Diagnostic antigen for quantitative detection of yersinia pestis antibodies

Abstract

The application discloses a diagnostic antigen for quantitatively detecting Yersinia pestis antibody. Specifically disclosed is a protein F1B139 with an amino acid sequence of SEQ ID No. 1 and application of the protein F1B139 as an antigen in detection, analysis or evaluation of Yersinia pestis antibody. Experiments show that the Yersinia pestis antigen has good sensitivity, can be recognized by rabbit anti-F1 polyclonal antibody, can be used as a quality control product of a Yersinia pestis antigen detection reagent, ensures batch stability of antibody raw material production, can be used as a raw material of an antibody detection reagent, the recombinant F1 antigen component is single, and the antibody can be accurately evaluated. The immunodetection method and product based on the antigen can realize quantitative detection of the antibody, have good sensitivity, can be widely used in detection, quality control and evaluation of the Yersinia pestis 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 prospects.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a diagnostic antigen for quantitatively detecting Yersinia pestis antibody. BACKGROUND

[0002] Yersinia pestis (Y. pestis) is the pathogen of the virulent zoonosis plague. Plague is a class A infectious disease in the People's Republic of China, and belongs to the virulent zoonosis. Plague has caused three pandemics in history, and the outbreak in the 14th century caused the death of one-third of the European population. There are many natural plague foci in the world, which are difficult to eliminate completely in a short period of time. The method of preventing and controlling plague from the source is to detect Y. pestis and monitor the natural foci, and early diagnosis is of great significance to reduce mortality.

[0003] At present, the methods for diagnosing plague infection mainly include nucleic acid detection method and immunodetection method. F1 capsular antigen is the most important surface antigen, which directly determines the antigenicity of Y. pestis, and is recognized by the World Health Organization (WHO) as a detection target. F1 antigen is highly conserved in Y. pestis, and is composed of linear fibers connected by Caf1 subunits, and the Caf1 subunit is a protein composed 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. Regardless of which mode, F1 antibody is an important raw material to ensure the sensitivity and specificity of the detection reagent, and therefore, it is crucial to accurately control and evaluate the F1 antibody.

[0004] Currently, the reagents for evaluating F1 antibody mainly include natural F1 antigen (Method for extracting and purifying natural F1 antigen of Yersinia pestis, application number 200810055697.7) and recombinant F1 (Tavares, D. H. C., et al. (2020). "A new recombinant F1 antigen as a cost and time-effective tool for plague diagnosis." J Microbiol Methods 172: 105903). Both natural F1 antigen and recombinant F1 exist in the form of a mixture of monomers and different numbers of caf1 protein polymers, and cannot quantitatively evaluate plague bacteria antibodies. Some scholars have tried to cut the natural F1 antigen into two fragments in the middle, and the cut fragments containing linear epitopes can be used for immunological analysis of antibody positive samples (Method for cutting Yersinia pestis F1 antigen and related applications, application number 200910083026.6), but if the linear epitope of the antibody is at the cutting site, the antibody cannot be analyzed. Therefore, there is still a need in the clinic for diagnostic antigens that can accurately and sensitively quantitatively analyze Yersinia pestis antibodies. SUMMARY

[0005] The technical problem to be solved by the present application is how to detect or quantitatively detect Yersinia pestis antibodies. The technical problem to be solved is not limited to the technical subject described, and other technical subjects not mentioned herein can be clearly understood by those skilled in the art through the following description.

[0006] To solve the above technical problems, the present application first provides a protein named F1B139, which can be any of the following:

[0007] A1) a protein with an amino acid sequence of SEQ ID No. 1;

[0008] A2) a protein with more than 80% identity to the protein of A1) and having the same function obtained by substitution, deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No. 1;

[0009] A3) a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0010] The protein F1B139 is a truncated F1 surface antigen protein of Yersinia pestis, also known as recombinant protein F1B139. Compared with the full-length F1 antigen (149 amino acids) without truncation, the recombinant protein F1B139 is truncated by 10 amino acids at the N-terminus, i.e. the last 139 amino acids of the full-length F1 antigen are selected as the new recombinant F1 antigen.

[0011] The full-length amino acid sequence of the F1 surface antigen protein of Yersinia pestis (referred to as F1 antigen) is: 5'-ADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ-3'.

[0012] The substitutions described herein can be conservative substitutions (also referred to as conservative replacements) or non-conservative substitutions outside the core functional region. As known to those skilled in the art, conservative substitutions or non-conservative substitutions outside the core functional region generally do not have a qualitative impact on the function of the protein.

[0013] In order to facilitate the purification or detection of the protein in A1), a tag protein can be connected to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing.

[0014] The tag described herein includes but is not limited to: a GST (glutathione S-transferase) tag protein, a His tag protein (His-tag), a MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, a HA tag protein, a Myc tag protein, an eGFP (enhanced green fluorescent protein), an eCFP (enhanced cyan fluorescent protein), an eYFP (enhanced yellow green fluorescent protein), an mCherry (monomeric red fluorescent protein), or an AviTag tag protein.

[0015] Those skilled in the art can easily mutate the nucleotide sequence encoding the recombinant protein F1B139 of the present application using known methods, such as directed evolution or point mutation methods. Those artificially modified nucleotides having 75% or more identity with the nucleotide sequence of the recombinant protein F1B139 isolated from the present application, as long as they encode the recombinant protein F1B139 and have the function of the recombinant protein F1B139, are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application.

[0016] The above-mentioned 75% or more identity can be 80%, 85%, 90%, or 95% or more identity.

[0017] Herein, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI home page website. For example, the value of the identity (%) can be obtained by performing a search in Advanced BLAST 2.1 using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and then calculating the identity of the amino acid sequence.

