N protein recombinant antigen for detecting novel bunyavirus antibody and application of N protein recombinant antigen

Abstract

The invention discloses an N protein recombinant antigen for detecting a new bunyavirus antibody and application of the N protein recombinant antigen. The N protein recombinant antigen comprises an N protein and a peptide fragment containing a linear epitope in the N protein. The N protein recombinant antigen completely covers SFTSV antibody recognition sites and has high activity, and the prepared protein is high in purity, stable in structure and good in batch-to-batch stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to N protein recombinant antigen for detecting novel bunyavirus antibody and use thereof, further relates to N protein recombinant antigen for detecting novel bunyavirus antibody, nucleic acid molecule encoding same, recombinant expression vector and recombinant cell comprising same, and use thereof. BACKGROUND

[0002] Novel bunyavirus (SFTSV) belongs to the family of Phenuiviridae and the genus of Phlebovirus.

[0003] The main transmission medium of SFTSV is ticks (such as Haemaphysalis longicornis), but it can also be transmitted between individuals by contact with the blood or body fluids of infected individuals. The virus has been reported in many countries, and even highly homologous virus strains have been isolated in the Americas, suggesting its global distribution and potential pandemic risk. Epidemiological data show that the incidence peak is highly consistent with the tick activity season (March to July), and the mortality rate is about 10%-30%.

[0004] The SFTSV genome consists of three segments, L, M, and S, which encode nucleocapsid protein (N protein), glycoprotein (Gn / Gc), and non-structural protein (NSs, etc.). Among them, the N protein is the core structural protein of virus replication and assembly. As the most conserved structural protein of SFTSV, the N protein is composed of an N-terminal RNA binding domain (NTD), a C-terminal dimerization domain (CTD), and a serine / arginine-rich functional domain (SRD), which is responsible for the packaging of viral RNA and the formation of nucleocapsid, protecting the stability of the genome. In addition to participating in virus assembly, the N protein mediates immune escape through mechanisms such as inhibiting the host interferon pathway and regulating apoptosis, directly affecting disease progression. Compared to the variable surface glycoprotein (Gn / Gc), the N protein antigen epitope is highly conserved, and high concentrations of expression can be detected in the blood in the early stages of infection, making it an ideal target for the development of serological detection reagents.

[0005] Currently, the diagnosis of SFTSV mainly relies on nucleic acid detection (such as RT-PCR) and immunological detection (such as ELISA). The existing ELISA kit has insufficient sensitivity (36%-80%) and specificity (47%-100%), and has a high rate of missed detection for low viral load samples, requiring secondary confirmation.

[0006] Traditional antigen detection uses inactivated virus or cell lysate prepared antigens, which are easily interfered by impurities, leading to false positives; and the production process involves live virus operation, with high biological safety risk.

[0007] The recombinant antigen expresses high-purity N protein antigen through genetic engineering, can avoid the risk of using live virus, improve antigen batch consistency, and enhance the binding efficiency with antibodies through optimized epitope design, to provide high-quality raw materials for high-sensitivity platforms such as chemiluminescence. Further development of recombinant antigens with higher activity suitable for SFTSV antibody detection by chemiluminescence is an important tool for realizing rapid and accurate clinical diagnosis of SFTSV.

[0008] Although the N protein of SFTSV has high immunogenicity, only a few of the antibodies screened by using the N protein have neutralizing activity, and the existing SFTSV-N protein recombinant antigen has the characteristics of incomplete antigen epitope and low activity, which cannot be well applied to the preparation of SFTSV antibody detection kits.

[0009] Therefore, there is an urgent need in the art to develop a new type of recombinant antigen. SUMMARY

[0010] Therefore, there is an urgent need in the art to develop a new type of recombinant antigen.

[0011] In a first aspect of the present application, a recombinant N protein antigen for detecting a novel bunyavirus antibody is provided, wherein the recombinant N protein antigen comprises a nucleocapsid protein (N protein) and a peptide segment comprising a linear epitope in the N protein.

[0012] The linear epitope comprises one or more of the following epitopes:

[0013] Epitope 1: derived from the N-terminal domain of the N protein;

[0014] Epitope 2: derived from the intermediate domain of the N protein; and,

[0015] Epitope 3: derived from the C-terminal domain of the N protein.

[0016] In some embodiments, the epitope 1 is located at positions 1-46 of the N protein.

[0017] In some embodiments, the epitope 2 is located at positions 132-142 of the N protein.

[0018] In some embodiments, the epitope 3 is located at positions 238-245 of the N protein.

[0019] In some embodiments, the amino acid sequence of the N protein is shown in SEQ ID NO: 2.

