CN112812192B

CN112812192B - ProA/G-dRep fusion protein serving as nucleic acid-antibody conjugate universal carrier and application thereof

Info

Publication number: CN112812192B
Application number: CN202110086733.1A
Authority: CN
Inventors: 邢航; 沈林; 聂勇
Original assignee: Hunan University
Current assignee: Hunan University
Priority date: 2021-01-22
Filing date: 2021-01-22
Publication date: 2022-05-20
Anticipated expiration: 2041-01-22
Also published as: CN112812192A

Abstract

The invention provides a ProA/G-dRep fusion protein serving as a nucleic acid-antibody conjugate universal carrier and application thereof, and particularly relates to the field of genetic engineering. The ProA/G-dRep fusion protein comprises a fusion protein of protein A/G, a flexible peptide and a duck circovirus replicase which are sequentially connected from an N end to a C end. The ProA/G-dRep fusion protein can accurately determine the connection quantity of the antibody and the single-chain DNA, has a simple synthesis method, and can be widely applied to the research of nucleic acid-protein conjugates.

Description

ProA/G-dRep fusion protein serving as nucleic acid-antibody conjugate universal carrier and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a ProA/G-dRep fusion protein serving as a nucleic acid-antibody conjugate universal carrier and application thereof.

Background

Nucleic acids and proteins are the two most important classes of biological macromolecules that form the basis of life. The current common methods for linking nucleic acid and antibody mainly rely on covalent linking methods of chemical coupling agents, and the common methods are as follows: 1. the method comprises the following steps of (1) coupling carboxyl and amino of an antibody and nucleic acid under the action of a chemical coupling agent EDC to complete the connection of the nucleic acid and the antibody, but the method cannot control the coupling position and can cause the inactivation of the antibody; 2. modifying nucleic acid with maleimide group, and coupling with free thiol on single-chain antibody to complete the connection between nucleic acid and antibody, wherein the method can not control the coupling position and coupling quantity; 3. commercial kit SiteClick using Thermofisiher^TMThe Antibody Azido Modification Kit performs azide on the sugar chain at the Fc end of the Antibody, and then performs coupling reaction with the nucleic acid modified with DBCO at the 5' end to complete the connection of the nucleic acid and the Antibody. Therefore, the methods in the prior art still have the disadvantages of high cost and incapability of determining the connection quantity of the antibody and the single-stranded DNA.

Disclosure of Invention

In order to solve the problems, the invention provides a ProA/G-dRep fusion protein serving as a universal carrier of a nucleic acid-antibody conjugate and application thereof, wherein the ProA/G-dRep fusion protein can accurately determine the connection quantity of an antibody and single-chain DNA, has a simple synthesis method, and can be widely applied to research of the nucleic acid-protein conjugate.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a ProA/G-dRep fusion protein serving as a universal carrier of a nucleic acid-antibody conjugate, which comprises a fusion protein of protein A/G, a flexible peptide and a duck circovirus replicase which are sequentially connected from an N end to a C end.

Preferably, the amino acid sequence of the ProA/G-dRep fusion protein is shown in SEQ ID No. 1.

Preferably, the amino acid sequence of the fusion protein of the protein A/G is shown as SEQ ID No. 2; the amino acid sequence of the flexible peptide is shown as SEQ ID No. 3; the amino acid sequence of the duck circovirus replicase is shown as SEQ ID No. 4.

The invention provides a recombinant expression vector for expressing the ProA/G-dRep fusion protein, which is characterized in that the basic plasmid of the vector is pET-28 a.

Preferably, the nucleotide sequence coding for the ProA/G-dRep fusion protein is inserted into the NdeI and XhoI enzyme cutting sites of the pET-28 a.

The invention provides an engineering bacterium containing the ProA/G-dRep fusion protein or the recombinant expression vector.

The invention provides application of the ProA/G-dRep fusion protein or the ProA/G-dRep fusion protein generated by the engineering bacteria in detecting myoglobin.

The invention provides a myoglobin detection reagent, and the effective components of the reagent comprise the ProA/G-dRep fusion protein or the ProA/G-dRep fusion protein produced by the engineering bacteria.

The invention provides a kit for detecting myoglobin, which comprises the ProA/G-dRep fusion protein or the ProA/G-dRep fusion protein generated by using the engineering bacteria.