[0018] Herein, the identity of 80% or more 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%.

[0019] The present application also provides a biological material, which can be any one of the following:

[0020] B1) a nucleic acid molecule encoding the protein F1B139;

[0021] B2) an expression cassette containing the nucleic acid molecule of B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0023] B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0024] B5) a recombinant host cell containing the nucleic acid molecule of B1), or a recombinant host cell containing the expression cassette of B2), or a recombinant host cell containing the recombinant vector of B3).

[0025] In the above biological material, the nucleic acid molecule of B1) can be any one of the following:

[0026] C1) a DNA molecule whose coding sequence is SEQ ID No. 2;

[0027] C2) a DNA molecule whose nucleotide sequence is SEQ ID No. 2.

[0028] Further, B2) the expression cassette, B3) the recombinant vector, B4) the recombinant microorganism and B5) the recombinant host cell can all express B1) the nucleic acid molecule.

[0029] The DNA molecule shown in SEQ ID No. 2 (the name can be F1B139 gene) encodes the recombinant protein F1B139 of the amino acid sequence of SEQ ID No. 1.

[0030] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA. The nucleic acid molecule can also include a nucleic acid molecule obtained by codon preference modification based on the nucleotide sequence shown in SEQ ID No. 2. Considering the degeneracy of codons and the preference of codons in different species, a person skilled in the art can use codons suitable for expression in a specific species as needed.

[0031] The vector described herein refers to a vector capable of carrying foreign DNA or a gene of interest into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmid, bacteriophage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e. cos plasmid), Ti plasmid, viral vector (such as retrovirus (including lentivirus), adenovirus, adeno-associated virus, etc.). In one or more embodiments of the present application, the vector is the vector pET-21a.

[0032] The microorganism described herein can be bacteria, fungi, actinomycetes, protozoa, algae or viruses. Among them, the bacteria can be from Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., Bacillus sp., etc. but not limited thereto, for example, the bacteria can be Escherichia coli, Bacillus subtilis or Bacillus pumilus. In one or more embodiments of the present application, the microorganism is Escherichia coli DH5α and / or Escherichia coli BL21(DE3).

[0033] The host cell (also referred to as a recipient cell) described herein can be a plant cell or an animal cell. The host cell can be understood to refer not only to the particular subject cell but also to the progeny of such a cell, and therefore can not necessarily be identical to the original parent cell as a result of natural, accidental or deliberate mutation and / or change, but still falls within the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell can be, but is not limited to, a plant cell of Arabidopsis thaliana, Nicotiana tabacum, Zea mays, Oryza sativa, Triticum aestivum, etc.; the animal cell can be, but is not limited to, a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), African green monkey kidney cell (Vero cell), baby hamster kidney cell (BHK cell), mouse mammary carcinoma cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast cell, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., Xenopus laevis cell or Andrias davidianus cell), fish cell (e.g., grass carp, carp, rainbow trout or catfish cell), insect cell (e.g., Sf21 cell or Sf-9 cell), etc.

[0034] The recombinant vector described herein refers to a recombinant DNA molecule constructed by connecting an exogenous target gene with a vector in vitro, which can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into a recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0035] The recombinant microorganism (or recombinant host cell) described herein refers to a recombinant microorganism (or a recombinant host cell) with changed function, which is obtained by operating and modifying the genes of a target microorganism (or a target host cell). For example, a recombinant microorganism (or a recombinant host cell) obtained by introducing an exogenous target 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 editing the endogenous genes of a target microorganism (or a target host cell). The recombinant microorganism (or the recombinant host cell) can be understood to refer not only to a specific recombinant microorganism (or a specific recombinant host cell), but also to the progeny of such a cell, and therefore can not necessarily be identical to the original parent cell as a result of natural, accidental or deliberate mutation and / or change, but still falls within the scope of the recombinant microorganism (or the recombinant host cell).

[0036] The recombinant vector of B3) can be the recombinant vector pET-21a / EV76-F1 11-149 .

[0037] The recombinant vector pET-21a / EV76-F1 11-149 is obtained by replacing the fragment (small fragment) between the recognition sites of BamH I and Xho I of the pET-21a vector with a DNA fragment of SEQ ID No. 2 in the sequence listing, while keeping other sequences of the pET-21a vector unchanged. The recombinant vector pET-21a / EV76-F1 11-149 contains the F1B139 gene shown in SEQ ID No. 2.

[0038] The recombinant microorganism of B4) can be the recombinant microorganism obtained by introducing the recombinant vector pET-21a / EV76-F1 11-149 into E. coli. Specifically, the recombinant microorganism can be the recombinant microorganism obtained by introducing the recombinant vector pET-21a / EV76-F1 11-149 into DH5α competent cells or BL21(DE3) competent cells.

[0039] The recombinant host cell of B5) can be the recombinant host cell obtained by introducing the recombinant vector pET-21a / EV76-F1 11-149 into a host cell.

[0040] The introduction can be any known transformation method, such as chemical transformation method (e.g. Ca 2+ induced transformation method, polyethylene glycol-mediated transformation method or metal cation-mediated transformation method), or electroporation transformation method, to transform the host bacteria with the vector carrying the DNA molecule of the present application; or can be the method of phage transduction to transduce the DNA molecule of the present application into the host bacteria. The introduction can also be any known transfection method, such as calcium phosphate coprecipitation method, liposome-mediated method, electroporation method or viral vector method, to transfect the host cell with the vector carrying the DNA molecule of the present application.