[0020] In some embodiments, the recombinant N protein antigen comprises one or more of the following peptide segments:

[0021] Peptide segment 1: an amino acid segment comprising an amino acid sequence as set forth in SEQ ID NO: 3;

[0022] Peptide segment 2: an amino acid segment comprising an amino acid sequence as set forth in SEQ ID NO: 4; and,

[0023] Peptide segment 3: an amino acid segment comprising an amino acid sequence as set forth in SEQ ID NO: 5.

[0024] In some embodiments, the N protein recombinant antigen comprises, from N-terminus to C-terminus, operatively linked peptide segment 1, N protein, peptide segment 2 and peptide segment 3.

[0025] In some embodiments, the operable linkage is by a linker peptide selected from the group consisting of (G4S)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)nand (A)n, wherein any n is independently an integer greater than or equal to 1 and less than or equal to 6.

[0026] In some embodiments, the N protein recombinant antigen comprises an amino acid segment having a sequence as set forth in SEQ ID NO: 8.

[0027] In some embodiments, the N protein recombinant antigen further comprises one or more of a purification tag and a fluorescent tag.

[0028] In some embodiments, the purification tag can include one or more of a His tag, a bio tag, an Avidin tag, a Flag tag and a GST tag.

[0029] In some embodiments, the N protein recombinant antigen has a His tag at the N-terminus and / or an Avidin tag at the C-terminus.

[0030] In some embodiments, the N protein recombinant antigen comprises an amino acid segment having an amino acid sequence as set forth in SEQ ID NO: 9.

[0031] In a second aspect of the present application, there is provided a kit for detecting antibodies against the novel Bunyavirus, comprising the N protein recombinant antigen as described in the first aspect.

[0032] In some embodiments, the kit further comprises one or more of a magnetic bead diluent, an acridinium diluent, a biotin diluent, an antibody-acridinium ester conjugate and a streptavidin conjugated magnetic bead.

[0033] In a third aspect of the present application, there is provided a method for detecting antibodies against the novel Bunyavirus using the N protein recombinant antigen as described in the first aspect or the kit as described in the second aspect, the method comprising:

[0034] contacting the N protein recombinant antigen with the sample;

[0035] detecting the contacting of the N protein recombinant antigen with the antibody.

[0036] In a fourth aspect of the present application, a nucleic acid molecule encoding the N protein recombinant antigen of the first aspect is provided.

[0037] In a fifth aspect of the present application, a recombinant expression vector comprising the nucleic acid molecule of the fourth aspect is provided.

[0038] In a sixth aspect of the present application, a recombinant cell expressing the N protein recombinant antigen of the first aspect is provided.

[0039] In some embodiments, it comprises one or more of the nucleic acid molecule of the fourth aspect and the recombinant expression vector of the fifth aspect.

[0040] In some embodiments, the host cell of the recombinant cell is a eukaryotic cell or a prokaryotic cell.

[0041] In a seventh aspect of the present application, a method for preparing the N protein recombinant antigen is provided, which comprises:

[0042] culturing the recombinant cell of the sixth aspect to obtain a culture;

[0043] isolating and purifying the N protein recombinant antigen from the culture.

[0044] In an eighth aspect of the present application, a method for preparing or screening a new bunyavirus antibody is provided, which comprises:

[0045] immunizing an animal with the N protein recombinant antigen of the first aspect;

[0046] preparing a hybridoma cell strain;

[0047] screening the hybridoma cell strain; and,

[0048] collecting and purifying the antibody.

[0049] The N protein recombinant antigen provided by an embodiment of the present application covers the SFTSV antibody recognition site completely, has high activity, and has high purity, stable structure and good batch stability. The prepared protein can be used for preparing an SFTSV antibody detection kit (chemiluminescence method), and the prepared SFTSV antibody detection kit (chemiluminescence method) can quickly and accurately detect SFTSV antibodies in serum samples, has high specificity and high sensitivity.

[0050] In some implementations, the recombinant N protein antigen is biotin-labeled, which can be directly used in kit preparation, reducing uncontrollable factors caused by the labeling process and better controlling batch-to-batch variations. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0052] Figure 1 This is a plasmid map of pET28a-His tag-P1-NP-P2-P3-Avi Tag in one embodiment of this application.

[0053] Figure 2 This is an SDS-PAGE image of the recombinant protein rSFTSV-NP in one embodiment of this application. Detailed Implementation

[0054] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0056] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0057] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0058] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0059] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0060] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it refers to either "with" or "without" a parallel solution. If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent. Unless otherwise specified, the descriptions such as "optionally include" and "optionally contain" in this application, taking "optionally include" as an example, mean "may include or not include."

[0061] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0062] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0063] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0064] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0065] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0066] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0067] Based on extensive research and investigation of existing technologies, the inventors initially concluded that the epitope design or purity of recombinant antigens may affect the effectiveness of antibodies, thereby impacting the sensitivity and specificity of detection reagents. Therefore, through inventive work and analysis of the SFTSV N protein epitope, the inventors superimposed its dominant epitopes on the SFTSV N protein sequence in sequence design, aiming to improve specificity and sensitivity.