Has the advantages that: the invention provides a ProA/G-dRep fusion protein serving as a universal carrier of a nucleic acid-antibody conjugate, which comprises a fusion protein of protein A/G, a flexible peptide and a duck circovirus replicase (namely the fusion protein of protein A/G-the flexible peptide-the duck circovirus replicase) which are sequentially connected from the N end to the C end. The ProA/G-dRep fusion protein is quickly connected with single-chain DNA through enzyme linking reaction and is quickly connected with IgG antibody through affinity reaction; the ProA/G-dRep fusion protein is used as a universal carrier of the single-chain deoxyribonucleic acid-antibody conjugate, can be used for quickly connecting any single-chain DNA (the 5' end comprises a sequence for specifically recognizing and connecting dRep) and any IgG antibody recognized and combined by ProA/G, accurately determines the connection quantity of the antibody and the single-chain DNA, is simple in synthesis method, and can be widely applied to research of the nucleic acid-protein conjugate. The ProA/G-dRep fusion protein can be used for myoglobin detection within the range of 0.1-5000 ng/ml, and has a correlation coefficient r of 0.9979 and high sensitivity.

Drawings

FIG. 1 is a schematic diagram of the structure of a recombinant fusion protein (ProA/G-dRep);

FIG. 2 is a diagram of single-stranded deoxyribonucleic acid-Antibody conjugate (ssDNA-Antibody conjugate);

FIG. 3 is an electrophoretogram of ligation reaction in example 3;

FIG. 4 is a graph showing the results of a western blot in example 4;

FIG. 5 is a standard graph of myoglobin in example 5.

Detailed Description

Unless otherwise required, the times employed in the embodiments of the present invention are those routinely purchased by those skilled in the art.

The invention provides a ProA/G-dRep fusion protein serving as a nucleic acid-antibody conjugate universal vector, the structure from N end to C end is shown in figure 1, and the fusion protein-flexible peptide-duck circovirus replicase comprises sequentially connected proteins A/G. In the present invention, the amino acids of the ProA/G-drp fusion protein are preferably as shown in sequence SEQ ID No. 1: VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPKGGGGSGGGGSGGGGSMAKSGNYSYKRWVFTINNPTFEDYVHVLEFCTLDNCKFAIVGEEKGANGTPHLQGFLNLRSNARAAALEESLGGRAWLSRARGSDEDNEEYCAKESTYLRVGEPVSKGRSSDLAEATSAV are provided.

The preparation method of the ProA/G-dRep fusion protein is not particularly limited, and the ProA/G-dRep fusion protein can be expressed by an expression vector or artificially synthesized, and preferably synthesized by entrusting Shanghai biology.

In the present invention, the amino acid sequence of the fusion protein of protein A/G is preferably as shown in SEQ ID No. 2: VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL AEAKKLNDAQAPK, respectively; the amino acid sequence of the flexible peptide is preferably shown as SEQ ID No. 3: GGGGSGGGGSGGS; the amino acid sequence of the duck circovirus replicase is preferably shown as SEQ ID No. 4: MAKSGNYSYKRWVFTINNPTFEDYVHVLEFCTLDNCKFAIVGEEKGANGTPHLQGFLNLRSNARAAALEESLGGRAWLSRARGSDEDNEEYCAKESTYLRVGEPVSKGRSSDLAEATSAV are provided.

The invention provides a recombinant expression vector for expressing the ProA/G-dRep fusion protein, and the basic plasmid of the vector is pET-28 a. In the present invention, the recombinant expression vector preferably has the nucleotide sequence encoding the proA/G-dRep fusion protein inserted into the NdeI and XhoI sites of pET-28 a. The invention has no special requirement on the type of the vector plasmid, and can insert the nucleotide sequence for coding the ProA/G-dRep fusion protein in a double digestion mode and finish the expression, and in the embodiment of the invention, the vector plasmid is preferably pET-28 a.

The invention provides an engineering bacterium containing the ProA/G-dRep fusion protein or the recombinant expression vector, namely BL21(DE3)/pET-28 a-ProA/G-dRep. In the invention, the basic bacterium of the engineering bacterium is BL21(DE 3).

The ProA/G-dRep fusion protein can be used for detecting myoglobin, and an antibody-nucleic acid conjugate constructed by the ProA/G-dRep fusion protein can be combined with myoglobin, so that the effect of detecting myoglobin is achieved.

The invention provides application of the ProA/G-dRep fusion protein or the ProA/G-dRep fusion protein generated by the engineering bacteria in detecting myoglobin. The ProA/G-dRep fusion protein can construct an antibody-nucleic acid conjugate, and is based on antigen immune-polymerase chain reaction to detect myoglobin within the range of 0.1-5000 ng/mL, the standard curve is y-640.77 x-101.2, the correlation coefficient is r-0.9979, wherein y represents fluorescence intensity, and x represents a concentration logarithm value.

