CN107354171A - Recombinate preparation method of the adalimumab Fab fragments in insect cell expression system - Google Patents
Recombinate preparation method of the adalimumab Fab fragments in insect cell expression system Download PDFInfo
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Abstract
The present invention relates to preparation method of the restructuring adalimumab Fab fragments in insect cell expression system, including：N-terminal is respectively provided with to the gene cloning of the light chain of the Fab fragments of signal peptide and the amino acid sequence of heavy chain in building recombinant expression carrier in the rhabdovirus expression vector with double-promoter；By the recombinant expression carrier swivel base of gained into competent cell, screening restructuring bacterial plaque, extract recombinant plasmid, and transfected into insect cell, obtain further amplicon virus after recombinant baculovirus, and the insect cell for amplifying culture is infected to express Fab albumen using virus, the supernatant product of the albumen containing Fab is collected by centrifugation；Gained insect cell expression supernatant is concentrated by ultrafiltration and replaced cushioning liquid first by film bag, then chromatography, you can obtain full human monoclonal antibody adalimumab Fab fragments.Show that purity is higher through analysis through the full human monoclonal antibody adalimumab Fab fragments that the inventive method obtains, and TNF α can be specifically bound.
Description
Technical field
The present invention relates to preparation side of the full people source adalimumab Fab fragments in Insect Cell/Baculovrius Expresion System expression system
Method, belong to biological technical field.
Background technology
Adalimumab is adalimumab, and trade name Humira (Xiu Meile) is best-selling biology global in recent years
Medicine, it is the first full human monoclonal antibody medicine for obtaining FDA approvals.Adalimumab is developed by Abbott Laboratories
(US6090382), now belong to AbbVie Corp., be the antigen-binding fragment (antigen-binding of IgG molecules
Fragments, Fab fragment).Adalimumab Fab fragments are human monoclonal D2E7 heavy chains and light chain through disulfide-bonded
Dimer, specifically it can be combined with TNF-α so as to block its phase interaction with the TNF-α acceptor of p55, p75 cell surface
With, for treating rheumatoid arthritis (rheumatoid arthritis, RA), psoriatic arthritis (psoriatic arthritis,
PA), ankylosing spondylitis (ankylosing spondylitis, AS), and Crohn disease (Crohn's disease, CD) etc.
Disease, and can alleviate modifying antirheumatic drug (DMARD) treatment it is invalid in severe RA patient S＆S.
Fab is the antigen-binding fragment of IgG molecules, is different two formed by Fd fragments and light chain by disulfide-bonded
Condensate, because its relative molecular mass is small, has integrality structure that height determines and the advantages such as immunogenicity is low to resist as IgG classes
The first choice of body production and transformation.Because Fab fragments do not have Fc sections, it is not necessary to the glycosylation and posttranslational modification of complexity are carried out,
So it need not be produced by mammalian cell system.And Bac-to-Bac insect cell expression systems and other eukaryotic expressions
System, which is compared, many advantages：Can high level expression foreign gene；The recombinant protein of most of expression is solvable；After being translated
Processing modification：Including glycosylation, phosphorylation, acylation, signal peptide excision etc.；It is easier to isolate and purify；Insect cell hangs
Floating life long easily amplification culture, is advantageous to express recombinant protein on a large scale.
The content of the invention
Coexpression is secreted it is an object of the invention to provide one kind in Insect Cell/Baculovrius Expresion System expression system to be recombinated
The preparation method of adalimumab Fab fragments.The present inventor it has been investigated that, by using insect cell as host carry out table
Reach, the restructuring adalimumab Fab albumen with bioactivity can be obtained.
