CN102234641A - Method for rapidly acquiring variable region gene sequence of specific human antibody - Google Patents
Method for rapidly acquiring variable region gene sequence of specific human antibody Download PDFInfo
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Abstract
The invention which belongs to the technical field of molecular biology concretely relates to a method for rapidly acquiring a variable region gene sequence of a specific human antibody, and a method for preparing a fully human antibody. The method provided by the invention has simple, rapid and effective processes, because the entire process of separating CD19 positive lymphocyte to acquiring the variable region gene sequence of the specific antibody consumes no more than 14 days. The invention provides a good technical platform, which can be widely applied to variable regions of heavy chains and light chains of a core component of the specific fully humanized antibody of various epidemic/infective and tumorous diseases, and autoimmune diseases especially some emergent and serious pandemic diseases such as SARS, bird flu and the like, and provides an effective means for fully humanized antibody medicament production.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of method of obtaining the method for specific human source antibody variable gene sequence fast and preparing total man source antibody.
Background technology
Along with the raising of expanding economy and medical level, overall morbidity infectious and communicable disease descends to some extent, but some infectivity or communicable disease remain the important factor that threatens human health.SARS large-scale outbreak from 2002, hand foot mouth disease to EV71 virus in 2008 initiation, the global flu outbreak that causes to Mexico H1N1virus in 2009 again, owing to can not find effective prevention and control way in the short period of time, in case break out they just very fast on a large scale in or the whole world spread.So burst, epidemic disease for this viroid causes pressed for and find effective prevention and methods of treatment in very short times very much.But the exploitation of conventional antiviral is a very long process, can't satisfy the demand at short notice; And conventional vaccine development needs the clear earlier antigen that separates, and detects, screens final formation vaccine product then, and this process needs the time of half a year at least.
Monoclonal antibody is the special antibody purification that produces at specific antigen, and it specifically has the height specificity, can take accurate aim, catch target, reacts with target specifically.Monoclonal antibody has become one of pillar product of world's biotech medicine product industry.Four-stage has been experienced in the monoclonal antibody drug development, be respectively: mouse monoclonal antibody, mosaic monoclonal antibody, Humanized monoclonal antibodies and full Humanized monoclonal antibodies, and in the monoclonal antibody market in the whole world, the full Humanized monoclonal antibodies of four-stage is its development in future direction.The mouse resource monoclonal antibody is the common monoclonal antibody drug with the mouse preparation, but it has tangible limitation: mouse monoclonal antibody and human complement component binding ability are low, a little less than the Fc receptor affinity of immunocyte such as NK surface, a little less than the ADCC effect of mediation, transformation period in people's circulation of blood is short, mouse monoclonal antibody has immunogenicity, the host easily produces anti-antibody and causes allergic reaction, often cause the untoward reaction of human body, thereby restricted its development greatly in clinical experiment and related application field.So-called humanized antibody exactly with antigen adsorption zone or variable region part (being Vh and Vl district) grafting to people's antibody, the outer derived components with on the reduction antibody reduces the immune side reaction that heterologous antibody causes human body.Humanized antibody comprises several classes such as chimeric antibody, reshaping antibody and full humanized antibody.Reach about 70% as chimeric antibody humanization degree, intactly kept the variable region of allos monoclonal antibody, kept its affine activity to greatest extent, reduced immunogenicity.But, can bring out the anti-mouse reaction of people HAMA because human mouse chimeric antibody has still kept 30% mouse source property.
Full humanized antibody and human body associativity are best, also are the safest and effective monoclonal antibodies.Full humanized antibody refers to all parts of antibody, comprises that the variable region part (being Vh and Vl district) and the constant region (being ch and cl district) that refer to antibody are coded by human antibody gene.Full humanized antibody is meant human antibody gene by transgenosis or transfection chromosome technology, the gene of human encoding antibody all is transferred in the genetic engineering modified animal or cell, make animal or cell expressing human antibodies, reach the complete humanized purpose of antibody.The research of full humanized antibody starts from nineteen nineties, and obtaining full humanization antibody method at present has antibody library triage techniques, genetically engineered mouse to prepare human antibody, transfection chromosome ox.
The antibody library triage techniques mainly comprises phage antibody library and ribosomal display technology.
Phage antibody library technique: from immunity or not by separation antibody variable region gene the B cell of immunity; The a complete set of gene fragment (as VH, VL) of pcr amplification antibody is cloned into respective carrier at random with VH, the VL gene fragment of amplification in vitro, forms combinatorial library; Assortment of genes library is inserted the gene III (g3) of phage encoded membranin or gene VIII (g8) guide's series near the downstream, make the polypeptide of exogenous gene expression be illustrated in the N end of coat protein gp III or gp VIII with the form of fusion rotein.Go out the antibody phage storehouse that expression specificity is good, avidity is strong with immobilization antigen through " affine combination-wash-out-amplification " several Cycle Screening.
