Recombinant antigens for american trypanosomiasis and methods of making and using the same

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a recombinant antigen of American trypanosomiasis and a preparation method and application thereof. The application provides a recombinant antigen of American trypanosomiasis, wherein a plurality of neutralizing epitopes and complete amino acid sequences of Trypanosoma cruzi antigens are connected according to a specific order to construct a recombinant antigen capable of covering all stages of the life cycle of Trypanosoma cruzi. The recombinant antigen is complete in Chagas antibody recognition site coverage, can recognize and combine all types of antibodies in the Chagas chronic and acute infection period, and is not easily interfered by other substances in serum, and is high in specificity.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to recombinant antigens of Chagas disease and their preparation methods and applications. Background Technology

[0002] Chagas disease is a zoonotic disease caused by the protozoan Trypanosoma cruzi. The pathogen was first discovered by Carlos Chagas in 1909, hence the name Chagas disease. As one of the most common "neglected tropical diseases," it affects approximately 10 million people worldwide with chronic infection, and causes 10,000 to 14,000 deaths annually.

[0003] Chagas disease can be transmitted through various routes, including via parasites (kiss bugs), blood transfusions, organ transplants, ingestion of food contaminated with the parasite, and mother-to-child transmission. Approximately 6 to 7 million people worldwide are infected with the disease, with the majority of cases concentrated in Latin America. In addition, about 70 million people (approximately 12% of the world's population) are at risk of infection.

[0004] Trypanosoma cruzie belongs to the phylum Sarcopodinae, subphylum Flagellates, class Kinostigonia, order Kinostigliophyta, and genus Trypanosoma. During its life cycle, it exhibits three morphologies depending on its parasitic environment: amastigote, epimastigote, and conoctigote. This worm is divided into two types: Trypanosoma cruzie type I and Trypanosoma cruzie type II. Both types are pathogenic, but type I primarily infects marsupials and participates in the wild transmission cycle; type II participates in the household transmission cycle. Trypanosoma cruzie type II is further divided into five subtypes: IIa, IIb, IIc, IId, and IIe. The CL Brener subgroup of type IIe has had its genome sequenced; its genome contains 60,372,297 bases, with a G+C content of 51%, and the haploid genome contains approximately 12,000 genes.

[0005] When a kissing bug ingests blood containing triatomine flagellates from a human or other mammal, the flagellates transform into amastigotes in the bug's gut and multiply rapidly by binary fission. They then transform into bulbocalli and enter the midgut, developing into epimastigotes. Epimastigotes also multiply by binary fission and develop into large epimastigotes. Around 3-4 days after feeding, epimastigotes appear in the rectum and attach to epithelial cells; after 5 days, they become rounded and develop into late-cycle triatomine flagellates. When the infected kissing bug feeds again, the triatomine flagellates are excreted with the bug's feces onto the skin surface at the bite site. Flagellates in fecal droplets can enter the human body through three routes: through bite wounds, through minor abrasions of nearby skin, or through the fingers to the eyes, mouth, or nose, invading the mucous membranes. Once inside the human body, the triatomine flagellates gradually transform into amastigotes and multiply rapidly by binary fission.

[0006] Chagas disease is typically divided into an acute phase and a chronic phase, with an asymptomatic latent period in between. The acute phase lasts from several weeks to 2-3 months and can begin immediately after infection; the pathogen is detectable in the blood. Inflammation occurs in the subcutaneous connective tissue at the site of trypanosome invasion, forming nodules at the bite site, known as "Chagas swelling." If the site of invasion is the conjunctiva, conjunctivitis, unilateral eyelid edema, and preauricular lymphadenitis (also known as Romania's sign) appear, which are typical characteristics of the acute phase. The chronic phase can last from several years to decades, or even a lifetime, and is typically characterized by organ failure, usually affecting the heart or digestive system. Most acute phase symptoms and signs resolve spontaneously within weeks to months. During the latent phase, there are no clinical symptoms or signs; Trypanosomes are difficult to detect in peripheral blood smears under a microscope, but specific antibodies are present.

[0007] Currently, serological diagnostic methods for detecting Chagas antibodies include immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and chemiluminescence immunoassay. Molecular biological diagnostic methods such as PCR have also been applied. The generally accepted gold standard method is blood inoculation of animals or trial inoculation diagnostic methods. This method has high accuracy and specificity, but it is difficult to operate and time-consuming. Each diagnostic method has its own advantages and disadvantages in terms of ease of operation, sensitivity, and specificity.