[0041] The present application also provides the protein F1B139, and / or any of the following applications of the biological material:

[0042] D1) application in detecting, analyzing or evaluating Yersinia pestis antibody (referred to as plague bacteria antibody) or preparing products for detecting, analyzing or evaluating Yersinia pestis antibody;

[0043] D2) application in quantitatively detecting, quantitatively analyzing or quantitatively evaluating Yersinia pestis antibody or preparing products for quantitatively detecting, quantitatively analyzing or quantitatively evaluating Yersinia pestis antibody;

[0044] D3) use in plague detection or in the preparation of products for plague detection;

[0045] D4) use in the preparation of a plague antigen detection product (such as a plague antigen detection kit) or a plague antibody detection product (such as a plague antibody detection kit);

[0046] D5) use in the preparation of a quality control product for plague antigen detection;

[0047] D6) use in the preparation or screening of a Yersinia pestis antibody;

[0048] D7) use in the preparation of a product for the diagnosis, auxiliary diagnosis, screening, efficacy observation, prognosis judgment or observation of the effect of vaccination for plague;

[0049] D8) use in the prevention and control of plague (such as epidemiological investigation of plague) or in the preparation of a product for the prevention and control of plague (such as a product for epidemiological investigation of plague).

[0050] The product described herein can be a reagent, a kit, a chip, a test paper or a detection card.

[0051] The product described herein can comprise the protein F1B139.

[0052] The detection, analysis or evaluation can include detection, analysis or evaluation of antibody titer, specificity and / or affinity.

[0053] Antigen-antibody reaction is the core and basis of immunological test technology. It is well known to those skilled in the art that based on the principle of specific binding of antigen and antibody, specific antibodies can be detected and analyzed using known antigens, which can be used to assist clinical diagnosis, efficacy observation, prognosis judgment and observation of the effect of vaccination, etc. It 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 test, fluorescence immunoassay, radioimmunoassay, enzyme immunoassay, chemiluminescence immunoassay, and POCT (point-of-care testing) related immunoassay techniques (such as colloidal gold immunoassay, fluorescence immunoassay chromatography) etc. Therefore, the recombinant protein F1B139 designed and developed by the present application can be applied in the applications described in D1) to D8).

[0054] The recombinant protein F1B139 can be used as a known detection antigen (or diagnostic antigen) in plague antigen detection methods or products for qualitative or quantitative detection of Yersinia pestis antibodies, and can also be used as a quality control product in plague antigen detection methods or products.

[0055] The present application also provides a reagent or a kit comprising the protein F1B139, which has at least one of the following uses:

[0056] E1) detecting, analyzing or evaluating Yersinia pestis antibodies;

[0057] E2) quantitatively detecting, quantitatively analyzing or quantitatively evaluating Yersinia pestis antibodies;

[0058] E3) for plague detection;

[0059] E4) for diagnosis, auxiliary diagnosis, screening, therapeutic observation, prognosis or prophylactic observation of plague;

[0060] E5) for plague prevention and control (such as epidemiological investigation of plague).

[0061] The kit can be a plague antigen detection kit or a plague antibody detection kit.

[0062] The kit can be an immunological detection kit.

[0063] Further, the kit can be an ELISA kit, an immunoblotting detection kit, an immunochromatographic detection kit, a flow cytometry kit or an immunohistochemical detection kit, but is not limited thereto.

[0064] Further, the detection sample of the reagent or kit can include a blood sample, a tissue sample, a saliva sample, a sputum sample, a body fluid sample or an environmental sample.

[0065] Further, the reagent or kit can be a plague antibody detection reagent or kit, and the detection sample thereof can include a blood sample (such as whole blood, plasma or serum).

[0066] Further, the reagent or kit can be a plague antigen detection reagent or kit, and the detection sample thereof can include an environmental sample, a tissue sample, a saliva sample, a sputum sample or a body fluid sample.

[0067] The various reagent components of the kit can be present in separate containers, or can be pre-combined into a reagent mixture in whole or in part.

[0068] The present application also provides any one of the following uses of the reagent or kit:

[0069] F1) detecting, analyzing or evaluating Yersinia pestis antibodies;

[0070] F2) quantitatively detecting, quantitatively analyzing or quantitatively evaluating Yersinia pestis antibodies.

[0071] The present application 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.

[0072] Further, the method for preparing the protein F1B139 can comprise the following steps:

[0073] G1) constructing a recombinant expression vector containing a nucleic acid molecule encoding the protein F1B139;

[0074] G2) introducing the recombinant expression vector into a microorganism of interest to obtain a recombinant microorganism;

[0075] G3) culturing the recombinant microorganism to obtain the protein F1B139 through separation and / or purification;

[0076] Further, the nucleic acid molecule encoding the protein F1B139 in G1) can be a DNA molecule (F1B139 gene) as shown in SEQ ID No. 2.

[0077] Further, the microorganism of interest in G2) can be E. coli, in particular E. coli BL21 (DE3) competent cells.

[0078] The present application also provides a method for detecting or quantitatively detecting Yersinia pestis antibodies, which can comprise detecting or quantitatively detecting by using the protein F1B139 or the reagent or kit.

[0079] In the above method, the detecting or quantitatively detecting by using the protein F1B139 or the reagent or kit can comprise performing through a precipitation reaction, an agglutination test, a fluorescence immunoassay, a radioimmunoassay, an enzyme immunoassay, a chemiluminescence immunoassay, a colloidal gold immunoassay or a fluorescence immunoassay chromatography.

[0080] Further, the method can be an ELISA detection method.

[0081] Further, the method comprises a step of coating the recombinant protein F1B139 on a solid phase carrier to obtain a coated antigen solid phase carrier. The coating refers to that the antigen is combined on the solid phase carrier through physical adsorption.

