CN106478824B - Accurate Fc site covalent coupling labeled biotinylated antibody - Google Patents

Accurate Fc site covalent coupling labeled biotinylated antibody Download PDF

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CN106478824B
CN106478824B CN201610897782.2A CN201610897782A CN106478824B CN 106478824 B CN106478824 B CN 106478824B CN 201610897782 A CN201610897782 A CN 201610897782A CN 106478824 B CN106478824 B CN 106478824B
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杨洪鸣
唐金宝
鲍如梦
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Abstract

The invention discloses a biotinylated antibody marked by precise Fc site covalent coupling, which uses genetic engineering technology to express Z affinity peptide-Avitag fusion protein with photosensitive group (Bpa) and uses BirA enzyme catalysis to complete biotin covalent fixed-point coupling, thus obtaining a biological reagent Z with precise Fc site couplingBpaAnd the Biotin is guided by the affinity peptide to Fc site for affinity coupling, and then the photosensitive group is excited by 365nm ultraviolet light to produce a covalent bond with the antibody, so that Biotin covalent coupling labeling of the Fc site is realized. The biotinylated antibody of the precise Fc site is an irreversible covalent coupling of the Fc site, is not influenced by pH, temperature, organic solvent and denaturant, can be used for fixing an IgG antibody on the surface of a carrier coated with avidin, is a three-dimensional directional IgG antibody with uniform Fab section fully exposed and high antigen binding activity, and breaks through the limitation that Z affinity peptide substances cannot be combined in an analyte in practical application.

Description

Accurate Fc site covalent coupling labeled biotinylated antibody
Technical Field
The invention relates to the field of genetic engineering and immunoassay, in particular to a labeling technology of biotin precise covalent coupling antibody Fc sites.
Background
The biotin-avidin system (BAS) is a new biological reaction amplification system applied to immunology in the later 70 s and developed rapidly. The affinity between avidin and biotin is extremely strong, and the affinity constant (Ka) of the two combinations is 1015mol-1Once bound, avidin and biotin are not affected by pH, temperature, organic solvents and denaturants (dissociation is only possible with denaturants at > 90 ℃). Because it has high affinity and multi-stage amplification effect between biotin and avidin, and is organically combined with immunological labeling techniques of fluorescein, enzyme and isotope, etc., the specificity and sensitivity of various immunological analyses can be further raised. The biotin coupling labeling antibody, namely the biotinylation antibody is the application premise of the BAS system, and the preparation technology of the biotinylation antibody adopted at present is based onIn the covalent coupling technology between various biotin derivatives (activated biotin) and amino acid residues (amino and carbonyl) in an antibody molecule, one antibody molecule can connect a plurality of biotin molecules, the biotin molecules cannot be specifically coupled to an Fc segment of the antibody due to the non-uniqueness of the number and the position of active amino acid residues in the antibody molecule, and when the biotin molecules are randomly coupled to a Fab segment of the antibody, the antigen binding of the antibody is hindered, so that the antigen binding activity of the antibody is reduced.
Indirect labeling of the Fc-terminus of antibodies can be achieved using certain antibody affinity proteins, such as staphylococcal protein a (spa) and its derivatives (ZZ affinity peptide), which bind to the Fc portion of various animal iggs via hydrophobic interactions, and which do not affect the immunological activity of the IgG Fab portion for specific binding to antigens. ZZ affinity peptides are repeats of the B domain derived from staphylococcal protein a, each ZZ affinity peptide molecule binding two IgG molecules, and a single Z sequence (Z affinity peptide) binding one heavy chain of an IgG molecule, and thus one IgG molecule binding two single Z sequences. The biotin affinity coupling of the Fc end of the antibody is realized by using a ZZ affinity peptide biotinylated at a fixed point (patent CN103694358B, site-specific biotin labeling recombination and application thereof in three-dimensional directional immobilization of IgG antibodies). However, the binding between the affinity peptide and the IgG is a reversible biological binding, and the antibody-directed immobilization technology is applied to immunoassay based on a chip technology, and the affinity peptide-IgG conjugate is dissociated during the regeneration of the chip and needs to be repeatedly bound with the capture antibody; more importantly, if the clinical sample analysis contains IgG class substances combined with the affinity peptide, the analysis result is seriously interfered, and the application range of the antibody directional fixing technology mediated by the affinity peptide is limited.
