CN113372448A - anti-CIB single-chain antibody and screening method and application thereof - Google Patents

Abstract

The invention discloses an anti-CIB single-chain antibody, a screening method and application thereof. The amino acid sequence of the single-chain antibody is shown in SEQ ID NO. 1; the nucleotide sequence of the gene for coding the single-chain antibody is shown in SEQ ID NO. 2. The invention constructs a CIB murine immunophage single-chain antibody library by using an antibody genetic engineering technology and a phage display technology, screens out ScFv of the CIB, and provides theoretical basis and material basis for establishing an immunological rapid detection method of the CIB in the later period.

Description

anti-CIB single-chain antibody and screening method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to an anti-CIB single-chain antibody, a screening method and application thereof.

Background

Currently, methods for detecting the sibutrol residues mainly focus on methods such as liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and Capillary Electrophoresis (CE). The methods are instrument verification methods, and the detection precision and accuracy are very reliable, but the methods have the problems of expensive instruments, complicated operation, need of professional personnel and the like, so that the requirement of daily rapid detection is difficult to meet. The immunochemical screening method based on antigen-antibody specificity recognition reaction can meet the requirement of daily rapid detection, and the core of the method is that stable, specific and sensitive antibodies must be obtained. At present, a report about a sibutro antibody is newly made, so that the development of a CIB antibody has important significance for constructing and perfecting a drug residue detection system in China.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides an anti-CIB single-chain antibody, a screening method and application thereof, and the developed anti-CIB single-chain antibody has excellent specificity and sensitivity and can be used for constructing the immunological detection of CIB.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

an anti-CIB single-chain antibody, the amino acid sequence of which is shown in SEQ ID NO. 1.

Further, the light chain variable region and the heavy chain variable region of the single chain antibody are linked by a linker polypeptide.

Further, the gene for connecting the polypeptides is a gene encoding 3 groups of repeated Gly₄A nucleotide sequence of Ser.

A gene for coding the anti-CIB single-chain antibody, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

An expression vector comprising the nucleotide.

A CIB inhibitor comprises the anti-CIB single-chain antibody.

A bacteriophage expressing the gene.

The anti-CIB single-chain antibody is applied to CIB immunological detection.

A kit for CIB immunological detection comprises the anti-CIB single-chain antibody.

A method for screening the anti-CIB single-chain antibody comprises the following steps:

(1) constructing a CIB complete antigen;

(2) extracting sample RNA after CIB complete antigen immunization, then respectively obtaining a light chain variable region gene and a heavy chain variable region gene through PCR amplification, and connecting by using a connecting fragment;

(3) cloning the gene obtained in the step (2) into a vector, then converting the vector into escherichia coli, obtaining a CIB phage single-chain antibody library after the phage rescue, and then obtaining the anti-CIB single-chain antibody through the complete antigen screening obtained by preparation.

Further, TBS containing excess carrier protein was used as a dilution of the CIB phage single-chain antibody library in the screening process of step (3).

Further, the vector used in step (3) was the phagemid vector pCANTAB5E, which presents an amber stop TAG between the single-stranded antibody and the PIII protein.

The invention has the beneficial effects that:

1. the invention utilizes antibody genetic engineering technology and phage display technology to construct a CIB murine immune phage single-chain antibody library, screens out ScFv of CIB, and provides theoretical basis and material basis for establishing an immunological rapid detection method of CIB in the later stage.

2. Transformation efficiency has an important impact on antibody library capacity. At present, chemical conversion and electroporation are two common methods. However, the transformation efficiency of the former transformation method is generally low, so that the transformation efficiency is usually ensured by the latter transformation method. Therefore, the present invention used electroporation to transform the recombinant vector into E.coli TG 1. Meanwhile, in order to ensure high activity of competence, competence is prepared in advance before transformation and can be immediately used for transformation after the preparation is finished in the invention, so that the high efficiency of transformation is ensured to the maximum extent.

3. In the subsequent screening process, TBS containing excessive carrier protein is used as the dilute solution of the antibody library, so that the specific recognition and adsorption of the carrier protein and the interference on screening are reduced.

Drawings

FIG. 1 is an identification chart of CIB-BSA 10% SDS-PAGE electrophoresis; wherein, M is a protein molecular weight standard (11-180 kDa); 1, BSA carrier protein; 2, a CIB-BSA coupling product;

FIG. 2 is a 10% SDS-PAGE electrophoretic identification chart of CIB-OVA; wherein, M: protein molecular weight standards (11-180 kDa); 1, an OVA carrier protein; 2, a CIB-OVA coupled product;

FIG. 3 is a CIB complete antigen ultraviolet scanning identification chart; wherein, A: ultraviolet scanning of a CIB-BSA coupling product; b: ultraviolet scanning of a CIB-OVA coupling product;

FIG. 4 is a complete antigen immunization effect evaluation; wherein, A: measuring the potency; b: mouse No.2 competition inhibition curve;

FIG. 5 is an electrophoretogram of mouse spleen RNA extraction;

FIG. 6 shows the amplification patterns of VH, VL and ScFv genes; wherein, A: m, DL2000 DNA marker; 1, VH gene amplification products; 2, VL gene amplification product; b: m, DL 2000; 1, ScFv gene amplification product;

FIG. 7 is the identification diagram of pCANTAB-5E vector by double digestion; wherein, M: DL5000 DNA marker; 1: double enzyme digestion of pCANTAB-5E vector; 2: pCANTAB-5E vector;

FIG. 8 shows overnight culture of transformant bacteria;

FIG. 9 shows the PCR identification result of bacterial liquid; wherein, M, DL2000 marker; 1-22, selecting colony PCR amplification result;

FIG. 10 shows the PCR results of the fourth round of panning colonies; wherein, M, DL2000 DNAmarker; 1-11, carrying out PCR amplification on the selected single colony;