[0082] Further, the solid phase carrier can be an enzyme-labeled plate, a membrane carrier, a microsphere, a biochip or a magnetic bead, but is not limited thereto.

[0083] 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.

[0084] The membrane carrier can be a nitrocellulose membrane, a glass cellulose membrane or a nylon membrane, but is not limited thereto.

[0085] Further, the coating concentration of the recombinant protein F1B139 can be 0.5-10 μg / mL, and specifically can be 1 μg / mL.

[0086] Further, the coating condition of the recombinant protein F1B139 can be coating at 4°C for 12 h.

[0087] In the above method, the detection sample for detection or quantitative detection can include a blood sample (such as whole blood, plasma or serum).

[0088] In an embodiment of the present application, the method for quantitatively detecting Yersinia pestis antibodies comprises the following steps:

[0089] (1) Coating: 1 μg / mL of purified recombinant protein F1B139 is coated on an enzyme-labeled plate, 100 μL per well, and placed at 4°C for 12 h.

[0090] (2) Blocking: 1.5% casein is diluted 15 times, 200 μL per well is added, and blocked at 37°C for 2 h.

[0091] (3) Incubation of primary antibody: 100 μL of rabbit anti-F1 polyclonal antibody with a concentration of 1 μg / mL is added per well, and incubated at 37°C for 30 min.

[0092] (4) Incubation of secondary antibody: 100 μL of 1:4000 diluted HRP-goat anti-rabbit IgG (product of Thermo Fisher Company, item number 65-6120) is added per well, and incubated at 37°C for 20 min.

[0093] (5) Color development: 100 μL of color developing solution is added per well, and incubated at 37°C for 10 min.

[0094] (6) Termination: 50 μL of termination solution is added per well, and the OD value is determined at 450 nm and 630 nm.

[0095] The preparation method of the rabbit anti-F1 polyclonal antibody can be as follows: 200, 400 and 800 μg of natural F1 antigen is injected subcutaneously into the groin of a rabbit in three times with an interval of 14 days, the immune serum is harvested, and the polyclonal antibody in the serum is extracted by n-octanoic acid-saturated ammonium sulfate method, thereby obtaining the rabbit anti-F1 polyclonal antibody.

[0096] The natural F1 antigen can be prepared according to the method described in the patent document (Extraction and purification method of natural F1 antigen of Yersinia pestis, application number 200810055697.7).

[0097] The purposes of the applications and methods described herein can be disease diagnosis purposes, disease prognosis purposes and / or disease treatment purposes, and their purposes can also be non-disease diagnosis purposes, non-disease prognosis purposes and non-disease treatment purposes; their direct purposes can be to obtain information of intermediate results of disease diagnosis results, disease prognosis results and / or disease treatment results, and their direct purposes can be non-disease diagnosis purposes, non-disease prognosis purposes and / or non-disease treatment purposes.

[0098] The method for detecting or quantitatively detecting Yersinia pestis antibodies provided by the present application can be a non-disease diagnosis and treatment method or a disease diagnosis and treatment method. Among them, the non-disease diagnosis and treatment method can detect Yersinia pestis antibodies when screening for plague in the population, or detect Yersinia pestis antibodies in epidemiological investigation of plague.

[0099] In this paper, the term ELISA (Enzyme Linked Immunosorbent Assay) refers to the adsorption of antigen or antibody on a solid carrier, and the detection process is to add the antibody (or antigen) to be detected and enzyme-labeled markers into the reaction system in turn, so that they react with the antigen (or antibody) on the solid carrier to form an antigen-antibody complex, then wash away the unbound free enzyme-labeled markers and free antigens (or antibodies), and then determine the enzyme activity of the bound enzyme-labeled markers, so as to determine the content of the antibody (or antigen) to be detected in the sample. ELISA includes direct method, indirect method, double antibody sandwich method, competition method and anti-enzyme antibody method, etc. Although the examples provided by the present application use ELISA indirect method to detect Yersinia pestis antibodies, the present application is not limited to this specific method. Any other immunodetection method can be used by those skilled in the art, as long as the method is based on the specific reaction of the antigen (recombinant protein F1B139) and the antibody of the present application to achieve the same technical effect as the present application, which does not deviate from the scope of the present application, and the present application should include these alternative methods.

[0100] The natural F1 antigen and the recombinant F1 exist in the form of a mixture of monomers and different numbers of Caf1 protein polymers, and can only be used for qualitative evaluation of antibodies, and cannot be used for quantitative evaluation of Yersinia pestis antibodies. Literature shows that the Caf1 protein forms fibrous F1 protein in a head-to-tail manner (Zavialov, A. V., et al. (2003). "Structure and biogenesis of the capsular F1 antigen from Yersinia pestis: preserved folding energy drives fiber formation." Cell 113 (5): 587-596). After extensive and in-depth research, the present application unexpectedly obtains a truncated F1 antigen capable of completely decomposing F1 polymers into monomers by cutting off several amino acids at the amino terminal or carboxyl terminal, and the F1 antigen existing in the form of monomers can be used for quantitative evaluation of antibodies. The advantage of this is that most of the linear epitope sites of the antibodies are retained, thereby realizing quantitative analysis of the antibodies.

[0101] The present application develops and prepares a recombinant diagnostic antigen for quantitative detection (evaluation) of Yersinia pestis antibodies, i.e., a recombinant protein F1B139. The recombinant protein F1B139 is a truncated Yersinia pestis F1 surface antigen protein, and the N-terminal is truncated by 10 amino acids compared with the full-length F1 antigen without truncation, i.e., 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 in SEQ ID No. 1, and the nucleotide sequence of the gene (F1B139 gene) encoding the recombinant protein F1B139 is shown in SEQ ID No. 2.

