The fusion protein of a kind of horseradish peroxidase and antibody fragment and applicationTechnical field
The invention discloses a kind of fusion protein and immunology application thereof, belong to genetic engineering and immunityField.
Background technology
Antibody (Antibody, Ab) refers to class shape after antigenic substance stimulates body immune systemBecome, have and corresponding antigens material occur specific binding reaction immunoglobulin.Antibody isThe important products of immunne response, is primarily present in blood, tissue fluid and exocrine secretion, therefore willAntibody-mediated immunity is referred to as humoral immunization.Antibody molecule is produced by bone-marrow-derived lymphocyte, and it is by twoIdentical heavy chain and two identical and mammal camber conservative light chain groupBecome.The molecular wt of IgG is about 160kDa, and it is a complicated molecule, H chain and L chainLaunch to be formed respectively 4 and 2 domains.Two light chains of immunoglobulin and two heavy chains byDisulfide bond connects one four peptide chain molecule of formation, constitutes the monomer of immunoglobulin molecules.Immunity ballIn protein monomer, the amino of four peptide chain two end dissociatives or the direction of carboxyl are consistent, are calledAminoterminal (N end) and c-terminus (C end).At the N end of Ig monomer molecule, the 1/2 of light chain with1/4 sequence of amino acid of heavy chain changes with antibody specificity difference, therefore this region is called canBecome district (Variable region, V district).This V district gives the antibody specificity with conjugated antigen.?3/4 part of the C end of Ig polypeptide chain, remaining of light chain 1/2 and heavy chain, aminoacid quantity, kind,Put in order and sugar content is the most more stable, therefore referred to as constant region (Constant Region, C district).Constant region can be used as the skeleton of Ig, also has other important biologic activity many.At VOn some ad-hoc location of district, the composition of its amino acid residue and putting in order than other positions in variable regionThe amino acid residue put is more volatile, therefore these positions are called hypervariable region (HypervariableRegion, HV district), such as the 26th～32,48～55,90～95 3 position of light chain, heavy chainThe the 31st～37,51～68,84～91,101～110 four positions amino acid residue change spyThe most violent.Position, hypervariable region on light chain, heavy chain is roughly the same.In variable region, other changes are lessPart be referred to as backbone amino acid residue (Framework Residues, FR district).Ig is anti-with specialThe position that former determinant combines, just in hypervariable region, is complementary determining region district so present also known as hypervariable region(Complementary Reterminant Region, CDR).The hypervariable region of antibody, antibody anti-The idiotypic determinant of former binding site and antibody is present in identical Ig structure, i.e. antibody moleculeThe stereochemical structure of variable region spherical tips depression.
Polyclonal antibody (Polyclonal Antibody, PcAb) is to be produced by polyclone B cell groupMixed antibody that give birth to, for multiple antigenic determinant.Because native antigen is to be divided by multiple antigenMolecular, every kind of antigen molecule contains again many antigenic determinants, and each antigenic determinant canActivate corresponding B cell clone, so differentiation, ripe and synthesize corresponding antibody.Monoclonal antiBody (monoclonal antibody, McAb) refers to be activated by a B cell, breed, broken upThe daughter cell clones secrete produced, for the antibody of an antigenic determinant, i.e. by single BThe homogeneous antibody of cell clone synthesis.A kind of Ig sequence of amino acid complete phase of monoclonal antibodyWith, the affinity that its antigenic specificity combines with corresponding antigens is the most identical.