[0008] Traditional techniques for detecting Chagas antibodies lack specificity because the antigens used do not cover all stages of the Chagas life cycle. Therefore, it is necessary to develop various recombinant chimeric antigen compositions to achieve more accurate and efficient Chagas antibody detection, thereby aiding in the diagnosis and treatment of the disease, controlling the prevalence and spread of Chagas disease, and reducing infection and mortality rates. Summary of the Invention

[0009] Based on this, one embodiment of this application provides a recombinant antigen of Chagas disease, its preparation method, and its application.

[0010] This application provides a recombinant antigen for Chagas disease, wherein the following elements are sequentially connected from the N-terminus to the C-terminus:

[0011] (a) Neutralizing epitopes of Trypanosoma cruzi B13 antigen;

[0012] (b) Neutralizing epitopes of Trypanosoma cruzi FcaBP antigen;

[0013] (c) Neutralizing epitopes of SAPA antigens in Trypanosoma cruzi;

[0014] (d) Complete amino acid fragment of Trypanosoma cruzi FRA antigen;

[0015] (e) Neutralizing epitopes of Trypanosoma cruzi FRA antigens;

[0016] (f) Complete amino acid fragments of Trypanosoma cruzi CRA antigen;

[0017] (g) Neutralizing epitopes of Trypanosoma cruzi CRA antigens;

[0018] (h) Complete amino acid fragments of Trypanosoma cruzi TcD antigen repeated 8-10 times;

[0019] (i) Complete amino acid fragments of Trypanosoma cruzi TcE antigen repeated 4-8 times.

[0020] In some embodiments, it further includes the following elements: (j) Avi tag; (k) His tag;

[0021] The elements (a) to (k) are arranged in the order (a)-(b)-(c)-(d)-(e)-(f)-(g)-(h)-(i)-(j)-(k), and a linking peptide is included between the following adjacent elements:

[0022] Between components (a) and (b), between components (b) and (c), between (c) and (d), between components (e) and (f), between components (g) and (h), between components (h) and (i), and between (i) and (j);

[0023] The remaining adjacent elements are directly connected by peptide bonds.

[0024] In some embodiments, the linker peptide is (GGGGS)n, where n is an integer from 1 to 5.

[0025] In some embodiments, the complete amino acid fragment of the Trypanosoma cruzi TcD antigen is repeated 9 times; and / or the complete amino acid fragment of the Trypanosoma cruzi TcE antigen is repeated 6 times.

[0026] In some embodiments, the lengths of the B13 neutralizing epitope, FcaBP neutralizing epitope, SAPA neutralizing epitope, and FRA neutralizing epitope are each independently 8 to 30 amino acids.

[0027] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi B13 antigen is shown in SEQ ID NO. 1.

[0028] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi FcaBP antigen is shown in SEQ ID NO.2.

[0029] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi SAPA antigen is shown in SEQ ID NO. 3.

[0030] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi FRA antigen is shown in SEQ ID NO.4.

[0031] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi FRA antigen is shown in SEQ ID NO. 5.

[0032] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi CRA antigen is shown in SEQ ID NO. 6.

[0033] In some embodiments, the neutralizing epitopes of the Trypanosoma cruzi CRA antigen are shown in SEQ ID NO.7.

[0034] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi TcD antigen is shown in SEQ ID NO.8.

[0035] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi TcE antigen is shown in SEQ ID NO.9.

[0036] In some embodiments, the amino acid sequence of the Avi tag is shown in SEQ ID NO.10.

[0037] In some of these embodiments, the amino acid sequence of the His tag is shown in SEQ ID NO.11.

[0038] In some embodiments, the amino acid sequence of the linker peptide is shown in SEQ ID NO.12.

[0039] In some of these embodiments, the amino acid sequence is shown in SEQ ID NO.14.

[0040] This application also provides a nucleic acid molecule that encodes the recombinant antigen of Chagas disease.

[0041] This application also provides a carrier comprising the aforementioned nucleic acid molecule.

[0042] Another aspect of this application provides a cell that expresses the recombinant antigen of Chagas disease; or, the cell comprises the nucleic acid molecule or the vector.

[0043] In some of these embodiments, the cells include mammalian cells.

[0044] This application also provides a method for constructing the aforementioned cells, the method comprising the step of introducing the aforementioned nucleic acid molecules or the aforementioned vector into a host cell.

[0045] This application also provides a method for preparing a recombinant antigen for Chagas disease, the method comprising: culturing the cells and isolating the recombinant antigen of Chagas disease from the resulting cells.

[0046] Another aspect of this application provides a Chagas disease antibody detection kit, which includes the Chagas disease recombinant antigen.

[0047] In some embodiments, the Chagas disease antibody detection kit is based on chemiluminescence immunoassay.

[0048] In some embodiments, the Chagas disease antibody detection kit further includes one or more of magnetic beads, luminescent markers, luminescent substrates, and detection antibodies.