[0102] The recombinant protein F1B139 of the present application can be used for evaluation of antibody raw materials in Yersinia pestis antigen immunoassay reagents, thereby realizing stable production of antigen reagents; and can also be directly used as raw materials of Yersinia pestis antibody immunoassay reagents. The present application provides a diagnostic antigen that can be quantitatively evaluated for Yersinia pestis antigen detection methods and antibody detection methods. Experiments prove that the Yersinia pestis antigen provided by the present application has good sensitivity and can be recognized by rabbit anti-F1 polyclonal antibodies, and can be used as a quality control product of Yersinia pestis antigen detection reagents, for precise quality control of antibody raw materials of Yersinia pestis antigen detection reagents, to ensure the batch-to-batch stability of antigen raw material production, and can also be directly used as raw materials of antibody detection reagents. The natural F1 or the full-length recombinant protein thereof exists in the form of a mixture of monomers and different numbers of Caf1 protein polymers, and the recombinant F1 antigen of the present application has a single composition, and can accurately evaluate antibodies.

[0103] The immunodetection method and product based on the recombinant F1 antigen (recombinant protein F1B139) of the application can realize quantitative detection of antibodies, has good sensitivity, can be widely applied to detection of antibodies in Yersinia pestis detection, quality control and evaluation, and provides help for clinical diagnosis, observation of therapeutic effect, prognosis judgment, observation of vaccination effect, epidemiological investigation of infectious diseases, and has very wide clinical application prospect and important significance. BRIEF DESCRIPTION OF DRAWINGS

[0104] Figure 1 The plasmid map of the recombinant vector pET-21a / EV76-F1. 11-149 The plasmid map of the recombinant vector pET-21a / EV76-F1.

[0105] Figure 2 The plasmid map of the recombinant vector pET-21a / EV76-F1. 13-149 The plasmid map of the recombinant vector pET-21a / EV76-F1.

[0106] Figure 3 The plasmid map of the recombinant vector pET-21a / EV76-F1. 1-139 The plasmid map of the recombinant vector pET-21a / EV76-F1.

[0107] Figure 4 It is the non-denaturing electrophoresis result of natural F1 in Example 2.

[0108] Figure 5 It is the non-denaturing electrophoresis result of full-length recombinant F1 in Example 2.

[0109] Figure 6 It is the non-denaturing electrophoresis result of recombinant protein F1B139 in Example 2. The first hole from left to right is marker, the second hole is natural F1, and the third hole is recombinant protein F1B139.

[0110] Figure 7 It is the WB result after denaturing electrophoresis of F1B139 (left) and F1B137 (right) and 5 monoclonal antibodies in Example 2. The 5 monoclonal antibodies are 1B6, 2E9, 4H2, 6E5 and 10E10 respectively.

[0111] Figure 8 It is the WB result after denaturing electrophoresis of F1B139 (left), F1B137 (middle) and F1A139 (right) and polyclonal antibody in Example 2. The polyclonal antibody is rabbit anti-F1. DETAILED DESCRIPTION

[0112] The application will be further described in detail below in conjunction with specific embodiments. The examples provided below are only for illustrating the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the application in any way.

[0113] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained commercially, unless otherwise specified.

[0114] The vector pET-21a used in the following examples is a product of Beijing Huayueyang Biotechnology Co., Ltd., and the item number is 4170.

[0115] Example 1, Design and preparation of recombinant protein F1B139

[0116] 1. Design of recombinant protein F1B139

[0117] The full-length plague F1 antigen is 149 amino acids, which exists in a multimeric form and can only be used for qualitative evaluation of antibodies, but cannot be used for quantitative evaluation of antibodies. Based on this, the present application designs a recombinant F1 antigen that exists entirely in a monomeric form. The advantage of the design is that most of the linear epitope sites of the antibodies are retained, and quantitative analysis of the antibodies can be achieved.

[0118] The recombinant F1 antigen designed in the present application is recombinant protein F1B139, which is a truncated plague F1 surface antigen protein. Compared with the full-length F1 antigen without truncation, the N-terminal is truncated by 10 amino acids, i.e., the last 139 amino acids of the full-length F1 antigen are selected as the new recombinant F1 antigen. The amino acid sequence of the recombinant protein F1B139 is shown in SEQ ID No. 1, and the nucleotide sequence of the gene (F1B139 gene) encoding the recombinant protein F1B139 is shown in SEQ ID No. 2.

[0119] Meanwhile, a recombinant protein (named as recombinant protein F1B137) selecting the last 137 amino acids of the full-length F1 antigen is designed for comparison experiments, which proves to be weaker in reaction with F1B139 and antibodies, especially for some monoclonal antibodies. Further, a recombinant protein (named as recombinant protein F1A139) selecting the first 139 amino acids of the full-length F1 antigen is selected for comparison experiments, which proves to be significantly weaker in reaction with F1B139 and antibodies.

[0120] The amino acid sequence of the recombinant protein F1B137 is shown in SEQ ID No. 3, and the nucleotide sequence of the gene (F1B137 gene) encoding the recombinant protein F1B137 is shown in SEQ ID No. 4.

[0121] The amino acid sequence of the recombinant protein F1A139 is shown in SEQ ID No. 5, and the nucleotide sequence of the gene (F1A139 gene) encoding the recombinant protein F1A139 is shown in SEQ ID No. 6.

[0122] The amino acid sequence of the full-length F1 antigen is as follows:

[0123] 5'-ADLTASTTATATLVEPARITLTYKEGAPITIMDNGNIDTELLVGTLTLGGYKTGTTSTSVNFTDAAGDPMYLTFTSQDGNNHQFTTKVIGKDSRDFDISPKVNGENLVGDDVVLATGSQDFFVRSIGSKGGKLAAGKYTDAVTVTVSNQ-3' (149 amino acids).