Within 1975, Koehler and Milstein uses cell-fusion techniques by mouse immune splenocyte with littleRat bone marrow tumour cell merges, and forms hybridoma.This hybridoma had both saved myelomaThe characteristic of cell Immortalization, has again immunized B cells synthesis and the ability of secreting specificity antibody.Then the technology such as limiting dilution assay are used can to pick out energy stably excreting antibody from hybridomaIndividual cells, promotes that its propagation becomes a cell clone, secrets out of the Dan Ke of homogeneity furtherGrand antibody.The hope that the foundation of hybridoma technology makes people produce a large amount of homogeneous monoclonal antibody becomesReality.Owing to monoclonal antibody has purity height, high specificity, titer height, can give birth in a large numberMany advantages such as product, have been widely used as diagnostic detection reagent, improve conventional immunological detection skillArt sensitivity, specificity, accuracy and detection speed in medical diagnosis on disease and analysis and research.Exempt fromEpidemiology detection method is exactly a series of mensuration antigens of applied immunology Design Theory, antibody, immunityThe experimental technique of the cytokine of cell and secretion thereof.Interpenetrate along with interdisciplinary, immunologyThe scope related to constantly expands, and new immunological detection method emerges in an endless stream.Answering of immunological methodBy scope also at expanding day, not only become the important method of various clinical medical diagnosis on disease, be also manyMulti-disciplinary research provides conveniently.For improving antigen and the sensitivity of antibody test, by known anti-The material of easily display on body or antigenic mark, by detection label, reflects anti-with or without antigen-antibodyShould, thus indirectly measure antigen or the antibody of trace.Conventional label has enzyme, fluorescein, putsInjectivity isotope, gold colloidal and electron dense substances etc., this antigen or antibody labeling show thingThe specific reaction carried out is referred to as immunolabelling technique (Immunolabelling Technique).Exempt fromEpidemic disease labelling not only substantially increases testability, if combining with light microscopic or electron microscopy, and can be rightTissue or intracellular test substance are accurately positioned, thus based on clinic study and examiningDisconnected offer is convenient.Immunolabelling technique is roughly divided into two big classes: a class belongs to immunohistochemistry skillArt, the location of antigen in tissue slice or other specimen.Another kind of referred to as immunoassay(Immunoassay), antigen or the mensuration of antibody, such as immunoenzyme technics in liquid sample(Immunoenzymatic Technique).The immunoenzyme technics applied the earliest is immunoenzyme systematismLearn dyeing, i.e. occur specific binding with the antibody of labelling with the antigen in specimen, when adding enzymeDuring substrate, under the effect of enzyme, produce coloring matter through a series of biochemical reactions, make by light microscopicLocation determination.At present, most widely used is elisa (Enzyme LinkedImmunosorbent Assay, ELISA).This method high specificity, sensitivity is high, both can detect anti-Body, can measure again soluble antigen.ELISA frequently with enzyme be horseradish peroxidase(Hosradish Peroxidase, HRP) or alkali phosphatase (Alkaline Phosphatase),Its substrate is diaminourea aniline (DAB) and 4-NPP salt (PNPP), the end respectivelyThing is decomposed, and develops the color, and can estimate or by microplate reader colorimetric.
Horseradish Peroxidase Conjugates, is by suitable with specific antibody warp for horseradish peroxidaseWhen method is formed by connecting.The quality of enzymic-labelled antibody depends primarily on the purity of enzyme, activity and antibodyAffinity, it is secondary good preparation method.At present, high-quality horseradish peroxidase,Domestic existing supply of commodities, is enzyme molecule isolated and purified from plant Radix Cochleariae officinalis.Horseradish peroxidaseEnzyme uses Over-voltage protection with antibody linked.Periodic acid method is first with NaIO4Enzyme is dividedThe glycan molecule of sub-surface is oxidized to aldehyde radical, combines with the amino on Ig the most again, obtained enzyme labellingThe productivity of antibody is up to 70%, and the antibody of 99% is combined with enzyme, is current most common method.?Needing in labeling process to use chemical reagent, can affect the biologic activity of enzyme and antibody, labelling is imitatedRate can not reach 100%, and wastes time and energy, and finally also needs to be purified enzyme labelled antibodyExcept unnecessary chemical reagent and unlabelled antibody and enzyme molecule, it is reacted to take pure from labellingThe enzyme labelled antibody changed needs the time at least 1 week.
