CN117304340A - Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate - Google Patents

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

Info

Publication number
CN117304340A
CN117304340A CN202311243546.5A CN202311243546A CN117304340A CN 117304340 A CN117304340 A CN 117304340A CN 202311243546 A CN202311243546 A CN 202311243546A CN 117304340 A CN117304340 A CN 117304340A
Authority
CN
China
Prior art keywords
fragment
antigen
seq
protein
fusion protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202311243546.5A
Other languages
Chinese (zh)
Other versions
CN117304340B (en
Inventor
孟媛
李俊
于秀玲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dongguan Pengzhi Biotechnology Co Ltd
Original Assignee
Dongguan Pengzhi Biotechnology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dongguan Pengzhi Biotechnology Co Ltd filed Critical Dongguan Pengzhi Biotechnology Co Ltd
Priority to CN202311243546.5A priority Critical patent/CN117304340B/en
Publication of CN117304340A publication Critical patent/CN117304340A/en
Application granted granted Critical
Publication of CN117304340B publication Critical patent/CN117304340B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a protein tag, fusion antigen or labeled conjugate containing the same and application thereof. The protein label improves the soluble expression quantity of the fusion expressed antigen, has excellent labelling performance, can ensure that the antigen has good solubility after being coupled with a label, and can keep the natural immunity of the antigen.

