CN114133433A

CN114133433A - Protein tag, fusion protein or labeled conjugate containing protein tag and application of protein tag

Info

Publication number: CN114133433A
Application number: CN202111382326.1A
Authority: CN
Inventors: 孟媛; 李俊; 于秀玲
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2021-11-22
Filing date: 2021-11-22
Publication date: 2022-03-04
Also published as: CN117304340A

Abstract

The invention relates to a protein tag, a fusion antigen or a labeled conjugate containing the protein tag and application of the fusion antigen or the labeled conjugate. The protein label improves the solubility expression quantity of the fusion expressed antigen, enables the fusion expressed antigen to have excellent markable performance, enables the antigen to have good solubility after being coupled with a marker, and keeps the natural immunity of the antigen, and when the marker conjugate is used for immunodetection, the sensitivity of the detection can be greatly improved.

Description

Protein tag, fusion protein or labeled conjugate containing protein tag and application of protein tag

Technical Field

The invention belongs to the field of immunodetection. More particularly, it relates to a protein tag, a fusion protein or a labeled conjugate containing the same, and uses thereof.

Background

Immunoassay (IA) is a method of measuring a specimen using an immunological technique. Antibodies or antigenic substances in body fluids are detected in clinical tests mainly by antigen-antibody reactions. The immunoassay is a sensitive measuring method, and the formed precipitate or turbidity is directly measured after antigen-antibody reaction, and the sensitivity can reach 5-10 mug/ml. However, in clinical tests, the content of some analytes in the specimen is far below this level, and therefore methods for increasing the sensitivity are sought. In the labeled immunoassay, an antigen or an antibody in a detection reagent is labeled with a measurable substance, and the sensitivity is increased by measuring the label. There are many types of labels, including enzymes, chemiluminescent materials, fluorescent materials, radioactive materials, colloids, and combinations thereof, which are used to increase the sensitivity of the immunoassay to a level that is distinguishable to the naked eye or readable by a machine.

In immunoassay, the double antigen sandwich method is widely applied to enzyme immunoassay, colloid-based assay, fluorescence assay and the like. The main principle of the double antigen sandwich method is as follows: coating antigen on the solid support, sealing, adding the sample to be detected, adding the conjugate after the second antigen labeling, combining the formed antigen-antibody complex with the conjugate to form an antigen-antibody-labeled antigen complex, and amplifying the signal by a certain means to obtain the final judgment result.

In immunoassay, the use of capture methods is also very common. Specific IgM in serum against certain antigens is often present in combination with specific IgG, which interferes with the detection of IgM antibodies. Therefore, the IgM antibody measuring multipurpose capture method is that all serum IgM (including specific IgM and non-specific IgM) is firstly fixed on a solid phase, and after IgG is removed, a specific antigen-marker is added for color development.

The competition method can be used for quantitative determination of antigens and haptens, and can also be used for determination of antibodies. Taking the measurement of antigen as an example, a specific antibody is adsorbed on a solid phase carrier, an antigen to be measured and a certain amount of known antigen-marker are added to enable the antigen to be measured and the known antigen-marker to be competitively combined with the solid phase antibody, and finally the antigen-marker combined with the solid phase is in negative correlation with the content of the antigen to be measured after washing and separation.

The labeled conjugate in the present invention refers to the antigen-label complex in the immunoassay mentioned above. The activity of the conjugate has a decisive influence on the sensitivity of the immunoassay, whereas highly active conjugates require two conditions: on the one hand, as many labels as possible are bound per antigen in the conjugate; on the other hand, the immunological activity of the antigen in the conjugate is as high as possible. Therefore, in the case of the limited labeling substance and labeling method, the activity of the prepared conjugate is determined by the characteristics of the antigen, and the antigen of the highly active conjugate needs to have the following conditions:

1. good solubility and correct folding conformation. The immunological activity of an antigen depends to a large extent on its spatial conformation, in particular on the conformational epitopes on the antigen. When expressed, antigens are not normally correctly folded if in inclusion body form. Such poorly soluble antigens generally have poor activity after labeling.

2. The antigen expression quantity is large, which is beneficial to mass production. As is well known to those skilled in the art.

Many antigens have low conjugate activity after conjugation to a label. The general solution is to fuse and express a protein tag on the antigen, so that the expressed fusion antigen has better performance in the above aspects, and has excellent marking performance.

Protein tags in the prior art include TRX, GST, DSBA and the like, but the labeling performance of fusion antigens co-expressed by the protein tags is poor.

In view of this, the invention is particularly proposed.

Disclosure of Invention

Through a large number of experiments, the inventor of the application unexpectedly discovers a protein tag, the protein tag improves the soluble expression quantity of fusion expressed protein, and the protein tag and the antigen of the invention are expressed through fusion, so that the marking performance of the fusion antigen is greatly improved. After the conjugate is coupled with a marker, the antigen can be well soluble, the natural immunity of the antigen is kept, and when the conjugate is used for immunodetection, the sensitivity, specificity and stability of the detection can be obviously improved.