Owing to conventional antibody molecule amount is big, reach 160kD, and light by 2 heavy chains and 2Chain is constituted, and after merging marker enzyme, molecular weight more than 200kD to be reached, the expression of antagonist can be producedRaw adverse influence, stability also can be deteriorated.Single-chain antibody is the one of genetic engineering antibody, isHeavy chain and the variable region of light chain of antibody by the one that flexible polypeptide is together in series, are had antigen and combineThe antibody fragment of activity.Being made up of single peptide chain, molecular weight is little, only about 30kD, Ke YiyongIn warm labelling horseradish peroxidase.ScFv fragment and complete antibody owing to obtaining have identicalAntigenic binding property, therefore, it can utilize the method labelling ScFv, exempted from by competitiveness enzyme-linkedEpidemic disease adsorption experiment, the concentration change of therapeutic antibodies in test experience animal or human serum, carry out medicineFor dynamic experiment.It addition, except traditional H in the serum of dromedary camel2L2Outside type IgG, alsoThere is a kind of natural antibody lacking light chain.This H2Type antibody homodimer is referred to as only heavy chainAntibody (HCAbs).The heavy chain of HCAbs can form three domains: N-end territory sequenceVariable region (VHH), hinge territory and two constant regions.With conventional antibodies in the heavy chain of H2 type antibodyThe part that first constant region (VH1) of heavy chain is of equal value has lacked, referred to as single domain antibody.GoExcept the variable region sequences behind constant region, molecular weight only has 15kD, the diameter of about 10 nanometers, quiltIt is referred to as nano antibody (Nbs).The similar functional antibodies lacking light chain is equally with the shape of mixtureState is present in nonmammalian such as shark and ratfish.With conventional antibodies fragment, such as Fab, ScFvEtc. comparing, Nbs is little because of its relative molecular mass, and has the character of some uniquenesses, such as immunogenThe property characteristic such as low, solubility good, stability is strong, penetrance is strong, easy expression.
There is labeling effciency based on prior art in labelling technique low, the biology of enzyme and antibody livesProperty because the impact of chemical reagent target the technical problem that declined, therefore, invention one can be protectedThe immunolabelling technique of the biologic activity holding enzyme and antibody has become the reality of the art to be neededAsk, to realize the most quickly immune detection.
It is an object of the invention to provide the biologic activity of a kind of enzyme and antibody not by chemical labelingImpact, can realize again the most immune labeled and detection function fusion protein and methods for using them.
Summary of the invention
Based on foregoing invention purpose, inventor is intended to use the mode of DNA recombinant expression, expresses anti-Body and the recombination fusion protein of marker enzyme, from culture medium supernatant or the broken engineering of engineering cellDirect purification antibody and the fusion protein of marker enzyme in Cytoplasm, it is thus achieved that enzyme labelled antibody, be used for being correlated withImmunology detection, specifically, it is simply that utilize the nano antibody of anti-hen egg-white lysozyme, preparationThe nano antibody marker enzyme fusion protein of detection hen egg-white lysozyme concentration, and it is applied to realityImmune detection in.
Therefore, present invention firstly provides a kind of fusion protein, described fusion protein is by anti-lysozymeAntibody or its variable region fragment are in series with horseradish peroxidase.Fusion egg of the present inventionIt is by the gene of encoding antibody or antibody variable region fragment and coding Radix Cochleariae officinalis in vain on DNA levelAfter the reporter gene fusion of peroxidase, by recombinant expressed method, as escherichia coli,The recombination fusion protein expressed in yeast, insect cell or mammalian cell.
Antibody or antibody fragment and the amalgamation mode of reporter gene protein, can be at antibody fragmentAminoterminal or c-terminus merge one or several reporter gene protein, it is also possible at antibody fragmentAminoterminal or c-terminus merge one or several reporter gene protein respectively, or at report baseBecause the aminoterminal of albumen or c-terminus merge the gene of one or several antibody fragments respectively.?Two ends are merged in the fusion protein of reporter gene protein or antibody fragment respectively, described fusion proteinIn reporter gene protein and antibody fragment can be different reporter gene protein or different antibodyFragment, is respectively directed to the different antigenic determinants of same antigen or different antigen molecules.
In a preferred embodiment, the aminoacid sequence of described nano antibody variable region is such asShown in SEQ ID NO:1.
In another preferred embodiment, the aminoacid sequence of described horseradish peroxidase is such asShown in SEQ ID NO:3.
Intervening sequence can be added between antibody fragment and horseradish peroxidase so that antibody fragment andReporter gene can each express and be formed the steric configuration of self, does not cause antibody fragment and reportGene protein sterically hindered, all has corresponding biologic activity, and intervening sequence can be(GGGGS)nFlexible polypeptide or other intervening sequence.