Description

Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate
RELATED APPLICATIONS
The application is aimed at the application number: 202111382326.1 (titled: a protein tag, fusion protein or labeled conjugate containing the same and use thereof, application date: 2021-11-22).
Technical Field
The invention belongs to the field of immunodetection. More particularly, it relates to a protein tag, fusion protein or tag 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 immune detection is a sensitive determination method, and the formed precipitate or turbidity is directly determined after antigen-antibody reaction, and the sensitivity can reach 5-10 mug/ml. However, in clinical tests, the level of some analytes in specimens is far below this level, and methods for increasing sensitivity have been sought. The labeled immunoassay is to label an antigen or antibody in a detection reagent with a substance capable of being measured in a minute amount, and to measure a label to thereby increase sensitivity. Labels are available in a wide variety of forms, including enzymes, chemiluminescent substances, fluorescent substances, radioactive substances, colloids, and combinations thereof, all of which function to increase the sensitivity of the immunoassay to a visually distinguishable or machine readable level.
In immunodetection, the double antigen sandwich method is widely applied to aspects of enzyme immunodetection, colloid-based detection, fluorescence detection and the like. The main principle of the double antigen sandwich method is as follows: the antigen is coated on the solid support, after the solid support is blocked, a sample to be detected is added, then a conjugate after the second antigen is marked is added, the antigen-antibody complex formed previously is combined with the conjugate to form an antigen-antibody-marked antigen complex, and then a signal is amplified by a certain means to obtain a final judging result.
The use of capture methods in immunoassays is also very common. Specific IgM against certain antigens in serum is often present together with specific IgG, which can interfere with the determination of IgM antibodies. Therefore, the multi-purpose capturing method for determining IgM antibody is to fix all serum IgM (including specific IgM and non-specific IgM) on a solid phase, add specific antigen-label after removing IgG, and develop color.
The competition method can be used for quantitative determination of antigen and hapten, and can also be used for determination of antibody. Taking the measurement of antigen as an example, adsorbing a specific antibody on a solid phase carrier, adding an antigen to be measured and a certain amount of known antigen-marker, combining the antigen and the marker competitively with the solid phase antibody, washing and separating, and finally, the antigen-marker combined with the solid phase is in negative correlation with the content of the antigen to be measured.
The term "label conjugate" as used herein refers to an antigen-label complex in the above-mentioned immunoassay. The activity of the conjugate has a decisive influence on the sensitivity of the immunoassay, whereas highly active conjugates need to have two conditions: in one aspect, as many labels as possible are bound to a unit antigen in the conjugate; on the other hand, the immunological activity of the antigen in the conjugate is as high as possible. Therefore, under the conditions defined by the markers and the marking method, the activity of the prepared conjugate is determined by the characteristics of the antigen, and the conditions required by the antigen of the conjugate with high activity are as follows:
1. good solubility and correct folding conformation. The immunological activity of an antigen is largely dependent on its spatial conformation, especially for conformational epitopes on top of the antigen. Antigens are expressed in the form of inclusion bodies, which are generally not properly folded. Such poorly soluble antigens generally have poor activity after labelling.
2. The antigen expression quantity is large, which is beneficial to mass production. This is well known to those skilled in the art.
Many antigens have very low activity of their conjugates after coupling to the label. The common solution is to fusion express a protein tag on the antigen, so that the expressed fusion antigen has better performance in the aspects, and has excellent marking performance.
The protein tags in the prior art include TRX, GST, DSBA, but the labeling performance of fusion antigens co-expressed by the protein tags is poor.
In view of this, the present invention has been made.
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 proteins, and the marking performance of fusion antigens is greatly improved through fusion expression of the protein tag and the antigens. After the conjugate is coupled with a marker, the antigen can be well soluble, the natural immunity of the antigen can be maintained, and when the conjugate is used for immunodetection, the sensitivity, the specificity and the stability of detection can be remarkably improved.
According to one aspect of the present invention, there is provided a protein tag comprising an X1 fragment, the X1 fragment amino acid sequence comprising A-G-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 a protein tag as described above and a protein moiety.