[0049] Another aspect of this application provides a method for detecting Chagas disease antibodies in a sample, the method comprising detecting Chagas disease antibodies in the sample to be tested using the Chagas disease recombinant antigen or the Chagas disease antibody detection kit described above.

[0050] This application provides a recombinant antigen for Chagas disease. By linking neutralizing epitopes and complete amino acid fragments of multiple Chagas antigens in a specific sequence, a recombinant antigen capable of covering all stages of the Chagas life cycle is constructed. This recombinant antigen has full coverage of Chagas antibody recognition sites, can recognize and bind to all types of antibodies during the chronic and acute infection phases of Chagas, and is not easily interfered with by other substances in the serum, exhibiting high specificity. Attached Figure Description

[0051] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0052] Figure 1 A map of the pPICZA expression cloning vector provided in an embodiment of this application;

[0053] Figure 2 An SDS-PAGE electrophoresis image of a recombinant protein provided in an embodiment of this application. Detailed Implementation

[0054] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0055] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0056] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0057] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0058] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0059] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0060] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0061] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0062] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0063] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0064] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0065] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0066] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0067] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0068] The term "neutralizing epitope" refers to a specific region on an antigen molecule that can be recognized and bound by specific antibodies, which neutralize the biological activity of pathogens. In this field, a neutralizing epitope is an immunogenic short peptide sequence on an antigen molecule, typically 8-30 amino acids in length. Neutralizing epitopes can induce the body to produce neutralizing antibodies, which can block the binding of pathogens to host cells or inhibit pathogen replication.

[0069] The term "linker peptide" refers to a short peptide composed of a specific amino acid sequence used to link two or more protein domains, allowing them to remain spatially independent while existing as a single molecule. In this field, linker peptides are typically composed of flexible amino acids such as glycine and serine. The small side chains of these amino acids provide the flexibility for linking, allowing the linked protein domains to have a degree of mobility. The main function of linker peptides is to prevent steric hindrance between domains, ensuring that each domain can fold correctly and perform its biological function.

[0070] Terminology: "Avi tag": A short peptide tag containing 15 amino acids. In this field, the Avi tag is the specific recognition site of the biotinylate ligase BirA, and can be biotinylated in vivo or in vitro. The biotinylated Avi tag can bind to streptavidin or streptavidin with high affinity, and this binding exhibits extremely high specificity and stability. The main function of the Avi tag is for the targeted immobilization, purification, and detection of proteins, playing an important role in the development of diagnostic reagents.

[0071] The term "His tag" is a short peptide tag consisting of 6-10 histidine residues, most commonly 6 histidine residues (HHHHHH). In this field, the His tag is one of the most commonly used tags in metal ion affinity chromatography, capable of binding with nickel ions (Ni... 2+ ), cobalt ions (Co) 2+His tags specifically bind to transition metal ions such as metal ions (MIGs). The main function of His tags is for the rapid purification of recombinant proteins. Recombinant proteins with His tags can be efficiently separated and purified using immobilized metal ion affinity chromatography (IMAC).

[0072] The term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be expressed in the host cells.

[0073] The term "protein tag" refers to a polypeptide or protein that is fused with a target protein using in vitro DNA recombination technology, in order to facilitate the expression, detection, tracing, and purification of the target protein.

[0074] The term "chemiluminescence detection" refers to the detection of target molecules based on chemiluminescence reactions, which involve the reaction of specific chemical substrates (such as luminol) with enzymes (such as horseradish peroxidase, HRP) to generate light signals.

[0075] This application provides a recombinant antigen for Chagas disease, which is sequentially linked from the N-terminus to the C-terminus as follows:

[0076] (a) Neutralizing epitope of Trypanosoma cruzi B13 antigen; (b) Neutralizing epitope of Trypanosoma cruzi FcaBP antigen; (c) Neutralizing epitope of Trypanosoma cruzi SAPA antigen; (d) Complete amino acid fragment of Trypanosoma cruzi FRA antigen; (e) Neutralizing epitope of Trypanosoma cruzi FRA antigen; (f) Complete amino acid fragment of Trypanosoma cruzi CRA antigen; (g) Neutralizing epitope of Trypanosoma cruzi CRA antigen; (h) Complete amino acid fragment of Trypanosoma cruzi TcD antigen (8-10 repetitions); (i) Complete amino acid fragment of Trypanosoma cruzi TcE antigen (4-8 repetitions).