In a preferred embodiment, the variable region of described nano antibody and reporter gene proteinBy connection peptides (GGGGS)nConnect, the wherein any integer in the 1-6 of n position
Preferably, the variable region of described nano antibody may be located at the aminoterminal of fusion protein, describedReporter gene protein is positioned at the c-terminus of fusion protein, n=3.
Preferably, the variable region of described nano antibody can also be positioned at the c-terminus of fusion protein, instituteState reporter gene protein and be positioned at the aminoterminal of fusion protein, n=3.
Currently preferred sequence is (GGGGS)3As antibody fragment and reporter gene proteinIntervening sequence.For the ease of purified fusion protein, can increase many in the optional position of fusion proteinPolyhistidyl tags, the recombination fusion protein after expression can use the side of nickel post metal chelate chromatographyMethod is purified.
Second, present invention also offers a kind of nucleotide sequence encoding above-mentioned fusion protein, describedNucleotide sequence is as shown in SEQ ID NO:5 or 6.
3rd, the invention provides a kind of carrier containing above-mentioned nucleotide sequence, described carrier ispCDNA3.1。
4th, present invention also offers a kind of cell containing above-mentioned carrier, described cell is 293TCell.
5th, the invention provides a kind of method preparing above-mentioned fusion protein, described method includes:
(1) variable region and the Radix Cochleariae officinalis of the nano antibody of the anti-lysozyme of pCDNA3.1 construction expression are usedThe fusion expression vector of peroxidase, the core of the nano antibody variable region of wherein said anti-lysozymeThuja acid coded sequence is SEQ ID NO:2, the nucleotide coding sequence of described horseradish peroxidaseFor SEQ ID NO:4；
(2) vector that step (1) obtains is entered host cell 293T；
(3) described fusion protein is gathered in the crops.
Finally, the invention provides above-mentioned fusion protein at the targeted lysozyme antigen of antibody fragmentVitro detection in application, as Enzyme-linked Immunosorbent Assay reaction (EIA), enzyme linked immunological double antibody folderHeart method (ELISA), SABC and immunofluorescence experiment or Western detection, and antibodyThe dynamic (dynamical) test experience of drug metabolism in vivo.
In traditional immunolabelling technique, horseradish peroxidase-labeled is residual at the lysine of albumenBase location.In the antigen binding domain of nano antibody, lysine is generally present in CDR region, is antigenCalmodulin binding domain CaM, therefore after labelling horseradish peroxidase, nano antibody and antigen may have been blockedCombination, cause developing the color, the most traditional labelling technique be difficult to apply to nano antibody thisOn novel antibodies.And the fusion protein that the present invention provides overcomes this technological deficiency, specific sequenceRow combination ensure that in fusion protein, nano antibody and horseradish peroxidase each defining space structureType so that it is each there is corresponding biologic activity.The fusion protein of the present invention has lysozyme and receivesRice antibody recognition and the ability of conjugated antigen, and the horseradish peroxidase merged can act on itSubstrate DAB develops the color, and the lysozyme nano antibody horseradish peroxidase fusion protein therefore obtained canUsing as the enzyme labelled antibody for lysozyme, for the immune detection of lysozyme concentration；Can also makeFor the competitive antibody of lysozyme nano antibody, for internal pharmacokinetic studies.The present invention willLysozyme nano antibody HRP fusion protein is the research of pharmacokinetics in nano antibody body,The standard curve of the nano antibody serum-concentration detection set up shows, in the concentration of 0.1-10ug/mlIn the range of, the concentration of absorbance and nano antibody is good dependency.Meanwhile, competitiveness enzyme-linkedThe detection display of immunoadsorption method, after injection lysozyme nano antibody, lysozyme in rat blood serumThe concentration of nano antibody is the concentration rapid decrease in serum after 30 minutes, substantially completely generation after 3 hoursThank.Demonstrate owing to the molecular weight of nano antibody is relatively low, there is shorter serum half-life shorter,This result meets the technique effect expection of nano antibody, the most fully demonstrates fusion protein of the present inventionProspect in actual applications.