Here, the present invention provides a kind of preparation method of full human monoclonal antibody adalimumab Fab fragments, including：
(a) N-terminal is respectively provided with the heavy chains (heavy chain, Hc) of the Fab fragments of signal peptide and light chain (light chain,
Lc the gene cloning of amino acid sequence) is in the rhabdovirus expression vector (being preferably pFastBacT1) with double-promoter
Build recombinant expression carrier；
(b) by the recombinant expression carrier swivel base of gained into competent cell, screening restructuring bacterial plaque, extract recombinant plasmid, and by its
Transfection obtains further amplicon virus after recombinant baculovirus, and use the insect of virus infection amplification culture into insect cell
The supernatant product of the albumen containing Fab is collected by centrifugation to express Fab albumen in cell；
(c) gained insect cell expression supernatant is concentrated by ultrafiltration and replaced cushioning liquid first by film bag, then chromatographic column is pure
Change, you can obtain full human monoclonal antibody adalimumab Fab fragments.
The present invention, will the Fab fragments with signal peptide (such as Ac NPV gp64) with technological means such as genetic engineerings
Heavy chain and light chain be cloned into respectively in baculovirus vector (such as pFastBacT1), recombinant plasmid is obtained, by recombinant plasmid
Swivel base obtains restructuring Bacmid DNA, transfection insect cell (such as Sf9) into competent cell (such as DH10Bac)
Recombinant baculovirus is obtained, using viral infected insect cell, secretion coexpression destination protein, is collected by centrifugation containing destination protein
Supernatant product.Chromatography is carried out again, obtains the adalimumab Fab albumen of bioactivity.Obtained through the inventive method
The full human monoclonal antibody adalimumab Fab fragments obtained show that purity is higher through analysis, and can specifically bind TNF-
α, its inhibition is similar to standard items Humira, illustrates that the inventive method Fab fragments remain standard items Humira and possessed
Antigen-binding and specificity；And the low easily targeting of the Fab fragments molecules amounts is in focus, insect cell suspension growth in addition
Easily amplification culture, the Fab fragments can in insect cell extensive Expression product, be easily isolated purifying.
In the present invention, expression is co-secreted from baculovirus insect cell system carrier pFastBacT1, macromolecular can be accommodated
Insert Fragment, expression various exogenous genes and can efficiently simultaneously mass expressing external gene.Can by adding outer secreting signal peptide
With secretion coexpression Fab albumen in insect cell supernatant, using extracellular oxidation environment, Fab heavy chains and light chain can be instructed
Between disulfide bond formation.
It is preferred that in step (a), the recombinant expression carrier is pFastBacT1, including：Two promoters, answer
System, polyclone enzyme enzyme site and two terminators.
It is preferred that in step (a), the signal peptide of the Lc and Hc of Fab fragments N-terminal is outer secreting signal peptide Ac
NPV gp64.It is preferred that the heavy chain of Fab fragments and the promoter of light chain are all polyhedrosis gene promoter (polyhedrin
(PH)promoter)。
It is preferred that in step (b), the competence is DH10Bac.It is preferred that swivel base bacterial plaque screening technique is indigo plant
Hickie screening method.
It is preferred that in step (b), the insect cell is Sf9 cells.It is preferred that transfection reagent can be liposome
Cellfection, in addition, used medium can be Sf-900 in transfection procedureTMII SFM culture mediums.
It is preferred that in step (b), the recombinant baculovirus P0 amplicon virus P1, i.e., 200 μ L are recombinated shaft-like
It is 2 × 10 that viral P0, which adds 100mL cell densities,6In cells/ml Sf9 insect cells, after 37 DEG C of shaking table culture 96h
Centrifugation obtains viral P1.
It is preferred that it is ESF AF culture mediums that amplification, which cultivates cell and expresses culture medium used, in step (b).
It is preferred that in step (c), the purifying includes successively：It is molten that buffering is concentrated by ultrafiltration and replaced using film bag
Liquid；And affinity chromatography and molecular sieve medium chromatography.
It is preferred that in step (c), the affinity chromatography medium of the affinity column chromatography is Protein L.
It is preferred that in step (c), the sieve chromatography medium of the molecular sieve medium chromatography is Superdex 75.