[0124] 2. Preparation of recombinant proteins

[0125] 2.1. Construction of recombinant plasmids

[0126] According to the conventional operation in the art, the genes encoding the three recombinant proteins (F1B139, F1B137 and F1A139) were constructed in the vector pET-21a, respectively, and the specific steps were as follows:

[0127] (1) The upstream primer was introduced with a BamH I restriction site, and the downstream primer was introduced with a Xho I restriction site. The upstream primer F1 (5'-CAG GGATCC GCAACTCTTGTTGAACCAGCCCGC-3', SEQ ID No. 7, the BamH I recognition site is underlined) and the downstream primer R1 (5'-GGG CTCGAG TTGGTT AGATACGGTTACGGTTAC-3', SEQ ID No. 8, the Xho I recognition site is underlined) were designed, and the DNA molecule (F1B139 gene) shown in SEQ ID No. 2 was used as a template for PCR amplification to obtain a PCR product (i.e., the target gene with the BamH I and Xho I restriction sites added at the 5' and 3' ends, respectively). The DNA fragment 1 was obtained by double digestion of the PCR product with BamH I and Xho I.

[0128] The expression vector pET-21a was double-digested with BamH I and Xho I to obtain the digested pET-21a vector fragment. The DNA fragment 1 and the digested pET-21a vector fragment were ligated to obtain a recombinant vector, which was named pET-21a / EV76-F1 11-149 (see the plasmid map in Figure 1 ).

[0129] The recombinant vector pET-21a / EV76-F1 11-149The recombinant expression vector is obtained by replacing the small fragment between the BamHI and XhoⅠ recognition sites of the pET-21a vector with the DNA fragment whose nucleotide sequence is SEQ ID No. 2 in the sequence listing, while keeping the other sequences of the pET-21a vector unchanged. The recombinant vector is pET-21a / EV76-F1. 11-149 It contains the F1B139 gene shown in SEQ ID No. 2.

[0130] (2) Introduce the BamHⅠ restriction site into the upstream primer and the XhoⅠ restriction site into the downstream primer to design the upstream primer F2(5'-CAG). GGATCC CTTGTTGAACCAGCCCGCATCACT-3' (SEQ ID No. 9, underlined is the BamHI recognition site) and downstream primer R1 (5'-GGG CTCGAG TTGGTT AGATACGGTTACGGTTAC-3', SEQ ID No. 8 (underlined is the XhoⅠ recognition site), was used as a template for PCR amplification of the DNA molecule (F1B137 gene) shown in SEQ ID No. 4 to obtain the PCR product (i.e., the target gene with BamHI and XhoⅠ restriction sites added to the 5' and 3' ends, respectively). The PCR product was then digested with BamHI and XhoⅠ to obtain DNA fragment 2.

[0131] The expression vector pET-21a was double-digested with BamHⅠ and XhoⅠ to obtain the digested pET-21a vector fragment. This DNA fragment was then ligated with the digested pET-21a vector fragment to obtain the recombinant vector, named pET-21a / EV76-F1. 13-149 (See plasmid map) Figure 2 ).

[0132] Recombinant vector pET-21a / EV76-F1 13-149 The recombinant expression vector is obtained by replacing the small fragment between the BamHI and XhoⅠ recognition sites of the pET-21a vector with the DNA fragment whose nucleotide sequence is SEQ ID No. 4 in the sequence listing, while keeping the other sequences of the pET-21a vector unchanged. The recombinant vector is pET-21a / EV76-F1. 13-149 It contains the F1B137 gene shown in SEQ ID No. 4.

[0133] (3) Introduce the BamHⅠ restriction site into the upstream primer and the XhoⅠ restriction site into the downstream primer to design the upstream primer F3(5'-CAG). GGATCCGCAGATTTAACTGCAAGCACCACT-3', SEQ ID No. 10, underlined is the BamH I recognition site) and the downstream primer R3 (5'-GGG CTCGAG AGTGT ATTTACCTGCTGCAAGTTT-3', SEQ ID No. 11, underlined is the Xho I recognition site), using the DNA molecule shown in SEQ ID No. 6 (F1A139 gene) as a template to perform PCR amplification, to obtain a PCR product (i.e. the target gene with the BamH I and Xho I enzyme digestion sites added at the 5' end and 3' end, respectively), and the DNA fragment 3 was obtained after double digestion of the PCR product with BamH I and Xho I.

[0134] The expression vector pET-21a was double-digested with BamH I and Xho I to obtain the digested pET-21a vector fragment, and the DNA fragment 3 was ligated with the digested pET-21a vector fragment to obtain a recombinant vector, designated as pET-21a / EV76-F1 1-139 (see plasmid map in Figure 3 ).

[0135] The recombinant vector pET-21a / EV76-F1 1-139 is a recombinant expression vector in which the fragment (small fragment) between the BamH I and Xho I recognition sites of the pET-21a vector is replaced by the DNA fragment with the nucleotide sequence shown in SEQ ID No. 6 in the sequence listing, while the other sequences of the pET-21a vector remain unchanged. The recombinant vector pET-21a / EV76-F1 1-139 contains the F1A139 gene shown in SEQ ID No. 6.

[0136] The three plasmids constructed in the above steps (1), (2) and (3) are identical in terms of enzyme digestion sites, etc., i.e. the target genes are obtained by PCR amplification, and the recombinant plasmids are obtained by ligation with the expression vector pET-21a after double digestion with BamH I and Xho I.