[0068] The N protein is the core protein of SFTSV, possessing strong immunogenicity and a stable antigenic determinant. Compared to the easily mutated surface glycoproteins (Gn / Gc), the N protein epitopes are highly conserved, and high concentrations of expression can be detected in the blood early in the infection process, making it an important biomarker for early diagnosis. In one embodiment of this application, the constructed SFTSV recombinant antigen uses the full-length N protein sequence (e.g., GenBank: AFN26700.1) and three additional linear epitope peptide segments, further ensuring the detection of SFTSV antibodies in human blood. The selection of the N protein sequence enables the detection of the pre-infection phase of SFTSV and improves reagent sensitivity while ensuring antigen epitope exposure (for example, the selected N protein fragment has 100% SFTSV homology). Based on this, three N protein linear epitope peptides are added (Peptide 1: aa1-46, Peptide 2: aa132-142, Peptide 3: aa238-245; hereinafter abbreviated as P1, P2, and P3). By repeating these sequences, the number and diversity of epitopes in the recombinant antigen are enhanced, which helps improve the detection sensitivity of the recombinant protein against SFTSV antibodies.

[0069] The recombinant N protein antigen obtained in the final preparation has complete epitopes, strong activity, and high affinity for human anti-SFTSV total antibody, and can be applied to the detection of SFTSV IgG or IgM antibodies.

[0070] One aspect of this application provides a recombinant N protein antigen for detecting antibodies against a novel Bunyavirus, the recombinant N protein antigen comprising a nucleocapsid protein (N protein) and a peptide comprising a linear epitope of the N protein.

[0071] Unless otherwise specified, the term "recombinant antigen" in this application refers to an antigen protein obtained by inserting a gene fragment encoding a specific antigen into an expression vector (such as a plasmid or virus) through genetic engineering technology, and then expressing and purifying it in a host cell (such as a prokaryotic cell like Escherichia coli, or a eukaryotic cell like yeast or non-human mammalian cells).

[0072] The inventors creatively combined the N protein with a peptide containing a linear epitope from the N protein, resulting in a recombinant N protein antigen that significantly improved the detection sensitivity of SFTSV antibodies.

[0073] Unless otherwise specified, the term "linear epitope" in this application refers to an antigenic determinant (i.e., a specific site recognized by an antibody or T-cell receptor) consisting of a sequence of consecutive amino acids in a protein (such as N protein) or polypeptide chain.

[0074] In some embodiments, the linear epitopes of the N protein are selected from the group consisting of epitope 1, epitope 2, and epitope 3; wherein:

[0075] Epitope 1: Derived from the N-terminal domain of the N protein;

[0076] Epitope 2: Derived from the intermediate domain of the N protein;

[0077] Epitope 3: C-terminal domain derived from N protein.

[0078] I do not wish to be limited by any theory, but I believe that the N-terminal domain (NTD) could be, for example, the first 100, 90, 80, 70, 60, 50, or 40 amino acids of the N protein.

[0079] In some embodiments, the N-terminal domain is the first to 100th amino acids of the N protein.

[0080] In some implementations, the N-terminal domain is the first to 50th amino acids of the N protein.

[0081] The C-terminal domain can be, for example, the last 70, last 60, or last 50 amino acids of the N protein. For instance, the C-terminal domain can be the 200th to 250th amino acids of the N protein.

[0082] The intermediate domain can be, for example, a domain of the N protein other than the N-terminal and C-terminal domains, such as, an amino acid fragment after position 100 and before position 200 of the N protein.

[0083] In some embodiments, epitope 1 is located at positions 1-46 of the N protein, or is composed of positions 1-46.

[0084] In some embodiments, epitope 2 is located at positions 132-142 of the N protein, or is constituted by positions 132-142.

[0085] In some embodiments, epitope 3 is located at positions 238-245 of the N protein, or is constituted by positions 238-245.

[0086] In some embodiments, the amino acid sequence of the N protein is shown in SEQ ID NO: 2.

[0087] In some embodiments, the N protein recombinant antigen comprises one or more of peptide 1, peptide 2, and peptide 3.

[0088] Peptide 1 contains an amino acid fragment with the amino acid sequence shown in SEQ ID NO: 3; peptide 2 contains an amino acid fragment with the amino acid sequence shown in SEQ ID NO: 4; and peptide 3 contains an amino acid fragment with the amino acid sequence shown in SEQ ID NO: 5.

[0089] In some embodiments, the N protein recombinant antigen, from the N-terminus to the C-terminus, sequentially comprises operably linked peptide 1, N protein, peptide 2, and peptide 3.