FIG. 11 shows the results of ELSIA assay for positive phage clones;

FIG. 12 shows the double digestion of high affinity recombinant phagemids; wherein, M, DL2000 plus marker; 1, a recombinant vector;

FIG. 13 shows the results of immunoblot analysis of CIB-ScFv; wherein, M is a protein molecular mass marker; 1, inducing expression by pCANTAB5E empty vector; 2, CIB-ScFv-pCANTAB5E induces expression;

FIG. 14 shows the result of SDS-PAGE electrophoresis of single-chain antibody induced expression; wherein, M is the protein molecular mass standard; 1, IPTG 0.1mmol/L, 30 ℃ and 6 h; 2, IPTG 0.25mmol/L, 30 ℃ and 6 h; 3, IPTG 0.5mmol/L, 30 ℃ and 6 h; 4, IPTG 0.1mmol/L, 30 ℃ and 10 h; 5, IPTG 0.25mmol/L, 30 ℃ and 10 h; 6, IPTG 0.5mmol/L, 30 ℃ and 10 h; 7, inducing expression by using an empty vector pCANTAB 5E; 8, CIB-ScFv-pCANTAB5E was not induced with IPTG;

FIG. 15 is a competition inhibition curve of 5-CIB-ScFv.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

Example 1 preparation and characterization of CIB complete antigen

1. Preparation of CIB complete antigen

(1) Diazotization of CIB

CIB was accurately weighed using a million position analytical balance and dissolved in 0.1moL/L pre-chilled hydrochloric acid. Slowly adding 10mg/mL precooled sodium nitrite solution (molar ratio is 1:1.2) in the ice water bath under the condition of low-speed slow stirring; starch-KI test paper was used to test whether the sodium nitrite solution was excessive, with the following criteria: the color of the test paper changed to grayish purple. And (4) keeping out of the sun, stirring for 1-2 hours at low temperature (0-5 ℃).

(2) Coupling to a Carrier protein

BSA and OVA (CIB to carrier protein molar ratio 30:1) were each accurately weighed and dissolved in 0.1moL/L sodium borate solution at pH 8.6, and stirred for 2h under ice bath conditions. Equally dividing the diazonium salt solution of CIB in the step (1) into two parts, and slowly adding the solution into the BSA/OVA solution. Using dilute NaOH solution to adjust the pH value of the solution to keep the pH value of the solution in the range of 7.5 to 8.5 all the time, stirring the solution at low temperature for overnight reaction.

(3) And (3) dialysis: the next day, after the reaction was complete, the bright yellow reaction product was collected, dialyzed against PBS at 4 ℃ for 3 days, and the solution was changed.

(4) Concentration measurement and storage

The concentration of the coupled product was measured using a Solebao Bradford protein concentration assay kit, and after adjusting the concentration to 1mg/mL, the concentration was dispensed into 1.5mLEP tubes at 0.5 mL/tube, and stored in a refrigerator at-80 ℃.

2. Identification of CIB complete antigen

(1) SDS-PAGE electrophoresis

Based on the results of concentration measurement, the amounts of BSA and CIB-BSA applied were adjusted to 2. mu.g, OVA and CIB-OVA to 6. mu.g, and the spotting volume was 20. mu.L. The concentration of the concentrated gel is 5 percent, and the concentration of the separation gel is 10 percent. During electrophoresis, the full-range voltage is 120V, after the electrophoresis is finished, Coomassie brilliant blue is used for dyeing for 2 hours, the destaining solution is used for destaining for 4 hours, and new destaining solution is replaced every 1 hour; the results are shown in FIGS. 1 and 2.

To identify whether the complete antigen was successfully coupled, the coupling product was first identified by SDS-PAGE electrophoresis. The migration rate of proteins in SDS-PAGE electrophoresis is only related to the size of molecular weight, and proteins with different molecular weights are trapped at different positions when the electrophoresis is finished. As shown in FIG. 1 and FIG. 2, migration rates of both the coupling products CIB-BSA and CIB-OVA were observed to be smaller than that of the original carrier protein, indicating that the molecular weights of both coupling products were increased. From the electrophoresis results, the successful coupling of CIB to carrier protein was demonstrated.

(2) Ultraviolet scanning

Firstly, PBS is used for accurately preparing BSA and OVA standard solutions, and the concentrations of CIB-BSA and CIB-OVA are adjusted. Then, an ultraviolet spectrophotometer is used, and a spectrum scanning mode is adopted to obtain the spectrogram of each solution in the range of 200-500 nm.

The carrier protein generally has a maximum absorption peak (about 280 nm) in an ultraviolet region, a new group is introduced to form a new substance after a new compound is coupled, the wavelength corresponding to the maximum absorption peak of the new substance can generate a red shift or blue shift phenomenon, and whether complete antigen coupling is successful or not can be identified by utilizing the phenomenon. From FIG. 3, it can be observed that the wavelength of the maximum absorption peak of the coupled product and the wavelength of the maximum absorption peak of the carrier protein are both red-shifted, which can prove that the coupling of CIB and the carrier protein is successful.

Example 2 evaluation of immune Effect

The prepared complete antigen CIB-BSA is used as immunogen to immunize 3 mice together, blood is collected after 10 days after four-immunization, and the titer of the antibody in the serum of the mice is measured by indirect ELISA, and the basic steps are as follows:

(1) coating: respectively coating the ELISA plate with detection antigen CIB-OVA and carrier proteins BSA and OVA, wherein the coating concentration is 2 mu g/mL and 50 mu L/hole, and placing the ELISA plate in a refrigerator for coating overnight at 4 ℃. The next day, wash the plate, repeat 5 times, each time standing for 3 min.