According to one aspect of the present invention, there is provided a protein tag comprising a fragment of X1, the amino acid sequence of the fragment of X1 comprising a-G-L-N-D-I-F-E-a-Q-K-I-E-W-H-E-D-T-G.

According to a second aspect of the present invention, there is provided a fusion protein comprising the protein tag and the protein portion as described above.

Further, the protein moiety is an antigen.

According to a third aspect of the present invention, there is provided a labeled conjugate comprising the fusion protein and a label, wherein the fusion protein is coupled with the label to form the labeled conjugate.

According to a fourth aspect of the present invention, there is provided a solid-phase conjugate comprising the fusion protein and a solid-phase carrier, wherein the fusion protein is coupled with the solid-phase carrier to form the solid-phase conjugate.

According to a fifth aspect of the present invention, there is provided a nucleic acid encoding the protein tag or fusion protein described above, a vector or a cell comprising the same.

According to a sixth aspect of the invention, there is provided a method of preparing a fusion protein and a marker conjugate.

According to a seventh aspect of the present invention, there is provided a reagent or kit.

Finally, the invention also relates to the application of the protein tag in improving the soluble expression quantity of the protein or improving the activity of the marker conjugate.

Drawings

FIG. 1 is a reducing SDS-PAGE result of x1 '-x 2-x 1' -HIV fusion antigen.

FIG. 2 shows the result of reducing SDS-PAGE of x1 '-x 2-x 1' -HIV fusion antigen after SAS treatment and purification.

FIG. 3 is a reducing SDS-PAGE result of x 1' -x2-HIV fusion antigen.

FIG. 4 shows the result of reducing SDS-PAGE of x 1' -x2-HIV fusion antigen after SAS treatment and purification.

FIG. 5 is a reducing SDS-PAGE result of x 1' -HIV fusion antigen.

FIG. 6 shows the result of reducing SDS-PAGE of x 1' -HIV fusion antigen after SAS treatment and purification.

FIG. 7 is a reducing SDS-PAGE result of x2-HIV fusion antigen.

FIG. 8 shows the result of reducing SDS-PAGE of x2-HIV fusion antigen after SAS treatment and purification.

Detailed Description

The protein tag is a functional unit, and in the present invention, the protein tag is a functional unit capable of increasing the soluble expression level of the fused protein moiety. The protein moiety may be an antigen or other protein, and in the present invention, the protein moiety is an antigen, and in the present invention, the protein tag is also referred to as an antigen tag. The fusion protein also becomes a fusion antigen.

In one aspect, the embodiments provide a protein tag comprising a fragment X1, wherein the amino acid sequence of the fragment X1 includes a-G-L-N-D-I-F-E-a-Q-K-I-E-W-H-E-D-T-G.

In alternative embodiments, the protein tag further comprises a fragment of X2, the X2 fragment amino acid sequence comprising SEQ ID NO 5;

in an alternative embodiment, the number of the X1 fragments is m, the number of the X2 fragments is n, the m is an integer greater than or equal to 1, and the n is an integer greater than or equal to 0;

in alternative embodiments, said m is taken from 1 or 2, said n is taken from 0, 1 or 2;

in alternative embodiments, said m + n is 1, 2 or 3;

1) when m + n ═ 1, the protein tag is: an X1 fragment;

2) when m + n is 2, the protein tag is selected from: (X1, X2) a combination fragment, (X2, X1) a combination fragment, or (X1, X1) a combination fragment;

3) when m + n is 3, the protein tag is selected from: (X1, X2, X1) combined segment, (X1, X1, X2) combined segment, (X2, X2, X1) combined segment, (X1, X2, X2) combined segment, (X2, X1, X1) combined segment or (X2, X1, X2) combined segment, which are arranged continuously or discontinuously in sequence from upstream (N-terminal) to downstream (C-terminal) of the sequence.

In alternative embodiments, m is 2, n is 1 or m is 1, n is 1.

The protein tag is selected from: (X1, X2) combined segment, (X2, X1) combined segment, (X1, X2, X1) combined segment, (X1, X1, X2) combined segment or (X2, X1, X1) combined segment, which are arranged continuously or discontinuously in sequence from upstream (N-terminal) to downstream (C-terminal) of the sequence.

In alternative embodiments, the X1 fragment is located upstream (N-terminus) and/or downstream (C-terminus) of the X2 fragment, and the X1 fragment is linked to the X2 fragment by a linker sequence.

Such Linker (Linker) sequences include, but are not limited to, linkers comprising one or more amino acids (e.g., Gly or Ser) or amino acid derivatives (e.g., Ahx, β -Ala, GABA or Ava), or PEG, and the like.