Ribosomal display technology: genotype and phenotype are linked together, the DNA of proteins encoded transcribes and translates external, because DNA has been carried out special processing and modification, as: remove 3 ' terminal terminator codon, when rrna is translated the mRNA end, owing to lack terminator codon, 3 ' the end that rests on mRNA does not break away from, thereby form protein-ribose-2mRNA tripolymer, with the specific aglucon immobilization of target protein, as: be fixed on ELISA micropore or magnetic bead surfaces, the rrna tripolymer that contains target protein is just can be in the elisa plate hole or on the magnetic bead screened to be gone out, the mixture that screening and separating is obtained decomposes, the mRNA that discharges carries out reversed transcriptive enzyme chain polymerization reaction (RT-PCR), the PCR product enters the next round circulation, through repeatedly circulation, makes the gene order of target protein and its coding obtain enrichment and separate.
The genetically engineered mouse prepares human antibody: the genetically engineered mouse of preparation human antibody comprises that human peripheral lymphocyte-Reconstruction in Sever Combined Immunodeciency Mice (hu-PBL-SCID mouse), transgenic mice and transchromosomic mice prepare people's antibody technique.
The Hu-PBL-SCID mouse is that the peripheral blood lymphocyte that produces certain immunoreactive donor or cancer patients's people is transplanted in Reconstruction in Sever Combined Immunodeciency Mice (SCID), can obtain human antibody behind antigen immune.
Transgenic mice: change people's antibody producing gene over to mouse, generate gene with the antibody of replacing mouse.The advantage that transgenic mice prepares people's antibody is that its effect is better than other and produces anti-human body protein monoclonal antibody technology.Weak point: (1) transgenosis has somatic mutation and other unique sequence usually, causes not human sequence very completely; (2) because antibody is to assemble in the mouse body, thereby the monoclonal antibody that produces has the mouse glycosylation pattern, so these monoclonal antibodies finally are not the total man's; (3) the people Ig diversity of transgenic mice expression is less, and can not produce each subclass of IgG in same mouse.
Transchromosomic mice: will produce the κ light chain segments transfection of 5～50Mb on the embryonal system fragment of IgH and No. 2 karyomit(e) on No. 14 karyomit(e)s of people to the ES cell by minicell mediated method (MMCT method), obtain mouse after the human serum albumin immunity, can produce the albuminous people Ig of AHS, immunity back IgM produces once more.By the mouse that the transfection chromosome technology obtains, each subclass of IgG of expressing in the amount of each subclass of human IgG of expression and the human serum roughly the same and can be expressed in a transgenic mice simultaneously; But though the people Ig segment that transchromosomic mice imports is bigger, the people Ig amount of its expression is lower.
The transfection chromosome ox: the building process that produces people Ig transfection chromosome ox still utilizes the MMCT method, contains the artificial karyomit(e) of the segmental people of people Ig weight chain gene (HAC) with what make up, imports the ox embryo fibroblast, further develops into the transfection chromosome ox.The structure characteristics that produce people Ig transfection chromosome ox are: (1) therefore imports the ox embryo fibroblast with the artificial karyomit(e) of people because the ox embryonic stem cell can not successfully be built and be; (2) the transfection chromosome ox is set up with the method for nuclear transplantation; (3) only can survive for 35 generations owing to the ox embryo fibroblast, after containing the minicell and the fusion of ox embryo fibroblast of people HAC, therefore the bovine fetal fibroblast 7d of can only surviving again must guarantee that HAC retains rate at the intravital height of transfection chromosome ox through 2 screenings.
But in the above-mentioned full humanization antibody technique, preparation and Production Flow Chart complexity, need time and cycle very long, can't be applicable to the research and development and the production of the antibody drug of some burst, epidemic disease, and some pathogenic agent or antigen variation aspect are fast seemed powerless.
Summary of the invention
Technical problem to be solved by this invention is at the existing existing problem of full humanization antibody technique, a kind of total man source antibody variable gene sequence acquisition methods of simple, quick, high specific is provided and has prepared the method for total man source antibody.
For this reason, the invention discloses a kind of method of obtaining specific human source antibody variable gene sequence fast, it comprises the following steps:
A) separation of C D19 male lymphocyte from donor peripheral blood or clone;
B) the external evoked differentiation of CD19 male lymphocyte;
C) detection specificity antibody and screening specific antibody male lymphocyte;
D) design degenerate primer group;
E) adopt the method for unicellular RT-PCR increase variable region gene and order-checking;
The external evoked differentiation step of wherein said CD19 male lymphocyte is for to cultivate with the individual cells form CD19 male lymphocyte body in the nutrient solution that has CD40L albumen situation, cytokine.