Further, the protein moiety is an antigen.
According to a third aspect of the present invention there is provided a label conjugate comprising a fusion protein as described above and a label, the fusion protein being coupled to the label to form the label conjugate.
According to a fourth aspect of the present invention there is provided a solid phase conjugate comprising a fusion protein as described above and a solid phase carrier, the fusion protein being coupled to 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 a protein tag or fusion protein as described above, a vector or cell comprising the same.
According to a sixth aspect of the present invention there is provided a method of preparing a fusion protein and a labelled conjugate.
According to a seventh aspect of the present invention there is provided a reagent or kit.
Finally, the invention also relates to the use of the protein tag in increasing the soluble expression level of the protein or increasing the activity of the labeled conjugate.
Drawings
FIG. 1 is a reducing SDS-PAGE result of x1 '-x2-x 1' -HIV fusion antigen.
FIG. 2 shows the results of reducing SDS-PAGE of the SAS treated and purified x1 '-x2-x 1' -HIV fusion antigen.
FIG. 3 is the result of reducing SDS-PAGE of x 1' -x2-HIV fusion antigens.
FIG. 4 shows the results of reducing SDS-PAGE of the x 1' -x2-HIV fusion antigen after SAS treatment and purification.
FIG. 5 is the result of reducing SDS-PAGE of x 1' -HIV fusion antigen.
FIG. 6 shows the results of reducing SDS-PAGE of the x 1' -HIV fusion antigen after SAS treatment and purification.
FIG. 7 shows the result of reducing SDS-PAGE of x2-HIV fusion antigen.
FIG. 8 shows the results of reducing SDS-PAGE of the x2-HIV fusion antigen after SAS treatment and purification.
Detailed Description
The protein tag means a functional unit, and in the present invention, the protein tag means a functional unit capable of increasing the soluble expression level of a fused protein moiety. The protein moiety may be an antigen or other protein, in the present invention the protein moiety is an antigen, in the present invention the protein tag is also referred to as an antigen tag. Fusion proteins also become fusion antigens.
In one aspect, embodiments of the present invention provide a protein tag comprising an X1 fragment, wherein the X1 fragment amino acid sequence comprises A-G-G-L-N-D-I-F-E-A-Q-K-I-E-W-H-E-D-T-G.
In an alternative embodiment, the protein tag further comprises an X2 fragment, the amino acid sequence of the X2 fragment 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, m is an integer greater than or equal to 1, and n is an integer greater than or equal to 0;
in alternative embodiments, m is taken from 1 or 2 and n is taken from 0,1 or 2;
in alternative embodiments, the m+n=1, 2 or 3;
1) When m+n=1, the protein tag is: an X1 fragment;
2) When m+n=2, the protein tag is selected from: (X1, X2) a combined fragment, (X2, X1) a combined fragment or (X1, X1) a combined fragment;
3) When m+n=3, the protein tag is selected from: (X1, X2, X1) combination fragment, (X1, X1, X2) combination fragment, (X2, X2, X1) combination fragment, (X1, X2, X2) combination fragment, (X2, X1, X1) combination fragment or (X2, X1, X2) combination fragment, the combination fragments are sequentially arranged continuously or discontinuously from the upstream (N end) to the downstream (C end) of the sequence.
In an alternative embodiment, m=2, n=1 or m=1, n=1.
The protein tag is selected from the group consisting of: (X1, X2) a combined fragment, (X2, X1) a combined fragment, (X1, X1, X2) a combined fragment or (X2, X1, X1) a combined fragment, which are arranged continuously or discontinuously in sequence from the upstream (N-terminal) to the downstream (C-terminal) of the sequence.
In alternative embodiments, the X1 fragment is located upstream (N-terminal) and/or downstream (C-terminal) of the X2 fragment, and the X1 fragment and the X2 fragment are linked 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, beta-Ala, GABA, or Ava), or PEG, etc.
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 an alternative embodiment, the protein tag is selected from the group consisting of: (X1-Linker-X2) combination fragment, (X2-Linker-X1) combination fragment, (X1-Linker-X2-X1) combination fragment, (X1-X2-Linker-X1) combination fragment, (X1-Linker-X1-X2) combination fragment, (X1-X1-Linker-X2) combination fragment, (X1-Linker-X2) combination fragment or (X2-Linker-X1-X1) combination fragment, (X2-X1-Linker-X1) combination fragment, (X2-Linker-X1) combination fragment, which are arranged sequentially from the upstream (N-terminus) to the downstream (C-terminus) of the sequence, said "-" representing a covalent linkage (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 an alternative embodiment, the protein tag is selected from the group consisting of: (X1-X2) a combined fragment, (X2-X1) a combined fragment, (X1-X1-X2) a combined fragment or (X2-X1-X1) a combined fragment, which are arranged in order from the upstream (N-terminal) to the downstream (C-terminal) of the sequence, said "-" representing a covalent linkage (peptide bond).
