CN115947803A - Diagnostic antigen for quantitatively detecting Yersinia pestis antibody - Google Patents

Abstract

The invention discloses a diagnostic antigen for quantitatively detecting Yersinia pestis antibodies. Specifically discloses a protein F1B139 with an amino acid sequence of SEQ ID No.1 and application thereof as an antigen in detection, analysis or evaluation of a plague bacillus antibody. Experiments show that the plague bacteria antigen has good sensitivity, can be identified by rabbit anti-F1 polyclonal antibody, can be used as a quality control product of a plague bacteria antigen detection reagent, ensures the batch stability of the production of antibody raw materials, can also be used as a raw material of the antibody detection reagent, has single component of the recombinant F1 antigen, and can accurately evaluate the antibody. The immunodetection method and the product based on the antigen can realize the quantitative detection of the antibody, have good sensitivity, can be widely used for the detection, quality control and evaluation of the plague bacillus antibody, provide help for clinical diagnosis, curative effect observation, prognosis judgment, epidemiological investigation of infectious diseases and the like, and have very wide clinical application prospect.

Description

Diagnostic antigen for quantitatively detecting Yersinia pestis antibody

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a diagnostic antigen for quantitatively detecting Yersinia pestis antibodies.

Background

Yersinia pestis (abbreviated as plague) is the pathogen of pestilence zoonosis infectious disease plague. Plague is an infectious disease class A specified in the infectious disease prevention and treatment Law of the people's republic of China, and belongs to a human and animal co-suffering severe disease. Plague historically caused three pandemics, the outbreak of which in the 14 th century caused one third of the population in europe to die. There are many natural plague areas all over the world, which are difficult to be eliminated completely in a short period. The method for preventing and controlling plague from the source is the detection of plague bacteria and the monitoring of natural plague source, and the early diagnosis has important significance for reducing the death rate.

The existing methods for diagnosing plague infection mainly comprise a nucleic acid detection method, an immunodetection method and the like. The F1 capsular antigen is the most main surface antigen, directly determines the antigenicity of plague bacteria, and is a detection target recognized by the World Health Organization (WHO). The F1 antigen is highly conserved in plague bacillus and consists of linear fibers connected by Caf1 subunits, wherein the Caf1 subunit is a protein consisting of 149 amino acids. The plague antigen detection reagent mainly adopts a double-antigen sandwich detection mode (such as an immunochromatographic test paper for detecting Yersinia pestis infection and a preparation method thereof, application number 200410103568.2) and an indirect detection mode. In any mode, the F1 antibody is an important raw material for ensuring the sensitivity and specificity of the detection reagent, so that the precise quality control and evaluation of the F1 antibody are important.

The current reagents for evaluating F1 antibodies mainly include natural F1 antigen (Yersinia pestis natural F1 antigen extraction and purification method, application No. 200810055697.7) and recombinant F1 (Tavares, D.H.C., et al. (2020), "A new recombiant F1 antigen as a cost and time-effective tool for developing diagnosis," JBiols Methods 172. Both the natural F1 antigen and the recombinant F1 exist in the form of a mixture consisting of monomers and different amounts of caf1 protein polymers, and the plague bacteria antibody cannot be quantitatively evaluated. Some researchers tried to cleave the natural F1 antigen into two fragments from the middle, and the cleaved fragment containing the linear epitope could be used for immunoassay of antibody positive samples (method for cleaving yersinia pestis F1 antigen and related applications, application No. 200910083026.6), but if the linear epitope of the antibody is at the cleavage site, the antibody could not be analyzed. Therefore, there is still a clinical need for diagnostic antigens that allow accurate and sensitive quantitative analysis of yersinia pestis antibodies.

Disclosure of Invention

The technical problem to be solved by the invention is how to detect or quantitatively detect Yersinia pestis antibodies. The technical problem to be solved is not limited to the described technical subject, and other technical subject not mentioned herein may be clearly understood by those skilled in the art through the following description.

In order to solve the technical problem, the invention firstly provides a protein, named as F1B139, wherein the protein can be any one of the following proteins:

a1 Protein of which the amino acid sequence is SEQ ID No. 1;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No. 1), has more than 80 percent of identity with the protein shown in A1) and has the same function;

a3 A fusion protein having the same function obtained by attaching a tag to the N-terminus and/or C-terminus of A1) or A2).

The protein F1B139 is a truncated plague bacillus F1 surface antigen protein, and is also called recombinant protein F1B139. Compared with the full-length F1 antigen without truncation (149 amino acids), the N-terminal of the recombinant protein F1B139 is truncated by 10 amino acids, namely, the last 139 amino acids of the full-length F1 antigen are selected as a new recombinant F1 antigen.

The full-length amino acid sequence of the plague bacterium F1 surface antigen protein (F1 antigen for short) is as follows: 5 '-ADLTASTTATLVEPARITLTYKEGAPITIMDNGNIDDLLVGTLTLTLGGYKTGTTS TSVNFTDAAGDPMYLTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDV VLATGSQDFFVRSIGGSKGGKLAAGKYTDTVSNQ-3'.

The substitutions described herein can be conservative substitutions (also referred to as conservative substitutions) or non-conservative substitutions of non-core functional regions. It is well known to those skilled in the art that conservative substitutions, or non-conservative substitutions in non-core functional regions, do not generally have a qualitative effect on the function of the protein.

In order to facilitate the purification or detection of the protein in A1), a tag protein may be attached to the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in SEQ ID No.1 of the sequence Listing.

Labels described herein include, but are not limited to: GST (glutathione mercaptotransferase) tag protein, his tag protein (His-tag), MBP (maltose binding protein) tag protein, flag tag protein, SUMO tag protein, HA tag protein, myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow green fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag protein.