[0077] In some embodiments, it further includes: (j) Avi tags; (k) His tags;

[0078] The elements (a) to (k) are arranged in the order (a)-(b)-(c)-(d)-(e)-(f)-(g)-(h)-(i)-(j)-(k), and there are connecting peptides between the following adjacent elements: between elements (a) and (b), between elements (b) and (c), between (c) and (d), between elements (e) and (f), between elements (g) and (h), and between elements (h) and (i), and between (i) and (j); the remaining adjacent elements are directly connected by peptide bonds.

[0079] In some embodiments, the linker peptide is (GGGGS)n, where n is an integer from 1 to 5; Linker linking is a technique that connects two proteins or protein domains by using specific amino acid sequences as linkers. These linkers are typically composed of small nonpolar amino acids (such as glycine) or polar amino acids (such as serine or threonine). The small size of these amino acids provides flexibility in the linker, allowing the linked protein domains to have a degree of mobility.

[0080] In some embodiments, the Trypanosoma cruzi TcD antigen is repeated 9 times; and / or the Trypanosoma cruzi TcE antigen is repeated 6 times;

[0081] In some embodiments, the B13 neutralizing epitope, FcaBP neutralizing epitope, SAPA neutralizing epitope, and FRA neutralizing epitope are antigenic epitopes of the corresponding antigens, with a length of 8-30 amino acids.

[0082] The recombinant antigens provided in this application for detecting human Chagas antibodies in isolated biological samples include: B13 (neutralizing epitope) - linker - FcaBP (neutralizing epitope) - linker - SAPA (neutralizing epitope) - FRA (complete amino acid sequence) - FRA (neutralizing epitope) - linker - CRA (extracellular segment of complete amino acid sequence) - CRA (complete amino acid sequence of neutralizing epitope) - linker - TcD (9 repeats of complete amino acid sequence) - linker - TcE (6 repeats of complete amino acid sequence) - AviTag - His tag.

[0083] Expression vectors B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His tag-pPICZA and birA-pPICZA were constructed, with the amino-terminal sequence shown below. B13, FcaBP, CRA, FRA, TcD, and TcE can detect antibodies in the chronic phase of Chagas disease, while SAPA can detect antibodies in the acute phase. CRA and FRA antigens are cytoplasmic and cytoskeletal proteins of *Trypanosoma cruzi*, respectively.

[0084] This application uses both the full-length protein sequence of the antigen and its antigen-neutralizing epitopes, ensuring reagent detection while improving reagent specificity.

[0085] Among them, B13, FcaBP, CRA, FRA, TcD, and TcE can detect antibodies in the chronic phase of Chagas disease, while SAPA can detect antibodies in the acute phase, achieving comprehensive coverage of both the acute and chronic phases of Chagas disease. CRA and FRA antigens are cytoplasmic and skeletal proteins of Trypanosoma cruzi, respectively. This application uses both the full-length protein sequence and the antigen-neutralizing epitopes of these antigens, ensuring both reagent detection and improved reagent specificity. FcaBP is a flagellated calcium-binding protein of Trypanosoma cruzi, exhibiting strong reactivity with Trypanosoma cruzi antibodies, making it suitable for use in combination with other recombinant proteins. B13 antigen is a dominant immunogenic antigen of Trypanosoma cruzi, but B13 has some cross-reactivity with human cardiac myosin; therefore, this application only selects the core conserved epitope of B13, which can improve the specificity of recombinant proteins and reduce false positives. TcD and TcE are identified dominant Chagas antigenic epitopes, which can improve the detection rate of recombinant proteins.

[0086] This application constructs a recombinant antigen covering all stages of the Trypanosoma cruzi life cycle by linking neutralizing epitopes and complete amino acid sequences of multiple Trypanosoma cruzi antigens in a specific order, enabling comprehensive detection of both the acute and chronic phases of Chagas disease. Secondly, this application utilizes both the full-length protein sequences of CRA and FRA antigens and their antigen-neutralizing epitopes, ensuring reagent detection while improving reagent specificity. Furthermore, this protocol selects only the core conserved epitope of B13, avoiding cross-reactivity with human cardiac myosin, thus improving the specificity of the recombinant protein and reducing false positives. This application also significantly improves the detection rate of the recombinant protein by repeatedly repeating the dominant TcD and TcE antigen epitopes, achieving 100% detection rate and specificity.

[0087] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi B13 antigen is shown in SEQ ID NO. 1.

[0088] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi FcaBP antigen is shown in SEQ ID NO.2.

[0089] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi SAPA antigen is shown in SEQ ID NO. 3.

[0090] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi FRA antigen is shown in SEQ ID NO.4.

[0091] In some embodiments, the amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi FRA antigen is shown in SEQ ID NO. 5.

[0092] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi CRA antigen is shown in SEQ ID NO. 6.