Accompanying drawing explanation
Fig. 1. the structural representation of fusion protein of the present invention；
The enzyme action of Fig. 2 .pCDNA-NHP1 and pCDNA-NHP2 identifies collection of illustrative plates；
The SDS-PAGE spectrum of Fig. 3 .NHP-1 and NHP-2 fusion protein；
Fig. 4. lysozyme nano antibody horseradish peroxidase fusion protein enzyme linked immunosorbent detection resultFigure；
Fig. 5. rat blood serum lysozyme nano antibody Concentration Testing canonical plotting；
Fig. 6. the pharmacokinetic of lysozyme nano antibody in rat blood serum.
Detailed description of the invention
Further describing the present invention, advantages of the present invention and feature below in conjunction with specific embodiment willCan be apparent along with description.But these embodiments are only exemplary, not to the present invention'sProtection domain constitutes any restriction.
Embodiment 1: preparation embodiment
1. according to aminoacid sequence and the SEQ ID of the lysozyme nano antibody of SEQ ID NO:1The aminoacid sequence of NO:3 horseradish peroxidase, optimizes to obtain and expresses in mammalian cellLysozyme and horseradish peroxidase cDNA sequence SEQ ID NO:2 and SEQ ID NO:4.
2. in lysozyme nano antibody and horseradish peroxidase sequence, add flexible peptide linker(GGGGS)3, the DNA sequence SEQ ID NO of acquisition expression NHP-1 and NHP-2 respectively:5 and SEQ ID NO:6.Two kinds of DNA sequence of synthetic.Fig. 1 shows described fusion eggThree kinds of white connected modes, the aminoacid at described fusion protein can also have signal peptide, be beneficial toThe secreting, expressing of fusion protein.
The most respectively NHP-1 and NHP-2 gene is cloned into carrier for expression of eukaryon pCDNA3.1'sIn EcoRI and HindIII restriction enzyme site, it is thus achieved that expression vector pCDNA-NHP1 andPCDNA-NHP2 (Fig. 2).In Fig. 2, the pCDNA-NHP1 of the most non-enzyme action；2.AflII/AvrIIPCDNA-NHP1 after double digestion；The pCDNA-NHP2 of the most non-enzyme action；4.AflII/AvrII is doublePCDNA-NHP2 after enzyme action.
The transfection of 4.293T cell: cultivated the 293T cell of 48 hours, discarded in culture dishCulture medium, wash twice by the PBS solution of 1ml.
5. with Tip head add 1ml Trypsin liquid, digest 1 minute (37 DEG C, 5%CO2)。Culture bottle wall is patted, it was observed that till cell splits away off completely from wall with hands.Add 1ml'sReaction is terminated containing blood serum medium.With Tip head repeatedly pressure-vaccum, cell is made to be completely dispersed.
6. loading in centrifuge tube by culture fluid, 1000rpm is centrifuged 5min.Resuspended carefully with culture fluidBorn of the same parents, select 0.8 × 10 after cell counting6Individual cell adds a 35mm culture dish.By suitable bodyLong-pending complete culture solution adds in centrifuge tube, adds gently in culture dish so that it is uniformly after mixing cellDistribution.Culture dish is proceeded to CO2Incubator is cultivated, transfection in second day.
7. the preparation of transfection reagent: 400ul goes nuclease water add in pipe, shakes 10 secondsClock, dissolves smectic thing.After concussion, reagent is placed on-20 DEG C of preservations, before using, also needs concussion.ChoosingSelect proper mixture ratio example (1:1-1:2/ liposome volume: DNA mass) and carry out transfectional cell.The serum-free medium of suitable volumes is added in a transfection pipe.Add the DNA of appropriate mass,Adding the transfection reagent of suitable volumes after concussion, again shaking.Mixed liquor is placed in room temperature10 15 minutes.
8. suck the culture medium in culture plate, clean once with PBS or serum-free medium.Add mixed liquor, cell is put back in incubator and cultivate one hour.After then, remove mixed liquor,Add afterwards and continue to cultivate 48-72 hour entirely without blood serum medium.