The invention also provides a myoglobin detection method based on the ProA/G-dRep fusion protein, which adopts myoglobin antigen immune-polymerase chain reaction and specifically comprises the following steps:

(1) after the ProA/G-dRep fusion protein reacts with ssDNA-1, washing off the ProA/G-dRep fusion protein which is not subjected to the connection reaction by using ssDNA-2 which is complementary to the ssDNA-1, carrying out unwinding treatment by using Tte UvrD helicase, eluting the ProA/G-dRep-ssDNA-1 conjugate and the ssDNA1 which is not subjected to the connection, carrying out affinity connection on the ProA/G-dRep-ssDNA-1 conjugate by using Ni-NTA chromatography, purifying to obtain a purified ProA/G-dRep-ssDNA-1 conjugate, and connecting the purified ProA/G-dRep-ssDNA-1 conjugate with a myoglobin monoclonal antibody 1 to obtain a myoglobin monoclonal antibody 1-single-chain deoxyribonucleic acid conjugate;

(2) centrifuging carboxyl magnetic beads to remove supernatant, mixing the supernatant with a myoglobin monoclonal antibody 2(Myo mAb2) antibody and MES, and performing coupling treatment to obtain a myoglobin monoclonal antibody 2(Myo mAb2) and magnetic bead conjugate;

(3) mixing Circular DNA with a corresponding Circular connecting sequence linker in equal volume, connecting, degrading the linker without connection reaction, purifying by ethanol precipitation to obtain Circular DNA, and adding Tris-Ac and MgAC₂、KAc、Tween、DTT、dNTP、

DNA polymerase and SYBGreen I to obtain a circular deoxyribonucleic acid reaction solution;

(4) dissolving the single-stranded deoxyribonucleic acid probe Signal DNA in TE buffer solution to obtain a single-stranded deoxyribonucleic acid probe;

(5) uniformly mixing a myoglobin antigen protein solution with a myoglobin monoclonal antibody 1-single-chain deoxyribonucleic acid conjugate, a myoglobin monoclonal antibody 2 and a magnetic bead conjugate respectively, mixing, removing a supernatant through magnetic separation, and repeatedly rinsing with a PBS solution to obtain a mixed component 1;

(6) adding the circular deoxyribonucleic acid reaction solution and the single-stranded deoxyribonucleic acid probe into the mixed component 1, uniformly mixing, performing amplification treatment, detecting fluorescence intensity and obtaining a standard curve;

the sequence of the steps (1), (2), (3) and (4) does not exist in time.

The purification in the step (1) of the invention comprises imidazole elution and G-25 desalination in sequence; the linked reaction system preferably comprises PBS, ProA/G-dRep-ssDNA-1 conjugate and Myo mAb 1; the temperature of the reaction is preferably 37 ℃ and the time is preferably 15 min.

The myoglobin monoclonal antibody 2 in the step (2) is a counterpart antibody of the monoclonal antibody 1.

The ligation in step (3) of the present invention is preferably carried out using T4 DNA ligase, and the system and method for the ligation can be referred to the instruction manual of T4 DNA ligase; after the connection, inactivation treatment is preferably further included; the temperature of the inactivation treatment is preferably 65 ℃ and the time is preferably 5 min. After the connection and inactivation treatment, degrading the linker without connection reaction, and preferably further comprising inactivation treatment after the degradation; the temperature of the inactivation treatment is preferably 85 ℃ and the time is preferably 20 min.

The dosage of the myoglobin antigen protein solution in the step (5) of the invention is preferably 10 mu l; the dosage of the myoglobin monoclonal antibody 1-single-chain deoxyribonucleic acid conjugate is preferably 50 mu l; the dosage of the myoglobin monoclonal antibody 2 and the magnetic bead conjugate is preferably 50 mu l.

The dosage of the circular deoxyribonucleic acid reaction solution in the step (6) in the invention is preferably 50 μ l; the amount of the single-stranded deoxyribonucleic acid probe is preferably 10. mu.l. In the present invention, the temperature of the amplification is preferably constant at 37 ℃; the time for the amplification is preferably 15 min.

For further illustration of the present invention, the following detailed description of the ProA/G-drp fusion protein and its application as a universal carrier for nucleic acid-antibody conjugates provided by the present invention will be made with reference to the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.

Example 1

1. And (3) selecting the amino acid sequence of the ProA/G-dRep fusion protein gene:

the amino acid sequence of Duck circovirus replicase (Duck circovirus Rep) (Unit Accession No. A7LI84, SEQ ID No.4) is as follows:

MAKSGNYSYKRWVFTINNPTFEDYVHVLEFCTLDNCKFAIVGEEKGANGTPHLQGFLNLRSNARAAALEESLGGRAWLSRARGSDEDNEEYCAKESTYLRVGEPVSKGRSSDLAEATSAV；

the amino acid sequence (SEQ ID No.2) of the fusion protein of protein A/G (ProA/G) is as follows:

VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK；

the amino acid sequence (SEQ ID No.3) of the flexible peptides (flexible peptides) is as follows:

GGGGSGGGGSGGGGS；

2. amino acid sequence design of ProA/G-dRep fusion protein

ProA/G is placed at the N terminal of ProA/G-dRep fusion protein, flexible peptide is placed between ProA/G and dRep, and dRep is placed at the C terminal of ProA/G-dRep fusion protein, so that the ProA/G-dRep fusion protein with the amino acid sequence shown in SEQ ID No.1 is formed as shown in figure 1.