[0090] Unless otherwise specified, the term "operably linked" in this application refers to the functional relationship between any two regions in the N protein recombinant antigen, such as peptide 2 and peptide 3, or peptide 1 and the N protein, wherein adjacent regions are linked to produce the N protein recombinant antigen.

[0091] For example, operative linking is performed via linkers (e.g., linking peptides).

[0092] In some implementations, operative linking is performed by selecting linker peptides from the group consisting of (G4S)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n, and (A)n, wherein any n is independently an integer greater than or equal to 1 and less than or equal to 6.

[0093] In some embodiments, the N protein recombinant antigen comprises an amino acid fragment with the sequence shown in SEQ ID NO: 8.

[0094] In some embodiments, the N protein recombinant antigen further comprises one or more of a purification tag and a fluorescent tag;

[0095] The purification label includes, for example, one or more of the following: His label, Bio label, Avidin label, Flag label, and GST label.

[0096] It should be understood that the Avidin tag allows for targeted biotinylation modification, which is helpful for the later development of highly stable diagnostic reagents.

[0097] Unless otherwise specified, the term "His tag" in this application refers to "histidine tag", which may be 6×His (HHHHHH), 8×His, or 10×His, etc.

[0098] Unless otherwise specified, the term "biolabel" in this application refers to "biotin label".

[0099] The common forms of the “Avidin tag” are NeutrAvidin or Streptavidin.

[0100] The sequence of the “Flag” is, for example, DYKDDDDK (SEQ ID NO: 10).

[0101] The full name of the “GST tag” is glutathione S-transferase tag, and its sequence is, for example, the full-length GST protein (~26 kDa).

[0102] In some embodiments, the N-terminus of the N-protein recombinant antigen is tagged with His.

[0103] In some embodiments, the C-terminus of the N protein recombinant antigen is tagged with Avidin.

[0104] In some embodiments, the N-terminus of the recombinant N-protein antigen is tagged with His, and the C-terminus is tagged with Avidin.

[0105] In some embodiments, the N protein recombinant antigen comprises an amino acid fragment with an amino acid sequence as shown in SEQ ID NO: 9.

[0106] Another aspect of this application provides a kit for detecting antibodies against the novel Bunyavirus, which contains the N protein recombinant antigen as described above.

[0107] In some embodiments, the kit further comprises one or more of the following: magnetic bead diluent, acridine diluent, biotin diluent, antibody acridine ester conjugate, and streptavidin-conjugated magnetic beads.

[0108] For example, the kit contains N protein recombinant antigen, magnetic bead diluent, acridine diluent, biotin diluent, antibody acridine ester conjugate, and streptavidin-conjugated magnetic beads.

[0109] The role of the magnetic bead diluent is at least to dilute the streptavidin-coupled magnetic beads in the preparation of the working solution. Its formulation can be, for example: 50 mM Tris, 0.9% NaCl, 0.5% BSA (bovine serum albumin), 0.05% Tween-20, 0.1% ProClin300, pH 7.5.

[0110] The purpose of the biotin diluent is at least to dilute the SFTSV recombinant antigen in the preparation of the biotin working solution. Its formulation, for example, can be 50 mM PB (phosphate buffer), 0.9% NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.0.

[0111] The role of acridine diluent is at least in diluting antibody-labeled conjugates to prepare acridine working solution. Its formulation, for example, can be 50 mM PB, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.5.

[0112] Another aspect of this application provides a method for detecting novel Bunyavirus antibodies using the N protein recombinant antigen or kit described above, the method comprising:

[0113] Contact the N protein recombinant antigen with the sample;

[0114] The contact between the N protein recombinant antigen and the antibody was detected.

[0115] In some implementations, detection is performed using a two-step indirect immunoassay with direct chemiluminescence technology.

[0116] In some implementations, methods for detecting antibodies against the novel Bunyavirus include:

[0117] Step 1 Incubation: Add sample, superparamagnetic microparticles, sample diluent and test diluent; the SFTSV IgG / IgM in the sample reacts with the SFTSV NP antigen coated on the superparamagnetic microparticles to form an antigen-antibody complex.

[0118] Cleaning: Under the action of a magnetic field, superparamagnetic particles are adsorbed onto the reaction tube wall, and unbound substances are washed away by the cleaning solution;

[0119] The second step is incubation: Acridine-labeled mouse anti-human IgG antibody (secondary antibody) is added to react and form an antigen-antibody-secondary antibody complex.

[0120] Wash again.

[0121] Another aspect of this application provides a nucleic acid molecule that encodes the N protein recombinant antigen as described above.

[0122] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffer solutions, or salts.

[0123] Another aspect of this application provides a recombinant expression vector comprising the nucleic acid molecules described above.