In certain embodiments, the lymphocytic method of separation of C D19 male is magnetic bead sorting method or stream type cell analyzer separating method among the described step a; Wherein preferred magnetic bead sorting method.
In certain embodiments, to have CD40L albumen situation described in the described step b be the medium that contains CD40L albumen, CD40L protein-crosslinking in the nutrient solution, one of express among the proteic trophocyte of CD40L situation, wherein the proteic trophocyte of preferred expression CD40L most preferably expresses the proteic CHO-K1 nurse cell of CD40L.
The proteic CHO-K1 nurse cell of described expression CD40L prepares through the following steps:
A) amplification of the method by RT-PCR CD40L gene;
B) again with this gene clone in carrier for expression of eukaryon;
C) transfection CHO-K1 cell strain, screening is to obtain stably express CD40L cell strain;
D) radiotreatment or mitomycin are handled the multiplication capacity that stops the CHO-K1 cell, promptly get and express the proteic CHO-K1 nurse cell of CD40L.
Described nurse cell is meant energy secretion or expressing protein or the factor for growth of purpose cell or differentiation, but self loses multiplication capacity but still the cell of retentive activity.
In certain embodiments, cytokine described in the described step b is IL-2 and/or IL-21, the IL-2 of wherein preferred 100 units/mL and 100ng/mL IL-21.
In certain embodiments, the method for detection specificity antibody described in the described step c one of can be in radioimmunology, ELISA method or the protein chip.
In one embodiment, described specific antibody is a Tetanus Antitoxin antibody.
In certain embodiments, degenerate primer group described in the described steps d is the primer group of variable region of heavy chain and variable region of light chain, wherein downstream primer is near the constant region of bordering on the variable region, upstream primer then according to existing other variable region sequences, consider the changeable characteristics of variable region sequences design degenerate primer group, and near 5 ' UTR district or initiator codons of variable region.
In one embodiment, the primer group of described variable region of heavy chain is shown in SEQ ID NO.1～SEQ ID NO.6, and the primer group of described variable region of light chain is shown in SEQ ID NO.7～SEQ ID NO.22.
In certain embodiments, unicellular RT-PCR step described in the described step e is carried out single stage method RT-PCR with this lysate as template, the variable region of increase respectively heavy chain and light chain again for earlier carrying out cracking with unicellular in different PCR pipes.
In one embodiment, described specific human source antibody variable gene sequence is a Tetanus Antitoxin specific human source antibody variable gene sequence.
On the other hand, the invention also discloses a kind of Tetanus Antitoxin specific human source antibody heavy chain variable region gene sequence as being shown in SEQ ID NO.23;
On the other hand, the invention also discloses a kind of Tetanus Antitoxin specific human source antibody chain variable region gene sequence as being shown in SEQ ID NO.24.
On the other hand, the invention also discloses the method for a kind of total man of preparation source antibody, comprise the following steps:
A) with above-mentioned described specific human source antibody variable gene sequence clone in the human constant region expression vector;
B) transfection and screening transformant;
C) expression and purifying total man source antibody.
Process involved in the present invention is simple, quick, effective, and to the variable region gene sequence that obtains specific antibody, whole process is no more than 14 days from separation of C D19 positive lymphocyte.The invention provides a good technical platform, can be widely used in the nucleus (variable region of heavy chain and light chain) that obtains specific full humanized antibody in multiple infectivity/infectivity, neoplastic disease, the autoimmune disorder, particularly some burst, great epidemic disease, as diseases such as SARS, bird flus, and the present invention provides an effective means for the production of full humanization antibody drug.
Description of drawings
Fig. 1 is the unicellular RT-PCR amplification Parameter Map of a specific embodiment.
Fig. 2 is the unicellular RT-PCR electrophorogram of a specific embodiment.
Embodiment
As do not have other explanations, before further describing the present invention, the term definition that uses among the present invention is as follows:
After " antibody " was meant B cell-specific identification antigen, proliferation and differentiation became plasmocyte, a class of institute's synthesis secretion can with corresponding antigens specificity bonded, sphaeroprotein with immunologic function.
" human antibody " is meant the antigen adsorption zone in the heterologous antibody or variable region part (being Vh and Vl district) grafting to people's antibody, to reduce the outer derived components on the antibody, reduces the immune side reaction that heterologous antibody causes human body.
" total man source antibody " is meant all parts of antibody, comprises that the variable region part (being Vh and Vl district) and the constant region (being ch and cl district) that refer to antibody are coded by human antibody gene.