3. ProA/G-dRep fusion protein gene synthesis

Sending the amino acid sequence to Shanghai life engineering for gene optimization and gene synthesis.

4. Construction of ProA/G-dRep fusion protein gene expression vector

The synthesized gene is designed and added with a restriction enzyme NdeI site at the 5 'end of a gene sequence, a terminator and a restriction enzyme XhoI site at the 3' end, and is subcloned into pET-28a expression plasmid after double digestion by the restriction enzyme NdeI/XhoI to obtain an expression vector pET-28a-ProA/G-dRep, and meanwhile, the N tail end of the fusion gene contains 6 × histidine tag when the fusion protein of the ProA/G-dRep is expressed.

Example 2

1. ProA/G-dRep fusion protein gene engineering bacteria preparation

(1) Adding 2 μ L of the dissolved plasmid into BL21(DE3) competent cells, and ice-cooling for 10 min;

(2) placing in 42 deg.C water bath, heat-shocking for 90s, immediately placing in ice bath for 5 min;

(3) adding 900 μ L of nonreactive LB culture medium, horizontally fixing in a shaking table, and shaking at 37 deg.C for 60 min;

(4) centrifuging to remove 800 mu L of supernatant, and uniformly coating the residual bacterium liquid after heavy suspension on an LB plate containing kanamycin at the concentration of 50 mu g/ml;

(5) the fusion protein is inverted and placed in a constant temperature incubator and cultured for 12h at 37 ℃ to obtain ProA/G-dRep fusion protein gene engineering bacteria BL21(DE3)/pET-28 a-ProA/G-dRep.

2. Expression by fermentation

(1) Taking a flat plate, picking well-grown monoclonal thallus into a test tube containing 5mL of LB liquid culture medium (50 mu g/mL kanamycin), and carrying out shake culture at 37 ℃ for 12 h;

(2) transferring the normally grown bacteria liquid into a shake flask containing 300mL LB liquid culture (50. mu.g/mL kanamycin), and carrying out shake culture at 37 ℃ for 8 hrs;

(3) the whole amount of the normally grown bacterial suspension was transferred to a flask containing 3000mL of LB liquid culture (50. mu.g/mL kanamycin), and cultured with shaking at 37 ℃ for 6hrs (OD)₆₀₀At 0.6-0.8), adding IPTG with the final concentration of 0.5mM for induction, and carrying out shaking culture at 30 ℃ for overnight;

(4) centrifuging the induced bacterial liquid at 4000rpm for 15min, removing supernatant, collecting bacteria, weighing wet bacteria, and freezing in a refrigerator at-30 deg.C.

3. Separating and purifying

(1) 5g of wet bacteria were weighed, resuspended in 1g:20mL of lysis buffer (50mM Tris, 350mM NaCl, 5 mM. beta. -ME, 10mM imidazole (imidazole), pH8.0), and disrupted using an ultrasonicator (parameters: probe direct 0.5cm, power 70%, ultrasonication 3s stop 2s) under ice bath conditions for 10 min. Centrifuging the crushed suspension at 9000rpm for 10min, separating the supernatant from the precipitate, performing sample electrophoresis for later use, and performing chromatography on the supernatant at 2-8 ℃;

(2) ni affinity chromatography column treatment: fixing a 5mL chromatographic column, connecting an upper pipeline and a lower pipeline (the upper pipeline is connected with a constant flow pump, and the lower pipeline is connected with an ultraviolet signal collector), washing 10 column volumes (removing 20% ethanol in the chromatographic column) by using purified water at the flow rate of 10mL/min, washing 20 column volumes by using an equilibrium buffer solution (50mM Tris, 1M NaCl, 5mM beta-ME, 10mM imidazole, pH8.0) at the flow rate of 10mL/min, balancing signals, and carrying out calibration treatment;

(3) loading a chromatographic column: taking the supernatant, sampling at the flow rate of 3mL/min (taking care not to generate bubbles), collecting the flow-through liquid at the same time, and taking a sample to be reserved as an electrophoresis sample (marked flow through);

(4) rinsing the chromatographic column: after the sample loading is finished, taking an equilibrium buffer solution (50mM Tris, 1M NaCl, 5mM beta-ME, 10mM imidazole (imidazole), pH8.0) for rinsing, rinsing signals to reach equilibrium according to the flow rate of 3mL/min, simultaneously collecting a rinsing solution, and sampling and reserving as an electrophoresis sample (marked wash);