After the Fab albumen for obtaining purifying, it can be used ELISA and the cell neutralization activity method validation point of TNF-α
Analysis.It is preferred that purifying Fab the cell neutralization activity of TNF-α is analyzed can be used l cell L929 cells and
Cell Counting Kit-8(CCK-8).Understood through analysis, the Fab albumen that the present invention obtains can be with antigen TNF-α specificity
With reference to and suppress its effect, the no significant difference compared with standard items Humira, illustrate that it possesses and standard items Humira identicals
Antibody characteristic and specificity.As the full human monoclonal antibody adalimumab Fab fragments prepared by the preparation method of the present invention
Mainly include rheumatoid arthritis and cancer available for treatment relevant disease as caused by raising TNF-α.
Brief description of the drawings
Fig. 1 is adalimumab Fab antibody fragment recombinant expression carrier collection of illustrative plates；
Fig. 2 is the Fab SDS-PAGEs after Protein L affinity purifications；Figure A therein is the electrophoresis under non reducing conditions
Acquired results, figure B are electrophoresis acquired results under reducing condition；Fab is IgG1 antigen binding domains Hc and Lc dimers, is gone back
It is two electrophoretic bands of 23kD under old terms, has single electrophoretic band at 47kD under non reducing conditions；
Fig. 3 is the Fab SDS-PAGEs of sieve chromatography after purification, and figure A therein is the electrophoresis institute under non reducing conditions
Result is obtained, figure B is electrophoresis acquired results under reducing condition；
Fig. 4 be sieve chromatography purify and concentrates after Fab SDS-PAGEs, show obtain Fab antibody fragment with compared with
High-purity；
Fig. 5 is the figure after Western Blot detection molecules sieve chromatographies are purified and concentrated；
Fig. 6 is that the adalimumab Fab antibodies fragment of ELISA measure purifying is lived with antigen TNF-α specific binding capacity and antibody
The figure of property；
Fig. 7 is the IC50 curves that expression and purification Fab albumen of the present invention is tested with Humira to the cell neutralization activity of TNF-α.
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and following embodiments, it should be appreciated that accompanying drawing and following embodiments are only
For illustrating the present invention, it is not intended to limit the present invention.
The present invention, by the use of baculoviral as expression vector, place is used as by the use of insect cell with means such as genetic engineerings
It is main, express adalimumab Fab fragments.In one example, preparation method of the invention includes：Ac will be respectively provided with
The heavy chain and light chain of the Fab fragments of NPV gp64 signal peptides are cloned into baculovirus vector pFastBacT1, structure restructuring table
Up to carrier, recombinant plasmid swivel base is obtained into restructuring Bacmid DNA into competent cell DH10Bac and transfected to Sf9 elder brothers
Recombinant baculovirus is obtained in worm cell, infects sf9 insect cells, secretion coexpression Fab destination proteins, centrifugation using virus
Obtain the supernatant product containing destination protein；Chromatography is carried out to the albumen of gained, obtains Adalimumab Fab albumen.
Hereinafter, the preparation method of the present invention is exemplarily illustrated.
(1) structure of recombinant expression carrier
The heavy chain of Fab fragments and the gene of light chain with signal peptide are respectively synthesized, is cloned in the pFastBacT1 with double-promoter
In carrier, adalimumab Fab antibody fragments recombinant expression carrier (Fab in pFastBacT1) is built.
Preferably, an outer secreting signal peptide Ac NPV gp64 is respectively added in Hc and Lc N-terminal.
In one example, signal peptide Ac NPV gp64 are added before Lc and forms AcNPV gp64+Lc in
pFastBacT1.Preferably, it is inserted in using 5'BamHI and 3'EcoRI after first promoter.
In another example, signal peptide Ac NPV gp64 formation AcNPV gp64+Hc in are added before Hc
pFastBacT1.Preferably, it is inserted in using 5'XhoI and 3'HindIII after second promoter.