Disclosure of Invention
Aiming at the prior art, the invention uses the genetic engineering technology to express the Z affinity peptide-Avitag fusion protein with photosensitive groups (4-benzoyl-L-phenylalanine, p-benzoylphenylalanine, Bpa for short) and uses the enzyme BirA to catalyze the biotin covalent fixed-point coupling to obtain the biological reagent Z with accurate Fc site couplingBpaBiotin, affinity by ZAfter the peptide is guided to the Fc site for affinity coupling, a photosensitive group is excited by 365nm ultraviolet light to produce a covalent bond with the antibody, so that the biotin covalent coupling labeled biotinylated antibody of the Fc site is realized. Because one IgG molecule can be combined with two single Z sequences, the biotinylated antibody of the precise Fc site provided by the invention is a Biotin molecule which can be quantitatively combined with two Biotin molecules by each IgG molecule, is an irreversible covalent coupling Biotin antibody, is not influenced by pH, temperature, organic solvent and denaturant, can realize that the IgG antibody fixed on the surface of a solid phase carrier is a three-dimensional directional IgG antibody which is uniform, fully exposes a Fab section and keeps high antigen binding activity, and breaks through the limitation that Z affinity peptide substances cannot be combined in an analyte in practical application.
The invention utilizes aminoacyl tRNA synthetase/amber suppressing tRNA (aminoacyl-tRNA synthetase/amber supressor tRNA, aaRS/supressor tRNA) technology to realize biosynthesis of target protein containing photosensitive group in the protein translation process.
The technical scheme adopted by the invention is as follows:
the first purpose of the invention is to provide a biological reagent (Z for short) for precisely guiding Fc site couplingBpaThe biological reagent comprises a Z affinity peptide, and is characterized in that 4-benzoyl-L-phenylalanine is connected to the α 1 domain of the Z affinity peptide, the carboxyl terminal residue of the Z affinity peptide is sequentially connected with an Avi-tag protein and a polyhistidine purification tag protein, and Biotin or a derivative thereof is connected to a lysine residue on the Avi-tag protein.
Preferably, the site of the α 1 domain to which the 4-benzoyl-L-phenylalanine is attached is at amino acid position 17 in terms of the effect of coupling to IgG antibodies after light irradiation, position 17 refers to a position 17 amino acid after the 16 th amino acid of the Z affinity peptide.
Preferably, the polyhistidine purification tag protein is 6 polyhistidine (6 × His).
The second purpose of the invention is to provide a preparation method of the biological reagent with the accurate Fc site coupling, which comprises the following steps:
(1) first, a recombinant plasmid pZ was constructedTAGThe synthesis of the target gene comprises the steps of firstly inserting amber codons (TAG) at required sites of α 1 domain sequences of Z-avidity peptide gene sequences according to the Z-avidity peptide gene sequences, then placing the Avitag gene sequences at the downstream tail ends of the Z-avidity peptide gene sequences and polyhistidine purification TAG gene sequences, finally respectively setting restriction enzyme cutting sites at the 5 'end and the 3' end of the gene sequences, and chemically synthesizing to obtain the target gene sequences;
(2) then, prokaryotic microorganism is utilized to express recombinant protein ZBpa-Avitag；
(3) Finally, biotin ligase (BirA enzyme) is used for recombining protein ZBpaBiotinylation of Avitag to give a biotinylated recombinant protein ZBpa-Avitag, i.e. the biological agent (Z)Bpa-Biotin)。
In step (1), the preparation of the recombinant plasmid expression vector is a conventional technical means known to those skilled in the art. The method comprises the following basic steps: after a target gene sequence is chemically synthesized, the target gene sequence is cloned into a plasmid, and a corresponding plasmid expression vector can be obtained. In a preferred embodiment of the present invention, the desired gene sequence is subcloned into pTBX1 plasmid to obtain recombinant plasmid pZTAG-an Avitag expression vector.
Preferably, the polyhistidine purification tag gene sequence is a 6-polyhistidine gene sequence.