(2) And (3) sealing: add 200. mu.L of blocking solution (5g skim milk powder in 100mL PBST) per well, block at 37 ℃ for 2h, repeat plate washing 5 times, each time standing for 3 min.

(3) Incubating the primary antibody: blood is collected from the tail of each mouse, added into PBS, centrifuged at 3000r/min for 5min, and the supernatant is taken. Diluting the serum with the dilution of 1:100 times in an enzyme-labeled hole according to the ratio of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 and 1:25600, wherein the reaction volume of each hole is 50 mu L, and incubating the serum at 37 ℃ for 60 min; the plate washing was repeated 5 times, each time for 3min, while the serum of the non-immunized mice was used as a negative control.

(4) Incubation of secondary antibody: diluting goat anti-mouse IgG marked by HRP (horse radish peroxidase) by using a confining liquid according to the working concentration of 1:5000 times, incubating at 37 ℃ for 25min, wherein the concentration of each hole is 50 mu L; the plate washing was repeated 5 times, each time for 3 min.

(5) Color development: adding 50 μ L/well of TMB color development solution, adding stop solution 50 μ L/well at 37 deg.C after 10min in dark.

(6) Reading: blank control for zero adjustment, enzyme labeling instrument for OD detection₄₅₀The value is obtained.

As shown in FIG. 4, 3 mice all produced antibodies, with mouse 1 and 2 multiple antisera reaching OD after dilution at 1:3200₄₅₀The value is still greater than 1.0, titer per mouse (P/N)>2.1) can reach more than 1: 10000.

While the titer is detected, carrier proteins BSA and OVA are respectively coated to detect the reactivity of the mouse multi-antiserum to the carrier proteins. As shown in Table 1, there was almost no cross-reaction between the mouse polyclonal antisera and the coating antigen carrier protein OVA, in addition to cross-reaction with the immunogen carrier protein BSA. This result is expected because the molecular weight of the carrier protein BSA is much greater than that of the hapten CIB, and there is a certain antigenic determinant in BSA itself that can be recognized by the body's immune system to induce an immune response. Although antibodies of BSA exist in mouse polyclonal serum, the detection procarrier protein adopted by the mouse is different from the immunogen carrier protein, so that the influence of carrier cross reaction on antibody detection can be effectively avoided.

TABLE 1 Cross-reactivity of mouse multiple antisera with Carrier proteins

The half Inhibitory Concentration (IC) of the antibody in the blood of the mice was then determined by indirect competitive ELISA₅₀) The basic steps are the same as the titer measurement, and only the sibutrol standard solution which is diluted in a gradient manner is added while the primary antibody is added.

Data processing: the OD of control wells without CIB standard (0ng/mL) was plotted on the abscissa in origine 9.0 software₄₅₀A value of B₀OD of CIB standard liquid well₄₅₀The value is B, B/B0 is used as a vertical coordinate for drawing, a Logitics four-parameter equation is adopted for fitting a curve, and IC is calculated₅₀。

Measurement ofAfter the titers, the sensitivity of the multiple antisera was determined by indirect competition ELISA on the basis of the titer of the mouse sera. The dilution factor of the polyclonal antibody is the dilution factor when the absorbance approaches 1.0, CIB is subjected to serial gradient dilution (3.9-1000ng/mL), data is fitted by using a four-parameter fitting function in origin9.0 software, and a competition inhibition curve is drawn by using the standard concentration as an abscissa and the absorbance B/B0 (B: absorbance of a competitor well; B0: absorbance of a non-competitor well) as an ordinate. IC of No.2 of 3 mice₅₀At the lowest, the half inhibitory concentration IC of No.2 mice is calculated₅₀62.107ng/mL, mouse No.1 IC₅₀443.084ng/mL, mouse No. 3 IC₅₀>1. mu.g/mL. The competitive inhibition curve for mouse 2 is shown in FIG. 4B.

Example 3 construction of CIB phage Single chain antibody library

1. Extraction of mouse spleen RNA

Based on the identification of the immune effect, mice # 2 were immunized a fifth time. 50 mu g of artificial complete antigen CIB-BSA is injected by adopting an intraperitoneal injection method, and no adjuvant is added in the hyperimmunization process. After 5 days, the serum was positive by orbital bleeding and the spleen was removed by killing the neck. RNA extraction was performed according to the protocol of the total RNA extraction kit from Baozbiol. And then immediately identified by electrophoresis on a 1.2% agarose gel.

As shown in FIG. 5, 28S and 16SRNA were detected, the bands were clear, and 5SRNA was partially degraded. Then, the identified RNA is reversely transcribed by RT-PCR to obtain cDNA, and then the cDNA is used as an amplification template for PCR amplification of VH and VL.

2. Reverse transcription to synthesize cDNA

After the RNA extraction is finished, reverse transcription is carried out immediately, and the components are mixed according to a reaction system of reverse transcription in the table 2, so that the operation is carried out at low temperature and the action is rapid. The reaction conditions were as follows: 37 deg.C, 15min, 85 deg.C, 5 s. After the reaction, the cDNA concentration was measured and stored at-20 ℃.

TABLE 2 reverse transcription reaction System

3. Amplification of antibody variable regions VH and VL

The VH and VL of an antibody consist of 4 relatively conserved Framework Regions (FR) and 3 antibody Complementary Determining Regions (CDR), respectively. The base composition and the arrangement sequence of the FR region are relatively conserved; the CDR region is a key region for the antibody to recognize antigen and specifically combine with the antigen, and the region has low conservation degree, variable base composition and arrangement sequence, and the antibody richness comes from the region. The primer design for amplifying VH and VL genes fully considers the characteristic of antibody genes, PCR primers are designed to begin to amplify from a framework region, the structural integrity of variable region genes is ensured, and degenerate bases are introduced into the primers to ensure the diversity of cloned genes. The primers were synthesized from Shanghai, and the specific sequence design is shown in Table 3. And (3) amplifying VH and VL genes by using a cDNA product obtained by reverse transcription in the step (2) as a template.