[0137] 2.2, Expression and purification of recombinant proteins

[0138] 2.2.1, Expression of recombinant proteins: the successfully constructed plasmids (i.e. the recombinant vectors pET-21a / EV76-F1 11-149 , pET-21a / EV76-F1 13-149 and pET-21a / EV76-F1 1-139) respectively into DH5a competent cells to prepare the clonal strains. The positive plasmids (the plasmids with correct sequencing) were transformed into BL21 (DE3) competent cells to prepare the expression strains. 5 mL of bacterial liquid (the culture bacterial liquid of the positive expression strain) was cultured at 37°C and 220 rpm for 3 h, and then was transferred to 1 L of 2YT culture medium. When the OD value of the bacterial liquid was about 0.6-0.8, 500 μL of IPTG was added, and the bacterial liquid was cultured at 18°C and 150 rpm for 20 h.

[0139] 2.2.2, Bacterial lysis and collection of supernatant: (1) Bacterial collection: the bacteria were collected by centrifugation at 4°C and 10000 x g for 15 min; (2) Bacterial resuspension: the bacteria were resuspended with about 40 mL of His binding buffer, and protease inhibitors were added at a volume ratio of 1:100; (3) Bacterial ultrasonic treatment: the resuspended liquid was placed in a 50 mL centrifuge tube in an ice box, and was ultrasonically treated for 50 min using a 6th gun head of an ultrasonic machine at 20% power, 2 s on and 3 s off; (4) Centrifugal collection of supernatant after lysis: the supernatant was obtained by centrifugation at 4°C and 9000 x g for 20 min.

[0140] 2.2.3, Purification of recombinant protein: the supernatant filtered through a 0.22 μm filter was added to a gravity chromatography column, and the supernatant was incubated with the nickel column at 4°C for 2 h. The opening valve of the gravity chromatography column was opened, 30 mL of His binding buffer was used for impurity flushing, 15-25 mL of 250 mM imidazole was used for elution of the target protein, and the target protein was collected. Further elution was performed using 30 mL of 400 mM imidazole to collect the target protein, thereby obtaining the purified recombinant proteins F1A139, F1B137 and F1A139.

[0141] Example 2, Functional identification of recombinant protein F1B137 as a diagnostic antigen

[0142] The antibodies used for detection in this example were derived from the following natural antigen sources:

[0143] Preparation method of rabbit anti-F1 polyclonal antibody: 200, 400 and 800 μg of natural F1 antigen were injected subcutaneously into the groin of a rabbit in three times with an interval of 14 days, and the immune serum was harvested. The polyclonal antibody in the serum was extracted using n-octanoic acid-saturated ammonium sulfate method, thereby obtaining the rabbit anti-F1 polyclonal antibody. The ELISA titer of the rabbit anti-F1 polyclonal antibody was about 31.25 ng / mL, which was detected using 1 μg / mL of natural F1 antigen to coat an ELISA plate.

[0144] Natural F1: natural F1 extracted according to the method described in the patent document (Method for extracting and purifying natural F1 antigen of Yersinia pestis, application number 200810055697.7).

[0145] Full-length recombinant F1: the preparation method is the same as F1B139 (the preparation method of the recombinant protein in step 2 in the implementation 1), that is, the gene encoding the full-length F1 protein is constructed in the vector pET-21a, and the full-length recombinant F1 is obtained after expression and purification.

[0146] 1. Native-PAGE non-denaturing electrophoresis and Western-blotting experiment:

[0147] The purified recombinant protein F1B139, native F1 and full-length recombinant F1 are subjected to Native-PAGE non-denaturing electrophoresis, and the steps are as follows: a 4%-15% polyacrylamide gel is configured, and Native-PAGE non-denaturing electrophoresis is performed at 4°C and 150V for 90 min.

[0148] After electrophoresis, the membrane is transferred at 90V for 45 min, and milk is blocked overnight. Rabbit anti-F1 polyclonal antibody is added for incubation, and Western-blotting is performed.

[0149] The results show that, after Native-PAGE non-denaturing electrophoresis, Western-blotting (WB) is performed using rabbit anti-F1 polyclonal antibody, it is found that the recombinant protein F1B139 and the native F1 and the full-length recombinant F1 are all specifically recognized by the rabbit anti-F1 polyclonal antibody, which indicates that the recombinant protein F1B139 has good reactivity (antigenicity), and can be used as a diagnostic antigen for detecting plague bacteria antibodies. The native F1 and the full-length recombinant F1 are composed of multiple bands, and are mostly concentrated in the upper half of the gel (as shown in Figure 4 and Figure 5 respectively), which proves that they are polymers. The composition of the band of the recombinant protein F1B139 (as shown in Figure 6 ) is relatively single, that is, it is composed of a monomer, which indicates that the recombinant protein F1B139 as a diagnostic antigen can realize accurate quantitative detection of antibodies, and can be further used to evaluate the antibody raw material of the plague antigen immunological detection reagent, and can be used as a quality control product of the plague bacteria antigen detection reagent, so as to realize stable production of the antigen reagent, and can also be directly used as a raw material of the plague antibody immunological detection reagent.

[0150] 2. Comparative experiment

[0151] The inventors of the present application have obtained the recombinant protein F1B139 through a large number of experiments, identification, screening and finally obtaining the recombinant protein F1B139. The recombinant protein F1B137 and the recombinant protein F1A139 prepared in the example 1 are used as a comparison verification in the comparative experiment, which indicates that the shorter recombinant protein and the recombinant protein selecting the first 139 amino acids of the full-length F1 antigen have weaker recognition ability with the plague antibody than the recombinant protein F1B139. The specific steps are as follows:

[0152] 1. Sample preparation

[0153] Samples were prepared at a concentration of 5 mg / mL. After adding 2× protein loading buffer, the protein concentration was 2.5 mg / mL.