[0093] In some embodiments, the neutralizing epitopes of the Trypanosoma cruzi CRA antigen are shown in SEQ ID NO.7.

[0094] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi TcD antigen is shown in SEQ ID NO.8.

[0095] In some embodiments, the complete amino acid sequence of the Trypanosoma cruzi TcE antigen is shown in SEQ ID NO.9.

[0096] In some embodiments, the amino acid sequence of the Avi tag is shown in SEQ ID NO.10.

[0097] In some of these embodiments, the amino acid sequence of the His tag is shown in SEQ ID NO.11.

[0098] In some embodiments, the amino acid sequence of the Linker is shown in SEQ ID NO.12.

[0099] In some embodiments, the amino acid sequence of the recombinant antigen of Chagas disease is shown in SEQ ID NO. 14.

[0100] B13 antigenic epitope: KPPPFGQAAAGDKPA; (SEQ ID NO.1)

[0101] FcaBP antigenic epitope: ITKRAFDKARALGSKLENKGSEDFV; (SEQ ID NO.2)

[0102] SAPA epitope: LCPSEPAHALAPGSSRVELFKRQNSTVPFEENGEVRQRVVHSFRLPA; (SEQ IDNO.3)

[0103] FRA antigen full amino acid sequence: MEQERRQLLEKDPRRNAKEIAALEESMNARAQELAREKKLADRAFLDQKPERVPLAD VPLDDDSDFVA (SEQ ID NO.4);

[0104] FRA antigenic epitope: FLDQKPERVPLADVPLDDDSDFVA; (SEQ ID NO.5)

[0105] Full amino acid sequence of CRA antigen: RVAEEKQRAAEATKVAEAEKQKAAEATKVAEAEKQRAAEATKVAEAEKQKAAEATKV AEAEKQKAAE; (SEQ ID NO.6)

[0106] CRA antigenic epitope: KQRAAEATKVAEAEKQKAAEATK; (SEQ ID NO.7)

[0107] TcD amino acid sequence: AEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKS; (SEQ IDNO.8)

[0108] TcE amino acid sequence: KAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPA; (SEQ IDNO.9)

[0109] Avi-tag amino acid sequence: GLNDIFEAQKIEWHE; (SEQ ID NO.10)

[0110] His-tag amino acid sequence: HHHHHH (SEQ ID NO.11)

[0111] Flexible linker amino acid sequence: GGGGSGGGGS; (SEQ ID NO.12)

[0112] birA: The sequence is: MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMA EVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK; (SEQ ID NO.13)

[0113] B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His-tag amino acid sequence:

[0114] KPPPFGQAAAGDKPAGGGGSGGGGSITKRAFDKARALGSKLENKGSEDFVGGGGSGGGGSLCPSEPAHALAPGSSRVELFKRQNSTVPFEENGEVRQRVVHSFRLPAGGGGSG GGGSMEQERRQLLEKDPRRNAKEIAALEESMNARAQELAREKKLADRAFLDQKPERVPLADVPLDDDSDFVAFLDQKPERVPLADVPLDDDSDFVAGGGGSGGGGSRVAEAEK QRAAEATKVAEAEKQKAAEATKVAEAEKQRAAEATKVAEAEKQKAAEATKVAEAEKQKAAEKQRAAEATKVAEAEKQKAAEATKGGGGSGGGGSAEPKSAEPKSAEPKSAEPK SAEPKSAEPKSAEPKSAEPKSAEPKSGGGGSGGGGSKAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPAGGGGSGGGGSGLNDIFEAQKIEWHEHHHHHH (SEQ ID NO.14)

[0115] This application, in another aspect, provides a nucleic acid molecule encoding the recombinant antigen of Chagas disease. The nucleic acid is typically RNA or DNA, and the nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked." For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence. DNA nucleic acid is preferred when it is ligated into a vector. Furthermore, the nucleic acid molecule can be codon-optimized for different host cells.

[0116] This application also provides a vector comprising the aforementioned nucleic acid molecule. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; bacteriophages; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0117] Another aspect of this application provides a cell that expresses the recombinant antigen of Chagas disease; or, the cell comprises the nucleic acid molecule or the vector.

[0118] In some embodiments, the cells comprise mammalian cells. Mammalian expression systems possess unique advantages in protein initiation signaling, processing, secretion, and glycosylation, making them suitable for expressing complete macromolecular proteins. They also significantly outperform prokaryotic expression systems and eukaryotic expression systems such as yeast and insect cells in terms of activity. The recombinant antigen of this application is expressed using a mammalian cell expression system, which possesses advanced post-translational modifications, improving the solubility of the recombinant protein and avoiding the limitations of simple modifications in E. coli expression systems, further enhancing the stability of the antigen.