In alternative embodiments, the linker may be a linker comprising G, GS, SG, GGGS, GGGGS basic building blocks, the number of which may be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In alternative embodiments, the protein tag is selected from the group consisting of: (X1-Linker-X2) combined segment, (X2-Linker-X1) combined segment, (X1-Linker-X2-X1) combined segment, (X1-X2-Linker-X1) combined segment, (X1-Linker-X2-Linker-X1) combined segment, (X1-Linker-X1-X2) combined segment, (X1-X1-Linker-X2) combined segment, (X1-Linker-X1-Linker-X2) combined segment or (X2-Linker-X1-X1) combined segment, (X2-X1-Linker-X1) combined segment, (X2-Linker-X1-Linker-X1) combined segment, the above combined fragments are arranged in sequence from the upstream (N-terminal) to the downstream (C-terminal) of the sequence, and the "-" represents a covalent bond (peptide bond).

In an alternative embodiment, the Linker is GGG.

In alternative embodiments, the X1 fragment is directly linked to the X2 fragment.

The direct connection refers to the connection that the C terminal carboxyl of the X1 fragment directly reacts with the N terminal amino of the X2 fragment to form a peptide bond.

In alternative embodiments, the protein tag is selected from the group consisting of: (X1-X2) combined fragment, (X2-X1) combined fragment, (X1-X2-X1) combined fragment, (X1-X1-X2) combined fragment or (X2-X1-X1) combined fragment, which are arranged in sequence from upstream (N-terminal) to downstream (C-terminal) of the sequence, wherein "-" represents a covalent bond (peptide bond).

In alternative embodiments, the X1 fragment has the amino acid sequence set forth in any one of SEQ ID NOs 1-4; or the amino acid sequence of the X2 fragment is shown as any one of SEQ ID NO 5-7; or the protein tag amino acid sequence is shown in any one of SEQ ID NO 1-4 or SEQ ID NO 8-13.

It is noted that in other embodiments, protein tags having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of the protein tag described above (SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) are also within the scope of the sequences protected by the present invention. Variants of the described sequences which are conceivable to the person skilled in the art are also within the scope of the sequences claimed in the present invention.

In another aspect, the embodiment of the present invention provides a fusion protein, including the protein tag and the protein portion described above.

The term "fusion protein" as used herein is a fusion expression product of a protein tag and a protein of interest.

In alternative embodiments, the protein moiety is an antigen.

The antigen refers to any substance that can bind to an antibody in an immune response. The antigen used in the present invention may be any antigen known in the art to be useful for immunodetection.

In alternative embodiments, the protein tag is located upstream of the antigen. The protein tag is indirectly connected with the antigen through a linker sequence or directly connected with the antigen.

In alternative embodiments, the antigen is an antigen associated with an infectious disease, endocrine, tumor, or drug.

In alternative embodiments, the antigen is an antigen associated with a viral or bacterial infectious disease.

In alternative embodiments, the antigen includes, but is not limited to, an HIV antigen, a hepatitis A virus antigen, a hepatitis B virus antigen, a hepatitis C virus antigen, a hepatitis D virus antigen, a hepatitis E virus antigen, a hepatitis G virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T-lymphocyte leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a West Nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an echovirus antigen, a coxsackie virus antigen, a encephalitis B virus antigen, a coxsackie virus antigen, an EB virus antigen, a mumps virus antigen, a treponema antigen, a virus antigen, a hepatitis B virus antigen, a hepatitis C virus antigen, a hepatitis D antigen, a hepatitis C virus antigen, a rubella virus antigen, a virus antigen, a virus antigen, a virus, a, A Borrelia burgdorferi antigen, a Chlamydia trachomatis antigen, a Chlamydia pneumoniae antigen, a Chlamydia psittaci antigen, a ureaplasma urealyticum antigen, a Mycoplasma pneumoniae antigen, a Mycobacterium tuberculosis antigen, a helicobacter pylori antigen, a gonococcus antigen, a Plasmodium antigen, a Trypanosoma cumini antigen and a Toxoplasma gondii antigen.

The gene of the antigen can be obtained by artificially synthesizing a nucleotide sequence, or can be amplified from the genome of Escherichia coli and the genome of other species, or can be obtained by modifying the sequence of a natural genome through genetic engineering. The invention preferably selects an artificial synthetic sequence, so that the gene can be synthesized according to more codons used by escherichia coli, thereby ensuring that the expression quantity of the fusion protein is not limited by the preference of the codons. The gene of the fusion protein of the invention is obtained by PCR amplification using Escherichia coli genome as a template, and the method is a method generally adopted in molecular biology and does not need to be described in detail.

In another aspect, the embodiment of the present invention provides a labeled conjugate, including the fusion protein and the label, where the fusion protein is coupled to the label to form the labeled conjugate.

The term "label" as used herein refers to a substance that can be detected in an immunoassay. The marker may be selected from any marker used in the art suitable for the present invention.

In alternative embodiments, the marker is selected from the group consisting of: an enzyme, a luminescent substance, a fluorescent substance, a colored substance, a radioactive substance, a colloid, or a combination thereof.