The nucleotide sequence of the invention coding for the recombinant protein F1B139 can easily be mutated by a person skilled in the art by known methods, for example directed evolution or point mutation. Those nucleotides which are artificially modified and have 75% or more than 75% identity with the nucleotide sequence of the recombinant protein F1B139 isolated by the present invention are derived from the nucleotide sequence of the present invention and are identical with the sequence of the present invention as long as they encode the recombinant protein F1B139 and have the function of the recombinant protein F1B139.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

Herein, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost, and the Lambda ratio are set to 11,1, and 0.85 (default values), respectively, and a search is performed to calculate the identity (%) of the amino acid sequence, and then the value (%) of identity can be obtained.

Herein, the 80% or greater identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

The invention also provides a biomaterial, which can be any one of the following:

b1 A nucleic acid molecule encoding said protein F1B139;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A recombinant host cell containing the nucleic acid molecule according to B1), or a recombinant host cell containing the expression cassette according to B2), or a recombinant host cell containing the recombinant vector according to B3).

In the above biological material, the nucleic acid molecule of B1) may be any one of:

c1 A DNA molecule with the coding sequence of SEQ ID No. 2;

c2 A DNA molecule whose nucleotide sequence is SEQ ID No. 2.

Further, the nucleic acid molecule of B1) may be expressed by all of the expression cassette of B2), the recombinant vector of B3), the recombinant microorganism of B4) and the recombinant host cell of B5).

The DNA molecule (the name can be F1B139 gene) shown in SEQ ID No.2 encodes recombinant protein F1B139 of which the amino acid sequence is SEQ ID No. 1.

The nucleic acid molecules described herein may be DNA, such as cDNA, genomic DNA or recombinant DNA. The nucleic acid molecule also can comprise a nucleic acid molecule obtained by codon preference modification on the basis of the nucleotide sequence shown in SEQ ID No. 2. In view of the degeneracy of the codons and the preference of codons for different species, one skilled in the art can use codons suitable for the expression of a particular species as needed.

The vector herein refers to a vector capable of carrying foreign DNA or a gene of interest into a host cell for amplification and expression, and may be a cloning vector or an expression vector, including but not limited to: plasmids, phages (e.g., lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), ti plasmids, viral vectors (e.g., retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of the invention, the vector is the vector pET-21a.

The microorganism described herein can be a bacterium, a fungus, an actinomycete, a protozoan, an algae, or a virus. Wherein the bacteria may be from the genera Escherichia (Escherichia sp.), erwinia (Erwinia sp.), agrobacterium (Agrobacterium sp.), flavobacterium (Flavobacterium sp.), alcaligenes (Alcaligenes sp.), pseudomonas (Pseudomonas sp.), bacillus (Bacillus sp.), etc., but not limited thereto, for example, the bacteria may be Escherichia coli (Escherichia coli), bacillus subtilis (Bacillus subtilis) or Bacillus pumilus (Bacillus pumilus). In one or more embodiments of the invention, the microorganism is escherichia coli DH5 α and/or escherichia coli BL21 (DE 3).

The host cell (also referred to as recipient cell) described herein can be a plant cell or an animal cell. Such host cells are understood to refer not only to the particular recipient cell but also to the progeny of such a cell, and such progeny may not necessarily be identical to the original parent cell, but are still included within the scope of the host cell, due to natural, accidental, or deliberate mutation and/or alteration. Suitable host cells are known in the art, wherein: the plant cell can be plant cells such as Arabidopsis thaliana (Arabidopsis thaliana), tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like, but is not limited thereto; the animal cell may be a mammalian cell (e.g., chinese hamster ovary cell (CHO cell), vero monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK 293 cell), human HeLa cell, fibroblast, myeloid cell line, T cell, or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., xenopus laevis (Xenopus laevis) cell or giant salamander (Andrias davidianus) cell), fish cell (e.g., grass carp, trout, or catfish cell), insect cell (e.g., sf21 cell or Sf-9 cell), and the like, but is not limited thereto.

The recombinant vector is a recombinant DNA molecule constructed by connecting an exogenous target gene and a vector in vitro, and can be constructed in any suitable manner as long as the constructed recombinant vector can carry the exogenous target gene into a receptor cell and provide the exogenous target gene with the capability of replication, integration, amplification and/or expression in the receptor cell.

The recombinant microorganism (or recombinant host cell) as used herein refers to a recombinant microorganism (or recombinant host cell) with altered function obtained by manipulating and modifying the gene of the microorganism (or host cell). For example, a recombinant microorganism (or a recombinant host cell) obtained by introducing an exogenous gene or a recombinant vector into a target microorganism (or a target host cell), or a recombinant microorganism (or a recombinant host cell) obtained by directly gene-editing an endogenous gene of a target microorganism (or a target host cell). The recombinant microorganism (or recombinant host cell) is understood to refer not only to the particular recombinant microorganism (or recombinant host cell), but also to the progeny of such a cell, and such progeny may not necessarily be identical to the original parent cell, due to natural, accidental, or deliberate mutation and/or alteration, but are still included within the scope of the recombinant microorganism (or recombinant host cell).

In the biological material, the recombinant vector B3) can be a recombinant vector pET-21a/EV76-F1 _11-149 。

Recombinant vector pET-21a/EV76-F1 _11-149 The recombinant expression vector can be obtained by replacing a fragment (small fragment) between BamHI and XhoI recognition sites of the pET-21a vector with a DNA fragment with a nucleotide sequence of SEQ ID No.2 in a sequence table and keeping other sequences of the pET-21a vector unchanged. Recombinant vector pET-21a/EV76-F1 _11-149 Contains F1B139 gene shown in SEQ ID No. 2.

In the above biological material, B4) the recombinant microorganism may be the recombinant vector pET-21a/EV76-F1 _11-149 Introducing into Escherichia coli to obtain recombinant microorganism. Specifically, the recombinant microorganism can be the recombinant vector pET-21a/EV76-F1 _11-149 Introduction of DH 5. Alpha. Competent cells or BL21 (DE 3) competent cellsAnd (4) a group of microorganisms.