In an alternative embodiment, the amino acid sequence of the X1 fragment is shown in any one of SEQ ID NOs 1 to 4; or the amino acid sequence of the X2 fragment is shown as any one of SEQ ID NO 5-7; or the amino acid sequence of the protein tag is shown as any one of SEQ ID NO 1-4 or SEQ ID NO 8-13.
In other embodiments, protein tags having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequences of the protein tags described above (SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13) are also within the scope of the present invention. Variations of the sequences described which are conceivable to the person skilled in the art are also within the scope of the sequences protected by the invention.
In another aspect, embodiments of the present invention provide a fusion protein comprising the protein tag and a protein portion described above.
The term "fusion protein" as used in the present invention is a fusion expression product of a protein tag and a protein of interest.
In an alternative embodiment, 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 immunoassays.
In an alternative embodiment, 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 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 b 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 epstein barr virus antigen, a coxsackie virus antigen, a encephalitis virus antigen, a coxsackie virus antigen, an epstein barr virus antigen, a treponema pallidum antigen, a bordetention spirochet antigen, a chlamydia antigen, a tuberculosis antigen, a mycobacterium antigen, a helicobacter pylori antigen, a coccoix antigen, a trypanosoma antigen, a toxoplasma antigen.
The antigen gene may be obtained by artificially synthesizing nucleotide sequence, or amplified from E.coli genome and genome of other species, or may be obtained by modifying the sequence of natural genome through genetic engineering. The invention preferably synthesizes the sequence artificially, can use more codon to synthesize genes according to escherichia coli, thereby ensuring that the expression quantity of the fusion protein is not limited by codon preference. The gene of the fusion protein is obtained by taking the escherichia coli genome as a template through PCR amplification, and the method is a method commonly adopted in molecular biology and does not need to be described in detail.
In yet another aspect, embodiments of the present invention provide a labeled conjugate comprising the fusion protein described above and a label, wherein 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 label may be selected from any label used in the art that is suitable for the present invention.
In an alternative embodiment, the label is selected from the group consisting of: enzymes, luminescent substances, fluorescent substances, colored substances, radioactive substances, colloids, or combinations 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 deoxygenase.
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, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lotensine and its derivatives, peroxyoxalate and its derivatives.
The fluorescent material used in the present invention may be any suitable fluorescent material including, but not limited to, fluorescein isothiocyanate, rhodamine and its derivatives, europium and its derivatives, quantum dots and its derivatives, rare earth complexes and its derivatives.
The radioactive material used in the present invention may be any suitable radioactive material including, but not limited to, actinium, thorium, uranium, plutonium, curium, and mium.
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 yet another aspect, embodiments of the present invention provide a solid phase conjugate comprising the fusion protein and a solid phase carrier described above, wherein 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 or membranes, for example the solid support may be magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.
In yet another aspect, embodiments of the present invention provide a nucleic acid encoding a protein tag as described above or a fusion protein as described above.
Nucleic acids are typically RNA or DNA, and nucleic acid molecules may 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 acids are used when they are incorporated into vectors.
In yet another aspect, embodiments of the present invention provide a vector comprising the nucleic acid described above.
In yet another aspect, embodiments of the present invention provide a cell comprising the nucleic acid described above or the vector described above.
In yet another aspect, embodiments of the present invention provide a method of preparing a fusion protein comprising culturing a cell as described above.
In yet another aspect, embodiments of the present invention provide a method of preparing a labeled conjugate, the method comprising coupling the fusion protein described above to a label in vitro.
The markers of the invention can be conjugated to the fusion antigen in any manner known in the art.
The label is preferably bound to the fusion antigen by means of chemical coupling.
In alternative embodiments, the coupling reaction employs a method selected from the group consisting of a mixed anhydride method, a carbodiimide method, a glutaraldehyde method, a glutaric anhydride method, a diazotization method, a succinic anhydride method, a carbonyldiimidazole method, and a sodium periodate method.