The enzyme used in the present invention may be any suitable enzyme including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, glucose-6-phosphate deoxyenzyme.

The luminescent material used in the present invention may be any suitable luminescent material including, but not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium ester and its derivatives, dioxane and its derivatives, loflunine and its derivatives, peroxyoxalate and its derivatives.

The fluorescent substance used in the present invention may be any suitable fluorescent substance, including, but not limited to, fluorescein isothiocyanate, rhodamine and derivatives thereof, europium and derivatives thereof, quantum dots and derivatives thereof, rare earth complexes and derivatives thereof.

The radioactive material used in the present invention can be any suitable radioactive material including, but not limited to, actinium, thorium, uranium, plutonium, curium, mendelevium.

The colloid used in the present invention may be any suitable colloid including, but not limited to, colloidal gold, colloidal selenium, colloidal silver, latex.

In another aspect, the embodiment of the present invention provides a solid-phase conjugate, including the fusion protein and a solid-phase carrier, where the fusion protein is coupled to the solid-phase carrier to form the solid-phase conjugate.

In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes, for example, the solid support can be magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.

In yet another aspect, the embodiments of the present invention provide a nucleic acid encoding the protein tag or the fusion protein.

Nucleic acids are typically RNA or DNA, and nucleic acid molecules can be single-stranded or double-stranded. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. DNA nucleic acid is used when it is ligated to a vector.

In yet another aspect, the embodiments provide a vector comprising the above-described nucleic acid.

In yet another aspect, the embodiments of the present invention provide a cell comprising the above-described nucleic acid or the above-described vector.

In another aspect, the present invention provides a method for preparing a fusion protein, comprising culturing the above-mentioned cells.

In still another aspect, the present invention provides a method for preparing a labeled conjugate, the method comprising coupling the fusion protein with a label in vitro.

The marker of the invention may be conjugated to the fusion antigen in any manner known in the art.

Preferably, the label is bound to the fusion antigen by chemical conjugation.

In alternative embodiments, the coupling reaction is carried out using a mixed anhydride method, a carbodiimide method, a glutaraldehyde method, a glutaric anhydride method, a diazotization method, a succinic anhydride method, a carbonyldiimidazole method, or a sodium periodate method.

Further preferably, the sodium periodate method is adopted, and an aldehyde compound formed by oxidizing the marker by NaIO4 can be connected with an amino group of the fusion antigen to form Schiff base, and the Schiff base can be further reduced by NaBH4 (or ethanolamine) to form a stable conjugate.

From the above description, one of ordinary skill in the art will readily appreciate that the label may be attached to any suitable residue of the fusion antigen, including but not limited to amino, carboxyl, hydroxyl, or sulfhydryl groups. Preferably, the label is attached to the amino group of the fusion antigen.

In yet another aspect, the embodiments of the present invention provide a reagent or a kit, comprising the above-described fusion protein and/or the above-described label conjugate and/or the above-described solid-phase conjugate.

Finally, the embodiment of the invention provides the application of the protein tag in improving the soluble expression quantity of the protein or improving the activity of the marker conjugate.

According to the description of the invention, the fusion protein has uniform distribution of acid-base amino acids, so that the expressed fusion protein has good hydrophilicity, the spatial conformation folding of the target antigen is closer to the natural antigen, and the fusion protein has moderate molecular weight and can not influence the biological activity of the target antigen due to overlarge molecular weight. The characteristics enable the expressed antigen to have high activity, and particularly, when the antigen is used for enzyme-labeled conjugates or colloid-labeled conjugates in immunoassay, the sensitivity, specificity, stability and the like are obviously improved.

Embodiments of the present invention will be described in detail with reference to examples. The examples are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available. The experimental procedures for which specific conditions are not specified in the following examples are generally performed according to conventional conditions, such as those described in Sambrook et al, handbook of molecular cloning laboratories (New York: Cold Spring Harbor Laboratory Press, 0989), or the method recommended by the manufacturer of the kit.

EXAMPLE 1 construction of vector P2-x2 with protein tag of x2(SEQ ID NO:5)

Referring to the protein sequence encoded by the Biotin purification tag DNA on PinPoint Xa-1 plasmid (Promega corporation, USA; V2020), GeneBank number U47628, a segment from amino acid 21 to amino acid 102 was selected, and primers for the segment were designed with the aid of Oligo software, and the primer sequence was: the upstream primer 5'-GGGAGATCTCACCATCACCATCACCATCACCATTCACACGAAAACCCGAT-3' has BglII site and three protecting bases at 5 'end, 8 His tags, downstream primer 5'-GGTGAATTCTTAGGATCCCTCAACCTTGCCGTCGGTG-3'has BamHI and EcoRI site at 5' end, a stop codon is added between the two sites, and three protecting bases at the extreme end.