The reaction procedure was as follows: 30s at 98 ℃; 1min at 98 ℃, 30s at 56 ℃ and 1min at 72 ℃ for 35 cycles; 10min at 72 ℃. The reaction system is shown in Table 4.

TABLE 3 amplification of Single chain antibody primers

Wherein, the italic part is a homologous sequence near a restriction enzyme cutting site of a vector pCANTAB-5E, and a sequence homologous to the vector can be amplified at two ends of a target gene; the cross line part is a restriction enzyme site; r is A/G; y is C/T; m is A/C; k is G/T; s is C/G; w is A/T; h is A/C/T; b is C/G/T; v is A/C/G; d is A/G/T.

TABLE 4 VH and VL gene PCR reaction systems

4. And (3) recovering and purifying a target gene: the amplified heavy chain and light chain genes were purified by agarose gel electrophoresis recovery, with the concentration of the agarose gel being 1.2%.

5. ScFv gene sequence splicing and amplification

And (3) taking the VH and VL obtained by amplification and purification in the steps as templates, splicing and assembling the VH, VL and Linker according to the form of VH-L-VL by adopting an SOE-PCR technology, and identifying PCR products by using 1% agarose gel electrophoresis. The PCR reaction conditions were as follows: 94 ℃ for 50s, 56 ℃ for 50s, 7 cycles; 10min at 72 ℃; the reaction system is shown in Table 5.

As shown in FIG. 6A, the sizes of VH and VL bands were about 350bp and 320bp, respectively, as expected. The VH, VL and Linker fragments were spliced into ScFv gene by SOE-PCR technique and identified by 1% agarose gel electrophoresis, as shown in FIG. 6B, there was a specific band of about 750bp, corresponding to the expected size of ScFc fragment.

TABLE 5 ScFv Gene PCR reaction System (step1)

The first round of PCR reaction products were divided into 5 aliquots and supplemented with upstream and downstream primers VHBACKSfi1 and VLFORNot1mix to amplify the ScFv genes followed by a second round of PCR amplification. The reaction conditions were as follows: 1min at 95 ℃; 94 ℃ for 50s, 56 ℃ for 50s, 72 ℃ for 50s, 35 cycles; the PCR reaction system is shown in Table 6, and the temperature is 72 ℃ for 10min and 4 ℃ for storage.

TABLE 6 ScFv Gene PCR reaction System (step2)

6. Construction and identification of single-chain antibody library

(1) ScFv Gene was ligated to pCANTAB5E vector

S1, recovering and purifying the ScFv gene by agarose gel electrophoresis.

S2, double enzyme digestion of the pCANTAB5E vector by using Not I and Sfi I endonucleases is carried out in 2 steps due to the large temperature difference of the two enzymes, the reaction system is shown in Table 7, and the pCANTAB5E gene is recovered and purified by agarose gel electrophoresis after the enzyme digestion is finished (shown in FIG. 7, the vector linearization is successful).

S3, connecting the ScFv fragment with a pCANTAB5E vector by using a homologous recombinase, wherein the reaction system is shown in Table 5, and connecting and transforming by using the homologous recombinase. The transformed cell suspension was plated on a plate and cultured overnight at 37 ℃ in an incubator (see FIG. 8).

TABLE 7 pCANTAB5E vector restriction system

(2) Preparation of competent cells

S1, streaking the frozen TG1 bacterial liquid on a 2 XYT plate, picking out colonies with good growth conditions the next day, inoculating the colonies into 10mL of 2 XYT liquid culture medium, and culturing at 37 ℃ and 200rpm overnight.

S2, the next day, 1:100 inoculating in 2 XYT liquid culture medium, culturing at 37 deg.C and 200rpm to logarithmic growth phase (OD)₆₀₀＝0.4)。

S3, stopping culturing, subpackaging in 50mL centrifuge tubes, carrying out ice bath for 40min, and centrifuging at 4 ℃ and 4500rpm for 20 min.

S4, leaving the precipitate, resuspending the precipitate with 10mL of precooled 10% glycerol, and centrifuging the suspension at 4 ℃ and 4000rpm for 25 min.

S5, repeating the step (4) for 2 times, respectively using 5mL of precooled 10% glycerol to resuspend and centrifuge.

And S6, finally, resuspending the precipitate by using 10% precooled glycerol to obtain competence, and placing the competence in crushed ice to be neutral, namely, the best effect is obtained.

(3) Electrotransformation of ligation products

S1. to a freshly prepared TG1 competent, 10. mu.L of ligation was added and gently mixed using a pipette gun, the ligation system is shown in Table 8.

And S2, adding the mixture into a pre-cooled sterile electric rotating cup, and immediately carrying out electric shock.

S3, adding 900 mu L of preheated SOB liquid culture medium at 37 ℃ into the shocking cup after shocking, culturing for 1h at 37 ℃ and 200r/min, and recovering the resistance of the carrier for selection and growth on a resistant plate.

S4.5000r/min, centrifuging for 3min, discarding the supernatant, and adding fresh 2 XYT liquid culture medium.

S5, sucking 100 mu L of bacterial liquid, coating the bacterial liquid on a 2 XYT-A plate, and taking the non-transformed TG1 competence as a negative control.

S6, the plate is inverted in a constant temperature incubator at 37 ℃ for overnight culture, and colonies are counted the next day to estimate the transformation efficiency.

S7, transferring all the ligation products into TG1 competent cells according to the steps, culturing overnight, sealing the plate, and storing at low temperature.