9. the SDS-PAGE electrophoresis detection of nano antibody horseradish peroxidase fusion protein: takeThe 293T cell culture medium supernatant that 10 microlitres transfect after cultivating 3 days, adds 10 microlitres reduction respectively and (containsBeta mercaptoethanol) and the sample-loading buffer of non-reduced electrophoresis (without beta mercaptoethanol), boilBoil 5 minutes, after ice bath cooling, the SDS-polyacrylamide gel of upper 15%, 120V electrophoresis 30-40Minute to bromophenol blue arrive glue bottom, take off gel, stained over night in Coomassie Brillant Blue solution,The expression of recombiant protein is observed after decolouring.(seeing Fig. 3).
The SDS-PAGE electrophoresis detection of nano antibody horseradish peroxidase fusion protein: take 10 micro-Rise the 293T cell culture medium supernatant of transfection after cultivating 3 days, add 10 microlitres respectively and reduce (containing betaMercaptoethanol) and the sample-loading buffer of non-reduced electrophoresis (without beta mercaptoethanol), boil 5Minute, after ice bath cooling, the SDS-polyacrylamide gel of upper 15%, 120V electrophoresis 30-40Minute to bromophenol blue arrive glue bottom, take off gel, stained over night in Coomassie Brillant Blue solution,The expression (seeing Fig. 3) of recombiant protein is observed after decolouring.In Fig. 3,1.293T cell conditioned medium(reduction)；2.293T cell conditioned medium (non-reduced)；3.NHP-1 transfects 293T cell conditioned medium(reduction)；4.NHP-1 transfectional cell supernatant (non-reduced)；5.NHP-2 transfectional cell supernatant is (alsoFormer)；6.NHP-2 transfectional cell supernatant (non-reduced).Tie from the SDS-PAGE electrophoresis of Fig. 3Fruit is it can be seen that NHP-1 and NHP-2 fusion protein has in the culture medium supernatant of 293T cellSignificantly secreting, expressing, purity is the highest, and the most non-reducing albumen mobility relatively reduces albumenMobility is higher, illustrates that in fusion protein, nano antibody and horseradish peroxidase each defining skyBetween configuration, it should there is corresponding biologic activity.
The detection of embodiment 2. fusion protein immunization activity
Take hen egg-white lysozyme, be configured to the solution of 10ug/ml with PBS, take 96 hole ELISA Plate,Every hole 100 microlitre is coated overnight.Within second day, rinse each hole with PBST, with the BSA solution of 0.5%Close 1 hour, take the 293T cell culture medium supernatant after 100 microlitre transfections, add in hand-hole, roomTemperature lucifuge is placed 1 hour, and PBST rinses 3 times, adds 100 microlitre DAB chromogenic substrates, room temperatureLucifuge is reacted 15 minutes, visual observations colour developing situation (seeing Fig. 4).In Fig. 4,1. blankNHP-1 transfects 293T cell conditioned medium；2. lysozyme be coated 4 × NHP-1 transfection 293T is thin in dilutionBorn of the same parents' supernatant；3. lysozyme is coated 10 × dilution NHP-1 and transfects 293T cell conditioned medium；4. lysozyme bag293T cell conditioned medium is transfected by 4 × dilution NHP-2；5. lysozyme is coated 10 × dilution NHP-2Rotaring redyeing 293 cell supernatant；6. blank NHP-2 transfects 293T cell conditioned medium.
Figure 4, it is seen that in the hole being coated lysozyme, have horseradish peroxidaseSpecific color, and in control wells, not colour developing, it was demonstrated that fusion protein has lysozyme nanometer and resistsBody identification and the ability of conjugated antigen, and the horseradish peroxidase merged can act on its substrateDAB develops the color, and the lysozyme nano antibody horseradish peroxidase fusion protein therefore obtained can be madeFor the enzyme labelled antibody for lysozyme, for the immune detection of lysozyme concentration.Can also be as moltenThe competitive antibody of bacterium enzyme nano antibody, for internal pharmacokinetic studies.