[0154] 2. SDS-PAGE

[0155] The samples were subjected to SDS-PAGE, followed by Western blotting with five F1 monoclonal antibodies (or polyclonal antibodies).

[0156] 3. Western blot (WB) experiment

[0157] After electrophoresis, transfer the membrane to the NC membrane. Incubate at 90V for 45 min. Block the membrane overnight in 5% skim milk on a shaker at 4°C, then wash with TBST. Incubate with primary antibody, five F1 monoclonal antibodies (1B6, 2E9, 4H2, 6E5, 10E10) or polyclonal antibodies on a shaker at 4°C for 4 h, then wash with TBST. Incubate with secondary antibody, goat anti-mouse (or goat anti-rabbit), on a shaker at room temperature in the dark for 1 h, then wash with TBST and observe the membrane.

[0158] The anti-plague monoclonal antibody was prepared and preserved in our laboratory (related literature: Zhang, P., et al. (2020). "Calibration of an Upconverting Phosphor-Based Quantitative Immunochromatographic Assay for Detecting Yersinia pestis, Brucella spp., and Bacillus anthracis Spores." Front Cell Infect Microbiol 10:147.)

[0159] Western blotting results are as follows: Figure 7 and Figure 8 As shown, F1B139 responds more strongly to monoclonal antibodies than F1B137, especially to 2E9 and 4H2. Regarding the strength of the response to polyclonal antibodies, F1B139 > F1B137 > F1A139.

[0160] Example 3: ELISA detection method for quantitative detection of Yersinia pestis antibodies

[0161] This embodiment establishes an ELISA method for the quantitative detection of Yersinia pestis antibodies based on the recombinant protein F1B139 prepared in Example 1 as the antigen protein. Taking the ELISA method as an example, it verifies that the recombinant protein F1B139 prepared in this invention can be used as a diagnostic antigen for the quantitative detection and analysis of Yersinia pestis antibodies. The ELISA detection experimental method is as follows:

[0162] (1) Coating: 1 μg / mL of purified recombinant protein F1B139 was coated on the ELISA plate; 1 μg / mL of native F1 antigen was coated on the ELISA plate, 100 μL per well, 4°C for 12 h.

[0163] (2) Blocking: 1.5% of dried casein was diluted 15 times, 200 μL per well, 37°C for 2 h.

[0164] (3) Incubation of primary antibody: 100 μL of 1 μg / mL rabbit anti-F1 polyclonal antibody was added to each well, 37°C for 30 min.

[0165] (4) Incubation of secondary antibody: 100 μL of 1:4000 diluted HRP-goat anti-rabbit IgG (thermofisher product, item number 65-6120) was added to each well, 37°C for 20 min.

[0166] (5) Color development: 100 μL of color developing solution was added to each well, 37°C for 10 min.

[0167] (6) Termination: 50 μL of termination solution was added to each well, and the OD value was determined at 450 nm and 630 nm. The results of ELISA detection are shown in Table 1:

[0168] Table 1, ELISA results of recombinant protein F1B139 and rabbit anti-F1 polyclonal antibody

[0169]

[0170] As can be seen from Table 1, F1B139 can achieve the same titer as native F1, both of which are 32.50 ng / mL. Because the components of native F1 and full-length recombinant F1 are complex, Figure 4 and 5 there is a mutual transformation of multimers and monomers, and the composition of each component under various conditions is not necessarily the same; and it is difficult to ensure batch-to-batch difference when different batches are prepared. Compared with native F1 and full-length recombinant F1, F1B139 has a single composition, Figure 2 and Figure 6 batch-to-batch difference is controllable, and sensitivity is higher, which is a better choice for quality control antibodies.

[0171] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A protein, characterized in that, The protein is any one of the following: A1) a protein having an amino acid sequence of SEQ ID No. 1 ; A2) a fusion protein having the same function obtained by connecting a tag to the N- terminal and / or C-terminal end of A1).

2. Biomaterial, characterized in that, The biological material is any one of the following: B1) a nucleic acid molecule encoding the protein of claim 1 ; B2) an expression cassette containing the nucleic acid molecule of B1); B3) a recombinant vector containing the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3); B5) a recombinant host cell containing the nucleic acid molecule of B1), or a recombinant host cell containing the expression cassette of B2), or a recombinant host cell containing the recombinant vector of B3).

3. The biomaterial of claim 2, wherein, The nucleic acid molecule of B1) is a DNA molecule of SEQ ID No.

2.

4. Use of the protein of claim 1, and / or the biological material of claim 2 or 3, for any one of the following: D1) in the preparation of a product for detecting, analyzing or evaluating antibodies against Yersinia pestis; D2) in the preparation of a product for quantitatively detecting, quantitatively analyzing or quantitatively evaluating antibodies against Yersinia pestis; D3) in the preparation of a quality control product for Yersinia pestis antigen detection reagents.

5. A reagent or kit characterized in that, The reagent or kit comprises the protein of claim 1, and has at least one of the following uses: E1) detecting, analyzing or evaluating antibodies against Yersinia pestis; E2) quantitatively detecting, quantitatively analyzing or quantitatively evaluating antibodies against Yersinia pestis.

6. The reagent or kit of claim 5, wherein, The sample to be detected by the reagent or kit comprises a blood sample, a tissue sample, a saliva sample, a sputum sample or an environmental sample.

7. A method of producing the protein of claim 1, characterized by, The method comprises expressing a nucleic acid molecule encoding the protein of claim 1 in a host cell to obtain the protein.

8. The method of claim 7, wherein, The host cell is a microorganism.

Images