TABLE 8 ScFv and vector pCANTAB5E ligation reaction System

(4) Recombinant plasmid transformation effect identification-bacterial liquid PCR identification

In order to verify the transformation effect, 22 single colonies were randomly picked from the transformed plate and subjected to PCR-based identification of the bacterial suspension, and the results are shown in FIG. 9. The reaction system was 9, and the procedure was the same as that for amplifying the ScFv gene. The primer sequences of pCANTAB5E-S1 and pCANTAB5E-S6 are shown in Table 10.

As shown in FIG. 9, 21 out of 22 clones amplified a band of about 750bp in size, with an insertion rate of 95.45%.

To further verify the correctness of the inserted sequence and the diversity of the sequence, the bacterial suspension (20) that was positive in the PCR detection of the above bacterial suspension was subjected to sequencing by the manufacturer. The sequencing results were analyzed by DNAMAN software, in which 17 inserted sequences were completely correct and the sequences were all heterogeneousThe same is true. Therefore, the accuracy of the inserted sequences in the antibody library was 77.27% (17/22), 1.5X 10⁵。

TABLE 9 PCR identification of bacterial solutions

TABLE 10 DNA sequencing primers

(5) Strain preservation of primary repertoire of single chain antibodies

Eluting the bacterial colony on the transformation plate by using 2 XYT-A nutrient medium, adding 50% glycerol into one part after the bacterial colony is blown and uniformly mixed, storing the part with the final concentration of 15% glycerol, then storing the part at minus 80 ℃, and using the other part for constructing a CIB phage antibody library.

(6) Preparation of helper phage M13K07

S1, selecting 1 single colony with good growth condition from a plate for culturing TG1, inoculating the single colony into 5mL of 2 XYT liquid culture medium, culturing overnight at 37 ℃ under the condition of 180r/min, adding the TG1 bacterial fluid 1:100 cultured overnight into 200mL of 2 XYT liquid culture medium the next day, and continuing culturing to logarithmic phase (OD)₆₀₀0.5).

S2, adding 10 mu L of the stored helper phage M13K07 into the bacterial liquid, after shaking for 1h at 37 ℃ at 100r/min, supplementing kanasin (Kan) according to the ratio of 1:1000, and then culturing overnight at 37 ℃ at 200 r/min.

S3, the next day, the overnight cultured bacterial liquid is centrifuged for 15min at the temperature of 4 ℃ and 8000r/min, the supernatant is collected and packed into a 50mL centrifuge tube, PEG/NaCl with the volume of 1/5 is added, and the mixture is fully mixed and then kept stand on ice for 4 h.

S4, centrifuging for 30min at 4 ℃ under the condition of 10000r/min, and then discarding the supernatant; resuspend the pellet with 1mL PBS, centrifuge at 13000r/min for 5min at 4 ℃, collect the supernatant, add 1/5 volumes of PEG/NaCl, mix well, then stand on ice for 1 h.

S5.4 ℃, 11000r/min for 15min, discarding the supernatant, wherein the precipitate is the phage, and after being resuspended by 200 mu L PBS, the phage is preserved at 4 ℃ for standby.

(7) Construction of CIB phage single-chain antibody library

S1, inoculating a bacterial liquid containing the transformed positive bacterial colony into 10mL of 2 XYT-A liquid culture medium, culturing at 37 ℃ at 200r/min until logarithmic phase, stopping culturing, and performing ice-water bath for 10-15min to enhance infection effect; adding 4X 10¹⁰pfu helper phage M13K 07.

S2.37 ℃, after continuing to culture for 1h at 180r/min, centrifuging for 10min at 4 ℃ at 7000r/min to precipitate bacteria, carefully removing supernatant, resuspending the precipitate with 20mL of 2 XYT-AK liquid medium, and continuing to culture for more than 12 h. The next day, at 4 deg.C, 8000r/min, centrifuge for 15min, carefully aspirate the supernatant. Recombinant phage were precipitated with 1/5 volumes of PEG/NaCl, as was done for helper phage preparation. And storing the obtained CIB phage single-chain antibody library in a refrigerator at 4 ℃ for next immunoaffinity screening and enrichment.

Example 4 screening and identification of CIB Single chain antibodies

1. Biopanning and enrichment of phage antibody libraries

Based on the phage antibody library constructed in the embodiment 3, CIB-BSA and CIB-OVA are used as coating antigens, and a solid phase screening method is adopted to carry out four-round adsorption-elution-amplification panning and enrichment on the CIB phage library. The specific process is as follows:

(1) coating: CIB-BSA was diluted to 50. mu.g/mL with ELISA coating solution and coated onto ELISA plates, 100. mu.L/well, overnight at 4 ℃. Plate washing for 5 times, 5 min/time

(2) And (3) sealing: adding 200 mu L of confining liquid per hole, and incubating for 2h at 37 ℃; washed three times with TBST solution, 5 min/time.

(3) Adding an antibody library: 50 μ L of phage single-chain antibody library was added to each well, followed by incubation at 37 ℃ for 1 h. Washed 10 times with 0.1% TBST, 3 min/time.

(4) Elution and neutralization: adding 200 μ L of eluent (pH2.2) into each well, shaking with constant temperature shaking table for 10min, immediately adding neutralizing solution (pH9.1) and 10 μ L/well to neutralize eluted phage solution, i.e. screening eluent of round 1.

(5) And (3) titer determination: taking 1 mu L of eluent to perform titer determination; the number of colonies on the plate was counted the next day and the recovered phage was calculated from the dilution factor.

(6) Amplification: 100 μ L of the acid-eluted and neutralized eluate was added to 5mL of 2 XYT-A medium and cultured to log phase (OD)₆₀₀0.4), ice-bath for 30min, add 2 × 10¹⁰And (3) standing the cfu helper phage M13K07 at room temperature for 30min, and then culturing at constant temperature of 180r/min for 1h at 37 ℃. Centrifuging at 8000r/min for 10min, and discarding the supernatant. 50mL of 2 XYT-AK culture medium were resuspended and incubated at 37 ℃ at 200r/min overnight.