" antibody variable region " is meant at immunoglobulin polypeptides chain aminoterminal (N end), light chain 1/2 with 1/4 zone of heavy chain in, amino acid whose kind, put in order very big with change of configuration, so be called the variable region.
" CDR " is meant antigen complementary determining region (complementary-determining region), three hypervariable regions of variable region of light chain (VH) and variable region of heavy chain (VL) are formed the antigen-binding site of immunoglobulin (Ig) jointly, this position forms one and epitope complementary surface, so the hypervariable region is called complementary determining region again.
" human donor " is meant from the people of the specific antigen of contacting, and can be detected specific antibody in the human donor peripheral blood of this class.
" magnetic bead sorting " is meant and utilizes the specific mark of cell surface, can combine with specific part (antibody), and this part (antibody) and magnetic bead mutually lotus root connect, under the effect that utilizes externally-applied magnetic field, those cells with cell specific marker are attracted on the magnetic field, do not have the cell of respective markers then to be eluted, thereby reach the purpose that purpose cell and non-purpose cell are separated.
" external evoked differentiation " is meant and adds various cytokines under the condition of vitro culture, impel pre-B lymphocyte differentiation and maturation justacrine antibody.
" cultivation altogether " is meant external and places same culture system to cultivate in dissimilar cells.
" ELISA " is that enzyme connects the abbreviation that immunosorbent is measured (Enzyme-Linked ImmunosorbnentAssay), it is based on immunological response, the very high experimental technique of a kind of susceptibility that the specific reaction of antigen, antibody and enzyme are combined to the efficient catalytic effect of substrate.
" RT-PCR " is meant reverse transcription PCR, claims that perhaps (reversetranscription-PCR RT-PCR), is a kind of of polymerase chain reaction (PCR) to reverse transcription PCR.In RT-PCR, a RNA chain is reversed record becomes complementary DNA, carries out DNA cloning as template by PCR again.Be transcribed into complementary DNA (cDNA) by a RNA single strand and be called " reverse transcription ", finish by the archaeal dna polymerase (reversed transcriptive enzyme) of dependenc RNA.Subsequently, another of DNA chain is finished by the archaeal dna polymerase of deoxynucleotide primer and dependence DNA, with each circulation multiplication, promptly common PCR.
" unicellular RT-PCR " is meant that the lysate with individual cells is the reverse transcription PCR of template.
" electrophoresis " is meant under the effect of external dc power supply, and DNA anode in Ago-Gel medium is done directed moving to reach the purpose that different big or small DNA are separated.
Proteic expression
The present invention includes carrier, transformant that code book is invented proteic DNA and contained these DNA.
Among the present invention, the term of use " transformant " (transformant) promptly has the host cell of allogeneic dna sequence DNA molecule.
The present invention also comprises by synthetic and recombinant technology and produces proteic method of the present invention.Can separate and purifying polynucleotide (DNA or RNA), carrier, transformant and organism by methods known in the art.
Being used for carrier of the present invention can be as phage, plasmid, clay, minichromosome, virus or retroviral vector.The carrier that can be used for cloning and/or express polynucleotide of the present invention is to duplicate and/or to express the carrier that duplicates and/or express polynucleotide in the host cell of polynucleotide at need.In general, polynucleotide and/or carrier can be used for any eucaryon or prokaryotic cell prokaryocyte, comprise mammalian cell (as people (as HeLa), monkey (as Cos), rabbit (as the rabbit reticulocyte), rat, hamster (as CHO, NSO and baby hamster kidney cell) or mouse cell (as the L cell)), vegetable cell, yeast cell, insect cell or bacterial cell (as intestinal bacteria).Relevantly be applicable to that the example of the suitable carrier of broad variety host cell can be referring to for example F.Ausubel et al., Current Protocolsin Molecular Biology.Greene Publishing Associates andWiley-Interscience (1992) and Sambrook et al. (1989).Can use the host cell that contains these polynucleotide to come great expression to can be used for for example protein of medicine, diagnostic reagent, vaccine and therapeutical agent.
Having developed several different methods is used for via the complementary sticky end polynucleotide being operated with carrier and links to each other.For example, can add complementary with the aggressiveness sequence fragment by the DNA section in desire is inserted carrier DNA.Then by the hydrogen bond connection carrier between the complementary homopolymeric tail and DNA section to form recombinant DNA molecules.