The restriction enzyme is not particularly limited and may be set as required. In a preferred embodiment of the invention, an NdeI restriction enzyme cutting site is arranged at the 5 'end of the target gene, and an XhoI restriction enzyme cutting site is arranged at the 3' end of the target gene; in a preferred embodiment of the present invention, the sequence of the target gene is:
Figure BDA0001131080570000031
Figure BDA0001131080570000032
as shown in SEQ ID NO. 1. Wherein, the bases underlined in double are amber codons, the sequences underlined in single are restriction sites, the bold sequence represents the 6-polyhistidine gene sequence, and the italic sequence represents the amino acid gene sequence of Avi-tag.
In step (2), the recombinant protein ZBpa-Avitag comprises a Z affinity peptide having 4-benzoyl-L-phenylalanine attached at the α 1 domain, the Z affinity peptide having an Avi-tag protein and a polyhistidine purification tag protein attached sequentially at the carboxy-terminal residue.
In order to realize the biological synthesis of target protein containing photosensitive group in the translation process of target protein, the expression vector in the step (1) and pEVOL-pBpF containing aminoacyl tRNA synthetase coding gene are co-transformed into prokaryote to obtain genetically engineered bacteria. The recombinant protein Z containing photosensitive group can be successfully expressed by utilizing the genetic engineeringBpa-Avitag. For successful synthesis of target proteins containing a photosensitive group, the aminoacyl tRNA synthetase/amber tRNA (aminoacyl-tRNAsynthitase/amber supressor tRNA) technique has been a relatively conventional technique. The aaRS/supppress RNA technology is utilized to translate the protein without stopping at amber codon (TAG) and translate into unnatural amino acid (generally, unnatural amino acid containing special group, such as Bpa contains photosensitive group), and the peptide chain continues to translate into the target protein.
The pEVOL-pBpF plasmid of the present invention is routinely obtained by those skilled in the art, and is commercially available, for example.
Culturing and activating the genetically engineered bacteria at 30-37 ℃ for 12-18 h, transferring the genetically engineered bacteria to a 2 XYT culture medium, culturing at 37 ℃ until OD600 is 0.6-0.8, adding 4-benzoyl-L-phenylalanine, continuously culturing for 0.5-2 h, adding IPTG (isopropyl- β -D-thiogalactoside) and arabinose, carrying out induction culture at 30-37 ℃ for 5-7 h, centrifugally collecting bacteria by the cultured engineered bacteria, carrying out resuspension by using a buffer solution, carrying out freeze thawing to crack the bacteria, centrifugally removing bacteria fragments, and purifying the target protein by using a chromatographic column.
Wherein the 2 XYT medium is a medium conventionally known in the art.
In the step (3), the birA enzyme in-vitro reaction system is utilized to carry out the reaction on the recombinant protein ZBpaBiotinylation by Avitag, and obtaining the biotinylated recombinant protein, namely Z, by chromatography column and ultrafiltration centrifuge tubeBpa-Biotin。
The BirA enzyme in-vitro reaction system comprises the following components: buffer A, buffer B, ZBpa-Avitag, biotin ligase, reacting for 5-7 h at 25-35 ℃; the buffer A is 0.5M bicine, pH 8.3; the buffer B is a solution with pH8.0, and contains 100mM ATP, 100mM magnesium acetate and 200mM biotin.
Preferably, the buffer A, the buffer B and the ZBpaThe volume ratio of Avitag is 1:4, and the addition amount of the BirA enzyme is 40-50U/mu L.
The third purpose of the invention is to provide a biotinylated antibody coupled with a precise guide Fc site, wherein the antibody is formed by connecting 4-benzoyl-L-phenylalanine guided by Z affinity peptide in the biological reagent with an IgG antibody Fc section through a covalent bond.
A fourth object of the present invention is a method for preparing the precisely targeted Fc site conjugated biotinylated antibody, comprising the steps of: and mixing the IgG antibody with the biological reagent, leading to the bioaffinity coupling of the Fc site, and then exciting a photosensitive group to generate a covalent bond with an amino group of an Fc segment of the IgG antibody under the irradiation of 365nm ultraviolet light to obtain the biotinylated antibody accurately leading to the Fc site coupling.