The x2 protein tag was PCR amplified using the PinPoint Xa-1 plasmid as template and the two primers designed above. The PCR conditions were: 94 ℃, 5 minutes × 1 cycle, (94 ℃, 30 seconds, 55 ℃, 30 seconds, 72 ℃, 30 seconds) × 30 cycles, 72 ℃, 10 minutes × 1 cycle. And (5) recovering a PCR product. Then, BglII and EcoRI are used for enzyme digestion simultaneously, and then the digestion product is recovered and is connected to the P2 vector after BamHI and EcoRI digestion, so that the modified vector P2-x2 is obtained.

EXAMPLE 2 construction of expression plasmid P2-x2-HIV containing x2-HIV fusion antigen

PCR amplified HIV env gene (GeneBank No. AF321145) 1486-1836bp DNA sequence, its upstream primer with BamHI site, downstream primer with EcoRI site and before EcoRI site with termination code TAA. The PCR fragment was cloned into P2-x2 vector after digestion with BamHI and EcoRI, and the resulting positive clone was P2-x 2-HIV.

By inserting an X1 gene sequence into an expression plasmid P2-X2-HIV, P2-X1 '-X2-X1' -HIV, P2-X1 '-X1' -X2-HIV, P2-X2-X1 '- (Linker) -X1' -HIV, P2-X1 '-X2-HIV and P2-X2-X1' -HIV expression plasmids described in Table 1 are formed; similarly, P2-HIV, P2-x1 '-HIV and P2-x1-x 2' -HIV expression plasmids were obtained by recombinant expression.

Example 3 expression and purification of HIV-containing fusion antigens

The expression plasmid P2-x1 '-x 2-x 1' -HIV transformed Escherichia coli ER2566, spread on LB plate containing 100ug/ml kanamycin sulfate, cultured overnight at 37 ℃, picked single clone, shake-cultured to OD6001.0 with 500ml LB medium containing the same concentration of kanamycin at 37 ℃, induced with IPTG at final concentration of 0.5mM, under the induction conditions: 37 ℃ at 200rpm for 4 hours. Centrifugation at 5000rpm for 20 minutes at 4 ℃ to collect the cells, centrifugation at 12000rpm for 20 minutes at 4 ℃ to resuspend the cells per liter of the lysate with 10ml of lysis buffer (50mM Tirs-HCl, pH8.0, 1mM EDTA, 100mM NaCl), ultrasonication, electrophoresis at 12000rpm for 20 minutes at 4 ℃, SDS-PAGE to identify most of the proteins in the lysate supernatant, collection of the supernatant, addition of 0.25 times the volume of the supernatant in saturated ammonium sulfate solution, centrifugation at 12000rpm for 20 minutes at 4 ℃, collection of the precipitate, use of 10ml of equilibration buffer (10mM Na2HPO4, 1.8mM KH2PO4, 140mM NaCl, 2.7mM KCl, 5mM imidazole, pH8.0) for solubilization, use of 10 times the bed volume of equilibration buffer in Ni-NTA affinity column (Qiagen, Cat # 30210), use of 10 times the medium volume of equilibration buffer to wash unbound proteins, use of 5 times the volume of equilibration buffer (50mM H2PO4, 300mM NaCl, 500mM imidazole, pH8.0), eluting the target protein, measuring the protein concentration, and storing at-20 ℃ for later use. The fusion antigen after purification of P2-x1 '-x 2-x 1' -HIV plasmid expression is abbreviated hereinafter as x1 '-x 2-x 1' -HIVAg, and so on.

The expression proteins of plasmids P2-x1 '-x 2-x 1' -HIV, P2-x1 '-x 2-HIV, P2-x 1' -HIV and P2-x2-HIV were subjected to reducing SDS-PAGE as shown in FIGS. 1 to 8. The expression of soluble protein in the four expression products is different from the expression of inclusion body, and the ratio is 9:1, 2:1 and 1:1 and 1: 1. The fact that the insertion of the X1 gene sequence into P2-X2-HIV can promote the soluble expression amount of protein is demonstrated, wherein the soluble expression amount of the protein of P2-X1 '-X2-X1' -HIV is 9 times that of P2-X2-HIV.

Example 4 labeling of HRP with fusion antigen to prepare HRP-containing conjugates

The conjugate was prepared using the NaIO4 oxidation method. Weighing 10mg of horseradish peroxidase, dissolving in 1ml of ultrapure water, slowly dropwise adding 5mg/ml NaIO4 solution freshly prepared from 1ml of ultrapure water, stirring gently for 40 minutes in the dark at room temperature, then adding 0.05ml of 20% glycol solution, and stirring for 40 minutes in the dark at room temperature. Then, the mixture was immediately dialyzed against 100mM, pH9.51 carbonate buffer for 2 hours, 2.5mg/ml of purified x1 '-x 2-x 1' -1 ml of HIVAg antigen, overnight at 4 ℃ against 100mM, pH9.51 carbonate buffer in the dark. The next day, 0.1ml of freshly prepared 4mg/ml NaBH4 solution was added dropwise to the mixture, mixed well and allowed to stand at 4 ℃ for 2 hours. The above solution was filled into a dialysis bag, dialyzed against PBS buffer (150mM, pH7.4) at 4 ℃ overnight. Adding enzyme protective agent and glycerol with final concentration of 50%, mixing, and storing at-20 deg.C in dark for use. The conjugate after x1 '-x 2-x 1' -HIVAg fusion antigen labeled HRP is referred to hereinafter in the text as x1 '-x 2-x 1' -HIVAg-HRP, and so on.