Constructed adalimumab Fab antibody fragment recombinant expression carrier collection of illustrative plates is as shown in Figure 1.As shown in Figure 1, should
Recombinant expression carrier includes：Two insect cell promoters, replicon, polyclone enzyme enzyme site and two terminators.
(2) expression of the Fab fragments in insect cell
The preparation of recombinant plasmid：By constructed Adalimumab Fab antibodies fragment recombinant expression carrier swivel base to competent cell
In (such as DH10Bac), screening restructuring bacterial plaque, recombinant plasmid is extracted.Wherein, swivel base bacterial plaque screening technique can be blue white
Spot screening method.
Bacmid transfection sf9 insect cells are recombinated, transfection reagent can be liposome cellfection, and culture medium can be Sf-
900TMII SFM culture mediums, obtain Fab-P0 generation viruses.Transfect the obtained further amplicon virus of P0 generation viruses and obtain P2.
In one example, amplification method is：It is 2 × 10 that 200 μ L recombinant baculovirus P0 are added into 100mL cell densities6
In cells/ml Sf9 insect cells, the viral P1 of centrifugation acquisition after 37 DEG C of shaking table culture 96h.It is thin using P2 infected insects
Cellular expression Fab albumen.
(3) Fab purifying
Fab albumen is expressed as the outer secreting, expressing of solubility, and it can be ESF AF culture mediums to express culture medium used, virus amplification sense
Contaminate cell culture to cell and cell suspension is collected when having infection sign and percentage of cells 80% or so, at 4 DEG C of centrifuge 3000g
10min is centrifuged, supernatant solution is collected, supernatant is concentrated using the cross-flow ultrafiltration film bags of Vivaflow 200 and replaces buffering
Liquid, facilitate follow-up isolation and purification.Then chromatography is carried out, obtains the Fab fragments of high-purity.Preferably, successively
Purified by affinity column chromatography and molecular sieve medium chromatography.In one example, the affinity chromatography medium of affinity column chromatography is
Protein L.In another example, the sieve chromatography medium of molecular sieve medium chromatography is Superdex 75.Purifying
In, SDS-PAGE electrophoretic analysis purity of protein can be used.30kD Milipore super filter tube protein concentrates can be used.
(analysis and detection)
SDS-PAGE electrophoretic analysis destination protein Fab fragments can be used.Fig. 2,3 are shown respectively through affinity column chromatography, molecular sieve
Medium chromatographs the SDS-PAGE of obtained albumen, to characterize the purity of albumen.Figure A in the two width figure is equal
For non reducing conditions, figure B is reducing condition.As shown in Figure 2, Fab is IgG1 antigen binding domains Hc and Lc dimerization
Body, have two electrophoretic bands at 23kD under reducing condition, from top to bottom respectively Hc and Lc, under non reducing conditions
It is Fab fragments to have single electrophoretic band at 47kD.Fig. 4 show sieve chromatography purify and concentrate after Fab albumen SDS-
PAGE electrophoretograms, the wherein sample of swimming lane 1 are to be handled under non reducing conditions, and swimming lane 2 is to be handled under reducing condition, can by Fig. 4
Know that the purity of the Fab antibody fragment after contracting is more than 98%.Fig. 5 shows that Western Blot detection molecules sieve chromatographies are purified and concentrated
Fab fragments afterwards, the anti-light chain antibody of goat-anti people lgk specificity of HRP marks, wherein sample in swimming lane 1 are used in detection
To be handled under reducing condition, sample is to be handled under non reducing conditions in swimming lane 2.
The adalimumab Fab fragments and antigen TNF-α specific binding capacity and resist that ELISA measure purifies can be used
Body activity.Fig. 6 shows the test result.It will be appreciated from fig. 6 that adalimumab Fab antibodies fragment can be special with antigen TNF-α
The opposite sex combines, and illustrates that the Fab fragments remain antibody characteristic and the specificity that standard items Humira possesses.Therefore, according to this
Fab fragments prepared by the preparation method of invention can be applied to prepare treatment and the relevant disease of TNF-α level rise (such as
Rheumatoid arthritis, cancer etc.) medicine.