[0124] The term "vector," also known as a "nucleic acid construct," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can integrate into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes to which they are effectively linked. Such vectors are referred to in this application as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included, which perform equivalent functions. In some embodiments, the vector is pET28a.

[0125] Another aspect of this application provides recombinant cells that express the N protein recombinant antigen as described above.

[0126] In some embodiments, it comprises one or more of the nucleic acid molecules and recombinant expression vectors as described above.

[0127] In some embodiments, the host cell of the recombinant cell is a prokaryotic cell or a eukaryotic cell.

[0128] For example, the prokaryotic cells include Escherichia coli, such as Escherichia coli BL21.

[0129] For example, the eukaryotic cell is a yeast cell or a non-human mammalian cell.

[0130] In some embodiments, the yeast includes Saccharomyces cerevisiae.

[0131] In some embodiments, the non-human mammalian cells are Chinese hamster ovary cells (CHO), HeLa cells, or monkey kidney cells.

[0132] In another aspect of this application, a method for preparing the aforementioned recombinant cells is provided. This includes the step of introducing the aforementioned or recombinant expression vector into target cells.

[0133] In one example, the import method uses transfection.

[0134] The term “transfection” refers to the process of introducing nucleic acids into cells, such as non-human mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0135] Another aspect of this application provides a method for preparing a recombinant N protein antigen, comprising:

[0136] The recombinant cells described above are cultured to obtain a culture.

[0137] The N protein recombinant antigen was isolated and purified from the culture.

[0138] Another aspect of this application provides a method for preparing or screening antibodies against novel Bunyaviruses, the method comprising:

[0139] Animals were immunized with the N protein recombinant antigen as described above.

[0140] Preparation of hybridoma cell lines;

[0141] Screening for hybridoma cell lines; and,

[0142] Collect and purify antibodies.

[0143] In some embodiments, a biotin tag is added to the C-terminus of the N protein recombinant antigen. The recombinantly expressed protein can be directly used in the preparation of SFTSV antibody detection kits (chemiluminescence assay), avoiding human differences caused by subsequent labeling and better controlling batch-to-batch variations. SFTSV antibody detection kits (chemiluminescence assay) containing this recombinant antigen can rapidly and accurately detect the content of SFTSV antibodies in serum isolated from the human body.

[0144] The following are some examples.

[0145] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0146] This application exemplarily discloses a method for preparing a recombinant antigen suitable for detecting SFTSV antibodies in isolated biological samples, and a method for detecting SFTSV antibodies using the recombinant antigen. Those skilled in the art can refer to the content of this invention and appropriately modify process parameters or change combinations according to the characteristics of the immune response to implement and apply the technology of this invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are all considered to be included in this invention.

[0147] Example 1: Preparation of SFTSV recombinant antigen

[0148] Plasmid construction: Construct pET28a-His tag-P1-NP-P2-P3-Avi-tag expression clone. See plasmid map below. Figure 1 (The orange arrow indicates the SFTSV recombinant protein -P1-NP-P2-P3-, which has a His-tag at the N-terminus and an Avi-tag at the C-terminus.) The NP sequence is the full length, ensuring detection of SFTSV antibodies in human blood. P1, P2, and P3 are three linear epitopes in NP (aa1-46, aa132-142, and aa238-245, respectively), repeatedly expressed to improve the detection sensitivity of the recombinant protein. The His-tag sequence can be used for subsequent protein purification. The Avi-tag sequence allows for targeted biotinylation modification, which is helpful for the later development of highly stable diagnostic reagents.

[0149] The amino acid sequence is shown below:

[0150] His-tag: HHHHHHHHHH; aa2-11 in SEQ ID NO: 9 (SEQ ID NO: 1).

[0151] NP (GenBank: AFN26700.1): aa72-316 (SEQ ID NO: 2) in SEQ ID NO: 9.

[0152] Peptide 1: The amino acid sequence is MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETG (SEQ ID NO: 3), aa12-56 in SEQ ID NO: 9; corresponding to positions 1-46 of the N protein as shown in SEQ ID NO: 2.

[0153] Peptide 2: The amino acid sequence is VMNLKVENYPP, aa332-342 in SEQ ID NO: 9 (SEQ ID NO: 4); the corresponding sequence is positions 132-142 of the N protein as shown in SEQ ID NO: 2.

[0154] Peptide 3: The amino acid sequence is AAAAYRNL, aa358-365 in SEQ ID NO: 9 (SEQ ID NO: 5); the corresponding sequence is positions 238-245 of the N protein as shown in SEQ ID NO: 2.

[0155] Flexible linker: The amino acid sequence is GGGGSGGGGSGGGGS, aa57-71, 317-331, 343-357, 366-380 in SEQ ID NO: 9; (SEQ ID NO: 6).

[0156] Avi-tag: The amino acid sequence is GLNDIFEAQKIEWHE, aa381-395 in SEQ ID NO: 9 (SEQ ID NO: 7).