B5 The recombinant host cell can be the recombinant vector pET-21a/EV76-F1 _11-149 A recombinant host cell obtained by introducing the host cell.

The introduction may be by chemical conversion (e.g. Ca) ²⁺ Induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation) or electroporation transformation) to transform the vector carrying the DNA molecule of the present invention into a host bacterium; the DNA molecules of the invention may also be transduced into host bacteria by means of phage transduction. The introduction may also be transfection of the host cells with the vector carrying the DNA molecule of the present invention by any known transfection method such as calcium phosphate co-precipitation, liposome-mediated, electroporation, or viral vector.

The invention also provides any one of the following applications of the protein F1B139 and/or the biological material:

d1 Application in detecting, analyzing or evaluating Yersinia pestis antibody (abbreviated as Yersinia pestis antibody) or preparing products for detecting, analyzing or evaluating Yersinia pestis antibody;

d2 Use in quantitative detection, quantitative analysis or quantitative evaluation of yersinia pestis antibodies or for the preparation of a product for quantitative detection, quantitative analysis or quantitative evaluation of yersinia pestis antibodies;

d3 Application in plague detection or preparation of products for plague detection;

d4 The application in preparing plague antigen detection products (such as plague antigen detection kits) or plague antibody detection products (such as plague antibody detection kits);

d5 Application in preparing quality control products for detecting plague antigens;

d6 Application in preparing or screening Yersinia pestis antibody;

d7 Use in the preparation of products for the diagnosis, auxiliary diagnosis, screening, observation of curative effect, prognosis judgment or observation of vaccination effect of plague;

d8 Use in the control of plague (such as epidemiological investigation of plague) or in the preparation of products for plague control (such as products for epidemiological investigation of plague).

The product described herein may be a reagent, kit, chip, strip or test card.

The products described herein may comprise the protein F1B139.

The detection, analysis or assessment may comprise detection, analysis or assessment of antibody titer, specificity and/or affinity.

Antigen-antibody reactions are the core and the basis of immunological testing techniques. It is well known to those skilled in the art that analysis of specific antibodies by known antigen detection based on the principle of antigen-antibody specific binding can be used to assist clinical diagnosis, observation of therapeutic effects, prognosis and observation of vaccination effects. Also has special and important significance in epidemiological investigation of infectious diseases. Methods for detecting antibodies using known antigens are well known to those skilled in the art, such as precipitation reaction, agglutination assay, fluorescence immunoassay, radioimmunoassay, enzyme immunoassay, chemiluminescence immunoassay, and POCT (point-of-care testing) related immunodetection techniques (e.g., colloidal gold immunoassay, fluorescence immunochromatography), etc. Therefore, the recombinant protein F1B139 designed and developed by the present invention can be applied to the applications described in D1) -D8) above.

The recombinant protein F1B139 can be used as a known detection antigen (or a diagnosis antigen) in a plague antigen detection method or a product, is used for qualitatively or quantitatively detecting a Yersinia pestis antibody, and can also be used as a quality control product in the plague antigen detection method or the product.

The invention also provides a reagent or a kit, wherein the reagent or the kit comprises the protein F1B139, and the reagent or the kit has at least one of the following uses:

e1 Detecting, analyzing or evaluating yersinia pestis antibodies;

e2 ) quantitatively detecting, quantitatively analyzing or quantitatively evaluating yersinia pestis antibodies;

e3 For plague detection;

e4 Used for the diagnosis, auxiliary diagnosis, screening, curative effect observation, prognosis judgment or vaccination effect observation of plague;

e5 For plague control (e.g., epidemiological investigation of plague).

The kit can be a plague antigen detection kit or a plague antibody detection kit.

The kit may be an immunoassay kit.

Further, the kit may be an ELISA kit, an immunoblot detection kit, an immunochromatography detection kit, a flow cytometry kit, or an immunohistochemical detection kit, but is not limited thereto.

Further, the detection sample of the reagent or the kit includes a blood sample, a tissue sample, a saliva sample, a sputum sample, a body fluid sample or an environmental sample.

Further, the reagent or the kit can be a plague antibody detection reagent or kit, and a detection sample of the plague antibody detection reagent or the kit can comprise a blood sample (such as whole blood, plasma or serum).

Further, the reagent or the kit can be a plague antigen detection reagent or kit, and a detection sample of the plague antigen detection reagent or the kit can comprise an environmental sample, a tissue sample, a saliva sample, a sputum sample or a body fluid sample.

The various reagent components of the kit may be present in separate containers or may be pre-combined in whole or in part into a reagent mixture.

The invention also provides any one of the following applications of the reagent or the kit:

f1 Detecting, analyzing or evaluating yersinia pestis antibodies;

f2 Quantitative detection, quantitative analysis or quantitative evaluation of yersinia pestis antibodies.

The invention also provides a method for preparing the protein F1B139, which can comprise expressing a nucleic acid molecule encoding the protein F1B139 in a microorganism or a host cell to obtain the protein F1B139.

Further, the method for preparing the protein F1B139 can comprise the following steps:

g1 Constructing a recombinant expression vector containing a nucleic acid molecule encoding said protein F1B139;

g2 Introducing the recombinant expression vector into a microorganism of interest to obtain a recombinant microorganism;

g3 Culturing said recombinant microorganism, and isolating and/or purifying to obtain said protein F1B139;

further, the nucleic acid molecule encoding the protein F1B139 in G1) may be a DNA molecule (F1B 139 gene) represented by SEQ ID No. 2.

Further, the microorganism of interest described in G2) may be Escherichia coli, and specifically may be Escherichia coli BL21 (DE 3) competent cells.

The invention also provides a method for detecting or quantitatively detecting Yersinia pestis antibodies, which can comprise detecting or quantitatively detecting the protein F1B139 or the reagent or the kit.