Example 5 HRP-containing conjugates for the detection of HIV antibodies by a double antigen sandwich ELISA

(1) Activity detection of labeled conjugates

Diluting HIV antigen (purchased from Fengpeng) with carbonate buffer solution (50mM, pH9.51) according to a certain proportion, adding 50 ul/hole of an enzyme label plate, coating for 24 hours at 4 ℃, washing the plate twice with PBST (10mM PB, 150mM NaCl, 0.05% Tween-20, pH7.4) washing solution on the next day, patting to dry, adding 30% newborn bovine serum, 8% sucrose, 5% casein, 1% o beta-mercaptoethanol, pH7.4 of 150mM NaCl, 10mM PB confining liquid into 120 ul/hole, confining for 2 hours at 37 ℃, throwing off liquid in the hole, patting to dry, and placing in a room with ventilation equipment for air drying at 20-25 ℃ and 55% -65% of humidity. Packaging in aluminum film bag with desiccant, and coating.

Adding 50ul of a sample to be detected, a negative reference sample (normal human negative serum) and a positive reference sample (HIV antibody positive serum) control into a coated enzyme label plate, and incubating for 30 minutes at 37 ℃; the plates were washed five times with PBST wash and patted dry. Then 50 ul/well 20mM PB buffer pH7.4 containing 20% newborn bovine serum, diluted in a certain ratio of x1 '-x 2-x 1' -HIVAg-HRP conjugate, incubated at 37 ℃ for 30 minutes; the plates were washed five times with PBST wash and patted dry.

50ul of each color developing agent A containing 0.5 per thousand of urea hydrogen peroxide, 4.76 per thousand of sodium acetate trihydrate and 0.9 per thousand of glacial acetic acid and color developing agents B containing 0.32 per thousand of TMB, 5mM of citric acid, 0.5mM of EDTA-2Na, 5% of methanol and 2 per thousand of dimethylformamide are added into each hole, and the mixture is shaded and developed for 10 minutes at 37 ℃. 50ul of stop solution containing 2M sulfuric acid was added to each well to terminate the reaction, and OD values were read after blank wells at a wavelength of 450nm (reference wavelength of 630nm) of the microplate reader were zeroed, as shown in Table 2. The results show that: compared with the control HIV-HRP, the labeling activity of the fusion antigen-conjugate expressed by fusion with the protein label is obviously higher than that of the control.

TABLE 1

TABLE 2

(2) Sensitive detection of labeled conjugates

The results of Table 3 were obtained by performing the assay on 100 serial dilutions of serum using the two-step sandwich method under the same environmental conditions of the reagents.

Cutoff (Cut off Value (COV)) calculation: the COV is multiplied by 2.0 (the negative control OD value is lower than 0.075 and higher than 0.075 according to the actual OD value), the OD value of the sample to be detected is positive when the COV is larger than or equal to the OD value, and the sample to be detected is negative when the OD value is smaller than the COV.

The results show that: compared with a control HIV-HRP, the sensitivity of the fusion antigen marked by the fusion expression with the protein label is obviously improved.

TABLE 3

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.

The application relates to an amino acid sequence:

SEQUENCE LISTING

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Glu Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val Ser Lys Ile Leu