(7) PEG/NaCl precipitation: the amplified phage is precipitated by a PEG/NaCl method, and the method is the same as the preparation of auxiliary phage. This is the secondary phage library after the first round of panning enrichment, and the next round of panning enrichment process is entered after titer determination. In order to apply a certain screening pressure during the screening process, the conditions of each round of panning were different, and the specific conditions were set as shown in table 11.

TABLE 11 different panning conditions for each panning round

Through the process of 4 rounds of 'adsorption-elution-amplification', as shown in table 12, the recovery rate of the phage is improved one round by one round, and the recovery rate of the phage in the 4 th round of panning is improved by about 140 times compared with that in the 1 st round of panning, which indicates that the specific phage is effectively enriched and lays a foundation for screening specific anti-CIB single-chain antibodies.

TABLE 12 efficiency of elutriation in each round

2. PCR identification of elutriated colonies

In order to check whether the panning phage vector still contains ScFv fragments, single colony was randomly picked after the 4 th panning for bacteria liquid PCR identification, the detection result is shown in FIG. 10, and the specific process is as follows:

(1) and re-infiltrating the fourth screening eluate into Escherichia coli TG1, coating a 2 XYT-A plate, culturing at 37 deg.C overnight, randomly selecting colony with good growth condition on the n mut mutext day, numbering in 1mL 2 XYT-A culture medium, and culturing at 37 deg.C for 2h at 200r/min constant temperature shaking table.

(2) Taking 5 mu L of the culture solution as a PCR template of the bacterial solution, adding 50% glycerol into the rest two parts, preserving at the temperature of minus 20 ℃, sending one part to be tested, and reserving the other part as a strain for a subsequent experiment.

(3) The PCR template of the bacterial liquid is the same as the sequencing primers S1 and S6, the specific reaction system is shown in Table 13, and the reaction program is as follows: 5min at 98 ℃; 1min at 98 ℃, 1min at 56 ℃, 1min at 72 ℃ and 25 cycles; 10min at 72 ℃.

TABLE 13 bacterial liquid PCR reaction system

As shown in FIG. 10, fragments of about 750bp were amplified from 11 randomly picked single colonies by PCR, indicating that the phagemid vector still retained ScFv fragments after panning and enrichment.

3. Positive phage phase-ELISA

On the basis of the identification, single colonies cultured by plate coating after the fourth round of screening are picked batch by batch to prepare phage, and phage subjected to 4 rounds of panning are subjected to specific screening by phase-ELSIA. After multiple rounds of phase-ELISA experiments, 10P/N (P: CIB-ScFv phage, N: helper phage) are screened out₄₅₀>0.5 of a positive bacteriophage. The results are shown in FIG. 11, in which the positive phage reactions numbered 5, 6, 7, 8 were strong (P/N)>3.0); the specific process is as follows:

(1) coating: the ELISA plates were coated with CIB-OVA, BSA, OVA, respectively, at concentrations of 10. mu.g/mL and 50. mu.L/well, and were coated overnight in a refrigerator at 4 ℃. The next day, wash the plate 5 times for 3 min/time.

(2) And (3) sealing: add 200. mu.L of blocking solution to each well, incubate for 2h at 37 ℃, wash the plate 5 times, 3 min/time.

(3) Adding a primary antibody: prepared phage were diluted to 1X 10 with blocking solution⁸cfu/mL, 50. mu.L/well, incubation at 37 ℃ for 1h, washing the plate five times, 3 min/time.

(4) Adding an enzyme-labeled secondary antibody: diluting Anti-M13 Antibody-HRP with blocking solution according to working concentration of 0.4 μ g/mL, 50 μ L/well, and incubating at 37 deg.C for 30 min; the plate was washed five times, 3 min/time.

(5) Color development: adding TMB single-component color developing solution 50 μ L into each well, developing at room temperature in dark for 10min, and adding stop solution 2mol/L H₂SO₄Solution, 50. mu.L/well.

(6) Reading: detection OD of enzyme-linked immunosorbent assay (OD)₄₅₀The value is obtained.

In addition, in order to identify the sensitivity of the positive phage single-chain antibody, the inhibition rate of the CIB standard substance concentration of 200ng/mL was determined by competitive inhibition ELISA. As shown in Table 14, inhibition was observed at a CIB standard concentration of 200ng/mL, with the phage inhibition rate of 5 being 60.84%, which was the best inhibition rate, and all subsequent experiments were carried out using the selected CIB-ScFv phage # 5.

TABLE 14 CIB-ScFv Positive phage inhibition ELISA assay results

4. Double enzyme digestion identification of CIB-ScFv positive phage

And (3) according to the phage which are screened out in the step 3 and have strong positive and inhibition, the bacterial liquid 1 preserved in the step 2: 100 were inoculated into 5mL of 2 XYT-A medium and numbered, and shake-cultured overnight at 37 ℃ and 180 r/min. The next day, the recombinant plasmid was extracted and double digested with Not I and Sfi I endonucleases, and the reaction components and procedures were specifically referred to the digestion reaction in step 6 of example 3.

As a result, as shown in FIG. 12, the recombinant phagemid vector was cleaved into two fragments by nucleic acid agarose gel electrophoresis under the action of NotI and SfiI fast-cutting enzymes, wherein the size of the 750bp fragment was consistent with the size of the ScFv fragment.