In the above method, the detecting or the quantitative detecting using the protein F1B139 or the reagent or the kit may include performing by a precipitation reaction, an agglutination test, a fluorescence immunoassay, a radioimmunoassay, an enzyme immunoassay, a chemiluminescence immunoassay, a colloidal gold immunoassay, or a fluorescence immunochromatography.

Further, the method may be an ELISA detection method.

Further, the method comprises the step of coating the recombinant protein F1B139 on a solid phase carrier to obtain an antigen-coated solid phase carrier. The coating means that the antigen is bound to the solid phase carrier by physical adsorption.

Further, the solid phase carrier may be an enzyme label plate, a membrane carrier, a microsphere, a biochip or a magnetic bead, but is not limited thereto.

The material of the solid phase carrier can be polystyrene, cellulose, cross-linked dextran, polyacrylamide, polyethylene, polypropylene, polyvinyl chloride, cross-linked dextran, glass, silicone rubber or agarose gel, but is not limited thereto.

The membrane carrier may be, but is not limited to, a nitrocellulose membrane, a glass cellulose membrane, or a nylon membrane.

Further, the coating concentration of the recombinant protein F1B139 can be 0.5-10 mug/mL, and specifically can be 1 mug/mL.

Further, the recombinant protein F1B139 can be coated for 12h at 4 ℃.

In the above method, the test sample for detection or quantitative detection may comprise a blood sample (e.g., whole blood, plasma or serum).

In one embodiment of the present invention, the method for quantitatively detecting yersinia pestis antibody comprises the following steps:

(1) Coating: the purified recombinant protein F1B139. Mu.g/mL is coated on an enzyme label plate, each well is 100. Mu.L, and the plate is placed at 4 ℃ for 12h.

(2) And (3) sealing: 1.5% casein was diluted 15-fold, 200. Mu.L per well, 37 ℃ and blocked for 2h.

(3) Incubating the primary antibody: mu.L of rabbit anti-F1 polyclonal antibody at a concentration of 1. Mu.g/mL was added to each well and incubated at 37 ℃ for 30min.

(4) Incubation of secondary antibody: add 100. Mu.L of 1: HRP-goat anti-rabbit IgG (product of Thermo Fisher Co., ltd., cat. No. 65-6120) at a dilution of 4000 was incubated at 37 ℃ for 20min.

(5) Color development: mu.L of developing solution was added to each well, and incubated at 37 ℃ for 10min.

(6) And (4) terminating: add 50. Mu.L of stop solution into each well, and measure OD values at both 450nm and 630 nm.

The preparation method of the rabbit anti-F1 polyclonal antibody comprises the following steps: injecting 200, 400 and 800 μ g natural F1 antigen into rabbit inguinal subcutaneous tissue for three times with interval of 14 days, harvesting immune serum, and extracting polyclonal antibody in the serum by using n-octanoic acid-saturated ammonium sulfate method, namely rabbit anti-F1 polyclonal antibody.

The natural F1 antigen can be produced by a method described in patent literature (Yersinia pestis natural F1 antigen extraction and purification method, application No. 200810055697.7).

The uses and methods described herein may be for disease diagnostic purposes, disease prognostic purposes, and/or disease treatment purposes, and may also be for non-disease diagnostic purposes, non-disease prognostic purposes, and non-disease treatment purposes; their direct purpose may be to obtain information on the outcome of a disease diagnosis, prognosis of a disease and/or intermediate outcome of a disease treatment, and their direct purpose may be non-disease diagnosis, non-disease prognosis and/or non-disease treatment.

The method for detecting or quantitatively detecting the Yersinia pestis antibody provided by the invention can be a non-disease diagnosis and treatment method and a disease diagnosis and treatment method. Wherein the non-disease diagnostic treatment method can detect Yersinia pestis antibodies, for example, when screening for plague in a human population, or in an epidemiological survey of plague.

Herein, the term ELISA (Enzyme Linked Immunosorbent Assay) refers to adsorbing an antigen or an antibody onto a solid-phase carrier, sequentially adding an antibody (or antigen) to be detected and an Enzyme label to a reaction system in the detection process, reacting the antibody (or antigen) and the Enzyme label with the antigen (or antibody) on the solid-phase carrier to form an antigen-antibody complex, washing away unbound free Enzyme label and free antigen (or antibody), and measuring the Enzyme activity of the bound Enzyme label, thereby determining the content of the antibody (or antigen) to be detected in a sample. ELISA includes direct method, indirect method, double antibody sandwich method, competition method and anti-enzyme antibody method. Although the examples provided herein employ an ELISA indirect method for detecting Yersinia pestis antibodies, the invention is not limited to this particular method. Any other immunoassay method can be used by those skilled in the art without departing from the scope of the present invention as long as the method achieves the same technical effect as the present invention based on the specific reaction between the antigen (recombinant protein F1B 139) of the present invention and the antibody, and the present invention shall include these alternative methods.

Both the natural F1 antigen and the recombinant F1 exist in the form of a mixture consisting of monomers and different amounts of caf1 protein polymers, and can only be used for qualitative evaluation of antibodies, but cannot quantitatively evaluate the plague bacillus antibodies. The literature shows that Caf1 protein is composed of fibrous F1 protein in an end-to-end manner (Zavialov, A.V., et al. (2003) 'Structure and biogenesis of the fibrous F1 antigen from Yersinia pestis.: preserved folding energy drive fiber formation.' Cell 113 (5): 587-596). The invention has been extensively and deeply studied to cleave several amino acids at the amino or carboxyl end, unexpectedly obtaining a truncated F1 antigen capable of achieving the complete decomposition of F1 multimers into monomers, whereas such F1 antigen, which is present in a completely monomeric form, can achieve the quantification of antibodies. This has the advantage that a substantial proportion of the linear epitope sites of the antibody are retained, allowing quantitative analysis of the antibody.