The synthetic linker that contains one or more restriction site provides the method for another kind of connection DNA section and carrier.Handle the DNA section that produces by the endonuclease restrictive diges-tion with phage T4DNA polysaccharase or e. coli dna polymerase I, described two kinds of polysaccharases with its 3 ', 5 '-exonucleolytic activity is removed outstanding γ-strand end, and mends flat 3 '-recessed end with its polymerization activity.Therefore, these active associatings have produced flush end DNA section.Then can catalysis the enzyme that connects of flush end dna molecular, as under the existence of phage T4 dna ligase the linkers of flush end section with molar excess being incubated.Therefore, reaction product is the DNA section that end carries the polylinker sequence.Use these DNA sections of suitable Restriction Enzyme cracking then, and be connected in the expression vector of using enzymatic lysis, described enzyme can produce and the compatible end of described DNA section.Can buy the synthetic linker that contains a plurality of restriction endonucleases site from a plurality of businessmans.
The polynucleotide inset should be operably connected to on the compatible suitable promotor of the host cell of expressing polynucleotide.Promotor can be strong promoter and/or inducible promoter.The example of some promotors of enumerating comprises phage PL promotor, intestinal bacteria lac, trP, phoA, tac promotor, SV40 is early stage and late promoter and retrovirus LTR promotor.Other suitable promotor is well known by persons skilled in the art.The express recombinant carrier further contains transcription initiation, termination site, and contains the ribosome bind site that is useful on translation at transcriptional domain.The encoding part of the transcript that recombinant vectors is expressed can comprise translation initiation codon that is positioned at the starting point place and the terminator codon that suitably is positioned at the end that is translated polypeptide (UAA, UGA or UAG).
As mentioned above, expression vector can comprise at least one selective marker.Described mark comprises Tetrahydrofolate dehydrogenase, G418, glutamine synthase or the neomycin resistance for eukaryotic cell culture; And the tsiklomitsin, kantlex or the ampicillin resistance gene that are used for intestinal bacteria and other microbial culture.Suitably host's representative example includes but not limited to: bacterial cell, as intestinal bacteria, streptomycete and salmonella typhimurium cell; The fungal cell is as yeast cell (as yeast saccharomyces cerevisiae or pichia pastoris phaff); Insect cell is as fruit bat S2 and noctuid SF9 cell; Zooblast, as CHO, COS, NSO, 293 and the Bowes melanoma cells; And vegetable cell.The appropriate culture medium of above-mentioned host cell and culture condition are known in the art.
For separation and purification effectively or secretion target protein, usually also can utilize label protein or the label polypeptide (Tag) of being convenient to separation and purification.Commonly used have glutathione-S-transferase (glutathioneS-transferase, GST), six polyhistidyl peptides (His.Tag), a-protein (protein A) and Mierocrystalline cellulose binding site (cellulose binding domain) etc.By the form of singularity albumen or polypeptide and target protein formation fusion rotein, utilize the special property of described label protein or label polypeptide to separate and purifying after the expression to target protein.Combine with Ni-ChelatingSepharose post specificity as His.Tag.Described label protein or label polypeptide can be removed fusion sequence with the locus specificity protease digestion behind purifying, as available zymoplasm, enteropeptidase and Xa factor etc., to obtain target protein.
The present invention also comprises the host cell that contains nucleotide sequence of the present invention, and described nucleotide sequence can be operated with one or more allos control region (as promotor and/or enhanser) through technology known in the art and link to each other.Can select to regulate the expression of the gene order of inserting, or can be according to the required particular form modification and the host strain of processed gene product.In the presence of some inductor, the expression that some promotor starts can raise; Therefore, can control polypeptide expression through genetic modification.In addition, different host cells have distinctive and special translation, translation post-treatment and the proteinic mechanism of modification (as phosphorylation, cracking).Can select suitable clone to guarantee that the exogenous protein of expressing is carried out desirable modification and processing.
By the transfection of calcium phosphate transfection, the mediation of DEAE-dextran, transfection, electroporation, transduction, infection or other method of cation lipid mediation, nucleic acid of the present invention and nucleic acid recombinant vectors can be imported host cell.Described method is described in the laboratory manual of a plurality of standards, as Davis et al., and BasicMethods In Molecular Biology (1986).
The described proteic polynucleotide of the present invention of encoding can be connected to breed in the host with the carrier that contains selective marker.In general, can be at throw out, import plasmid vector in the mixture as calcium phosphate precipitation thing or itself and charged lipids.If carrier is a virus, can use suitable packing cell to tie up to and external it be packed, transduce to host cell again.
Can identify by successful cell transformed by well-known technology, promptly contain the cell of DNA recombinant vectors of the present invention.For example, can cultivate the cell of importing express recombinant carrier gained to produce required polypeptide.Collect and lysing cell, use as Southem (1975) J.Mol.Biol.95,503 or Berent et al (1985) Biotech.3,208 described methods detect the existence of DNA in its DNA content.Perhaps, use proteinic existence in the antibody test supernatant liquor.