[0119] This application also provides a method for constructing cells, the method comprising the step of introducing the nucleic acid molecule or the vector into a host cell.

[0120] This application also provides a method for preparing a recombinant antigen for Chagas disease, the method comprising:

[0121] The cells were cultured and the recombinant antigen of Chagas disease was isolated from the resulting cells.

[0122] Another aspect of this application provides a Chagas disease antibody detection kit, which includes the Chagas disease recombinant antigen.

[0123] In some embodiments, the Chagas disease antibody detection kit is based on chemiluminescence immunoassay.

[0124] In some embodiments, the Chagas disease antibody detection kit further includes one or more of magnetic beads, luminescent markers, luminescent substrates, and detection antibodies.

[0125] This application also provides a method for detecting Chagas disease antibodies in a sample. The method includes detecting Chagas disease antibodies in the sample using the described Chagas disease recombinant antigen or the described Chagas disease antibody detection kit. It is understood that this detection can be for disease diagnosis and treatment purposes or for non-disease diagnosis and treatment purposes, including laboratory research or ecological surveys.

[0126] 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 specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0127] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0128] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0129] Example 1

[0130] This embodiment provides a recombinant antigen for Chagas disease and its preparation method.

[0131] 1. Construction of the expression carrier

[0132] Construct B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His tag-pPICZA and birA-pPICZA expression vectors. The plasmid map of the pPICZA expression cloning vector is shown below. Figure 1 As shown.

[0133] The synthesized sequences of each antigen's complete amino acid fragment and antigenic epitope fragment are shown below:

[0134] B13 antigenic epitope: KPPPFGQAAAGDKPA; (SEQ ID NO.1).

[0135] FcaBP antigenic epitope: ITKRAFDKARALGSKLENKGSEDFV; (SEQ ID NO.2).

[0136] SAPA epitope: LCPSEPAHALAPGSSRVELFKRQNSTVPFEENGEVRQRVVHSFRLPA; (SEQ IDNO.3).

[0137] FRA antigen full amino acid sequence: MEQERRQLLEKDPRRNAKEIAALEESMNARAQELAREKKLADRAFLDQKPERVPLAD VPLDDDSDFVA (SEQ ID NO.4).

[0138] FRA antigenic epitope: FLDQKPERVPLADVPLDDDSDFVA; (SEQ ID NO.5).

[0139] The full amino acid sequence of CRA antigen: RVAEEKQRAAEATKVAEAEKQKAAEATKVAEAEKQRAAEATKVAEAEKQKAAEATKV AEAEKQKAAE; (SEQ ID NO. 6).

[0140] CRA antigenic epitope: KQRAAEATKVAEAEKQKAAEATK; (SEQ ID NO.7).

[0141] TcD amino acid sequence: AEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKS; (SEQ ID NO. 8).

[0142] TcE amino acid sequence: KAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPA; (SEQ ID NO. 9).

[0143] Avi-tag amino acid sequence: GLNDIFEAQKIEWHE; (SEQ ID NO.10).

[0144] His-tag amino acid sequence: HHHHHH (SEQ ID NO.11).

[0145] Flexible Linker amino acid sequence: GGGGSGGGGS; (SEQ ID NO.12).

[0146] BirA: The sequence is: MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK; (SEQ ID NO.13).

[0147] The amino acid sequence of B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His-tag:

[0148] KPPPFGQAAAGDKPAGGGGSGGGGSITKRAFDKARALGSKLENKGSEDFVGGGGSGGGGSLCPSEPAHALAPGSSRVELFKRQNSTVPFEENGEVRQRVVHSFRLPAGGGGSGGGGSMEQERRQLLEKDPRRNAKEIAALEESMNARAQELAREKKLADRAFLDQKPERVPLADVPLDDDSDFVAFLDQKPERVPLADVPLDDDSDFVAGGGGSGGGGSRVAEAEKQRAAEATKVAEAEKQKAAEATKVAEAEKQRAAEATKVAEAEKQKAAEATKVAEAEKQKAAEKQRAAEATKVAEAEKQKAAEATKGGGGSGGGGSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSAEPKSGGGGSGGGGSKAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPAKAAAAPAGGGGSGGGGSGLNDIFEAQKIEWHEHHHHHH (SEQ ID NO.14).

[0149] 2. Conversion

[0150] The vectors B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His-tag-pPICZA and birA-pPICZA were mixed in a 1:1 ratio, linearized with SalI, and transformed into competent cells of GS115 strain by electroporation to co-express the recombinant protein B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His-tag. Screening was performed using YPD solid plates containing bleomycin, and after single colonies grew, they were inoculated into YPD liquid medium containing bleomycin.