The invention develops and prepares a recombinant diagnostic antigen for quantitatively detecting (evaluating) the plague bacillus antibody, namely recombinant protein F1B139. The recombinant protein F1B139 is a truncated plague bacillus F1 surface antigen protein, compared with the full-length F1 antigen without truncation, the N end is truncated by 10 amino acids, namely, the last 139 amino acids of the full-length F1 antigen are selected as a new recombinant F1 antigen. The amino acid sequence of the recombinant protein F1B139 is shown as SEQ ID No.1, and the nucleotide sequence of the gene (F1B 139 gene) for coding the recombinant protein F1B139 is shown as SEQ ID No. 2.

The recombinant protein F1B139 disclosed by the invention can be used for evaluating an antibody raw material in a plague antigen immunoassay reagent, so that the stable production of the antigen reagent is realized; can also be directly used as a raw material of a plague antibody immunodetection reagent. The invention provides a diagnostic antigen capable of being quantitatively evaluated for a plague bacteria antigen detection method and an antibody detection method, and experiments prove that the plague bacteria antigen provided by the invention has good sensitivity, can be identified by rabbit anti-F1 polyclonal antibody, can be used as a quality control product of a plague bacteria antigen detection reagent, is used for accurately controlling an antibody raw material of the plague bacteria antigen detection reagent, ensures the batch stability of antigen raw material production, and can also be directly used as a raw material of the antibody detection reagent. While native F1 or its full-length recombinant protein exists as a mixture of monomers and varying amounts of polymers of the caf1 protein, the recombinant F1 antigen of the invention is single in composition and allows for the precise evaluation of antibodies.

The immunodetection method and the product based on the recombinant F1 antigen (recombinant protein F1B 139) can realize the quantitative detection of the antibody, have good sensitivity, can be widely applied to the detection, quality control and evaluation of the antibody in the detection of plague bacillus, provide help for clinical diagnosis, curative effect observation, prognosis judgment, observation of vaccination effect, epidemiological investigation of infectious diseases and the like, and have very wide clinical application prospect and important significance.

Drawings

FIG. 1 shows recombinant vector pET-21a/EV76-F1 _11-149 The plasmid map of (1).

FIG. 2 shows a recombinant vector pET-21a/EV76-F1 _13-149 The plasmid map of (1).

FIG. 3 shows the recombinant vector pET-21a/EV76-F1 _1-139 The plasmid map of (3).

FIG. 4 shows the results of native F1 native electrophoresis in example 2.

FIG. 5 shows the results of native electrophoresis of the full-length recombinant F1 of example 2.

FIG. 6 shows the results of non-denaturing electrophoresis of the recombinant protein F1B139 in example 2. Wherein, the first hole on the left is marker, the second hole is natural F1, and the third hole is recombinant protein F1B139.

FIG. 7 shows the results of denatured electrophoresis of F1B139 (left panel), F1B137 (right panel) and 5 monoclonal antibodies of example 2, and then obtained as WB. The 5 monoclonal antibodies are 1B6, 2E9, 4H2, 6E5 and 10E10 respectively.

FIG. 8 shows the WB results after denaturing electrophoresis of F1B139 (left panel), F1B137 (middle panel) and F1A139 (right panel) and multiple antibodies of example 2. The polyclonal antibody is rabbit anti-F1.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The vector pET-21a in the examples described below is manufactured by Biotech, inc., beijing Huayuyo, inc., cat # 4170.

Example 1 design and preparation of recombinant protein F1B139

1. Design of recombinant protein F1B139

The overall length of the plague bacterium F1 antigen is 149 amino acids, which exists in a polymer form, and can only be used for qualitative evaluation of the antibody, but can not quantitatively evaluate the antibody. Based on this, the invention designs the recombinant F1 antigen existing in a monomer form, and the design has the advantages that the linear epitope sites of most antibodies are reserved, and the quantitative analysis of the antibodies can be realized.

The recombinant F1 antigen designed by the invention is recombinant protein F1B139, the recombinant protein F1B139 is a truncated plague bacillus F1 surface antigen protein, and compared with the non-truncated full-length F1 antigen, the N-terminal is truncated by 10 amino acids, namely the last 139 amino acids of the full-length F1 antigen are selected as a new recombinant F1 antigen. The amino acid sequence of the recombinant protein F1B139 is shown as SEQ ID No.1, and the nucleotide sequence of the gene (F1B 139 gene) for coding the recombinant protein F1B139 is shown as SEQ ID No. 2.

Meanwhile, the recombinant protein (named as recombinant protein F1B 137) of the last 137 amino acids of the full-length F1 antigen is designed and selected as a comparison for experiment, and the experiment proves that the recombinant protein has weaker reaction than that of F1B139 and an antibody, especially when aiming at certain monoclonal antibodies. And further selecting the first 139 amino acid recombinant proteins (named as recombinant proteins F1A 139) of the full-length F1 antigen to carry out a comparison experiment, and proving that the first 139 amino acid recombinant proteins are obviously weaker than the F1B139 and the antibody in reaction.

The amino acid sequence of the recombinant protein F1B137 is shown as SEQ ID No.3, and the nucleotide sequence of the gene (F1B 137 gene) for coding the recombinant protein F1B137 is shown as SEQ ID No. 4.

The amino acid sequence of the recombinant protein F1A139 is shown as SEQ ID No.5, and the nucleotide sequence of the gene (F1A 139 gene) for coding the recombinant protein F1A139 is shown as SEQ ID No. 6.

The amino acid sequence of the full-length F1 antigen is shown below:

5 '-ADLTASTTATLVEPARITLTYKEGAPITIMDNGNIDTLLVGTLTLTLGGYK TGTTSTSTSYVNFTDAAGDPMYLTSQDGHQFTTKVIGKDSRDFDISPKVNGENL VGDDVVALATGSQDFFVRSIGSKGGKLAAGKYTDAVTVSNQ-3' (149 amino acids).