[0157] Recombinant protein rSFTSV-NP amino acid sequence:

[0158] MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGGGGSGGGGSGGGGSMSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALII KKLKETGGDDWVKDTK FIIVFALTRGNKIVK ASGKMSNSGSKRL MALQEKYGLV ERAETRL SITPVRVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTA GVS EATT KTLMEAYSLWQDAFTKTINV KMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNLGGGGSGGGGSGGGGSVMN LKVENYPP GGGGSGGGGSGGGGSAAAAYRNL (SEQ ID NO: 8) (underlined to match the 6 linear epitopes mentioned in patent CN201510866489.5).

[0159] Recombinant protein rSFTSV-NP amino acid sequence (including fusion tag):

[0160] MHHHHHHHHHHSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGGGGSGGGGSGGGGSMSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVKDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERAETRLSITPVRVAQSLPTWTCAAAAALKEYLPV GPAMNLKVENYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNLGGGGSGGGGSGGGGSVMNLKVENYPPGGGGSGGGGSGGGGSAAAAYRNLGGGGSGGGGSGGGGSGLNDIFEAQKIEWHE (SEQ ID NO: 9).

[0161] Plasmid transformation: The extracted recombinant plasmid was transformed into competent Escherichia coli BL21 containing the ligase BirA. The addition of IPTG (0.5 mmol / L) can induce the co-expression of SFTSV recombinant protein and BirA, thus achieving biotinylation.

[0162] Inoculation: Inoculate Escherichia coli BL21 containing the recombinant plasmid into 5 mL LB liquid medium (containing 50 μg / mL kanamycin) and incubate overnight at 37°C with shaking.

[0163] Induction: Dilute overnight bacteria 1:100, incubate at 37°C with shaking at 200 rpm, and continuously monitor the OD of the bacterial culture. 600 When OD 600When the concentration of 0.4 mM (Blank: ddH2O) was reached, the shake flask was removed, and 1 M IPTG was added in a clean bench to bring the final IPTG concentration to 0.5 mM. The shaker temperature was set to 16°C and the rotation speed to 200 rpm, and the culture was continued for 24 h. Shaking culture at 16°C induced BL21 protein expression to a great extent maintained the stability of the protein structure, increased the proportion of soluble recombinant antigen, and reduced the proportion of inclusion bodies.

[0164] Protein purification: Prepare an NTA chromatography column. Resuspend the bacterial cell pellet in 5 mL of 1.5% Triton X-405 lysis buffer. Sonicate the bacterial cells to lyse, and gently mix the sample with a shaker for 60 minutes. Centrifuge at 12000 rpm for 30 minutes at 4°C, and discard the pellet. Load the supernatant onto a NiTA column at a flow rate of 15 mL / h. 2+ -NTA column, collect the eluent. Wash the column with washing buffer at a flow rate of 15 mL / h, OD 280 The eluent was collected at a concentration of 0.01 for 3 hours. The column was washed with eluent, and fractions were collected in 1 mL increments. 10 μL of each collected fraction was subjected to SDS-PAGE for verification. The purified SFTSV antigen recombinant protein (molecular weight approximately 41.34 kDa) was obtained. SDS-PAGE results are shown below. Figure 2 .

[0165] Example 2: Application of SFTSV recombinant antigen detection

[0166] The SFTSV antibody detection kit (chemiluminescence method) has the following specific steps:

[0167] 1. Prepare the magnetic bead diluent with the following formula: 50 mM Tris, 0.9% NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.5;

[0168] 2. Prepare acridine dilution solution with the following formula: 50 mM PB, 0.9% NaCl, 1% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.5;

[0169] 3. Prepare a biotin dilution solution with the following formula: 50 mM PB, 0.9% NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.0;

[0170] 4. Acridinium ester was labeled onto mouse anti-human IgG antibody to prepare antibody-labeled conjugates;

[0171] 5. Preparation of streptavidin-coupled magnetic beads (SA beads);

[0172] 6. Prepare acridine working solution by diluting the antibody-labeled conjugate with acridine diluent; prepare magnetic bead working solution by diluting SA magnetic beads with magnetic bead diluent; prepare biotin working solution by diluting SFTSV recombinant antigen with biotin diluent, and assemble the SFTSV antibody detection kit.

[0173] The kit uses a two-step indirect immunoassay with direct chemiluminescence technology for detection. The specific steps are as follows:

[0174] Step 1 Incubation: Add sample, superparamagnetic microparticles, sample diluent, and test diluent. The SFTSV IgG / IgM in the sample reacts with the SFTSV NP antigen coated on the superparamagnetic microparticles to form an antigen-antibody complex.

[0175] Cleaning: Under the influence of a magnetic field, superparamagnetic particles are adsorbed onto the reaction tube wall, and unbound substances are washed away by the cleaning solution.