(5) primary elution of the chromatographic column: after the completion of the rinsing, eluent 1(50mM Tris, 350mM NaCl, 5 mM. beta. -ME, 50mM imidazole, pH8.0) was eluted at a flow rate of 6mL/min while collecting sample eluent 1 until the signal reached equilibrium, and a sample was taken and left as an electrophoretic sample (marker E1);

(6) and (3) secondary elution of the chromatographic column: after the initial washing, eluent 2(50mM Tris, 350mM NaCl, 5mM beta-ME, 100mM imidazole, pH8.0) is taken to elute at the flow rate of 6mL/min, and simultaneously, sample eluent 2 is collected until the signals reach the equilibrium, and a sample is taken and reserved as an electrophoresis sample (marker E2);

(7) three times of elution of the chromatographic column: after the second washing, eluent 3(50mM Tris, 350mM NaCl, 5mM beta-ME, 250mM imidazole, pH8.0) is taken to elute at the flow rate of 6mL/min, and simultaneously, sample eluent 3 is collected until the signals reach the equilibrium, and a sample is taken and reserved as an electrophoresis sample (marker E3);

(8) four elutions of the column: after the third washing, eluent 4(50mM Tris, 350mM NaCl, 5mM beta-ME, 500mM imidazole, pH8.0) is taken to elute at the flow rate of 6ml/min, and simultaneously, sample eluent 4 is collected until the signals reach the equilibrium, and a sample is taken and reserved as an electrophoresis sample (marker E4);

(9) and after the chromatography is finished, performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on the elution sample, and observing the elution solution of the target protein after the Ni-NTA purification and the purity of the target protein, wherein the purity requirement is more than 95%.

4. Desalination

(1) G-25 desalting column treatment: fixing 150mL G-25 desalting column, connecting upper and lower pipelines (the upper pipeline is connected with a constant flow pump, and the lower pipeline is connected with an ultraviolet signal collector), washing 10 column volumes with purified water at a flow rate of 10mL/min (removing 20% ethanol in the chromatographic column), washing 20 column volumes with exchange buffer (50mM Hepes, 350mM NaCl, pH7.5) at a flow rate of 6mL/min to balance signals, and performing calibration treatment.

(2) Loading a chromatographic column: sampling the eluent of the target protein after Ni-NTA purification at the flow rate of 3mL/min (taking care not to generate bubbles);

(3) eluting the chromatographic column: after the sample loading is finished, taking an exchange buffer solution (50mM Hepes, 350mM NaCl, pH7.5) for elution at the flow rate of 6ml/min, and simultaneously collecting a sample eluent according to a signal value;

(4) after the chromatography is finished, the eluted sample is subjected to SDS-PAGE electrophoresis, and the purity of the target protein after G-25 desalination is observed.

5. Concentrating by ultrafiltration

(1) Taking the G-25 desalted target protein solution, and selecting an ultrafiltration tube with the interception specification of 10KDa according to the molecular weight (25kDa) of the target protein for ultrafiltration concentration;

(2) the ultrafiltration parameter is 12000rpm, and the centrifugation is carried out for 20mins at 4 ℃;

(3) after concentration by ultrafiltration, the protein concentration was measured using BCA protein concentration measurement kit (from Solebao, cat # PC0020) and the protein concentration was 3.0 mg/ml.

Example 3

Test for connecting single-stranded deoxyribonucleic acid (DNA) with ProA/G-dRep fusion protein

1. Preparation of single-stranded deoxyribonucleic acid

(1) According to the literature (J.Am.chem.Soc.2017, 139, 7030-7035), the dRep recognizes the sequence of the specific single-stranded deoxyribose nucleic acid as follows: 5' -AAGTATTACCAGAAA-3' (SEQ ID No.5, abbreviated as dRep ssDNA, underlined represents dRep specific recognition cleavage and ligation of the single stranded deoxyribonucleic acid site, molecular weight about 4.655 kD);

(2) the DNA sequence is synthesized in Shanghai, and then dissolved in 100mM NaCl solution to 100 μ M concentration, and frozen at-30 deg.C for use.

2. Single-stranded deoxyribonucleic acid ligation assay

(1) Taking the ProA/G-dRep solution and dRep ssDNA to perform a ligation reaction according to the following table, wherein the ligation reaction system is shown in table 1:

TABLE 1 ligation reaction System

(2) After the reaction, SDS-PAGE 5 Xloading buffer was added, and after mixing, SDS-PAGE electrophoresis was performed, and the ligation test was judged by observing the molecular weight difference between ProA/G-dRep (molecular weight 25kD) and ProA/G-dRep-ssDNA conjugate (molecular weight about 30 kD). As shown in FIG. 3, after reaction, ProA/G-dRep-ssDNA-conjugate can be generated, and the ProA/G-dRep fusion protein of the present invention can be linked with single-stranded deoxyribonucleic acid.