[0151] 3. Expression purification

[0152] After culturing the GS115 strain containing the target gene for 5 days, the fermentation broth in the shake flask was centrifuged. The first centrifugation was at 4000-5000 rpm for 10 minutes to remove cells and obtain the supernatant. The second centrifugation was at 12000 rpm for 30 minutes to remove large aggregates or broken cells to prevent clogging of the filter column. The supernatant was concentrated and passed through an equilibrated Ni affinity chromatography column, washing away impurities before elution. The SDS-PAGE electrophoresis image of the B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His-tag recombinant protein is shown below. Figure 2 As shown.

[0153] Example 2

[0154] Using the Chagas recombinant antigen prepared in this application, a Chagas disease antibody detection kit (chemiluminescence method) was prepared, as follows:

[0155] 1. Preparation of diluents: The magnetic bead diluent formulation is 50 mM Tris buffer pair, 0.9 w / v% NaCl, 0.5 w / v% BSA, 0.05 w / v% Triton-X405, 0.1 w / v% ProClin300, pH 7.5; the acridine diluent formulation is 50 mM PB buffer pair, 0.9 w / v% NaCl, 0.5 w / v% BSA, 0.5 w / v% Tween-20, 0.1 w / v% ProClin300, pH 7.5;

[0156] The biotin diluent was formulated with 50 mM PB buffer, 0.9 w / v % NaCl, 0.5 w / v % BSA, 0.5 w / v % Tween-20, and 0.1 w / v % ProClin300, pH 7.0.

[0157] 2. The B13-FcaBP-SAPA-FRA-CRA-TcD-TcE-Avi-tag-His-tag antigen does not require labeling and is used directly as a biotin component. Mouse anti-human IgG is labeled with acridine ester as an acridine component. Commercially available streptavidin-conjugated magnetic beads (SA beads) purchased from Thermo Fisher Scientific Inc. are used as magnetic bead components.

[0158] 3. 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 Chagas recombinant antigen with biotin diluent, and assemble the Chagas antibody detection kit.

[0159] 4. Using the principle of the indirect method, after adding the sample, magnetic bead component and biotin component and incubating for a period of time, magnetic separation and washing are performed. Then, acridine component is added and incubated for a period of time. After magnetic separation and washing, excitation solution and pre-excitation solution are added to measure the luminescence value.

[0160] Result verification:

[0161] I. Verification of batch-to-batch differences in biotin components

[0162] 1. A kit for detecting Chagas antibodies was prepared by simultaneously assembling recombinant antigens from batches 1, 2, and 3 of Pichia pastoris to express three different batches of antigen compositions. Samples with different COI values ​​were prepared to verify the batch-to-batch differences.

[0163] 2. Add the sample, along with R1 working solution containing magnetic beads and R3 working solution containing biotin, to the reaction vessel. After incubation for a certain period, wash with washing buffer (PBS buffer) and perform magnetic separation. Add R2 working solution containing acridinium ester and incubate for a certain period, then wash again with washing buffer (PBS buffer) and perform magnetic separation. Add activation and pre-activation solutions to detect the signal. A COI value ≥ 1 is considered positive, and a COI value < 1 is considered negative. The results are shown in Table 1.

[0164] Table 1

[0165]

[0166] II. Verification reagent detection rate

[0167] Fifteen acute-phase samples, 20 chronic-phase samples (abbot valued samples from Latin America, clinically diagnosed as acute or chronic Chagas infection), and a transformation disc (10 points) were tested. The Chagas test kit had a detection rate of 100%. Specific data are shown in Tables 2-4 below.

[0168] Table 2

[0169]

[0170] Table 3

[0171]

[0172] Table 4

[0173]

[0174] III. Verification of reagent specificity

[0175] One hundred samples from different populations (random physical examination blood, pregnant women's blood, elderly blood and children's blood) (physical examination samples collected from hospitals, with clinical gender, age and other information) were tested, and no cases were detected. The Chagas test kit has a specificity of nearly 100%. Specific data are shown in Table 5.

[0176] Table 5

[0177]

[0178] The above are examples of the application of Chagas recombinant antigen in Chagas antibody detection kits (chemiluminescence method). The Chagas recombinant antigen and its adapted detection method involved in this patent can shorten the Chagas antibody detection time to within 25 minutes, with a detection rate and specificity of 100%, and can be widely used for Chagas patient screening.