The fifth purpose of the invention is to provide the application of the biological reagent in three-dimensional directional immobilization of IgG antibodies. The application method comprises the following steps: the 4-benzoyl-L-phenylalanine in the biological reagent is connected with the Fc segment of the IgG antibody through a covalent bond, and the IgG antibody is fixed in a three-dimensional orientation manner through the action of biotin-avidin.
The method comprises the following specific steps:
mixing IgG antibody and the biological reagent according to the molecular number ratio of 1: 4-6, oscillating at room temperature for 20-40 min, placing the reaction system on ice, irradiating for 2-4 h by 365nm ultraviolet, and ultrafiltering the irradiated productCentrifuging the tube to remove excess ZBpaAnd a Biotin molecule, namely obtaining the biotinylated antibody coupled with the precisely oriented Fc site.
The room temperature is 18-37 ℃.
The sixth purpose of the invention is to provide an immune sensor chip, which comprises a substrate slice, wherein avidin is fixed on the substrate slice, and the avidin is combined with biotin in the biotinylated antibody.
The avidin immobilized on the substrate sheet is common knowledge in the art, and is not described herein again. The substrate sheet is not particularly limited, and one embodiment of the present invention is a CM5 chip, which is conventionally available. In a specific embodiment of the invention, the avidin is Streptavidin (SA).
The chip can be used for analyzing and measuring biological samples, and mainly refers to detecting relevant antigens, wherein the relevant antigens refer to substances capable of being specifically combined with IgG antibodies, such as: carcinoembryonic antigen (CEA), Alpha Fetoprotein (AFP) and other disease markers.
It is known from the background art that one antibody molecule can link a plurality of biotin molecules based on covalent coupling technology between various biotin derivatives (activated biotin) and amino acid residues (amino groups and carbonyl groups) in the antibody molecule, but biotin and active groups in the antibody molecule are in a random combination mode, when the amino acid residues of the reactive groups are positioned at or near the Fab of the antibody, the Fc end of the immobilized biotinylated antibody is outwards caused to cover the Fab section, and the adsorbed antibody cannot be guaranteed to be a directionally immobilized antibody.
The invention uses gene engineering technology to express Z affinity peptide-Avitag fusion protein with photosensitive group and BirA enzyme catalysis to complete ZBpa-biotin covalent site-directed coupling of the Avitag fusion protein itself to obtain a Z capable of precisely directing Fc site couplingBpaBiotin, followed by a Biotin covalently labeled antibody of the Fc site under 365nm uv excitation. The biotinylated antibody of the precise Fc site provided by the invention is an irreversible covalent coupling of the Fc site, is not influenced by pH, temperature, organic solvent and denaturant, and canThe IgG antibody fixed on the surface of the avidin-coated carrier is a three-dimensional directional IgG antibody with the advantages of uniformity, fully exposed Fab section and high antigen binding activity, and the limit that Z affinity peptide substances cannot be bound in an analyte is broken through in practical application.
Drawings
FIG. 1ZBpa-Biotin-mediated precise Fc site biotinylated antibody schematic.
FIG. 2ZBpa-Biotin precision-conjugated antibody Fc-site electrophoresis and Western analysis.
FIG. 3 is a schematic diagram of the three-dimensional directional immobilization and application of photo-biotinylated IgG.
FIG. 4 schematic representation of three-dimensional directional immobilization and application of NHS-biotinylated IgG.
FIG. 5A comparison of the assay performance of immobilized photo-biotinylated IgG and NHS-biotinylated IgG.
Detailed Description
Unless otherwise specified, the reagents and the test method are conventional reagents and conventional methods.
Interpretation of terms in the present invention:
fab section: antigen-binding fragments (Fab), corresponding to the two arms of an antibody molecule, consist of one complete light and heavy chain VHAnd CH1Domain composition.
Fc fragment/Fc: a crystallizable fragment (Fc) corresponds to the C of IgH2And CH3The domain is the site of interaction of Ig with effector molecules or cells.
ZZ affinity peptide: is two artificially synthesized repeated sequences derived from staphylococcal protein A and can be combined with an IgG antibody Fc segment.
Z affinity peptide: is a single sequence artificially synthesized from staphylococcal protein A and can be combined with an IgG antibody Fc segment.
The α 1 domain of the Z affinity peptide refers to the 4 th to 18 th positions of the amino acid sequence of the Z affinity peptide.