35 40 45

Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr Val Leu Val Leu

50 55 60

Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro Thr Asp Gly Lys

65 70 75 80

Val Glu

<210> 6

<211> 130

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 6

Met Lys Leu Lys Val Thr Val Asn Gly Thr Ala Tyr Asp Val Asp Val

1 5 10 15

Asp Val Asp Lys Ser His Glu Asn Pro Met Gly Thr Ile Leu Phe Gly

20 25 30

Gly Gly Thr Gly Gly Ala Pro Ala Pro Ala Ala Gly Gly Ala Gly Ala

35 40 45

Gly Lys Ala Gly Glu Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val

50 55 60

Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr

65 70 75 80

Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro

85 90 95

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

100 105 110

Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly Asp Leu Glu Leu Ile Glu

115 120 125

Gly Gly

130

<210> 7

<211> 83

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 7

Met Ser His Glu Asn Pro Met Gly Thr Ile Leu Phe Gly Gly Gly Thr

1 5 10 15

Gly Gly Ala Pro Ala Pro Ala Ala Gly Gly Ala Gly Ala Gly Lys Ala

20 25 30

Gly Glu Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val Ser Lys Ile

35 40 45

Leu Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr Val Leu Val

50 55 60

Leu Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro Thr Asp Gly

65 70 75 80

Lys Val Glu

<210> 8

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 8

Met Ala Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp

1 5 10 15

His Glu Asp Thr Gly Gly Ser Ser His Glu Asn Pro Met Gly Thr Ile

20 25 30

Leu Phe Gly Gly Gly Thr Gly Gly Ala Pro Ala Pro Ala Ala Gly Gly

35 40 45

Ala Gly Ala Gly Lys Ala Gly Glu Gly Glu Ile Pro Ala Pro Leu Ala

50 55 60

Gly Thr Val Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys Ala

65 70 75 80

Gly Gln Thr Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu Ile

85 90 95

Asn Ala Pro Thr Asp Gly Lys Val Glu Met Ala Gly Gly Leu Asn Asp

100 105 110

Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp Thr Gly Gly Ser

115 120 125

<210> 9

<211> 128

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 9

Met Ala Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp

1 5 10 15

His Glu Asp Thr Gly Gly Ser Met Ala Gly Gly Leu Asn Asp Ile Phe

20 25 30

Glu Ala Gln Lys Ile Glu Trp His Glu Asp Thr Gly Gly Ser Ser His

35 40 45

Glu Asn Pro Met Gly Thr Ile Leu Phe Gly Gly Gly Thr Gly Gly Ala

50 55 60

Pro Ala Pro Ala Ala Gly Gly Ala Gly Ala Gly Lys Ala Gly Glu Gly

65 70 75 80

Glu Ile Pro Ala Pro Leu Ala Gly Thr Val Ser Lys Ile Leu Val Lys

85 90 95

Glu Gly Asp Thr Val Lys Ala Gly Gln Thr Val Leu Val Leu Glu Ala

100 105 110

Met Lys Met Glu Thr Glu Ile Asn Ala Pro Thr Asp Gly Lys Val Glu

115 120 125

<210> 10

<211> 131

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 10

Ser His Glu Asn Pro Met Gly Thr Ile Leu Phe Gly Gly Gly Thr Gly

1 5 10 15

Gly Ala Pro Ala Pro Ala Ala Gly Gly Ala Gly Ala Gly Lys Ala Gly

20 25 30

Glu Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val Ser Lys Ile Leu

35 40 45

Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr Val Leu Val Leu

50 55 60

Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro Thr Asp Gly Lys

65 70 75 80

Val Glu Met Ala Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile

85 90 95

Glu Trp His Glu Asp Thr Gly Gly Ser Gly Gly Gly Met Ala Gly Gly

100 105 110

Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Asp Thr

115 120 125

Gly Gly Ser

130

<210> 11

<211> 105

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 11

Met Ala Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp

1 5 10 15

His Glu Asp Thr Gly Gly Ser Ser His Glu Asn Pro Met Gly Thr Ile

20 25 30

Leu Phe Gly Gly Gly Thr Gly Gly Ala Pro Ala Pro Ala Ala Gly Gly

35 40 45

Ala Gly Ala Gly Lys Ala Gly Glu Gly Glu Ile Pro Ala Pro Leu Ala

50 55 60

Gly Thr Val Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys Ala

65 70 75 80

Gly Gln Thr Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu Ile

85 90 95

Asn Ala Pro Thr Asp Gly Lys Val Glu

100 105

<210> 12

<211> 105

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 12

Ser His Glu Asn Pro Met Gly Thr Ile Leu Phe Gly Gly Gly Thr Gly

1 5 10 15

Gly Ala Pro Ala Pro Ala Ala Gly Gly Ala Gly Ala Gly Lys Ala Gly

20 25 30

Glu Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val Ser Lys Ile Leu

35 40 45

Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr Val Leu Val Leu

50 55 60

Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro Thr Asp Gly Lys

65 70 75 80

Val Glu Met Ala Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile

85 90 95

Glu Trp His Glu Asp Thr Gly Gly Ser

100 105

<210> 13

<211> 153

<212> PRT

<213> Artificial Sequence

<220>

<223> Artificial sequence

<400> 13

Ala Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His

1 5 10 15

Glu Asp Thr Gly Met Lys Leu Lys Val Thr Val Asn Gly Thr Ala Tyr

20 25 30

Asp Val Asp Val Asp Val Asp Lys Ser His Glu Asn Pro Met Gly Thr

35 40 45

Ile Leu Phe Gly Gly Gly Thr Gly Gly Ala Pro Ala Pro Ala Ala Gly

50 55 60

Gly Ala Gly Ala Gly Lys Ala Gly Glu Gly Glu Ile Pro Ala Pro Leu

65 70 75 80

Ala Gly Thr Val Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys

85 90 95

Ala Gly Gln Thr Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu

100 105 110

Ile Asn Ala Pro Thr Asp Gly Lys Val Glu Lys Val Leu Val Lys Glu

115 120 125

Arg Asp Ala Val Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly Asp Leu

130 135 140

Glu Leu Ile Glu Gly Gly

145 150

Claims

1. A protein tag comprising a fragment X1, wherein the amino acid sequence of the fragment X1 comprises a-G-L-N-D-I-F-E-a-Q-K-I-E-W-H-E-D-T-G.