By well-known method from the reconstitution cell culture, reclaim and purifying described albumen of the present invention comparatively favourable, described method comprise sulfuric acid by or ethanol sedimentation, acid extraction, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, dewatering electric charge effect chromatography and lectin chromatography.In some embodiments, can use high performance liquid chromatography (HPLC) to carry out purifying.
In some embodiments, can use above-mentioned one or more chromatography method purifying described albumen of the present invention.In other embodiments, can use following one or more chromatography column purifying described fusion rotein of the present invention, described chromatography column has: Q sepharose FF post, SP sepharose FF post, Qsepharose High Performance post, Blue sepharose FF post, Blue post, PhenylSepharose FF post, DEAE Sepharose FF, Ni-Chelating Sepharose FF post or Methyl post etc.
In addition, can use the method purifying albumen of describing among the international publication number WO00/44772 (listing this paper in as a reference in full) of the present invention.Those skilled in the art can easily change wherein said method to be used for purifying albumen of the present invention.Can be from comprising that for example the protokaryon or the eucaryon host of bacterium, yeast, higher plant, insect and mammalian cell reclaim albumen of the present invention through the product that recombinant technology produces.
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is usually according to the normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
In the following embodiments, following shortenings is represented following meaning, and the shortenings that does not add definition uses its common acceptable definition:
℃=degree centigrade
Min=minute
Hr=hour
The M=mole
MM=person of outstanding talent mole
The mL=milliliter
The ng=nanogram
μ L=microlitre
The PBS=phosphoric acid buffer
The FBS=foetal calf serum
The PBMC=peripheral blood lymphocytes
IL-2=human recombinant interleukin-22
IL-21=human recombinant interleukin-22 1
IMDM=Iscove ' s Modified Dulbecco ' s Medium substratum
The HRP=horseradish peroxidase
The BSA=bovine serum albumin
TMB=3 ', 4 ', 5 '-TMB
DNTP=triphosphoric acid dezyribonucleoside
Materials and methods
Substratum
The substratum of Shi Yonging in this application, if there are not other explanations, the general 10% foetal calf serum (GIBCO that adopts, cat#89-4901DJ), the glutamine (GIBCO of 200mM, cat#25030-081), the nonessential amino acid (GIBCO of 10mM, cat#11140-050), the Sodium.alpha.-ketopropionate (GIBCO of 100mM, cat#11360-070), the beta mercaptoethanol (GIBCO of 55mM, cat#21985-023), the penicillin of 1X and Streptomycin sulphate (GIBCO, cat#15750-053) IMDM (GIBCO, cat#12440-046) substratum.
The human peripheral blood mononuclear cell
From U.S. Stemcell Technologies Inc company (cat#PB005F).In the peripheral blood of this lymphocyte strain donor, be detected Tetanus Antitoxin antibody.
The lymphocytic magnetic bead sorting of CD19+
PBMC earlier connects the magnetic bead of anti human CD 19 antibody with lotus root, and (Stemcell Technologies Inc cat#18054) is hatched, and (Stemcell Technologies Inc cat#18000) separates to use the magnet separator column again.Briefly, lymphocyte is by marked by magnetic bead, and cell is by a separator column that places permanent high-intensity magnetic field after the mark.In this high-intensity magnetic field, what be labeled is adsorbed and is retained on the tube wall with magnetic bead bonded positive cell, and the negative cells that combines with magnetic bead antibody is not then in solution, and when toppling over when in the pipe solution, negative cells is separated goes out.Thereby reach isolating purpose.
The CHO-K1 cell strain preparation of stably express CD40L
The total length (from the initiator codon to the terminator codon) of the method amplification CD40L gene by RT-PCR, again with this gene clone in the plasmid of carrier for expression of eukaryon pcDNA 3.1, after sequence verification,, use G418 drug screening to obtain stably express CD40L cell strain with this recombinant plasmid transfection CHO-K1 cell strain.Before the CHO-K1 cell strain of stably express CD40L is cultivated altogether as trophoderm and lymphocyte, need to stop the multiplication capacity of CHO-K1 cell.The invention provides corresponding method, promptly adopt the method for Gamma radiation exposure.The growth curve of irradiating and detecting cell has been lost the ability of propagation with clear and definite its.Postradiation cell can directly be cultivated as trophoderm and lymphocyte altogether, or in the future standby in frozen and the liquid nitrogen.
The result: the present invention's human CD40L full length gene that successfully increases, and successfully being cloned in the plasmid of carrier for expression of eukaryon pcDNA3.1, at transfection CHO-K1 cell and successfully obtain the cell strain of stably express CD40L after 4 week of G418 drug screening.In the method that adopts the Gamma radiation exposure, promptly utilize the irradiation dose irradiation of 3000Rad after, the CHO-K1 cell is promptly lost multiplication capacity, but still retentive activity.