Example 4

Antibody (IgG) affinity ligation assay

Western blot test

(1) Carrying out SDS-PAGE electrophoresis on the ProA/G protein, wherein the sample loading amount is 60 mu G;

(2) film transfer: taking down the electrophoresis gel, removing lamination glue and redundant parts, and soaking in a membrane transfer buffer solution; 4 pieces of filter paper and 1 piece of PVDF film with the same size as the glue are cut, and a pencil is used for marking one corner of the film. The PVDF membrane was sequentially soaked in 100% methanol for about 1min, ddH₂Soaking in O for 2min, and soaking in membrane transfer buffer. Flatly placing the negative plate of the transfer deviceThe layer is made of sponge soaked by the membrane transferring buffer solution, two layers of filter paper soaked by the membrane transferring buffer solution are placed on the sponge in order, and bubbles are expelled by a glass rod. The gel was laid flat on filter paper, and the filter paper was precisely aligned to drive out the air bubbles. The treated PVDF membrane was placed on the gel with the label side facing down. The remaining two soaked filter papers were placed neatly on the PVDF membrane, again ensuring that there were no air bubbles. Finally, a layer of sponge soaked in the membrane conversion buffer solution is placed, the anode plate is covered on the sponge, and the clamp is clamped. The device is placed in an electric rotating tank, a precooled membrane rotating buffer solution is filled, the power supply is switched on, and 200mA electricity is rotated for 1.5 h.

(3) And (3) sealing: the electroporated PVDF membrane was removed and washed once with TBST buffer and then blocked with a 5% mil/TBST shaker at room temperature for 1 h.

(4) Washing: rinse with TBST for 2 min.

(5) Primary antibodies (mouse IgG1, from pecan, cat # a 7028; rabbit IgG, from wuhanpu jia, cat # ATA 8001; sheep IgG, from ABCam, cat # ab 6795; human IgG, from ABCam, cat # ab91102) bind to the target protein: primary antibody was diluted with 1% BSA/PBST (sigma) at the recommended dilution ratio (1:1000), and the membrane was blocked with hybridization bags and refrigerated overnight at 4 ℃.

(6) Washing: wash 3 times with PBST for 10min each.

(7) Secondary antibodies (HRP-labeled goat-anti-mouse secondary antibody, HRP-labeled goat-anti-rabbit secondary antibody, HRP-labeled rabbit-anti-sheep secondary antibody, HRP-labeled goat-anti-human secondary antibody, all purchased from wuhan sanying) and primary antibody incubation: PVDF membrane was placed in horseradish peroxidase-labeled secondary antibody (1:1000) diluted with 5% mil/PBST (sigma) and incubated for 1h in a shaker at room temperature.

(8) Washing: wash 3 times with PBST for 10min each.

(9) Color development: equal amounts of Enhanced Luminol Reagent and Oxidizing Reagent (thermolisher) were taken and mixed with 5ml of 30% ddH₂Diluting with O, mixing, and dripping onto sealing film. And (3) contacting the front side of the PVDF film with a luminescent reagent downwards, developing for 1.5-2.0 min, turning over the PVDF film, and observing the result by using a gel imaging system. As shown in FIG. 3, ProA/G-dRep is capable of binding to IgG of mouse, rabbit, sheep and human originUnder the same condition, the affinity is close, and the ProA/G-dRep fusion protein can be connected with an antibody.

Example 5

Application of immune-polymerase chain reaction to myoglobin detection

The components are as follows: construction of myoglobin monoclonal antibody 1-single-stranded deoxyribonucleic acid conjugate

(1) ProA/G-dRep fusion protein is taken to react with ssDNA-1(AAGTATTACCAGAAACATCCATCCTTATCAACTA, SEQ ID No.6) to generate ProA/G-dRep-ssDNA-1 conjugates, and the reaction system is shown in Table 1.