The horseradish peroxidase chemical labeling of embodiment 3. nano antibody
5mg horseradish peroxidase is dissolved in 0.5ml 0.1mol/L NaHCO3In solution；Add0.5ml 10mmol/L NaIO4Solution, mixing, cover tightly bottle stopper, room temperature lucifuge effect 2 hours.Add0.75ml 0.1mol/L Na2CO3Mixing, is subsequently adding the anti-lysozyme nano antibody of 0.75ml purification(15mg/ml), mixing.Weigh Sephadex G25 dry powder 0.3g, add an end opening pad glassIn cotton 5ml syringe urceolus；Subsequently above-mentioned cross-linking agent is moved into syringe jacket；Cover tightly, roomTemperature effect (lucifuge) 3 hours or 4 DEG C is overnight.With a little PBS, cross-linking agent is all washed out, collectEluate, adds 1/20 volume freshly prepared 5mg/ml NaBH4 solution, mixing, room temperature effect30 minutes；Add 3/20NaBH4Solution, mixing, room temperature effect 1 hour (or 4 DEG C overnight).WillCross-linking agent proceeds in the super filter tube that 30kD molecular weight retains, and 4 DEG C 3000 leaves the heart 30 minutes, addEqual-volume PBS, continues centrifugal 3 times, removes labelled reagent and unlabelled nano antibody, it is thus achieved thatThe lysozyme nano antibody of horseradish peroxidase-labeled.
The enzyme-linked immunosorbent assay of the lysozyme nano antibody of horseradish peroxidase chemical labeling:Method as described in step 10, becomes the solution of 10ug/ml, often with PBS preparation hen egg-white lysozymeHole 100 microlitre is coated and takes 96 hole ELISA Plate overnight.After washing is closed, each hole adds 1:10 respectively, and 1:HRP marking nano antibody 100 microlitre after purification of 100 and 1:1000 dilutions, room temperature lucifugePlacing 1 hour, PBST rinses 3 times, adds 100 microlitre DAB chromogenic substrates, and room temperature lucifuge is anti-Answer 15 minutes, visual observations colour developing situation.
Each hole is showed no significantly colour developing.Owing to HRP is the lysine residue position being marked at albumenPutting, in the antigen binding domain of nano antibody, lysine is generally present in CDR region, is the knot of antigenClose region, therefore after labelling HRP, the combination of nano antibody and antigen may have been blocked, the most notCan colour developing.
In embodiment 4. lysozyme nano antibody HRP fusion protein medicine generation in nano antibody body, is dynamicThe research of mechanics
Take rat blood serum, be separately added into 0.1, the bacteriolyze of 0.5,2,10,50 and 200ug/ml concentrationEnzyme nano antibody.Take hen egg-white lysozyme, be configured to the solution of 10ug/ml with PBS, take 96 holesELISA Plate, every hole 100 microlitre is coated overnight.Within second day, rinse each hole with PBST, with 0.5%BSA solution is closed 1 hour, after being blended with the rat blood serum dilution 50 times of lysozyme nano antibody,Taking 50 microlitres respectively and join in 96 orifice plates, each concentration repeats 3 holes, in each hole,Add 50 microlitres and contain 10ug/ml lysozyme nano antibody horseradish peroxidase fusion proteinPBS, incubated at room 2 hours.After PBST washs 96 orifice plate 3 times, add DAB and showColor, after 0.2M sulphuric acid terminates reaction, reads absorbance under 450nm wavelength condition, and foundation is receivedThe standard curve (seeing Fig. 5) of rice antibody sera concentration detection, from figure 5 it can be seen thatIn the concentration range of 0.1-10ug/ml, the concentration of absorbance and nano antibody is good dependency.
Take the Wistar rat of body weight about 200 grams, according to the dosage of 10mg/kg body weight, pass throughThe method of tail vein injection, injects lysozyme nano antibody to rat.Blood is taken once every 30 minutes,Take 8 times altogether.According to lysozyme in aforesaid competitiveness enzyme-linked immunoadsorption method detection rat blood serumThe concentration of nano antibody.Owing to the molecular weight of nano antibody is relatively low, can be fallen by renal excretion,Therefore its serum half-life is shorter, the concentration rapid decrease in serum, base after 3 hours after 30 minutes(seeing Fig. 6) is fallen in this metabolism completely.