CD19
+Lymphocytic vitro culture and induction
Be inoculated into 384 orifice plates after the CHO-K1 cell strain recovery with stably express CD40L frozen behind the Gamma ray.Again the CD19 positive cell that is separated at first is diluted to 5 * 10
3Individual cell/mL, further be diluted to 500 cell/mL again, get 2000 cell dilution to 25 cell/mL, and add an amount of IL-2 (Roche company, Cat#11011456001) and IL-21 (PROSPEC Tany TechnoGeneLtd company, Cat#CYT-408), at 5 384 well culture plates (each culture hole is inoculated 30,000 CHO-K1 cells) of the CHO-K1 cell of cell inoculation after inoculated the Gamma radiation exposure that will dilution, finally in each culture hole of each culture plate, inoculate 1 lymphocyte.The final culture system of each culture hole is 60 μ L, and culture plate is placed 37 ℃, cultivates ELISA detection after 10 days in the incubator of 5%CO2.
ELISA detects
From above-mentioned 5 384 culture plates, take a sample, shift 40 μ L nutrient solutions in each culture hole and to 384 new orifice plates, be used for the ELISA detection, and in original culture hole, adding the sterile glycerol of 10 μ L 20%, 384 orifice plates that will contain cell immediately are frozen in-80 ℃ refrigerator.Will be with coating buffer (yellow soda ash of 1.59g/L, the sodium bicarbonate of 2.93g/L, 0.02% sodium azide solution) dilution Tetanus Antitoxin (Astarte Biologics LLC company, the U.S., Cat#1002) to 0.1 μ g/ μ L, be added into 96 orifice plates (Sigma Aldrich, the U.S., Cat#CLS3590), 25 μ L/ holes, 4 ℃ are spent the night.Second day with washings (the PBS solution that contains 0.02% polysorbas20) washed twice.Add 100 μ L confining liquids (the PBS solution that contains 3%BSA) room temperature sealing 1hr; To add in 96 orifice plates every hole 20 μ L from the culture supernatant of 384 orifice plates to.Hatch 1hr for 37 ℃, washings washing 2 times; (1hr is hatched for 37 ℃ in abcam company, cat#ab6759) (1: 10000), washings washing 2 times to dilute horseradish peroxidase (HRP) lotus root rabbit anti-human igg's antibody even with confining liquid; Every hole add 50 μ L tmb substrates (Sigma Aldrich, the U.S., Cat#T0440), room temperature reaction 30min, 50 μ L 1MHCL termination reactions are added in every hole; Detect A450 on the microplate reader.
The degenerate primer group
The primer group of synthetic respectively heavy chain of according to the form below and light chain.
| |
?SEQIDNO.13 | ?5′-GGTCTCCTCAGCYTGTGCTG-3 |
| Upstream primer | ||
| 8 | ?SEQIDNO.14 | ?5′-GTTCTTCCAATTTATGCTG-3′ |
| Upstream primer 9 | ?SEQIDNO.15 | ?5′-GGTCCAATTCYCAGTGGTG-3′ |
| Upstream primer 10 | ?SEQIDNO.16 | ?5′-GAGTGGATTCTCTGTGGTG-3′ |
| Upstream primer 11 | ?SEQIDNO.17 | ?5′-CCTCTCCTCCTCACCTCCT-3′ |
| Upstream primer 12 | ?SEQIDNO.18 | ?5′-CTCCTCACTCAGGCACA-3′ |
| Upstream primer 13 | ?SEQIDNO.19 | ?5′-ATGGCCTGGAYCTCTCC-3 |
| Downstream primer | ||
| 1 | ?SEQIDNO.20 | ?5′-GTTTCTCGTAGTCTTGCTCA-3 |
| Downstream primer | ||
| 2 | ?SEQIDNO.21 | ?5′-CACCAGTGTCCTTTGGCTTG-3 |
| Downstream primer | ||
| 3 | ?SEQIDNO.22 | ?5′-AGCTCCTCGAGAGGGYGG-3′ |
Unicellular RT-PCR and order-checking
(1MTris pH8.0 is from Ambion company, Cat#AM9855G to configure cell pyrolysis liquid (the RNA enzyme inhibitors of the TRIS of 10mM, 3 units/mL) earlier; The RNase inhibitor is from Fisher company, cat#PR-N2515); 384 orifice plates of-80 ℃ of preservations are thawed on ice, in every hole, add 20 μ L cell pyrolysis liquids, and abundant mixing, as the RT-PCR template.According to following configuration reaction system (QIAGENOneStep RT-PCR Kit, cat#210210):
RNase-free water 27.3 μ L
5x RT-PCR damping fluid 10.0 μ L
DNTP mixture (concentration of every kind of dNTP is 10mM) 2.0 μ L
Mix primer (heavy chain or light chain) 6.6 μ L (every kind of primer amount is 0.6 μ M)
Rnase inhibitor 0.1 μ L
Cell lysate 2.0 μ L
RT-PCR enzyme mixture 2.0 μ L
Total system 50 μ L
Adopt the parameter of Fig. 1 to carry out the amplification of variable region gene then.Amplified production adopts 2% agarose gel electrophoresis detection, and the result is the 1-4 swimming lane as shown in Figure 2: heavy chain product band; The 5-8 swimming lane: light chain product band, positive findings are then directly inspected by ready samples and are checked order and analyze, and obtain Tetanus Antitoxin specific human source antibody heavy chain variable region gene sequence as being shown in SEQ ID NO.23; Tetanus Antitoxin specific human source antibody chain variable region gene sequence is as being shown in SEQ ID NO.24.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating all respects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.Every piece of reference mentioned above is listed this paper in as a reference all in full.