2. The protein tag of claim 1, further comprising a fragment of X2, wherein the fragment of X2 comprises the amino acid sequence of SEQ ID No. 5.

3. The protein tag according to claim 1 or 2, wherein the number of the X1 fragments is m, the number of the X2 fragments is n, the m is an integer greater than or equal to 1, and the n is an integer greater than or equal to 0;

optionally, said m is taken from 1 or 2, said n is taken from 0, 1 or 2;

optionally, said m + n is 1, 2 or 3;

optionally, said m-2 and n-1; or m is 1 and n is 1.

4. The protein tag according to any one of claims 1 to 3, wherein the X1 fragment is located upstream and/or downstream of the X2 fragment, the X1 fragment is linked to the X2 fragment via a linker sequence, or the X1 fragment is directly linked to the X2 fragment;

alternatively, the linker may be a linker comprising G, GS, SG, GGGS, GGGGS basic building blocks, and the number of basic building blocks may be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

5. The protein tag as claimed in any one of claims 1 to 4, wherein the amino acid sequence of the fragment X1 is represented by any one of SEQ ID NO 1-4; or the amino acid sequence of the X2 fragment is shown as any one of SEQ ID NO 5-7; or the protein tag amino acid sequence is shown in any one of SEQ ID NO 1-4 or SEQ ID NO 8-13.

6. A fusion protein comprising the protein tag of any one of claims 1 to 5 and a protein moiety;

optionally, the protein moiety is an antigen;

optionally, the protein tag is located upstream of the antigen;

optionally, the antigen is an antigen associated with an infectious disease, endocrine, tumor, or drug;

optionally, the antigen is an antigen associated with a viral or bacterial infectious disease;

optionally, the antigen includes, but is not limited to, an HIV antigen, a hepatitis A virus antigen, a hepatitis B virus antigen, a hepatitis C virus antigen, a hepatitis D virus antigen, a hepatitis E virus antigen, a hepatitis G virus antigen, a rubella virus antigen, a human cytomegalovirus antigen, a herpes simplex virus type 1 antigen, a herpes simplex virus type 2 antigen, a rabies virus antigen, a human T-lymphocyte leukemia virus antigen, a dengue virus antigen, a human papilloma virus antigen, a West Nile virus antigen, a forest encephalitis virus antigen, a measles virus antigen, an influenza virus antigen, a parainfluenza virus antigen, a varicella virus antigen, an echovirus antigen, a coxsackie virus antigen, an encephalitis B virus antigen, a coxsackie virus antigen, an EB virus antigen, a mumps virus antigen, a treponema antigen, a borrelia antigen, Chlamydia trachomatis antigen, Chlamydia pneumoniae antigen, Chlamydia psittaci antigen, ureaplasma urealyticum antigen, Mycoplasma pneumoniae antigen, Mycobacterium tuberculosis antigen, helicobacter pylori antigen, gonococcus antigen, Plasmodium falciparum antigen, Trypanosoma cruzi antigen, Toxoplasma gondii antigen.

7. A label conjugate comprising the fusion protein of claim 6 and a label, the fusion protein coupled to a label to form the label conjugate;

optionally, the label is selected from an enzyme, a luminescent substance, a fluorescent substance, a colored substance, a radioactive substance, a colloid, or a combination thereof.

8. A solid-phase conjugate comprising the fusion protein of claim 6 and a solid-phase carrier, the fusion protein being conjugated to a solid-phase carrier to form the solid-phase conjugate;

alternatively, the solid support is selected from the group consisting of microspheres, plates, and membranes, for example, the solid support can be magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon, and nitrocellulose membranes.

9. A nucleic acid encoding the protein tag of any one of claims 1 to 5 or the fusion protein of claim 6.

10. A vector comprising the nucleic acid of claim 9.

11. A cell comprising the nucleic acid of claim 9 or the vector of claim 10.

12. A method of producing a fusion protein comprising culturing the cell of claim 11.

13. A method for preparing a label conjugate, the method comprising subjecting the fusion protein of claim 6 to a coupling reaction with a label;

alternatively, the coupling reaction is carried out by a mixed acid anhydride method, a carbodiimide method, a glutaraldehyde method, a glutaric anhydride method, a diazotization method, a succinic anhydride method, a carbonyl diimidazole method, or a sodium periodate method.

14. A kit comprising the fusion protein of claim 6 and/or the label conjugate of claim 7 and/or the solid phase conjugate of claim 8.

15. Use of the protein tag according to any one of claims 1 to 5 for increasing the soluble expression level of a protein.

16. Use of a protein tag according to any one of claims 1 to 5 for increasing the activity of a labeled conjugate.