The present invention has the advantage that compared with prior art：
(1) the Fab fragments molecules amounts prepared by the present invention are small, therefore penetration power is big during tumour target site is reached；
(2) the Fab fragments prepared by the present invention remain high specific and high-affinity of the whole antibody to TNF-α, and it partly declines
Phase is grown compared with similar drugs, it is possible to reduce the access times of medicine；
(3) recombinant protein of insect cell system expression has complete biological function, can correctly fold and to form Fab intermolecular
Important disulfide bond, expression product is in structure and function close to native protein；
(4) the processing modification after being translated：Including glycosylation, phosphorylation, acylation, signal peptide excision etc.；
(5) compared with other eukaryotic expression systems, baculovirus expression system can be high with efficiently expressing exogenous gene, expression quantity；
(6) Insert Fragment of macromolecular can be accommodated：Baculoviral virion can expand, and can pack big genetic fragment and can be in elder brother
Various exogenous genes are expressed in worm cell；
(7) in purge process, the antibody protein that purity is more than 98% is obtained using affinity chromatography-molecular sieve chromatography purifying, is a kind of
It is simple to operate, low cost, the antibody fragment purification preparation method of high production；
(8) the invention provides a kind of preparation method of adalimumab Fab antibodies fragment, to obtain the monoclonal antibody of high quality
Fab fragments are laid a good foundation.
Embodiment is enumerated further below to describe the present invention in detail.It will similarly be understood that following examples are served only for this hair
It is bright to be further described, it is impossible to be interpreted as limiting the scope of the invention, those skilled in the art is according to the present invention's
Some nonessential modifications and adaptations that the above is made belong to protection scope of the present invention.Following specific technique ginsengs of example
Number etc. is also only an example in OK range, i.e. those skilled in the art can be done in suitable scope by this paper explanation
Selection, and do not really want to be defined in the concrete numerical value of hereafter example.The test method of unreceipted actual conditions in the following example, lead to
Often according to normal condition, or according to the condition proposed by manufacturer.
(1) structure of recombinant plasmid
Gene A cNPV gp64+Lc are cloned into carrier by amplification gene Lc first, addition signal peptide AcNPV gp64
PFastbacT1, structure plasmid AcNPV gp64+Lc in pFastBacT1.Then from template plasmid Fab in pETDuet1
Amplification gene Hc, signal peptide AcNPVgp64,3 ' terminator codon TAA are added, Gene A cNPV gp64+Hc are cloned
To carrier pFastbacT1, structure plasmid AcNPVgp64+Hc in pFastBacT1.Finally from template plasmid Ac NPV
Amplification gene PH promoter+Ac NPV gp64+Hc in gp64+Hc in pFastBacT1, using the method for restructuring by gene
PH promoter+Ac NPV gp64+Hc are cloned into carrier AcNPV gp64+Lc in pFastBacT1, structure plasmid Ac
NPV gp64+Lc and Ac NPV gp64+Hc in pFastBacT1。
(2) expression of the Fab in Sf9 insect cells
Sf9 insect cells are at 27 DEG C, Shaking culture under conditions of 90rpm.When Sf9 insect cell concentration reaches 2.0 × 106
During cells/ml, adding P2 generations virus with the ratio that infection multiplicity (MOI) is 1pfu/cell, (i.e. every liter of cell adds 20ml
P2 viruses), in carrying out viral infection in 27 DEG C of incubators.Fab albumen is expressed as the outer secreting, expressing of solubility, disease
Poison amplification infection cell culture to cell collects cell suspension, centrifuge when having infection sign and percentage of cells 80% or so
4 DEG C of centrifugation 10min of 3000g, collect cell conditioned medium.
(3) Fab expresses concentration and the buffer exchange of supernatant
Fab albumen is expressed as the outer secreting, expressing of solubility, the supernatant solution being collected by centrifugation, uses the cross-flow ultrafiltrations of Vivaflow 200
Film bag is concentrated and replaces buffer solution, facilitates follow-up isolation and purification.