Further preferably, sodium periodate is used, and the aldehyde formed by oxidation of the label with NaIO4 may be linked to the amino group of the fusion antigen to form a Schiff base which may be further reduced with NaBH4 (or ethanolamine) to form a stable conjugate.
From the above description, one of ordinary skill in the art will readily appreciate that the tag may be attached to any suitable residue of the fusion antigen, including but not limited to amino, carboxyl, hydroxyl, or thiol. Preferably the tag is attached to the amino group of the fusion antigen.
In a further aspect, embodiments of the present invention provide a reagent or kit comprising a fusion protein as described above and/or a labelled conjugate as described above and/or a solid phase conjugate as described above.
Finally, the embodiment of the invention provides application of the protein tag in improving the protein soluble expression level or improving the activity of the labeled conjugate.
According to the description of the invention, the acid-base amino acid of the fusion protein is uniformly distributed, so that the expressed fusion protein has good hydrophilicity, the spatial conformation of the target antigen is folded to be more similar to that of a natural antigen, and the molecular weight of the fusion protein is moderate and cannot be excessively large so as to influence the biological activity of the target antigen. The characteristics enable the expressed antigen to have high activity, and especially the sensitivity, the specificity, the stability and the like are obviously improved when the antigen is used for enzyme-labeled conjugate or colloid-labeled conjugate in immunodetection.
Embodiments of the present invention will be described in detail below with reference to examples. The examples are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified. The experimental procedures, which do not address the specific conditions in the examples below, are generally followed by conventional conditions, such as those described in Sambrook et al, molecular cloning, A laboratory Manual (New York: cold Spring Harbor Laboratory Press, 0989), or the recommendations of the kit manufacturer.
EXAMPLE 1 construction of vector P2-x2 carrying the x2 (SEQ ID NO: 5) protein tag
Referring to the Biotin purification tag DNA encoded protein sequence on the PinPoint Xa-1 plasmid of GeneBank No. U47628 (promega corporation, cat# V2020), a segment from amino acid 21 to amino acid 102 was selected and primers for this segment were computer-aided designed using Oligo software, with the primer sequences: the upstream primer 5'-GGGAGATCTCACCATCACCATCACCATCACCATTCACACGAAAACCCGAT-3' has BglII cleavage site and three protecting bases at the 5 'end, 8 His labels, the downstream primer 5'-GGTGAATTCTTAGGATCCCTCAACCTTGCCGTCGGTG-3'has BamHI and EcoRI cleavage sites at the 5' end, a stop codon is added between the two cleavage sites, and three protecting bases are at the 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 min x1 cycles, (94 ℃,30 seconds, 55 ℃,30 seconds, 72 ℃,30 seconds) x 30 cycles, 72 ℃,10 min x1 cycles. And (5) recovering PCR products. Then, the vector P2-x2 was obtained by simultaneously digesting with BglII and EcoRI, recovering the digested product, and ligating the digested product to the P2 vector digested with BamHI and EcoRI.
EXAMPLE 2 construction of expression plasmid P2-x2-HIV containing x2-HIV fusion antigen
The DNA sequence of 1486-1836bp in env gene of HIV (GeneBank No. AF 321145) was amplified by PCR, the upstream primer with BamHI site, the downstream primer with EcoRI site and before EcoRI site with termination codon TAA. The PCR fragment was cloned into the P2-x2 vector after BamHI and EcoRI cleavage, and the positive clone obtained was P2-x2-HIV.
By inserting the X1 gene sequence on the expression plasmid P2-X2-HIV, forming P2-X1 ' "-X2-X1 '" -HIV, P2-X1 ' "-X2-HIV, P2-X2-X1 '" - (Linker) -X1 ' "-HIV, P2-X1 '" -X2-HIV, and P2-X2-X1 ' "-HIV expression plasmids as set forth in table 1; 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 fusion antigen
E.coli ER2566 was transformed with the expression plasmid P2-x1 '-x2-x 1' -HIV, spread on LB plates containing 100ug/ml kanamycin sulfate, cultured overnight at 37℃to pick up a monoclonal, shake-cultured with 500ml LB medium containing the same concentration of kanamycin at 37℃to about OD6001.0, and induced with IPTG at a final concentration of 0.5mM under the following conditions: 37℃at 200rpm for 4 hours. The cells were collected by centrifugation at 5000rpm for 20 minutes at 4℃and resuspended in 10ml lysis buffer (50 mM Tirs-HCl, pH8.0,1mM EDTA,100mMNaCl) per liter of cells, sonicated, centrifuged at 12000rpm for 20 minutes at 4℃and the majority of the protein was distributed in the lysis supernatant after SDS-PAGE electrophoresis identification, the supernatant was collected, 0.25 fold supernatant volume of saturated ammonium sulfate solution was added, the pellet was collected by centrifugation at 12000rpm for 20 minutes at 4℃and dissolved in 10ml equilibration buffer (10mM Na2HPO4,1.8mM KH2PO4, 140mM NaCl,2.7mM KCl,5mM imidazole, pH 8.0), ni-NTA affinity column (Qiagen, cat# 30210) was equilibrated with 10 column bed volumes of equilibration buffer, protein samples were added, unbound protein was washed out with 10 volumes of equilibration buffer, 5 volumes of elution buffer (50mM NaH2PO4, 300mM NaCl,500mM imidazole, pH 8.0), protein concentration was measured and the protein was stored at-20℃for further use. P2-x1 '-x2-x 1' -HIV plasmid expression after purification the fusion antigen is hereinafter abbreviated as x1 '"-x 2-x 1'" -HIVAg, other names and so on.
