anti-folates (e.g. aminopterin and methotrexate), antimetabolites (e.g.
  • daunorubicin (formerly daunomycin) and doxorubicin, adriamycin, idarubicin, morpholinodoxorubicin, epirubicin, doxorubicin hydrazides), antibiotics (e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins, CC- 1065, enediyenes, neocarzinostatin), and anti-mitotic agents (e.g. vincristine and vinblastine). See Garnett, 2001, Advanced drug Delivery Reviews 53 : 171 -216 for further details .
  • antibiotics e.g. dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins, CC- 1065, enediyenes, n
  • an antibody fusion protein may facilitate depletion or purification of a polypeptide as described herein, increase half-life in vivo, and enhance the delivery of an antigen across an epithelial barrier to the immune system.
  • the fusion protein is an antibody fragment linked to an effector or reporter molecule
  • this may be prepared by standard chemical or recombinant DNA procedures.
  • a preferred effector group is a polymer molecule, which may be attached to the modified Fab fragment to increase its half-life in vivo.
  • the polymer molecule may, in general, be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide.
  • Particular optional substituents which may be present on the above-mentioned synthetic polymers include one or more hydroxy, methyl or methoxy groups.
  • synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol) or derivatives thereof.
  • Particular naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof.
  • Derivatives as used above is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like.
  • the reactive group may be linked directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.
  • the size of the polymer may be varied as desired, but will generally be in an average molecular weight range from 500Da to 50000Da 5 preferably from 5000 to 40000Da and more preferably from 25000 to 40000Da. The polymer size may in particular be selected on the basis of the intended use of the product.
  • the product is intended to leave the circulation and penetrate tissue, for example for use in the treatment of a tumour
  • a small molecular weight polymer for example with a molecular weight of around 5000Da
  • a higher molecular weight polymer for example having a molecular weight in the range from 25000Da to 40000Da.
  • Particularly preferred polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 25000Da to about 40000Da.
  • a polyalkylene polymer such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 25000Da to about 40000Da.
  • Each polymer molecule attached to the modified antibody fragment may be covalently linked to the sulphur atom of a cysteine residue located in the fragment.
  • the covalent linkage will generally be a disulphide bond or, in particular, a sulphur-carbon bond.
  • An activated polymer may be used as the starting material in the preparation of polymer-modified antibody fragments as described above.
  • the activated polymer may be any polymer containing a thiol reactive group such as an ⁇ -halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide.
  • a thiol reactive group such as an ⁇ -halocarboxylic acid or ester, e.g. iodoacetamide, an imide, e.g. maleimide, a vinyl sulphone or a disulphide.
  • Such starting materials may be obtained commercially, for example from Nektar Therapeutics, Inc (Huntsville, AL) or may be prepared from commercially available starting materials using conventional chemical procedures.
  • Standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector or reporter molecule either before or after reaction with the activated polymer as appropriate may be used.
  • Particular chemical procedures include, for example, those described in WO 93/06231, WO 92/22583, WO 90/09195, WO 89/01476, WO 99/15549 and WO03/031581.
  • the effector or reporter molecule is a protein or polypeptide the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86/01533 and EP 0392745.
  • a modified Fab fragment is PEGylated, i.e. has PEG (poly(ethyleneglycol)) covalently attached thereto, e.g. according to the method disclosed in EP 0948544 [see also "Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York, “Poly(ethyleneglycol) Chemistry and Biological Applications", 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. Aslam and A.
  • a PEG modified Fab fragment has a maleimide group covalently linked to a single thiol group in a modified hinge region.
  • a lysine residue may be covalently linked to the maleimide group.
  • To each of the amine groups on the lysine residue may be attached a methoxypoly(ethyleneglycol) polymer having a molecular weight of approximately 20,000 Da. The total molecular weight of the entire effector molecule may therefore be approximately 40,000 Da.
  • the invention includes an antibody or fragment thereof isolated according to any one of the methods described, above. Repertoires of such antibodies, or fragments thereof, are also useful in bacteriophage libraries. Such antibodies are particularly useful as therapeutic agents, hi one embodiment, antibodies isolated using the methods of the invention are humanised (see, for example, Adair et al., 1992, Immunol Rev. 130:5-40 and WO91/09967). The antibodies of the invention are also of use as diagnostic and prognostic reagents. Thus, further provided is the use of an antibody for the manufacture of a medicament for the treatment and/or prophylaxis of a disease involved in aberrant expression or activity of an antigen recognised by said antibody.
  • Antibodies isolated according to the methods of the invention are also useful in diagnosis.
  • a method of screening for and/or diagnosis or prognosis of a disease in a subject, and/or monitoring the effectiveness of therapy for said disease which comprises the step of detecting and/or quantifying in a biological sample obtained from said subject, the expression of an antigen recognised by an antibody isolated according to the methods of the invention.
  • the step of detecting comprises contacting the sample with the antibody and detecting whether binding has occurred between the antibody and the antigen in the sample.
  • cDNA was prepared by reverse transcription using a SuperscriptTM III Reverse Transcriptase kit (Invitrogen cat.# 18080-044) and RNasin, an RNAase Inhibitor (Promega cat. # N2511). The following master mix (20 ⁇ l) was added directly to the selected single mouse B cell:
  • Oligo dTVx primers were as follows: ttttttttttttttttttttttttttttttttva (SEQ ID NO:213), tttttttttttttttttttttvg (SEQ ID NO:214), tttttttttttttttttttttvc (SEQ ID NO:215), and tttttttttttttttttttttttttttvt (SEQ ID NO:216), where v is a, g, or t.
  • the reverse transcription reaction was performed at 50°C for 60min followed by 70 0 C for 15min.
  • Primary PCR fragments were prepared by adding 2 ⁇ l of the cDNA and the appropriate primary primers in conjunction with a TaqPlus Precision PCR system (Stratagene Cat. No. # 600211) mix in a total volume of 50 ⁇ l as described below: dH 2 O 37.5 ⁇ l 1 OX Precision PCR buffer 5 ⁇ l
  • Secondary PCR fragments were prepared by adding 2 ⁇ l of the primary PCR product and the appropriate primary primers in conjunction with a TaqPlus Precision PCR system (Stratagene cat No. # 600211) mix in a total volume of 50 ⁇ l as described below: dH 2 O ' 33.5 ⁇ l
  • variable region (V H and V L ) of the antibody recognising antigen Y was confirmed by running a 5 ⁇ l sample of the product on a 2%w/v agarose gel.
  • V H and V L The DNA product present in each case (V H and V L ) was approximately 450bp and was cloned into an expression vector using the Hind Ill/Xho I/Bsi WI restriction sites.