(4) chromatography
First step Protein L affinity chromatographys
1) chromatographic column：Affinity chromatography medium：Protein L are affine filler；
2) preparation of Protein L affinity columns
3) sample treatment：The supernatant solution centrifuging and taking supernatant of concentration and displacement cushioning liquid will be completed；
4) buffer solution configures：Buffer A：50mmol/L Tris-HCl pH7.2,150mmol/L NaCl, 10%Glycerol,
Buffer B：0.1mol/L Glycine,pH 2.5；
5) purge process：With buffer A pre-balance filler, pillar is balanced with buffer A after sample loading, then use buffer solution
B is eluted, and Ep pipes are collected and neutralize the Fab albumen under elution to pH 7.0 or so；
6) result：Target protein peak is collected, protein concentration is surveyed and calculates the rate of recovery, SDS-PAGE analyzing proteins purity (Fig. 2).
As a result it is more than 90% to show the purity of protein.
Second step sieve chromatography
1) chromatographic column：HiLoad 16/60Superdex 75prep grade, GE companies of the U.S.；
2) sample treatment：The ultrafiltration concentration that molecular cut off is 30KD is utilized by the target protein that Protein L affinity chromatographys elute
Pipe is concentrated into 2-4ml；
3) buffer solution configures：Buffer solution is 50mmol/L Tris-HCl pH7.0,150mmol/L NaCl, 10%Glycerol；
4) purge process：With buffer solution pre-balance molecular sieve, loading, eluted with 1ml/min；
5) result：Target protein peak is collected, sample carries out SDS-PAGE electrophoresis (Fig. 3).As a result show that the purity of protein is more than
98%.
Finally, purified product is concentrated by ultrafiltration using Millipore ultrafiltration systems, the regeneration that molecular cut off is 30KD is fine
Tie up plain film bag and desalination and concentration are carried out to protein solution after purification, whole purifying combination is reasonable, and simple to operate, technique is steady
It is fixed.SDS-PAGE analyzing proteins purity (Fig. 4).Result is extracted in Western Blot analyses, and SDS- is carried out under constant pressure
PAGE, glue is unloaded into carry out transferring film after terminating, 60min takes out film after terminating from electric turn trough, is immersed in confining liquid and delays
Slowly 1h is swayed, then jog is incubated at room temperature with the confining liquid of the anti-light chain antibody of goat-anti people lgk specificity marked containing HRP
1h, after incubation terminates, embathed again 3 times after rinsing film with PBST, 5~10min, is developed the color using ECL methods afterwards every time
(Fig. 5).As a result show adalimumab the Fab antibody Hc and Lc that secreting, expressing in insect cell be present, and light chain with
Heavy chain forms the heterodimer of disulfide-bonded.
(5) indirect elisa method analysis adalimumab Fab antigen binding performance
Antigen TNF-α is coated in 96 hole elisa Plates, 4 DEG C of refrigerators are stood overnight.1%BSA confining liquids are added after washing, are used
The Fab albumen and Hurmia of purifying are made same gradient dilution by dilution, per the μ L of hole 100, set secondary orifices to ensure to test
As a result accuracy, adds the anti-light chain antibody of HRP- goat-anti people lgk specificity, and TMB (3,3', 5,5'- tetramethyl benzidine) makees
Developed the color for substrate, 450nm-650nm reading is recorded using SpectraMax M5 ELIASAs, data use GraphPad
Prism5 softwares are handled (Fig. 6).As a result adalimumab Fab fragments energy and the antigen obtained by expression and purification are shown
TNF-α is specifically bound.