Claims (10)
1. method of obtaining specific human source antibody variable gene sequence fast, it comprises the following steps:
A) separation of C D19 male lymphocyte from donor peripheral blood or clone;
B) the external evoked differentiation of CD19 male lymphocyte;
C) detection specificity antibody and screening specific antibody male lymphocyte;
D) design degenerate primer group;
E) adopt the method for unicellular RT-PCR increase variable region gene and order-checking;
The external evoked differentiation step of wherein said CD19 male lymphocyte is for to cultivate with the individual cells form CD19 male lymphocyte body in the nutrient solution that has CD40L albumen situation, cytokine.
2. the method for claim 1 is characterized in that the lymphocytic method of separation of C D19 male is magnetic bead sorting method or stream type cell analyzer separating method among the described step a.
3. the method for claim 1, to it is characterized in that existing described in the described step b CD40L albumen situation be the medium that contains CD40L albumen, CD40L protein-crosslinking in the nutrient solution, one of express among the proteic trophocyte of CD40L situation.
4. the method for claim 1 is characterized in that cytokine described in the described step b is IL-2 and/or IL-21.
5. method as claimed in claim 4 is characterized in that described cytokine is IL-2 and the 100ng/mL IL-21 of 100 units/mL.
6. the method for claim 1, the method that it is characterized in that detection specificity antibody described in the described step c one of can be in radioimmunology, ELISA method or the protein chip.
7. the method for claim 1, it is characterized in that the primer group of degenerate primer group described in the described steps d for variable region of heavy chain and variable region of light chain, wherein downstream primer is near the constant region of bordering on the variable region, upstream primer then according to existing other variable region sequences, consider the changeable characteristics of variable region sequences design degenerate primer group, and near 5 ' UTR district or initiator codons of variable region.
8. method as claimed in claim 7, the primer group who it is characterized in that described variable region of heavy chain is for shown in SEQ ID NO.1～SEQ ID NO.6, and the primer group of described variable region of light chain is shown in SEQ ID NO.7～SEQ ID NO.23.
9. the method for claim 1, it is characterized in that unicellular RT-PCR step described in the described step e is for earlier carrying out cracking with unicellular, carry out single stage method RT-PCR with this lysate as template again, the variable region of in different PCR pipes, increase respectively heavy chain and light chain.
10. a method for preparing total man source antibody comprises the following steps:
A) the specific human source antibody variable gene sequence clone that the described method of claim 1 is obtained is in the human constant region expression vector;
B) transfection and screening transformant;
C) expression and purifying total man source antibody.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106086009A (en) * | 2016-06-17 | 2016-11-09 | 中国农业科学院哈尔滨兽医研究所 | A kind of method utilizing single B cell round pcr to produce full pig resource monoclonal antibody |
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| EP1222929A2 (en) * | 2001-01-11 | 2002-07-17 | D. Collen Research Foundation vzw | Method and pharmaceutical composition for preventing and/or treating systemic inflammatory response syndrome |
| WO2009072660A1 (en) * | 2007-12-03 | 2009-06-11 | Kabushiki Kaisya Advance | Method for production of antibody |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106086009A (en) * | 2016-06-17 | 2016-11-09 | 中国农业科学院哈尔滨兽医研究所 | A kind of method utilizing single B cell round pcr to produce full pig resource monoclonal antibody |
| CN106086009B (en) * | 2016-06-17 | 2020-03-17 | 中国农业科学院哈尔滨兽医研究所 | Method for producing all-pig-derived monoclonal antibody by using single B cell PCR technology |
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