[0176] The second step is incubation: Acridine-labeled mouse anti-human IgG antibody (secondary antibody) is added to react and form an antigen-antibody-secondary antibody complex.

[0177] Wash again.

[0178] Excitation and reading: The chemiluminescent reaction was measured by relative luminescence intensity (RLU) after adding pre-excitation and excitation solutions to the reaction complex.

[0179] The amount of SFTSV IgG / IgM antibody in the sample was positively correlated with the relative luminescence intensity (RLU) measured by the instrument.

[0180] The instrument automatically compares the relative luminescence intensity (RLU) generated by the sample with the cutoff value calculated by the SFTSV IgG / IgM antibody calibrator.

[0181] Comparison of detection performance of different SFTSV recombinant antigens:

[0182] 1. Construct pET28a-NP-Avi-Tag and pET28a-P1-NP-P2-P3-Avi-Tag plasmid vectors, transform and induce host cells to express proteins, and purify the corresponding biotinylated modified proteins.

[0183] 2. Assemble the SFTSV recombinant antigen into an SFTSV-IgG antibody detection kit (chemiluminescence method) according to the method in Example 1.

[0184] 3. Thirty clinically positive samples and 100 clinically negative samples were tested. The test results of the three SFTSV recombinant antigens in clinically positive samples are shown in Table 1, and the test results of the three SFTSV recombinant antigens in clinically negative samples are shown in Table 2.

[0185] Among them, the clinically positive samples came from SFTS-positive patients, whose infection was confirmed by SFTSV nucleic acid testing and whose clinical symptoms were consistent.

[0186] Experimental results show that:

[0187] (1) Compared with pET28a-NP-Avi-Tag, pET28a-P1-NP-P2-P3-Avi-Tag has higher detection sensitivity and is more sensitive to the detection of serum IgG antibodies in SFTSV nucleic acid positive samples. The addition of three antigenic epitopes increases the sensitivity from 72% to 90%.

[0188] (2) Neither pET28a-NP-Avi-Tag nor pET28a-P1-NP-P2-P3-Avi-Tag showed false positives, and their specificity reached 100%.

[0189] Table 1. Detection results of clinically positive samples of two types of SFTSV recombinant antigens

[0190]

[0191]

[0192] Note: The unit of concentration is AU / mL; a measured value ≥20AU / mL is considered positive.

[0193] Table 2. Detection results of clinically negative samples of two types of SFTSV recombinant antigens.

[0194]

[0195]

[0196] Note: The unit of concentration is AU / mL; a value <20AU / mL is considered negative.

[0197] Inter-batch variation verification of recombinant antigen rSFTSV-NP:

[0198] 1. Plasmid Transformation and Amplification: 1 μL of the constructed pET28a-His tag-P1-NP-P2-P3-AviTag plasmid vector (≥100 ng / mL) was added to 100 μL of competent DH5α cells. The cells were incubated on ice for 30 minutes, heat-shocked at 42℃ for 90 seconds, and then immediately incubated on ice for 5 minutes. 1 mL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37℃ with shaking at 200 rpm for 40 minutes. The cells were centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL of LB medium (containing 50 μg / mL kanamycin). The suspension was then plated onto LB solid medium (containing 50 μg / mL kanamycin). The cells were incubated overnight at 37℃ with the medium inverted. Single colonies were picked and added to 5 mL of LB liquid medium (containing 50 μg / mL kanamycin), and the cells were incubated at 37℃ with shaking at 200 rpm for 12 hours.

[0199] 2. Plasmid extraction: Extract plasmids using a plasmid mini-prep kit.

[0200] 3. The plasmids transformed, amplified and extracted from different batches were transformed into competent Escherichia coli BL21 containing an IPTG-inducible plasmid encoding the ligase BirA, and the expression of biotin-tagged SFTSV recombinant antigen was induced.

[0201] 4. Assemble the SFTSV antibody detection kit (chemiluminescence method) using different batches of SFTSV recombinant antigen according to the method in Example 1.

[0202] 5. Test the batch-to-batch difference of SFTSV antibody. The test methodology is as follows: Take 10 μL of the sample to be tested, add 50 μL of magnetic bead working solution and 50 μL of biotin working solution, incubate for 10 minutes, perform magnetic separation, add 100 μL of acridine working solution, incubate for 10 minutes, perform magnetic separation, add activation solution and pre-activation solution and take the reading.

[0203] 6. The deviation between different batches is within ±5%, which can effectively control the inter-batch difference. See the attached table for specific data.