The cell neutralization activity experiment of TNF-α
By mouse into fiber L929 cells with 2 × 103Individual cell per well 100ul spreads 96 orifice plates, is positioned over 37 DEG C of CO2Trained in incubator
Fab samples and Hurmia foster to stay overnight, that gradient dilution purifies, the certain density TNF-αs of 50 μ L of addition and 50 μ L various concentrations
Fab samples, standard items are positive controls (Hurmia+ culture mediums) and TNF-α control group (TNF-α+culture medium),
37℃CO2CCK-8 dyeing 1-4h are added after 2d is incubated in incubator, are absorbed at ELIASA measure wavelength 450nm-650nm
It is worth (Fig. 7), data are handled using GraphPad Prism5 softwares, as a result shown, the Fab albumen energy that the present invention obtains
Specifically bound with antigen TNF-α and suppress its effect, and the no significant difference compared with standard items Humira.
Result above is shown, the higher adalimumab Fab antibody fragments of purity are prepared, and can be with antigen TNF-α
Specific binding, illustrates that the Fab fragments remain antibody characteristic and the specificity that Hurmia possesses.
Claims (10)
- A kind of 1. method using Insect Cell/Baculovrius Expresion System expression system Prepare restructuring adalimumab Fab fragments, it is characterised in that including：（a）N-terminal is respectively provided with to the gene cloning of the light chain of the Fab fragments of signal peptide and the amino acid sequence of heavy chain in building recombinant expression carrier in the rhabdovirus expression vector with double-promoter；（b）By the recombinant expression carrier swivel base of gained into competent cell, screening restructuring bacterial plaque, extract recombinant plasmid, and transfected into insect cell, obtain further amplicon virus after recombinant baculovirus, and the insect cell for amplifying culture is infected to express Fab albumen using virus, the supernatant product of the albumen containing Fab is collected by centrifugation；（c）Gained insect cell expression supernatant is concentrated by ultrafiltration and replaced cushioning liquid first by film bag, then chromatography, you can obtain full human monoclonal antibody adalimumab Fab fragments.
- 2. preparation method according to claim 1, it is characterised in that in step（a）In, the signal peptide of the heavy chain of Fab fragments and the N-terminal of light chain is all outer secreting signal peptide Ac NPV gp64.
- 3. preparation method according to claim 1 or 2, it is characterised in that in step（a）In, the heavy chain of Fab fragments and the promoter of light chain are all polyhedrosis gene promoter.
- 4. preparation method according to any one of claim 1 to 3, it is characterised in that in step（a）In, the recombinant expression carrier is pFastBacT1, including：Two promoters, replicon, polyclone enzyme enzyme site and two terminators.
- 5. preparation method according to any one of claim 1 to 4, it is characterised in that in step（b）In, the competence is DH10Bac, and swivel base bacterial plaque screening technique is blue hickie screening method.
- 6. preparation method according to any one of claim 1 to 5, it is characterised in that in step（b）In, the insect cell is Sf9 cells.
- 7. preparation method according to any one of claim 1 to 6, it is characterised in that transfection reagent is liposome cellfection, and used medium is Sf-900 in transfection procedureTMII SFM culture mediums.
- 8. preparation method according to any one of claim 1 to 7, it is characterised in that in step（b）In, the recombinant baculovirus P0 amplicon virus P1, i.e., it is 2 × 10 200 μ L recombinant baculovirus P0 to be added into 100 mL cell densities6In cells/ml Sf9 insect cells, the viral P1 of centrifugation acquisition after 37 DEG C of h of shaking table culture 96.
- 9. preparation method according to any one of claim 1 to 8, it is characterised in that in step（b）In, the culture medium used in amplification culture cell and expression is ESF AF culture mediums.
- 10. preparation method according to any one of claim 1 to 9, it is characterised in that in step（c）In, the purifying includes：Film bag is concentrated by ultrafiltration and replaced cushioning liquid；And affinity chromatography and molecular sieve medium chromatography；The affinity media of wherein described affinity column chromatography is Protein L；The sieve chromatography medium of the molecular sieve medium chromatography is SuperdexTM 75。
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