2. Preparation of recombinant proteins

2.1 construction of recombinant plasmids

Genes encoding three recombinant proteins (F1B 139, F1B137 and F1A 139) are respectively constructed in a vector pET-21a according to the conventional operation in the field, and the specific steps are as follows:

(1) Introducing a BamH I restriction site at an upstream primer, introducing an Xho I restriction site at a downstream primer, and designing an upstream primer F1 (5' -CAG)GGATCCGCAACTCTCTTGTTGAACCAGCCGC-3 ', SEQ ID No.7, underlined BamHI recognition site) and the downstream primer R1 (5' -GGG)CTCGAGTTGGTT AGATACGGTTACGGTTAC-3', SEQ ID No.8, underlined is an XhoI recognition site), DNA molecules (F1B 139 gene) shown by SE Q ID No.2 are used as a template for PCR amplification to obtain PCR products (namely target genes of which 5' ends and 3' ends are respectively added with BamH I and XhoI enzyme cutting sites), and the PCR products are subjected to double enzyme cutting by BamH I and XhoI to obtain a DNA fragment 1.

The expression vector pET-21a is subjected to double enzyme digestion by BamH I and Xho I to obtain an enzyme digested pET-21a vector fragment, and the DNA fragment 1 is connected with the enzyme digested pET-21a vector fragment to obtain a recombinant vector named pET-21a/EV76-F1 _11-149 (plasmid map see FIG. 1).

Recombinant vector pET-21a/EV76-F1 _11-149 The recombinant expression vector is obtained by replacing a fragment (small fragment) between BamHI and XhoI recognition sites of a pET-21a vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.2 in a sequence table and keeping other sequences of the pET-21a vector unchanged. Recombinant vector pET-21a/EV76-F1 _11-149 Contains F1B139 gene shown in SEQ ID No. 2.

(2) Introducing a BamH I restriction site at an upstream primer, introducing an Xho I restriction site at a downstream primer, and designing an upstream primer F2 (5' -CAG)GGATCCCTTGTTGAACCAGCCGCATCACT-3 ', SEQ ID No.9, underlined BamHI recognition site) and the downstream primer R1 (5' -GGG)CTCGAGTTGGTT AGATACGGTTACGGTTAC-3', SEQ ID No.8, with Xho I recognition site underlined), using DNA molecule (F1B 137 gene) shown in SE Q ID No.4 as template for PCR amplification to obtainObtaining PCR products (namely target genes of BamH I restriction enzyme sites and Xho I restriction enzyme sites are respectively added at the 5 'end and the 3' end), and obtaining a DNA fragment 2 after double restriction enzyme digestion of the PCR products by BamH I restriction enzyme and Xho I restriction enzyme.

The expression vector pET-21a is subjected to double enzyme digestion by BamH I and Xho I to obtain an enzyme digested pET-21a vector fragment, and the DNA fragment 2 is connected with the enzyme digested pET-21a vector fragment to obtain a recombinant vector which is named as pET-21a/EV76-F1 _13-149 (plasmid map see FIG. 2).

Recombinant vector pET-21a/EV76-F1 _13-149 The recombinant expression vector is obtained by replacing a fragment (small fragment) between BamHI and XhoI recognition sites of a pET-21a vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.4 in a sequence table and keeping other sequences of the pET-21a vector unchanged. Recombinant vector pET-21a/EV76-F1 _13-149 Contains F1B137 gene shown in SEQ ID No. 4.

(3) Introducing a BamH I restriction site at an upstream primer, introducing an Xho I restriction site at a downstream primer, and designing an upstream primer F3 (5' -CAG)GGATCCGCAGATTTAACTGCAAGCACCAT-3 ', SEQ ID No.10, underlined BamHI recognition site) and the downstream primer R3 (5' -GGG)CTCGAGAGTGT ATTTACCTGCGCTGCAAGTTT-3 ', SEQ ID No.11, underlined is XhoI recognition site), DNA molecule (F1A 139 gene) shown in SEQ ID No.6 is taken as a template to carry out PCR amplification, a PCR product (namely target gene with BamHI and XhoI enzyme cutting sites added at 5' end and 3' end respectively) is obtained, and the PCR product is subjected to double enzyme cutting by BamHI and XhoI to obtain DNA fragment 3.

The expression vector pET-21a is subjected to double enzyme digestion by BamH I and Xho I to obtain an enzyme digested pET-21a vector fragment, and the DNA fragment 3 is connected with the enzyme digested pET-21a vector fragment to obtain a recombinant vector which is named as pET-21a/EV76-F1 _1-139 (plasmid map see FIG. 3).

Recombinant vector pET-21a/EV76-F1 _1-139 The recombinant expression vector is obtained by replacing a fragment (small fragment) between BamHI and XhoI recognition sites of a pET-21a vector with a DNA fragment of which the nucleotide sequence is SEQ ID No.6 in a sequence table and keeping other sequences of the pET-21a vector unchanged. Recombinant vector pET-21a/EV76-F1 _1-139 Contains F1A139 gene shown in SEQ ID No. 6.

The restriction enzyme sites of the three plasmids constructed in the steps (1), (2) and (3) are the same, namely, the target gene is obtained through PCR amplification, and is connected with an expression vector pET-21a into a recombinant plasmid after double restriction enzyme digestion by BamH I and Xho I.

2.2 expression and purification of recombinant proteins

2.2.1, recombinant protein expression: successfully constructed plasmids (i.e., recombinant vector pET-21a/EV 76-F1) _11-149 、pET-21a/EV76-F1 _13-149 And pET-21a/EV76-F1 _1-139 ) The respective strains were transformed into DH 5. Alpha. Competent cells to prepare clonal strains. Expression strains were prepared by transforming positive plasmids (correctly sequenced plasmids) into BL21 (DE 3) competent cells. 5mL of the bacterial liquid (culture liquid of positive expression strain) is cultured for 3h at 37 ℃ and 220rpm, then transferred to 1L 2YT culture medium, 500 microliter IPTG is added when the OD value of the bacterial liquid is about 0.6-0.8, and the bacterial liquid is cultured for 20h at 18 ℃ and 150 rpm.