(2) After the reaction was completed, magnetic beads (ThermoFisher Dynabeads) coated with ssDNA-2(TAGTTGATAAGGATGGATGTTTCTGGTAATACTT, SEQ ID No.7) having a sequence complementary to ssDNA-1 were used^TMM-270 Carboxylic Acid) and using Tte UvrD helicase (from NEB, cat # M1202S) to unwind complementary duplexes, eluting the ProA/G-drp-ssDNA-1 conjugate and unligated ssDNA 1;

(3) then, carrying out affinity connection on the ProA/G-dRep-ssDNA-1 conjugate by utilizing Ni-NTA chromatography, removing ssDNA-1 and Tte UvrD helicase which do not have a connection reaction, eluting by using 500mM high-concentration imidazole and desalting by using G-25 to obtain a purified ProA/G-dRep-ssDNA-1 conjugate;

(4) carrying out affinity reaction on the purified ProA/G-dRep-ssDNA-1 conjugate and myoglobin monoclonal antibody 1(Myo mAb1, purchased from Fipeng organisms, Cat. No. MYO-03) according to the following table to prepare and obtain the myoglobin monoclonal antibody 1-single-chain deoxyribonucleic acid conjugate, namely, the Myo mAb1-ssDNA-1 conjugate, wherein the affinity reaction system is shown in table 2:

TABLE 2 affinity reaction systems

And (2) component: coupling method of myoglobin monoclonal antibody 2(Myo mAb2) and magnetic beads

(1) mu.L of carboxyl magnetic beads (ThermoFisher Dynabeads) was taken^TMM-270 Carboxylic Acid) was added to 1 centrifugation of 2mLMagnetically separating in tube for 3min, and removing supernatant;

(2) adding 2 times of SDS cleaning solution into the centrifuge tube, vibrating and mixing uniformly, magnetically separating out supernatant, and cleaning for 2 times;

(3) adding 100 mu L of 50mg/mL EDC and 100 mu L of 50mg/mL NHS into the centrifuge tube at the same time, and shaking and mixing uniformly;

(4) two centrifuge tubes were placed on a homogenizer for 30min of activation. After activation, washing twice with 2 volumes of 0.02M MES, and removing the supernatant;

(5) mu.g of Myo mAb2, which is the partner for Myo mAb1 in fraction 1, 300. mu.L of 0.02M MES, was added to the centrifuge tube and coupled for 2 h. After coupling, the cells were washed 2 times with 2 volumes of magnetic bead blocking solution and the supernatant was removed.

(6) And adding 2 times of magnetic bead sealing solution into the centrifuge tube, shaking and uniformly mixing for 30min, and removing the supernatant.

(7) Washing the solution once with 2 times of volume of magnetic bead preservation solution, removing supernatant, and then transferring the solution to 30mL of magnetic bead preservation solution for preservation for later use.

And (3) component: method for preparing circular deoxyribonucleic acid reaction liquid

(1) Dissolving a single-stranded DNA sequence (TAGTTGATAAGGAATCAAAGCAGCCAGGAGCAAACTCTTGGTACAGC, SEQ ID No.8) synthesized into Circular DNA by Shanghai and a corresponding Circular connecting sequence linker (TCCTTATCAACTAGCTGTACCA, SEQ ID No.9) in TE buffer (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA) respectively;

(2) respectively mixing Circular DNA and linker with the same volume, heating at 95 ℃ for 5min, and cooling at room temperature;

(3) t4 DNA ligase and ligase buffer (Takara) were added to the mixture, and the mixture was left at 37 ℃ for reaction for 2 hours. Heating at 65 deg.C for 5min after reaction to inactivate T4 DNA ligase;

(4) ExoI (Takara) and ExoIII (Takara) were added to the reaction mixture and left to react overnight at 37 ℃ to degrade the linker which had not undergone ligation. Heating at 85 deg.C for 20min to inactivate ExoI and ExoIII;

(5) finally, purifying by an ethanol precipitation method to obtain Circular deoxyribonucleic acid (DNA), and preparing reaction liquid according to the following table 3:

TABLE 3 reaction solution composition

And (4) component: single-stranded deoxyribonucleic acid probe synthesis for signal export

Single-stranded deoxyribonucleic acid probe Signal DNA (GGAGCAAACTCTTGGTCTTC, SEQ ID No.10) for Signal output was synthesized by Shanghai and dissolved in TE buffer.

The Signal DNA can be complemented with long-chain DNA amplified by a rolling circle and combined to form double-chain DNA, and SYBGreen I can be embedded into the double-chain DNA structure and shows fluorescence.

Myoglobin antigen detection method

(1) Mixing 10 μ l of myoglobin antigen (0.1, 1, 5, 10, 50, 100, 500, 1000, 5000ng/ml) protein solution with 50 μ l of component 1 and 50 μ l of component 2, reacting at 37 deg.C for 5min, magnetically separating to remove supernatant, and repeatedly rinsing with PBS solution for 3 times;

(2) respectively adding 50 mul of the component 3 and 10 mul of the component 4, uniformly mixing, performing constant-temperature amplification for 15min at 37 ℃, detecting the fluorescence intensity and obtaining a standard curve, wherein the result is shown in a table 4 and a figure 5.