[0179] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. 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 scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A recombinant antigen of Chagas disease, characterized in that, The components are connected sequentially from the N terminal to the C terminal as follows: (a) Neutralizing epitopes of Trypanosoma cruzi B13 antigen; (b) Neutralizing epitopes of Trypanosoma cruzi FcaBP antigen; (c) Neutralizing epitopes of SAPA antigens in Trypanosoma cruzi; (d) Complete amino acid fragment of Trypanosoma cruzi FRA antigen; (e) Neutralizing epitopes of Trypanosoma cruzi FRA antigens; (f) Complete amino acid fragments of Trypanosoma cruzi CRA antigen; (g) Neutralizing epitopes of Trypanosoma cruzi CRA antigens; (h) Complete amino acid fragments of Trypanosoma cruzi TcD antigen repeated 8-10 times; (i) Complete amino acid fragments of Trypanosoma cruzi TcE antigen repeated 4-8 times.

2. The recombinant antigen for Chagas disease according to claim 1, characterized in that, It also includes the following elements: (j) Avi tag; (k) His tag; The elements (a) to (k) are arranged in the order (a)-(b)-(c)-(d)-(e)-(f)-(g)-(h)-(i)-(j)-(k), and a linking peptide is included between the following adjacent elements: Between components (a) and (b), between components (b) and (c), between (c) and (d), between components (e) and (f), between components (g) and (h), between components (h) and (i), and between (i) and (j); The remaining adjacent elements are directly connected by peptide bonds.

3. The recombinant antigen for Chagas disease according to claim 1, characterized in that, The linker peptide is (GGGGS)n, where n is an integer from 1 to 5; Optionally, the complete amino acid fragment of the Trypanosoma cruzi TcD antigen is repeated 9 times; and / or the complete amino acid fragment of the Trypanosoma cruzi TcE antigen is repeated 6 times; Optionally, the lengths of the B13 neutralizing epitope, FcaBP neutralizing epitope, SAPA neutralizing epitope, and FRA neutralizing epitope are each independently 8-30 amino acids.

4. The recombinant antigen for Chagas disease according to any one of claims 2 to 3, characterized in that, The amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi B13 antigen is shown in SEQ ID NO.1; and / or The amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi FcaBP antigen is shown in SEQ ID NO.2; and / or The amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi SAPA antigen is shown in SEQ ID NO.3; and / or The complete amino acid sequence of the Trypanosoma cruzi FRA antigen is shown in SEQ ID NO.4; and / or The amino acid sequence of the neutralizing epitope of the Trypanosoma cruzi FRA antigen is shown in SEQ ID NO. 5; and / or The complete amino acid sequence of the Trypanosoma cruzi CRA antigen is shown in SEQ ID NO. 6; and / or The neutralizing epitopes of the Trypanosoma cruzi CRA antigen are shown in SEQ ID NO. 7; and / or The complete amino acid sequence of the Trypanosoma cruzi TcD antigen is shown in SEQ ID NO. 8; and / or The complete amino acid sequence of the Trypanosoma cruzi TcE antigen is shown in SEQ ID NO. 9; and / or Optionally, the amino acid sequence of the Avi tag is as shown in SEQ ID NO.10; and / or Optionally, the amino acid sequence of the His tag is as shown in SEQ ID NO.11; and / or Optionally, the amino acid sequence of the linker peptide is shown in SEQ ID NO.

12.

5. The recombinant antigen for Chagas disease according to claim 4, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

14.

6. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant antigen of Chagas disease as described in any one of claims 1 to 5.

7. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 6.

8. A cell, characterized in that, The cells express the recombinant antigen of Chagas disease according to any one of claims 1 to 5; or, the cells comprise the nucleic acid molecule according to claim 6 or the vector according to claim 7; The cells include mammalian cells.

9. The method for constructing cells according to claim 8, characterized in that, The construction method includes the step of introducing the nucleic acid molecule of claim 6 or the vector of claim 7 into a host cell.

10. A method for preparing recombinant antigen, used to prepare the recombinant antigen of Chagas disease according to any one of claims 1 to 5, characterized in that, The preparation method includes: Culturing the cells of claim 8 and isolating the recombinant antigen of Chagas disease from the resulting cells.

11. A test kit for Chagas disease antibody detection, characterized in that, The Chagas disease antibody detection kit includes the Chagas disease recombinant antigen as described in any one of claims 1 to 5.

12. The Chagas disease antibody detection kit according to claim 11, characterized in that, The Chagas disease antibody detection kit is based on chemiluminescence immunoassay. Optionally, the Chagas disease antibody detection kit may further include one or more of magnetic beads, luminescent markers, luminescent substrates, and detection antibodies.

13. A method for detecting Chagas disease antibodies in samples, characterized in that, The detection method includes detecting Chagas disease antibodies in the sample to be tested using the Chagas disease recombinant antigen of any one of claims 1 to 5 or the Chagas disease antibody detection kit of any one of claims 11 to 12.

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