2.2.2, cracking thalli and collecting supernatant: (1) collecting thalli: centrifuging at 4 deg.C and 10000 Xg for 15min to collect thallus; (2) cell resuspension: the cells were resuspended using approximately 40mL of His binding buffer, in a volume ratio of 1:100 adding protease inhibitor; (3) ultrasonic treatment of the thallus: placing the resuspended liquid in a 50mL centrifuge tube, placing in an ice box, and performing ultrasound for 50min by using a No.6 gun head of an ultrasound machine with 20% power and 2s on and 3s off; (4) centrifuging after crushing and collecting supernatant: centrifugation was carried out at 9000 Xg for 20min at 4 ℃ to obtain a supernatant.

2.2.3, recombinant protein purification: the supernatant after filtration through a 0.22 μm filter was added to a gravity chromatography column and the supernatant was incubated with a nickel column at 4 ℃ for 2h. Opening an opening valve of the gravity chromatographic column, washing impurities by using 30mL of His binding buffer, eluting target protein by using 15-25 mL of 250mM imidazole and collecting the target protein, and further eluting by using 30mL of 400mM imidazole and collecting the target protein to obtain purified recombinant proteins F1A139, F1B137 and F1A139.

Example 2 functional characterization of recombinant protein F1B139 as a diagnostic antigen

The antibodies and natural antigens used in the assays of this example were derived as follows:

the preparation method of the rabbit anti-F1 polyclonal antibody comprises the following steps: injecting 200, 400 and 800 μ g natural F1 antigen into rabbit inguinal subcutaneous tissue for three times with interval of 14 days, harvesting immune serum, and extracting polyclonal antibody in the serum by using n-octanoic acid-saturated ammonium sulfate method, namely rabbit anti-F1 polyclonal antibody. The ELISA plate was coated with 1. Mu.g/ml of the native F1 antigen, and the ELISA titer of the rabbit anti-F1 polyclonal antibody was determined to be about 31.25ng/ml.

Natural F1: natural F1 was extracted according to the method described in the patent document (Yersinia pestis natural F1 antigen extraction and purification method, application No. 200810055697.7).

Full-length recombinant F1: the preparation method is the same as F1B139 (the same as the recombinant protein preparation method in step 2 of embodiment 1), namely, the gene coding the full-length F1 protein is constructed in a vector pET-21a, and the full-length recombinant F1 is obtained after expression and purification.

1. Native-PAGE non-denaturing electrophoresis and Western-blotting experiments:

the purified recombinant protein F1B139, the natural F1 and the full-length recombinant F1 are subjected to Native-PAGE (Native-PAGE) non-denaturing electrophoresis, and the steps are as follows: preparing 4-15% polyacrylamide gel, and performing Native-PAGE (Native-PAGE electrophoresis) at 4 ℃ and 150V for 90 min.

After electrophoresis, the membrane was transferred at 90V for 45min and blocked with milk overnight. Adding rabbit anti-F1 polyclonal antibody for incubation, and performing Western-blot detection.

The result shows that Western-blotting (WB) is carried out by using rabbit anti-F1 polyclonal antibody after the negative-PAGE non-denaturing electrophoresis, and the recombinant protein F1B139, the natural F1 and the full-length recombinant F1 are both specifically identified with the rabbit anti-F1 polyclonal antibody, which indicates that the recombinant protein F1B139 has good reactogenicity (antigenicity) and can be used as a diagnostic antigen for detecting the plague bacillus antibody. Native F1 and full-length recombinant F1 consisted of multiple bands and were mostly concentrated in the upper half of the gel (as shown in fig. 4 and 5, respectively), demonstrating that they are multimers. The components of the band (figure 6) of the recombinant protein F1B139 are relatively single, namely the band is composed of monomers, which shows that the recombinant protein F1B139 can realize accurate quantitative detection of antibodies when being used as a diagnostic antigen, and further can be used for evaluating antibody raw materials of plague antigen immunodetection reagents, can be used as quality control products of plague bacteria antigen detection reagents, thereby realizing stable production of the antigen reagents, and can also be directly used as raw materials of plague antibody immunodetection reagents.

2. Comparative experiment

The inventors of the present application identified, screened and finally obtained the recombinant protein F1B139 of the present invention through a large number of experiments. This comparative experiment, using the recombinant protein F1B137 and the recombinant protein F1a139 prepared in example 1 as comparative validation, showed that the recognition ability of shorter recombinant proteins and the selection of the first 139 amino acids of the full-length F1 antigen with pestis antibodies was weaker than that of the recombinant protein F1B139. The method comprises the following specific steps:

1. sample preparation

Samples were prepared at 5mg/mL, and after 2 Xprotein loading buffer was added, the protein concentration was 2.5mg/mL.

2、SDS-PAGE

The samples were subjected to SDS-PAGE and then subjected to WB assay with 5F 1 monoclonal antibodies (or polyclonal antibodies)

3. WB experiment

And (4) carrying out NC membrane transfer after electrophoresis. 90V,45min.5% skim milk was shaken overnight at 4 ℃ and washed with TBST. Primary antibody incubation, 5F 1 mAbs (1B 6, 2E9, 4H2, 6E5, 10E 10) or multiple antibodies were incubated with shaking at 4 ℃ for 4H and the membranes were washed with TBST. Incubating the secondary antibody, resisting the goat or the rabbit, shaking the membrane at normal temperature in a dark place for 1h, washing the membrane by TBST, and observing the membrane on a machine.

Monoclonal antibodies against plague bacteria were prepared and stored in the laboratory (related articles: zhang, P., et al. (2020). "Calibration of an upper converting Phosphor-Based immunological Assay for Detecting Yersinia pestis, brucella sp., and Bacillus anthracensis Spors." FrontCell Infect Microbiol 10:147.)。

As shown in FIGS. 7 and 8, the Western-blotting identification results showed that F1B139 reacted more strongly to the mAb than F1B137, especially 2E9 and 4H2. And the strength of the multiresistance reaction, F1B139> F1B137> F1A139.