TABLE 4 plotting Standard Curve raw data

As shown in table 4 and fig. 5, the Antibody-nucleic acid conjugate (ssDNA-Antibody conjugate, fig. 2) constructed by using the ProA/G-drp fusion protein of the present invention can be used to detect myoglobin at a concentration range of 0.1 to 5000ng/ml, and the standard curve is: y 640.77x 101.2, and r 0.9979.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> university of Hunan

<120> ProA/G-dRep fusion protein serving as universal carrier of nucleic acid-antibody conjugate and application thereof

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 193

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Gly Gly Gly Gly Ser Gly

50 55 60

Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Lys Ser Gly Asn Tyr

65 70 75 80

Ser Tyr Lys Arg Trp Val Phe Thr Ile Asn Asn Pro Thr Phe Glu Asp

85 90 95

Tyr Val His Val Leu Glu Phe Cys Thr Leu Asp Asn Cys Lys Phe Ala

100 105 110

Ile Val Gly Glu Glu Lys Gly Ala Asn Gly Thr Pro His Leu Gln Gly

115 120 125

Phe Leu Asn Leu Arg Ser Asn Ala Arg Ala Ala Ala Leu Glu Glu Ser

130 135 140

Leu Gly Gly Arg Ala Trp Leu Ser Arg Ala Arg Gly Ser Asp Glu Asp

145 150 155 160

Asn Glu Glu Tyr Cys Ala Lys Glu Ser Thr Tyr Leu Arg Val Gly Glu

165 170 175

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

180 185 190

Val

<210> 2

<211> 58

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

<210> 3

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 4

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Ala Lys Ser Gly Asn Tyr Ser Tyr Lys Arg Trp Val Phe Thr Ile

1 5 10 15

Asn Asn Pro Thr Phe Glu Asp Tyr Val His Val Leu Glu Phe Cys Thr

20 25 30

Leu Asp Asn Cys Lys Phe Ala Ile Val Gly Glu Glu Lys Gly Ala Asn

35 40 45

Gly Thr Pro His Leu Gln Gly Phe Leu Asn Leu Arg Ser Asn Ala Arg

50 55 60

Ala Ala Ala Leu Glu Glu Ser Leu Gly Gly Arg Ala Trp Leu Ser Arg

65 70 75 80

Ala Arg Gly Ser Asp Glu Asp Asn Glu Glu Tyr Cys Ala Lys Glu Ser

85 90 95

Thr Tyr Leu Arg Val Gly Glu Pro Val Ser Lys Gly Arg Ser Ser Asp

100 105 110

Leu Ala Glu Ala Thr Ser Ala Val

115 120

<210> 5

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

aagtattacc agaaa 15

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aagtattacc agaaacatcc atccttatca acta 34

<210> 7

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tagttgataa ggatggatgt ttctggtaat actt 34

<210> 8

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tagttgataa ggaatcaaag cagccaggag caaactcttg gtacagc 47

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tccttatcaa ctagctgtac ca 22

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ggagcaaact cttggtcttc 20

Claims

1. A ProA/G-dRep fusion protein as a universal carrier of nucleic acid-antibody conjugates is characterized by comprising a fusion protein of protein A/G, a flexible peptide and duck circovirus replicase which are sequentially connected from N end to C end.

2. The ProA/G-drp fusion protein according to claim 1, wherein the amino acids of the ProA/G-drp fusion protein are represented by the sequence SEQ ID No. 1.

3. The ProA/G-drp fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein of protein a/G is shown in SEQ ID No. 2; the amino acid sequence of the flexible peptide is shown as SEQ ID No. 3; the amino acid sequence of the duck circovirus replicase is shown as SEQ ID No. 4.

4. A recombinant expression vector for expressing the ProA/G-drp fusion protein of any one of claims 1 to 3, wherein the base plasmid of the vector is pET-28 a.

5. The recombinant expression vector according to claim 4, wherein the nucleotide sequence encoding the ProA/G-dRep fusion protein is inserted into the NdeI and XhoI sites of pET-28 a.

6. An engineered bacterium comprising a ProA/G-drp fusion protein according to any one of claims 1 to 3 or a recombinant expression vector according to claim 4.

7. Use of the ProA/G-drp fusion protein according to any one of claims 1 to 3 or the ProA/G-drp fusion protein produced by the engineered bacterium according to claim 6 for detecting myoglobin.

8. A reagent for detecting myoglobin, wherein the active ingredient of the reagent comprises the ProA/G-dRep fusion protein according to any one of claims 1 to 3 or the ProA/G-dRep fusion protein produced by the engineering bacterium according to claim 6.

9. A kit for detecting myoglobin, which comprises the ProA/G-dRep fusion protein according to any one of claims 1 to 3 or the ProA/G-dRep fusion protein produced by the engineering bacterium according to claim 6.