[0204] Table 3. Validation of batch-to-batch differences in SFTSV recombinant antigen rSFTSV-NP

[0205]

[0206] The above are examples of the application of recombinant SFTSV antigen in SFTSV antibody detection kits (chemiluminescence method). The recombinant antigen rSFTSV-NP involved in this application has high sensitivity and excellent batch-to-batch stability. Its compatible detection method can shorten the SFTSV antibody detection time to within 40 minutes, with no false positives or missed detections. Recombinant antigen rSFTSV-NP promotes the product development, upgrading, and application of SFTS detection; it can be widely used in SFTSV patient screening and ultimately support the prevention and treatment of SFTS.

[0207] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0208] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A recombinant N protein antigen for detecting antibodies against the novel Bunyavirus, characterized in that, The N protein recombinant antigen comprises a nucleocapsid protein (N protein) and a peptide containing a linear epitope from the N protein; The linear tabletop includes one or more of the following tabletops: Epitope 1: Derived from the N-terminal domain of the N protein; Epitope 2: an intermediate domain derived from the N protein; and, Epitope 3: C-terminal domain derived from N protein; Optionally, the linear epitope satisfies one or more of the conditions shown in 1) to 3) below: 1) Epitope 1 is located at positions 1-46 of the N protein; 2) Epitope 2 is located at positions 132-142 of the N protein; and, 3) Epitope 3 is located at positions 238-245 of the N protein.

2. The N protein recombinant antigen as described in claim 1, characterized in that, The amino acid sequence of the N protein is shown in SEQ ID NO: 2; Optionally, the N protein recombinant antigen comprises one or more of the following peptides: Peptide 1: Contains an amino acid fragment with an amino acid sequence as shown in SEQ ID NO: 3; Peptide 2: Contains an amino acid fragment with the amino acid sequence shown in SEQ ID NO: 4; and, Peptide 3: Contains an amino acid fragment with an amino acid sequence as shown in SEQ ID NO: 5; Optionally, from the N-terminus to the C-terminus, the N protein recombinant antigen sequentially comprises operably linked peptide 1, N protein, peptide 2, and peptide 3; optionally, operably linked by linking peptides selected from the group consisting of (G4S)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n, and (A)n, wherein any n is an integer greater than or equal to 1 and less than or equal to 6. Further, optionally, the N protein recombinant antigen comprises an amino acid fragment with the sequence shown in SEQ ID NO:

8.

3. The N protein recombinant antigen as described in claim 2, characterized in that, The N protein recombinant antigen also includes one or more of a purification tag and a fluorescent tag; The purification label may optionally include one or more of the following: His label, bio label, Avidin label, Flag label, and GST label; Optionally, the N-terminus of the recombinant N-protein antigen is tagged with His, and / or the C-terminus is tagged with Avidin; Further, optionally, the N protein recombinant antigen comprises an amino acid fragment with an amino acid sequence as shown in SEQ ID NO:

9.

4. A kit for detecting antibodies against the novel Bunyavirus, characterized in that, It contains the N protein recombinant antigen as described in any one of claims 1 to 3; Optionally, the kit may further comprise one or more of the following: magnetic bead diluent, acridine diluent, biotin diluent, antibody acridine ester conjugate, and streptavidin-conjugated magnetic beads.

5. A method for detecting antibodies against the novel Bunyavirus using the N protein recombinant antigen as described in any one of claims 1 to 3 or the kit as described in claim 4, characterized in that, The method includes: Contact the N protein recombinant antigen with the sample; The contact between the N protein recombinant antigen and the antibody was detected.

6. A nucleic acid molecule, characterized in that, It encodes the N protein recombinant antigen as described in any one of claims 1 to 3.

7. A recombinant expression vector, characterized in that, It comprises the nucleic acid molecule as described in claim 6; Optionally, the backbone of the recombinant expression vector includes pET28a.

8. Recombinant cells, characterized in that, It expresses the N protein recombinant antigen as described in any one of claims 1 to 3; Optionally, it comprises one or more of the nucleic acid molecules as described in claim 6 and the recombinant expression vectors as described in claim 7; Optionally, the host cell of the recombinant cells is a prokaryotic cell or a eukaryotic cell; Optionally, the prokaryotic cells include Escherichia coli, such as Escherichia coli BL21; Optionally, the eukaryotic cells include yeast and non-human mammalian cells; wherein the yeast may optionally include Saccharomyces cerevisiae; and the non-human mammalian cells include Chinese hamster ovary cells (CHO), HeLa cells, and monkey kidney cells.

9. A method for preparing recombinant N protein antigen, characterized in that, It includes: Culture the recombinant cells as described in claim 8 to obtain a culture; The N protein recombinant antigen was isolated and purified from the culture.

10. A method for preparing or screening antibodies against novel Bunyaviruses, characterized in that, The method includes: Animals were immunized with the N protein recombinant antigen as described in any one of claims 1 to 3. Preparation of hybridoma cell lines; Screening for hybridoma cell lines; and, Collect and purify antibodies.

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