5. Gene sequence analysis of CIB-ScFv-positive phage

The preserved bacterial liquid is subjected to amplification culture, then the No. 5 positive bacterial liquid is subjected to Shanghai engineering sequencing, and then the DNA MAN comparison analysis shows that the size of the nucleotide sequence of the single-chain antibody is 738bp (SEQ ID NO.2), and the coded amino acid sequence is shown as SEQ ID NO. 1; the size of the VH segment is 366bp, 122 amino acids are coded, the size of the VL segment is 327bp, 109 amino acids are coded, the size of the Link segment is 45bp, and 15 amino acids are coded. By NCBI BLAST alignment analysis, the VH Sequence has 92% homology with the Sequence of the murine antibody (Sequence ID: JF690472.1), and the VL has 94% homology with the Sequence of the murine antibody (Sequence ID: AB 435431.1).

By further using Ig BLAST alignment analysis, VH and VL have obvious 3 CDR region, 4 FR region, amino acid sequence of CDR region, FR region. The amino acid sequence in front of the Linker is a VH amino acid sequence, and the amino acid sequence in back of the Linker is a VL amino acid sequence. From the result of the alignment analysis of the Ig BLAST, it can be found that cysteine exists in the amino acid sequences of VH and VL, which accords with the structural characteristics of the antibody and can play a role in maintaining the stability of the antibody.

6. Soluble CIB single chain antibody immunoblot analysis

In order to determine whether the soluble CIB-ScFv recombinant protein is expressed, the strong positive phage 5 invades the amber terminator uninhibited strain HB2151 for induced expression and is analyzed by an immunoblotting method, and the specific process is as follows:

(1) electrophoresis: the electrophoresis sample is periplasmic cavity extract supernatant expressed by CIB-ScFv-pCANTAB5E and pCANTAB5E under no-load induction at 30 ℃, IPTG concentration of 0.1mmol/L and induction time of 8 h.

(2) Transfer printing: and immediately performing wet transfer printing after electrophoresis, wherein the transfer printing film is PVDF, the PVDF film is treated by methanol for 30-60s before transfer printing, and the transfer printing condition is constant current of 300mA for 90 min.

(3) And (3) sealing: 5% skimmed milk powder, sealed at 37 deg.C for 2 hr, and washed with 1% Tween 20PBS on decolorizing shaker for 5 times, each for 5 min.

(4) A first antibody: Anti-E-tag (HRP) with 1:3000 times of primary antibody, incubating for 2h in a thermostat at 37 ℃, washing the membrane with PBST for 5 times, and placing on a decolorizing shaker for 5min each time.

(5) And (3) developing: and (3) dripping a proper amount of DAB color developing solution on the PVDF membrane to develop color at room temperature in a dark place.

As shown in FIG. 13, the periplasmic cavity extract induced by CIB-ScFv-pCANTAB5E vector had a protein of about 28kDa, while pCANTAB5E had no recombinant protein expression in E.coli HB2151 carrying the E-tag. It was confirmed that the CIB-ScFv-pCANTAB5E vector induced expression of the soluble CIB-ScFv recombinant protein in HB 2151.

7. Soluble expression of CIB Single chain antibodies

In order to discuss the expression condition of soluble CIB-ScFv under different conditions, different IPTG concentrations at different times are set to induce the expression of the CIB-ScFv, and SDS-PAGE electrophoresis is adopted to detect; the specific process is as follows:

adding 10 mu L of the amplified CIB positive phage into 100 mu L of a culture medium containing logarithmic phase Escherichia coli HB2151, carrying out static culture in a constant temperature box for 30min, carrying out constant temperature shaking table culture at 180rpm for 15min, coating all bacterial liquid on a 2 XYT-A plate, and carrying out overnight culture at 37 ℃.

The n mutext day, single colonies on the plates were picked, inoculated into 10mL of 2 XYT-A liquid medium, and cultured overnight at 37 ℃ and 200 rpm. On the third day, the overnight culture 1:100 was inoculated into 250mL of 2 XYT-A medium, cultured with shaking at 30 ℃ and 250rpm until the logarithmic phase, and IPTG was added to induce mutexpression. The final concentration of IPTG is 0.1mmol/L, the culture temperature is 30 ℃, and the induction time is 8 h. After the induction is stopped, collecting bacterial liquid, centrifuging at 10000rpm for 20min, collecting thalli, fully suspending with 2mL of precooled TBS, standing at-20 ℃ for 20min, standing at room temperature for 20min, centrifuging at 10000rpm for 20min, and treating the thalli with a hypertonic buffer solution and a hypotonic buffer solution in sequence to extract soluble protein in a periplasmic cavity.

After the soluble CIB single-chain antibody is determined to be expressed, the IPTG concentration is set to be 0.1, 0.25 and 0.5mmol/L, the induction time is set to be 8 and 12h for inducing soluble expression, and the expression condition under different conditions is analyzed by SDS-PAGE detection.

As shown in FIG. 14, soluble CIB-ScFv was expressed at 30 ℃ and different IPTG concentrations (0.1, 0.25, 0.5mmol/L) and different induction times (8, 12h), and the expression effect was the best when IPTG concentration was 0.1mmol/L and induction was performed at 30 ℃ for 12h (lane 4).

8. Sensitivity and specificity analysis of CIB single-chain antibody

(1) Firstly, measuring the titer of a soluble single-chain antibody by adopting an indirect ELISA (enzyme-Linked immuno sorbent assay) experiment, and selecting a proper working concentration, specifically P/N>2.1，OD₄₅₀The value is between 1.0 and 1.2. The negative control was pCANTAB-5E/HB 2151.

(2) According to the working titer selected in the above steps, indirect competition ELSIA is adopted to determine affinity and specificity, the specific steps are the same as example 2, wherein the free competitors are CIB, CL, CIM, RAC and ZIL in the beta-receptor agonist respectively; the concentration gradient of the Chinese and western butenediol is 1000, 500, 250, 100, 50, 10, 5, 1 and 0mg/mL, and the concentration gradient of other competitors is 2000, 1000, 500, 100, 50, 10 and 0 ng/mL.