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Abstract

L'invention concerne des ensembles d'amorces pour isoler des séquences de zones variables d'anticorps de souris. La présente invention porte également sur des anticorps comprenant ces zones, qui sont utiles au développement d'agents thérapeutiques, en particulier, au développement d'anticorps humanisés.
PCT/GB2006/001251 2005-04-06 2006-04-05 Amorces de souris Ceased WO2006106331A2 (fr)

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GB0507046.1 2005-04-06
GB0507046A GB0507046D0 (en) 2005-04-06 2005-04-06 Primers

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WO2006106331A2 true WO2006106331A2 (fr) 2006-10-12
WO2006106331A3 WO2006106331A3 (fr) 2007-03-29

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6291161B1 (en) * 1989-05-16 2001-09-18 Scripps Research Institute Method for tapping the immunological repertiore
JP2996864B2 (ja) * 1994-03-30 2000-01-11 寳酒造株式会社 抗体可変領域ｄｎａ
DE69936927T2 (de) * 1998-10-21 2008-05-15 Altor Bioscience Corp., Miramar Polyspezifische bindemoleküle und deren verwendung
EP1456237A2 (fr) * 2001-12-21 2004-09-15 Vlaams Interuniversitair Instituut voor Biotechnologie vzw. Procede de clonage de sequences de domaines variables

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