Example 3 ELISA detection method for quantitative detection of plague bacteria antibodies

In this example, an ELISA detection method for quantitatively detecting an antibody against plague bacteria was established based on the recombinant protein F1B139 prepared in example 1 as an antigen protein. By taking an ELISA detection method as an example, the recombinant protein F1B139 prepared by the invention can be used as a diagnostic antigen to carry out quantitative detection and analysis on the plague bacillus antibody. The ELISA detection experiment method comprises the following steps:

(1) Coating: the purified recombinant protein F1B139 mu g/mL coats the ELISA plate; the natural F1 antigen 1 ug/mL is coated on the enzyme label plate, each well is 100 ul, and the plate is placed at 4 ℃ for 12h.

(2) And (3) sealing: 1.5% casein was diluted 15-fold, 200. Mu.L per well, 37 ℃ and blocked for 2h.

(3) Incubating the primary antibody: mu.L of rabbit anti-F1 polyclonal antibody at a concentration of 1. Mu.g/mL was added to each well and incubated at 37 ℃ for 30min.

(4) Incubation of secondary antibody: add 100. Mu.L of 1: HRP-goat anti-rabbit IgG (product of therofisher Co., ltd., cat. No. 65-6120) at a dilution of 4000 was incubated at 37 ℃ for 20min.

(5) Color development: mu.L of developing solution was added to each well, and incubated at 37 ℃ for 10min.

(6) And (4) terminating: add 50. Mu.L of stop solution to each well and measure OD at 450nm and 630 nm. The results of the ELISA assay are shown in table 1:

TABLE 1 ELISA results for recombinant protein F1B139 and rabbit anti-F1 polyclonal antibody

As can be seen from Table 1, F1B139 achieved the same potency as native F1, both 32.50ng/mL. Because the components of native F1 and full-length recombinant F1 are complex (fig. 4 and 5), there is interconversion of multimers and monomers, and the composition of each component is not necessarily the same under various conditions; and the batch-to-batch difference is difficult to guarantee when different batches are prepared. Compared with the natural F1 and the full-length recombinant F1, the F1B139 has a single component (shown in figures 2 and 6), controllable batch-to-batch difference and higher sensitivity, and is a better choice for quality control antibodies.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims (10)

1. A protein, wherein the protein is any one of:

a1 Protein of which the amino acid sequence is SEQ ID No. 1;

a2 Protein which is obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No. 1), has more than 80 percent of identity with the protein shown in A1) and has the same function;

a3 A fusion protein having the same function obtained by attaching a tag to the N-terminus and/or C-terminus of A1) or A2).

2. A biomaterial, characterized in that the biomaterial is any one of the following:

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule according to B1);

b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);

b5 A recombinant host cell containing the nucleic acid molecule according to B1), or a recombinant host cell containing the expression cassette according to B2), or a recombinant host cell containing the recombinant vector according to B3).

3. The biomaterial according to claim 2, wherein B1) the nucleic acid molecule is any one of the following:

c1 A DNA molecule whose coding sequence is SEQ ID No. 2;

c2 A DNA molecule having the nucleotide sequence of SEQ ID No. 2.

4. The protein of claim 1, and/or the biomaterial of claim 2 or 3 for any one of the following uses:

d1 Use in detecting, analyzing or evaluating yersinia pestis antibodies or for the manufacture of a product for detecting, analyzing or evaluating yersinia pestis antibodies;

d2 Use in quantitative detection, quantitative analysis or quantitative evaluation of yersinia pestis antibodies or for the preparation of a product for quantitative detection, quantitative analysis or quantitative evaluation of yersinia pestis antibodies;

d3 Application in plague detection or preparation of products for plague detection;

d4 Application in preparing pestis antigen detection products or pestis antibody detection products;

d5 Application in preparing quality control products for detecting plague antigens;

d6 Application in preparing or screening Yersinia pestis antibody;

d7 Use in the preparation of products for the diagnosis, auxiliary diagnosis, screening, observation of curative effect, prognosis judgment or observation of vaccination effect of plague;

d8 Application in plague control or preparation of products for plague control.

5. A reagent or kit comprising a protein according to claim 1, wherein the reagent or kit has at least one of the following uses:

e1 Detecting, analyzing or evaluating yersinia pestis antibodies;

e2 Quantitative detection, quantitative analysis or quantitative evaluation of yersinia pestis antibodies;

e3 For plague detection;

e4 Used for the diagnosis, auxiliary diagnosis, screening, curative effect observation, prognosis judgment or vaccination effect observation of plague;

e5 For plague control.

6. The reagent or kit according to claim 5, wherein the test sample of the reagent or kit comprises a blood sample, a tissue sample, a saliva sample, a sputum sample, a body fluid sample, or an environmental sample.

7. The reagent or kit of claim 5 or 6 for use as any one of:

f1 Detecting, analyzing or evaluating yersinia pestis antibodies;

f2 ) quantitatively detecting, quantitatively analyzing or quantitatively evaluating yersinia pestis antibodies.

8. A method for producing the protein of claim 1, which comprises expressing a nucleic acid molecule encoding the protein of claim 1 in a microorganism or a host cell to obtain the protein.

9. A method for detecting or quantitatively detecting Yersinia pestis antibodies, comprising detecting or quantitatively detecting the protein of claim 1 or the reagent or kit of claim 5.

10. The method of claim 9, wherein the detection or quantitative determination using the protein of claim 1 or the reagent or kit of claim 5 comprises a precipitation reaction, an agglutination assay, a fluorescence immunoassay, a radioimmunoassay, an enzyme immunoassay, a chemiluminescent immunoassay, a colloidal gold immunoassay, or a fluorescence immunochromatographic technique.

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