Plasmid P2-x1 '-x2-x 1' -HIV P2-x1 '-x2-HIV the expression proteins of P2-x 1' -HIV and P2-x2-HIV were subjected to reducing SDS-PAGE, as shown in fig. 1-8. The expression of the soluble protein and the expression of the inclusion body in the four expression products are inconsistent, and the ratio is 9:1, 2:1 and 1 respectively: 1 and 1:1. It was demonstrated that insertion of the X1 gene sequence into P2-X2-HIV promotes the soluble expression level of the protein, wherein the soluble expression level of the protein of P2-X1 '-X2-X1' -HIV is 9 times that of P2-X2-HIV.
EXAMPLE 4 labelling of HRP with fusion antigen to prepare HRP-containing conjugates
Conjugates were prepared using the NaIO4 oxidation method. 10mg of horseradish peroxidase is weighed and dissolved in 1ml of ultrapure water, then 1ml of 5mg/ml NaIO4 solution freshly prepared by ultrapure water is slowly added dropwise, the mixture is gently stirred at room temperature for 40 minutes in a dark place, then 0.05ml of 20% glycol solution is added, and the mixture is stirred at room temperature for 40 minutes in a dark place. Immediately after this, 1ml of purified x1 '-x2-x 1' -HIVAg antigen, which had been dialyzed against 100mM, pH9.51 carbonate buffer for 2 hours at 4℃and 2.5mg/ml, was added and dialyzed overnight against 100mM, pH9.51 carbonate buffer at 4 ℃. The next day, 0.1ml of freshly prepared 4mg/ml NaBH4 solution was added dropwise to the mixture, and the mixture was stirred well and allowed to stand at 4℃for 2 hours. The solution was packed in a dialysis bag, dialyzed against PBS buffer (150 mM, pH 7.4) and at 4℃overnight. Adding enzyme protectant and glycerol with final concentration of 50%, mixing, and keeping at-20deg.C in dark place. Conjugate after HRP is marked by x1 '-x2-x 1' -HIVAg fusion antigen in the following text, is referred to as x1 '-x2-x 1' -HIVAg-HRP, other names and so on.
EXAMPLE 5 HRP-containing conjugate for double antigen sandwich ELISA detection of HIV antibodies
(1) Activity detection of labeled conjugates
HIV antigen (from Fei Peng) was diluted in a proportion with carbonate buffer (50 mM, pH 9.51), 50 ul/well was added to the ELISA plate, coated at 4℃for 24 hours, washed twice with PBST (10mM PB,150mM NaCl,0.05%Tween-20, pH 7.4) wash next day, patted dry, 120 ul/well was added with pH7.4 containing 30% newborn calf serum, 8% sucrose, 5%o casein, 1%o beta-mercaptoethanol, 150mM NaCl, 10mM PB blocking solution, blocked at 37℃for 2 hours, the in-well liquid was thrown off, patted dry, and air dried in a room with ventilation equipment at room temperature 20-25℃humidity 55% -65%. Packaging in aluminum film bag with desiccant, and coating.
Firstly, 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) into a coated ELISA plate, and incubating for 30 minutes at 37 ℃; the plates were washed five times with PBST wash and patted dry. A further 50 ul/well of 20mM PB buffer pH7.4 containing 20% neonatal bovine serum, diluted in a proportion of x1 '-x2-x 1' -HIVAg-HRP conjugate, was added and incubated for 30 minutes at 37 ℃; the plates were washed five times with PBST wash and patted dry.
Each well was charged with 50ul of a color developer A containing 0.5% hydrogen peroxide urea, 4.76% sodium acetate trihydrate, 0.9% glacial acetic acid, and a color developer B containing 0.32% TMB, 5mM citric acid, 0.5mM EDTA-2Na, 5% methanol, 2% dimethylformamide, respectively, and developed at 37℃for 10 minutes in the absence of light. Each well was quenched with 50ul of a stop solution containing 2M sulfuric acid, and the OD values were read after zeroing the blank wells at a wavelength of 450nm (reference wavelength 630 nm) of the microplate reader, as shown in Table 2. The results show that: the labelling activity of the fusion antigen-conjugate expressed in fusion with the protein tag was significantly higher than that of the control, compared to the control HIV-HRP.
TABLE 1
TABLE 2
(2) Sensitive detection of labeled conjugates
The results of Table 3 were obtained by testing 100 parts of serum in serial dilution ratios using the two-step sandwich method under conditions of reagents unified in the same environment.
Cut Off Value (COV)) calculation: COV=average OD of negative control×2.0 (negative control OD lower than 0.075 calculated as 0.075 and higher than 0.075 calculated as actual OD), the OD of the sample to be tested is greater than or equal to COV positive, and the OD of the sample to be tested is less than COV negative.
The results show that: compared with the control HIV-HRP, the sensitivity of the fusion antigen marked by fusion expression with the protein tag is obviously improved.
TABLE 3 Table 3
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
The amino acid sequence referred to in the present application:

Claims (18)

1. a fusion protein comprises a protein tag and a protein part, and is characterized in that the protein tag comprises an X1 fragment, and the amino acid sequence of the X1 fragment is shown as any one of SEQ ID NO. 2-4.
2. The fusion protein of claim 1, wherein the protein tag further comprises an X2 fragment, and wherein the amino acid sequence of the X2 fragment is any one of SEQ ID NOs 5 to 7.
3. A fusion protein comprises a protein tag and a protein part, and is characterized in that the protein tag comprises an X2 fragment, and the amino acid sequence of the X2 fragment is shown in any one of SEQ ID NO. 6-7.
4. The fusion protein of claim 3, wherein the protein tag further comprises an X1 fragment, and the amino acid sequence of the X1 fragment is any one of SEQ ID NOs 1 to 4.
5. A fusion protein comprising a protein tag and a protein portion, the protein tag comprising:
1) An X2 fragment shown in SEQ ID NO. 6 and an X1 fragment shown in SEQ ID NO. 1;
2) An X2 fragment shown in SEQ ID NO. 6 and an X1 fragment shown in SEQ ID NO. 2;
3) An X2 fragment shown in SEQ ID NO. 6 and an X1 fragment shown in SEQ ID NO. 3;
4) An X2 fragment shown in SEQ ID NO. 6 and an X1 fragment shown in SEQ ID NO. 4;
5) An X2 fragment shown in SEQ ID NO. 7 and an X1 fragment shown in SEQ ID NO. 1;
6) An X2 fragment shown in SEQ ID NO. 7 and an X1 fragment shown in SEQ ID NO. 2;
7) An X2 fragment shown in SEQ ID NO. 7 and an X1 fragment shown in SEQ ID NO. 3;
8) An X2 fragment shown in SEQ ID NO. 7 and an X1 fragment shown in SEQ ID NO. 4;
9) An X2 fragment shown in SEQ ID NO. 5 and an X1 fragment shown in SEQ ID NO. 1;
10 An X2 fragment shown in SEQ ID NO. 5 and an X1 fragment shown in SEQ ID NO. 2;
11 An X2 fragment shown in SEQ ID NO. 5 and an X1 fragment shown in SEQ ID NO. 3; or (b)
12 An X2 fragment shown in SEQ ID NO. 5 and an X1 fragment shown in SEQ ID NO. 4.
6. The fusion protein of any one of claims 1-5, wherein the number of X1 fragments is m, the number of X2 fragments is n, m is an integer greater than or equal to 1, and n is an integer greater than or equal to 0;
alternatively, m is taken from 1 or 2 and n is taken from 0,1 or 2;
optionally, the m+n=1, 2 or 3;
alternatively, the m=2 and n=1; or m=1 and n=1;
optionally, the protein tag is selected from the group consisting of: (X1, X2) a combined fragment, (X2, X1) a combined fragment, (X1, X2, X1) a combined fragment, (X1, X2) a combined fragment, (X2, X1) a combined fragment, (X1, X2) a combined fragment, (X2, X1) a combined fragment or (X2, X1, X2) a combined fragment.
7. The fusion protein of any one of claims 1 to 6, wherein the fragments are linked by a linker sequence or the fragments are linked directly;
optionally, the protein tag is selected from the group consisting of: (X1-Linker-X2) combination fragment, (X2-Linker-X1) combination fragment, (X1-Linker-X2-X1) combination fragment, (X1-X2-Linker-X1) combination fragment, (X1-Linker-X1-X2) combination fragment, (X1-X1-Linker-X2) combination fragment, (X1-Linker-X2) combination fragment or (X2-Linker-X1-X1) combination fragment, (X2-X1-Linker-X1) combination fragment;
alternatively, the linker may be a linker comprising G, GS, SG, GGGS, GGGGS basic structural units, the number of which may be an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more;
optionally, the protein tag is selected from the group consisting of: (X1-X2) a combination fragment, (X2-X1) a combination fragment, (X1-X1-X2) a combination fragment or (X2-X1-X1) a combination fragment.
8. The fusion protein of any one of claims 1 to 7, wherein the protein moiety is an antigen;
alternatively, the protein tag is linked to the antigen via a linker sequence or directly;
optionally, the protein tag is located upstream of the antigen;
alternatively, the antigen is an antigen associated with an infectious disease, endocrine, tumor, or drug;
alternatively, the antigen is an antigen associated with a viral or bacterial infectious disease;
alternatively, 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 b 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 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 virus antigen, an EB virus antigen, a mumps virus antigen, a treponema pallidum antigen, a borrelia antigen, a chlamydia trachomatis antigen, a chlamydia pneumoniae antigen, a ureaplasma urealyticum antigen, a helicobacter pylori antigen, a gonococcus antigen, a toxoplasmodium antigen, a trypanosoma antigen.
9. A label conjugate comprising the fusion protein of any one of claims 1-8 and a label, the fusion protein being coupled to a label to form the label conjugate;
optionally, the label 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.
10. A solid phase conjugate comprising the fusion protein of any one of claims 1-8 and a solid phase carrier, the fusion protein being coupled to the solid phase carrier to form the solid phase conjugate;
alternatively, the solid support is selected from microspheres, plates or membranes, for example the solid support may be magnetic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.
11. A nucleic acid encoding the fusion protein of any one of claims 1 to 8.
12. A vector comprising the nucleic acid of claim 11.
13. A cell comprising the nucleic acid of claim 11 or the vector of claim 12.
14. A method of preparing a fusion protein comprising culturing the cell of claim 13.
15. A method of preparing a label conjugate, the method comprising coupling the fusion protein of any one of claims 1-8 to a label;
alternatively, the coupling reaction adopts a method of mixed anhydride method, carbodiimide method, glutaraldehyde method, glutaric anhydride method, diazotization method, succinic anhydride method, carbonyl diimidazole method or sodium periodate method.
16. A kit comprising the fusion protein of any one of claims 1-8 and/or the label conjugate of claim 9 and/or the solid phase conjugate of claim 10.
17. Use of a protein tag in a fusion protein according to any one of claims 1 to 8 for increasing the soluble expression level of a protein.
18. Use of a protein tag in a fusion protein according to any one of claims 1 to 8 for increasing the activity of a labelled conjugate.
CN202311243546.5A 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate Active CN117304340B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311243546.5A CN117304340B (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111382326.1A CN114133433A (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing protein tag and application of protein tag
CN202311243546.5A CN117304340B (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN202111382326.1A Division CN114133433A (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing protein tag and application of protein tag