(3) Data processing: the OD of control wells without CIB standard (0ng/mL) was plotted on the abscissa in origine 9.0 software₄₅₀A value of B₀OD of CIB standard liquid well₄₅₀The value is B, B/B0 is used as a vertical coordinate for drawing, a four-parameter equation is adopted to fit a curve, and IC is calculated₅₀。

To determine the sensitivity of soluble expressed CIB-ScFv, the potency of the selected CIB-ScFv was first analyzed using an indirect ELISA assay. As shown in Table 14, the indirect ELISA detection result shows that the titer of the obtained CIB-ScFv can reach 1:160(P/N > 2.1).

To further identify the affinity of the selected CIB-ScFv, the dilution factor 1:40 was used as the working titer of soluble ScFvs in an indirect competition ELISA experiment. FIG. 15 shows the competitive inhibition curve of CIB-ScFv, whose IC was calculated from origin9.0₅₀＝164.197ng/mL，IC₈₀965.67 ng/mL. The results show that the obtained CIB-ScFv has good sensitivity and affinity.

In order to further identify and screen the specificity of the CIB-ScFv, the cross-reactivity of the CIB-ScFv with 6 beta-receptor agonists CL, SAL, CIM, ZIL, RAC and TBL is respectively determined. As shown in Table 15, the cross reaction rate of the selected sample with clenbuterol hydrochloride was 26.12%. Except for certain cross reaction with CL (26.12%), the cross reaction rate with other detected beta-receptor stimulant small molecules SAL, CIM, RAC, TBL and ZIL is less than 0.1%.

TABLE 155-CIB-ScFv Cross-reactivity with its beta-receptor agonist

In conclusion, the invention successfully screens 1 strain of strong positive phage with higher reactivity with complete antigen CIB-OVA, and the cross reactivity of the phage and carrier protein is the lowest. The sequencing result shows that the whole length of the single-chain antibody gene is 738bp, the size of VH is 366bp, and the size of VL is 327bp, which is consistent with the expectation. After CIB-ScFv strong positive phage is infected with expression strain HB2151, inducing expression to obtain soluble expression ScFv recombinant protein, and its identification shows that it has good binding activity with CIB and IC₂₀＝9.938ng/mL，IC₅₀＝164.197ng/mL，IC₈₀965.67 ng/mL. The specific identification result shows that the cross reaction with clenbuterol hydrochloride is only 26.12 percent; the single-chain antibody can provide theoretical basis and material basis for establishing an immunological rapid detection method of CIB.

Sequence listing

<110> college of New county

<120> anti-ICB single-chain antibody, and screening method and application thereof

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Gln Val Gln Leu Gln Glu Ser Gly Ala Asp Leu Val Arg Pro Gly Thr

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Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

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Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

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Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser

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Ser Ala Thr Ser Ser Leu Ser Ser Asn Tyr Leu His Trp Tyr Gln Gln

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Ala Ser Gly Val Pro Thr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser

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tcctgcaagg cttctggata cgccttcact aattacttga tagagtgggt aaagcagagg 120

cctggacagg gccttgagtg gattggagtg attaatcctg gaagtggtgg tactaactac 180

aatgagaagt tcaagggcaa ggccacactg actacagaca aatcctccag cacagcctac 240

atgcagctca acagcctgac atctgaggat tctgcagtgt atttctgtaa aagacctcct 300

atgattacga cgaggaggac cggctttgac tactggggcc aagggaccac ggtcaccgtc 360

tcctcaggtg gaggcggttc aggcggaggt ggctctggcg gtggcggatc ggacattgag 420

ctcacccagt ctccaaccac catggctgca tctcccgggg agaagatcac tatcacctgc 480

agtgccacct caagtttaag ttccaattac ttgcattggt atcagcagaa gccaggattc 540

tcccctaaac tcttgattta taggacatcc aatctggctt ctggagtccc aactcgcttc 600

agtggcagtg ggtctgggac ctcttactct ctcacaattg gcaccatgga ggctgaagat 660

gttgccactt attactgcca ccagtatcat cgttccccat tcacgttcgg ctcgggcacc 720

aagctggaaa taaaacgg 738

Claims (10)

1. An anti-CIB single-chain antibody is characterized in that the amino acid sequence of the single-chain antibody is shown as SEQ ID NO. 1.

2. The anti-CIB single chain antibody according to claim 1, wherein the light chain variable region and the heavy chain variable region of said single chain antibody are linked by a linking polypeptide.

3. A gene encoding the anti-CIB single-chain antibody according to claim 1 or 2, wherein the nucleotide sequence of the gene is represented by SEQ ID No. 2.

4. An expression vector comprising the nucleotide of claim 3.

5. A CIB inhibitor comprising the anti-CIB single-chain antibody according to claim 1 or 2.

6. A bacteriophage comprising the nucleotide according to claim 3.

7. Use of the anti-CIB single chain antibody of claim 1 or 2 in an immunological detection of CIB.

8. A kit for conducting an immunological test for CIB, comprising the anti-CIB single-chain antibody according to claim 1 or 2.

9. A method for screening the anti-CIB single chain antibody of claim 1 or 2, comprising the steps of:

(1) constructing a CIB complete antigen;

(2) extracting sample RNA after CIB complete antigen immunization, then respectively obtaining a light chain variable region gene and a heavy chain variable region gene through PCR amplification, and connecting by using a connecting fragment;

(3) cloning the gene obtained in the step (2) into a vector, then transforming the vector into escherichia coli, obtaining a CIB phage single-chain antibody library after phage rescue, and then screening the prepared complete antigen to obtain the anti-CIB single-chain antibody.

10. The method of claim 9, wherein the step (3) of screening uses TBS with excess carrier protein as a dilution of the CIB phage single chain antibody library.

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