The Avi-tag is a short peptide tag consisting of 15 amino acid residues, and biotin can be connected to a lysine residue by biotin ligase in vivo or in vitro, so that biotinylation of the protein is realized.
The present invention will be further described with reference to the following examples.
As shown in FIG. 1, a precisely oriented Fc site coupled biological reagent (Z for short)BpaThe biological reagent comprises a Z affinity peptide, and is characterized in that 4-benzoyl-L-phenylalanine is connected to the α 1 domain of the Z affinity peptide, the carboxyl terminal residue of the Z affinity peptide is sequentially connected with an Avi-tag protein and a polyhistidine purification tag protein, wherein Biotin or a derivative thereof (the derivative thereof also refers to activated Biotin) is connected to a lysine residue on the Avi-tag protein.
EXAMPLE 1 recombinant plasmid pZTAGConstruction of Avitag expression vector
In order to realize biosynthesis of target protein containing photosensitive groups in the translation process of the protein by utilizing an aaRS/supressor tRNA technology and realize covalent coupling of biotin at specific sites of the target protein by utilizing a BirA/Avitag technology:
firstly, according to the gene sequence of the Z affinity peptide, an amber codon (TAG) is inserted into the α 1 domain sequence of the Z affinity peptide by replacing the appropriate site through analysis optimization design.
The invention realizes the covalent coupling of biotin at a specific site of a target protein by utilizing a BirA/Avitag technology, and an Avitag gene sequence is placed at the downstream of a Z affinity peptide gene sequence and at the upstream of a 6 XHis purification tag.
The 5 'end of the target gene sequence is provided with NdeI restriction endonuclease cutting site, the 3' end is provided with XhoI restriction endonuclease cutting site, and the chemical synthesis method is used for synthesizing Z containing 6 XHis purification tagTAG-Avitag gene sequence (shown as SEQ ID NO. 1), subcloning target gene sequence into NdeI/XhoI position of pTBX1 plasmid to obtain recombinant pZTAG-Avitag plasmid expression vector.
Example 2 recombinant protein ZBpaExpression and purification of Avitag
(1) Recombinant expression plasmid pZTAGColi BL21(DE3) and its application in the transformation of competent bacteria E.coli BL21 by the Avitag and plasmid pEVOL-pBpF calcium chloride methodLB solid culture plate containing 100 mug/mL of ampicillin and 50 mug/mL of chloramphenicol, overnight culture at 37 ℃, screening positive clone to obtain gene engineering bacteria;
(2) picking the monoclonal colony to 10mL liquid LB culture medium (ampicillin 100 mug/mL, chloramphenicol 50 mug/mL), culturing at 37 ℃ overnight, activating;
(3) the activated cells were transferred to 50mL of 2 XYT medium and cultured at 37 ℃ until OD600 was 0.6-0.8; adding Bpa with final concentration of 300 μ M, and continuing to culture for 1 h; then adding IPTG with final concentration of 0.5mM and arabinose with final concentration of 0.2% respectively, and carrying out induced culture at 37 ℃ for 6 h;
(4) centrifuging the cultured genetically engineered bacteria at 4 deg.C and 6000rpm for 15min, collecting thallus, resuspending with 5mL of 20mM PBS (containing 20mM imidazole, 150mM NaCl, pH8.0) buffer solution, and freeze thawing in refrigerator at-70 deg.C for 5 times to lyse cells; centrifuging at 8000rpm for 15min to remove thallus debris; after the supernatant was filtered through a 0.45 μm filter, the objective protein was purified by HisTrap column chromatography. The elution was carried out with 20mM PBS (containing 250mM imidazole, 150mM NaCl, pH8.0) buffer and the eluted fractions were collected.
(5) Centrifugal desalting and concentrating at 8000rpm with Millipore ultrafiltering centrifuge tube (molecular weight cut-off is 10kDa) to appropriate volume, freeze drying, weighing, and purifying to obtain about 10mg of target protein from 1L of thallus fermentation broth.
Example 3 recombinant protein ZBpaPreparation of Avitag biotinylation in vitro
In this example, the BirA in vitro reaction System was used to target recombinant protein ZBpaBiotinylation by Avitag, and obtaining of the biotinylated recombinant protein Z by HisTrap chromatography column and Millipore Ultrafiltration centrifuge tubeBpa-Avitag。
The method comprises the following steps:
(1) the reaction system adopting the BirA enzyme is as follows: 25 μ L buffer A, 25 μ L buffer B, 100 μ L ZBpa-Avitag, 45U/. mu.l BirA, 250. mu.l reaction volume 30 ℃ reaction for 6 h;
(2) the mixture after the above reaction was subjected to HisTrap column chromatography and HisTrap column chromatography according to the steps (4) and (5) of example 2
Figure BDA0001131080570000072
Ultra-4 (NMWL: 10kDa) ultrafiltration centrifugal tube to obtain biotinylation recombinant protein ZBpa-Avitag i.e. ZBpa-Biotin and removal of excess Biotin and BirA enzyme;
(3) biotinylation product ZBpaAfter 15% SDS-PAGE separation, Biotin is transferred to an NC membrane, after membrane transfer is finished, the membrane is blocked by 5% BSA solution for 30min, after TBST (containing 0.1M Tris pH 7.5 and 0.05% Tween20) washing, the membrane is immersed in SA-HRP (horse radish peroxidase labeled streptavidin, 1: 1000) solution, incubated and combined for 30min at 37 ℃, TBST is washed for 3 times, 5min each time, and a chromogenic substrate TMB (TMB is a conventional chromogenic solution) is added for color development.
(4) Recombinant protein ZBpa-biotinylation efficiency assay of Avitag: the HABA method is used for detecting the biotin labeling effect, and the detection result shows that 98 percent of protein Z is obtained after the in vitro biotinylation is carried out by the BirA enzymeBpaAvitag binds biotin, i.e.ZBpa-Biotin。
The buffer A was 0.5M bicine (pH 8.3); the buffer B is a solution with pH8.0, and contains 100mM ATP, 100mM magnesium acetate and 200mM biotin.
Example 4ZBpa-Biotin-mediated precision biotinylated Fc-site antibody
This embodiment is based on ZBpaBiotin can be directionally combined with IgG through the characteristic of bioaffinity, and as Bpa is a photosensitive group, under the 365nm ultraviolet irradiation condition, activated Bpa is promoted to form a covalent bond with an amino group of adjacent IgG, so that the Fc site of Biotin precision covalent coupling antibody can be realized (shown in figure 1).
The method comprises the following steps:
(1) IgG antibodies and ZBpaBiotin was mixed in a quartz cuvette at a ratio of 1: 5 molecules and gently shaken at room temperature for 30 min.
(2) Placing the quartz vessel on ice, irradiating with 365nm ultraviolet for 3 hr, and collecting the product
Figure BDA0001131080570000071
Ultra-0.5 (NMWL: 100kDa) ultrafiltration centrifuge tubes to remove excess ZBpaBiotin molecules, the end product being BiotinThe chemoattractant antibody was named photo-biotinylated IgG.
(3) The photo-biotinylated IgG was first analyzed by 12% non-denaturing SDS-PAGE and denaturing SDS-PAGE, followed by confirmation of Fc site coupling: pepsin was dissolved in hydrolysis (50mM sodium acetate, 10mM EDTA, pH 3.3) buffer to a final concentration of 100. mu.g/mL; mu.L of pepsin solution was mixed with 100. mu.L of photo-biotinylated IgG and reacted at 37 ℃ for 2 h; separating by 15% SDS-PAGE, transferring to NC membrane, after the membrane is transferred, blocking for 30min by 5% BSA solution, washing by TBST (containing 0.1M Tris pH 7.5 and 0.05% Tween20), immersing the membrane in SA-HRP (1: 1000) solution, incubating and combining for 30min at 37 ℃, washing for 3 times by TBST, 5min each time, and adding a chromogenic substrate TMB for color development. The results showed that the 12KD band after photo-biotinylated IgG water was clearly colored, indicating that ZBpaV of Biotin not associated with the heavy chainH，CH1And hinge region, but to the Fc portion of IgG, and 90% of IgG molecules are covalently coupled to ZBpaBiotin (FIG. 2).
Example 6ZBpaApplication of-Biotin mediated accurate Fc site biotinylated antibody
This example is based on CEA (carcinoembryonic antigen) as a biomarker for clinical diagnosis of various cancers, and indirectly evaluates the application of targeted immobilized photo-biotinylated IgG in biosensing by detecting CEA, and uses a randomly conjugated biotinylated antibody as a control (NHS-biotinylated-rabbit anti-CEAIgG).
The method comprises the following steps:
(1) rabbit anti-CEA IgG with excess ZBpaBiotin is incubated for 30min at room temperature and irradiated for 3h by ultraviolet light to obtain the product photo-biotinylated-rabbit anti-CEA IgG,
Figure BDA0001131080570000081
ultra-0.5 Ultra-filtration centrifugal tube centrifugation, taking acetic acid buffer (10mM, pH 3.2) as washing liquid during centrifugation, and separating and removing redundant ZBpaBiotin and non-covalently bound rabbit anti-CEAIgG.
(2) A5 μ M of photo-biotinylated-rabbit anti-CEA IgG was bound to the SA immobilized CM5 chip by Streptavidin (SA) -biotin interaction at a flow rate of 30 μ L/min, PBS (10mM, pH8.0) buffer as mobile phase, resulting in a directionally immobilized photo-biotinylated IgG immunosensor chip, as shown in FIG. 3.
(3) NHS-biotinylated IgG control by chemical covalent coupling, 5 μ M of NHS-biotinylated-rabbit anti-CEA IgG at 30 μ L/min flow rate, PBS (10mM, pH8.0) buffer as mobile phase, was bound to the SA immobilized CM5 chip by Streptavidin (SA) -biotin interaction to produce a targeted immobilized NHS-biotinylated IgG immunosensor chip, as shown in FIG. 4.
(5) Various serial concentrations of mouse anti-CEA diluted 0-200 ng/mL were used as test samples, PBS (10mM, pH8.0) buffer was used as mobile phase, and acetic acid buffer (10mM, pH 3.2) was used to regenerate the chip surface. The result shows that the linear detection range of the photo-biotinylated IgG immunosensor chip is 2-100ng/mL, and the LOD value is 2 ng/mL; the linear detection range of the NHS-biotinylated IgG immunosensor chip is 10-80ng/mL, and the LOD value is 10ng/mL, as shown in FIG. 5.
(6) The method comprises the steps of diluting human serum by 100 times with PBS (10mM, 0.05% Tween20, pH8.0), adding CEA with different amounts to make the final concentrations of the CEA respectively 10ng/mL, 20ng/mL, 40ng/mL and 80ng/mL, analyzing the samples by using a photo-biotinylated IgG immunosensor chip, wherein the recovery rate is 103.40% -109.53% and the RSD is 4.51% -13.50%, and the result shows that endogenous antibodies in the serum do not interfere the determination of the photo-biotinylated IgG immunosensor chip on the CEA, thereby indicating that the immunosensor chip can be used for analyzing and determining biological samples.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Figure IDA0001131080640000011

Claims (14)

1. A biological reagent which is precisely directed to an Fc site and can realize covalent coupling comprises a Z affinity peptide with a photosensitive active group, and is characterized in that 4-benzoyl-L-phenylalanine is connected to the α 1 domain of the Z affinity peptide, the position where 4-benzoyl-L-phenylalanine is connected to the α 1 domain is the 17 th position of the amino acid of the Z affinity peptide, the carboxyl terminal residue of the Z affinity peptide is sequentially connected with an Avi-tag protein and a polyhistidine purification tag protein, wherein biotin is connected to a lysine residue on the Avi-tag protein, and the gene sequence of the Z affinity peptide is shown as SEQ ID No. 1.
2. The method of claim 1 for preparing a precision-directed Fc site covalently coupled bioreagent comprising the steps of:
(1) first, a recombinant plasmid pZ was constructedTAGThe synthesis of the target gene comprises the steps of firstly inserting amber codons at the 17 th position of α 1 structural domain sequence Z affinity peptide amino acids of the Z affinity peptide gene sequence according to the Z affinity peptide gene sequence, then placing the Avitag gene sequence and a polyhistidine purification tag gene sequence at the downstream end of the Z affinity peptide gene sequence, finally respectively setting restriction enzyme cutting sites at the 5 'end and the 3' end of the gene sequence, and chemically synthesizing to obtain the target gene sequence;
(2) then the expression vector in the step (1) utilizes prokaryotic microorganisms to express the recombinant protein ZBpa–Avitag；
(3) Finally, biotin ligase is used for recombining protein ZBpa-Avitag biotinylation, resulting in a biotinylated recombinant protein: zBpa-Biotin, i.e. the biological agent; the sequence of the target gene is shown as SEQ ID NO. 1.
3. The method of claim 2, wherein: in the step (1), the target gene sequence is subcloned into pTBX1 plasmid to obtain recombinant plasmid pZTAG-an Avitag expression vector.
4. The method of claim 3, wherein: the polyhistidine purification tag gene sequence is a 6-polyhistidine gene sequence.
5. The method of claim 3, wherein: the 5 'end of the target gene is provided with an NdeI restriction endonuclease cut site, and the 3' end of the target gene is provided with an XhoI restriction endonuclease cut site.
6. The method of claim 2, wherein: in the step (2), the expression vector and pEVOL-pBpF containing aminoacyl tRNA synthetase coding gene are co-transformed into prokaryotes to obtain genetically engineered bacteria.
7. The preparation method of claim 6, wherein the genetically engineered bacteria are cultured and activated at 30-37 ℃ for 12-18 h, transferred to a 2 XYT medium, cultured at 37 ℃ until OD600 is 0.6-0.8, added with 4-benzoyl-L-phenylalanine, continuously cultured for 0.5-2 h, added with isopropyl- β -D-thiogalactoside and arabinose, induced and cultured at 30-37 ℃ for 5-7 h, centrifugally collected, resuspended in buffer solution, lysed by freeze thawing, centrifugally removed of bacterial debris, and purified with a chromatographic column.
8. The method of claim 2, wherein: in the step (3), the birA enzyme in-vitro reaction system is utilized to carry out the reaction on the recombinant protein ZBpaBiotinylation by Avitag, and then obtaining biotinylation recombinant protein through a chromatographic column and an ultrafiltration centrifugal tube, and accurately guiding the biotinylation recombinant protein to a biological reagent coupled with the Fc locus.
9. The method of claim 8, wherein: the BirA enzyme in-vitro reaction system comprises the following components: buffer A, buffer B, ZBpa-Avitag, biotin ligase, reacting for 5-7 h at 25-35 ℃; the buffer A is 0.5M bicine, pH 8.3; the buffer B is a solution with pH8.0, and contains 100mM ATP, 100mM magnesium acetate and 200mM biotin.
10. Use of the biological agent of claim 1 for the three-dimensional directional immobilization of IgG antibodies.
11. A biotinylated antibody coupled to a precisely oriented Fc site, comprising: the antibody is a biotinylated antibody which is formed by that a Z affinity peptide in a biological reagent of claim 1 leads an IgG antibody Fc segment to be bound on an IgG antibody Fc site through biological affinity, and then ultraviolet irradiation excites 4-benzoyl-L-phenylalanine in the biological reagent of claim 1 to form covalent bond connection with adjacent Fc segment amino acid, thereby realizing Fc site covalent coupling.
12. The method of claim 11 for the preparation of a precisely targeted Fc site conjugated biotinylated antibody comprising: mixing the IgG antibody with the biological reagent of claim 1, leading to the bioaffinity coupling of the Fc site, and then exciting a photosensitive group to generate a covalent bond with the adjacent amino group of the Fc segment of the IgG antibody under the irradiation of 365nm ultraviolet light, thereby obtaining the biotinylated antibody accurately leading to the Fc site coupling.
13. The method of claim 12, wherein: mixing the IgG antibody and the biological reagent according to the molecular number ratio of 1: 4-6, oscillating at room temperature for 20-40 min, placing the reaction system on ice, irradiating for 2-4 h by 365nm ultraviolet, and removing excessive Z from the irradiated product by using an ultrafiltration centrifugal tubeBpaAnd a Biotin molecule, namely obtaining the biotinylated antibody coupled with the precisely oriented Fc site.
14. An immunosensing chip, which is characterized in that: the immuno chip includes a substrate sheet having avidin immobilized thereon which binds biotin in the biotinylated antibody of claim 11.
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