Publications (2)

Publication Number Publication Date
CN117304340A true CN117304340A (en) 2023-12-29
CN117304340B CN117304340B (en) 2024-09-20

Family

ID=80390914

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202111382326.1A Pending CN114133433A (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing protein tag and application of protein tag
CN202311243546.5A Active CN117304340B (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202111382326.1A Pending CN114133433A (en) 2021-11-22 2021-11-22 Protein tag, fusion protein or labeled conjugate containing protein tag and application of protein tag

Country Status (1)

Country Link
CN (2) CN114133433A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5723584A (en) * 1993-07-30 1998-03-03 Affymax Technologies N.V. Biotinylation of proteins
CN101403746A (en) * 2008-07-18 2009-04-08 深圳市菲鹏生物股份有限公司 Conjugate used for immunity detection

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9678080B2 (en) * 2013-06-14 2017-06-13 Pacific Biosciences Of California, Inc. Bis-biotinylation tags

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5723584A (en) * 1993-07-30 1998-03-03 Affymax Technologies N.V. Biotinylation of proteins
CN101403746A (en) * 2008-07-18 2009-04-08 深圳市菲鹏生物股份有限公司 Conjugate used for immunity detection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
S. SALMAN ASHRAF等: "A novel multi-affinity tag system to produce high levels of soluble and biotinylated proteins in Escherichia coli", PROTEIN EXPRESSION & PURIFICATION, vol. 33, 31 December 2004 (2004-12-31) *

Also Published As

Publication number Publication date
CN117304340B (en) 2024-09-20
CN114133433A (en) 2022-03-04

Similar Documents

Publication Publication Date Title
CA2195753C (en) Determination of a specific immunoglobulin using multiple antigens
US20080193955A1 (en) Kit and Method for the Detection of Anti-Hepatitis C Virus (Hcv) Antibodies
EP1371983B1 (en) Immunoassay, reagent for immunoassay, and production method of the same
CN109324191A (en) The antibody test prize law and its kit of indirectly marked with nanometer granule
US6274324B1 (en) Specific binding reagent comprising a variable domain protein linked to a support or tracer
JP2001500723A (en) Multiple epitope fusion protein
CN113087792B (en) Canine distemper virus nano antibody and application thereof
US7824875B2 (en) Recombinant antigens for the detection of Coxiella burnetii
CN109613240A (en) It is a kind of for detecting the kit of HIV
JPH06505639A (en) Recombinant FIV glycoprotein 160 and p24GAG protein
US20100279432A1 (en) Indirectly labelled assay conjugates and methods of preparing and using same
CN101403746B (en) Conjugate used for immunity detection
CN110996986B (en) Multi-epitope fusion proteins of HCV antigens and uses thereof
CN117304340B (en) Protein tag, fusion protein or labeled conjugate containing same and application of fusion protein or labeled conjugate
AU650857B2 (en) Antibody variable domain conjugates
Witkowski et al. Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein
CN116699130A (en) Emulsion immunochromatography test strip for rapidly detecting transmissible gastroenteritis coronavirus and preparation method thereof
CN113721023A (en) Biological immunosensing system based on Barnase/barstar and application
JP3404035B2 (en) Cysteine thiol protected peptides for use in immunoassays
JP2010122002A (en) Antibody detecting method and reagent kit used therein
US20090111123A1 (en) Compositions for detecting antibodies to babesia microti and methods of uses thereof
WO2018119868A1 (en) Hiv recombinant antigen, expression gene, expression vector, and hiv test kit
US20240280575A1 (en) Compositions and Methods for the Detection of Mycoplasma
AU2002251445B2 (en) Method and kit for quantitation of histidine-tagged polypeptides
ES2256862T3 (en) HAPTEN-PEPTIDE CONJUGATES IMPROVED TO DETECT ANTI-HIV-1 OR ANTI-HIV-2 ANTIBODIES.

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant