Hepatitis D virus recombinant antigen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a hepatitis delta virus recombinant antigen and a preparation method and application thereof. According to the hepatitis D virus recombinant antigen constructed by the invention, the S-HDAg protein sequence full length and repeated three linear epitope peptide fragments are used, HDV antibody recognition sites are completely covered, the activity is relatively high, 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 belongs to the field of biotechnology, and specifically relates to a recombinant hepatitis D virus antigen and a preparation method and application thereof. Background Art

[0002] HDV is a defective virus that must rely on hepatitis B virus (HBV) or other hepatotropic DNA viruses to acquire an envelope to complete its life cycle and maintain infectivity. It is primarily transmitted through blood, mother-to-child transmission, and sexual contact, but can also be transmitted through damaged skin and mucous membranes. Globally, 15 to 20 million people are infected with HDV; however, its diagnosis rate is exceptionally low, particularly in areas with high HDV prevalence. Therefore, the global number of HDV infections may be severely underestimated. Compared with HBV infection alone, HDV / HBV co-infection accelerates liver disease progression and leads to more severe liver damage, such as chronic active hepatitis, cirrhosis, and severe hepatitis. Therefore, hepatitis D is considered the most severe viral hepatitis and has become a major public health issue.

[0003] HDV is approximately 1.7 kb in length and 35-37 nm in diameter. Its core contains a single-stranded, negative-strand covalently closed circular RNA and hepatitis delta antigen (HDAg). The outer membrane is coated with HBsAg. HDV utilizes the pre-S1 domain of the large HBsAg (HBsAg-L) to bind to the NTCP receptor on the host cell surface, thereby entering the hepatocyte and replicating its RNA genome in the nucleus. The HDV genome contains only one open reading frame (ORF), encoding two forms of hepatitis delta antigen: small HDAg (S-HDAg, (195 nucleotides)) and large HDAg (L-HDAg, (214 nucleotides)). L-HDAg is produced through host cell ADAR1-mediated RNA editing, which changes the amber / stop codon in the s-HDAg ORF to a tryptophan codon. L-HDAg is synthesized by adding 19 C-terminal amino acids. Therefore, L-HDAg contains the S-HDAg sequence and a C-terminal prenylation motif required for linking the HDV ribonucleoprotein to HBsAg during viral particle assembly. S-HDAg is essential, produced early in infection, and crucial for viral RNA replication; L-HDAg is typically synthesized later, blocking replication and promoting envelope maturation for viral assembly.

[0004] Hepatitis D testing involves both screening and confirmatory methods. First, serum anti-HDV antibodies are screened, and positive results are then verified for HDV RNA. HDV antibody levels are typically high in active HDV infection, and qualitative antibody testing yields highly consistent results. Therefore, the production and application of recombinant HDV antigens are crucial for improving the accuracy and efficiency of HDV infection detection.

[0005] Traditional techniques still lack homology between HDV recombinant proteins and HDV, and the recombinant antigen has low activity. This makes it difficult to accurately detect HDV antibodies in specific serological tests, posing a risk of missed detection. Furthermore, the use of existing HDV recombinant antigens in chemiluminescence systems requires additional magnetic particle coating or luminescent labeling, introducing complex processes and poor control of batch-to-batch variation, making it difficult to develop kits for detecting HDV antibodies. Summary of the Invention

[0006] Based on this, an embodiment of the present application provides a hepatitis D virus recombinant antigen and a preparation method and application thereof.

[0007] On the one hand, the present application provides a recombinant hepatitis D virus antigen, the amino acid sequence of which is shown in SEQ ID NO.8.

[0008] In one embodiment, one or both of the C-terminus and the N-terminus of the recombinant antigen are linked to a tag protein. In one embodiment, the tag protein is independently selected from one of a His tag, an HA tag, a c-Myc tag, an AviTag tag, a SNAP tag, and a Flag tag.

[0009] On the other hand, the present application provides a nucleic acid molecule containing the compound encoding the above-mentioned hepatitis D virus recombinant antigen.

[0010] On the other hand, the present application provides a vector comprising the above-mentioned nucleic acid molecule.

[0011] In one embodiment, the vector comprises pET28a.

[0012] On the other hand, the present application provides a cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0013] In one embodiment, the cell comprises a prokaryotic cell.

[0014] In one embodiment, the cell comprises Escherichia coli.

[0015] In one embodiment, the Escherichia coli includes Escherichia coli BL21.

[0016] On the other hand, the present application provides a method for preparing the above-mentioned cells, which comprises the step of introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into the target cell.

[0017] In one embodiment, the method for preparing the above-mentioned hepatitis D virus recombinant antigen comprises: culturing the cells as described above, and isolating the hepatitis D virus recombinant antigen from the obtained culture.

[0018] In one embodiment, the culture conditions include: a temperature of 14° C. to 18° C., an oscillation speed of 180 rpm to 220 rpm, and a culture time of 22 h to 26 h.

[0019] In one embodiment, the separation step includes: collecting cells in the culture, adding a lysis solution to disrupt the cells, and performing protein purification on the obtained cell lysis products.

[0020] In one embodiment, the lysis solution comprises Triton X-405 at a final concentration of 1 w / v% to 3 w / v%.

[0021] On the other hand, the present application provides an anti-HDV detection product, which uses the above-mentioned HDV recombinant antigen as a detection antigen.

[0022] On the other hand, the present application provides a method for detecting hepatitis D virus, wherein the method uses the hepatitis D virus recombinant antigen to prepare a detection antibody, and determines whether the sample to be tested contains hepatitis D virus through an immune binding reaction.

[0023] On the other hand, the present application provides the use of the above-mentioned hepatitis D virus recombinant antigen, the above-mentioned nucleic acid molecule, the above-mentioned vector or the above-mentioned cell in the preparation of a hepatitis D virus detection product.

[0024] The details of one or more embodiments of the present application are set forth in the description below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0026] Figure 1 This is the plasmid map of pET28a-His tag-S-HDAg-P1-P2-P3-Avi Tag in one embodiment of the present application;

[0027] Figure 2 This is an SDS-PAGE image of the recombinant protein HDV-SE in one embodiment of the present application. DETAILED DESCRIPTION

[0028] Below in conjunction with embodiment and example, the application is described in further detail.Should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description hereinafter, a large amount of specific details are given in order to provide a more complete understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0029] 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.

[0030] the term

[0031] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0032] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or 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 of 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, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0033] In this application, "plurality", "multiple", "multiple times", "multiples", 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.

[0034] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0035] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0036] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0037] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0038] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, 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 included therein.

[0040] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0041] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0042] All documents mentioned in this application are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited in their entirety and for all purposes. When cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be adaptively amended according to the description in this application.

[0043] The term "linker" refers to fusion protein linkers. As an indispensable component of fusion protein recombinant engineering, linkers play a crucial role in constructing stable, biologically active fusion proteins. Linkers are amino acid chains that connect two fusion proteins, possessing a degree of flexibility to allow the proteins on either side to perform their independent functions. Protein linkers are generally categorized into three types: flexible linkers, rigid linkers, and cleavable linkers.

[0044] The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can be introduced into a host cell through transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell.

[0045] The term "tag protein" refers to a polypeptide or protein that is fused with a target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and purification of the target protein.

[0046] On the one hand, the present application provides a recombinant hepatitis D virus antigen, the amino acid sequence of which is shown in SEQ ID NO.8.

[0047] The recombinant hepatitis D virus antigen constructed in this application uses the full-length S-HDAg protein sequence and three repeated linear epitope peptides to ensure the detection of HDV antibodies in human blood. S-HDAg is necessary to initiate and maintain HDV RNA replication and plays a role in the early stages of viral infection; while L-HDAg is usually synthesized in the later stages of infection; and HDV key sequence variations are mainly concentrated at the C-terminus of L-HDAg. Therefore, the selection of the full S-HDAg sequence in this application can monitor the early stages of HDV infection and improve the sensitivity of the reagent while ensuring the exposure of the antigenic epitope.

[0048] In addition, the S-HDAg fragment selected in this application has 100% homology to HDV. On this basis, three peptide segments of HDAg linear epitopes are added (Peptide 1: aa59-84, Peptide 2: aa92-114, Peptide 3: aa149-172; hereinafter abbreviated as P1, P2, P3). The number and diversity of epitopes of the recombinant antigen are enhanced by repeating sequences, which helps to improve the detection sensitivity of the recombinant protein for HDV antibodies.

[0049] Furthermore, this recombinant antigen covers all the HDV antibody recognition sites, has high activity, and the prepared protein has high purity, stable structure, and good inter-batch stability; the HDV antibody detection kit (chemiluminescence method) prepared using the recombinant antigen HDV-SE can quickly and accurately detect HDV antibodies in serum samples with high sensitivity, reducing false positives and missed detections.

[0050] In some embodiments, the C-terminus of the recombinant antigen is linked to a tag protein. A protein tag refers to a protein or polypeptide that is fused to the target protein during recombinant protein production. The tag protein improves the solubility and stability of the target protein, facilitates its detection, and facilitates its purification.

[0051] In some embodiments, the tag protein is a His tag, an HA tag, a c-Myc tag, an AviTag tag, a SNAP tag, or a Flag tag.

[0052] Another aspect of the present application provides a nucleic acid molecule comprising a nucleic acid encoding the above-mentioned hepatitis D virus recombinant antigen. 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. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is operably linked to the coding sequence. DNA nucleic acids are preferably used when incorporated into a vector. In addition, the nucleic acid molecule can be codon-optimized for different host cells.

[0053] On the other hand, the present application provides a vector comprising the above-mentioned nucleic acid molecule. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phages or M13 phages, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0054] In some embodiments, the vector described in the present application contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or polyadenylation signals and poly-U sequences, etc.).

[0055] In some embodiments, the vector includes pET28a. pET28a is one of the pET series expression vectors, widely used for recombinant protein expression in Escherichia coli. It is based on the T7 promoter system and can efficiently drive the expression of exogenous genes.

[0056] On the other hand, the present application provides a cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0057] Optionally, the cells are mammalian cells. Mammalian expression systems offer unique advantages in protein initiation signals, processing, secretion, and glycosylation, making them suitable for expressing intact macromolecular proteins. They also far outperform prokaryotic expression systems and eukaryotic expression systems such as yeast and insect cells in terms of activity. This application utilizes a mammalian cell expression system for recombinant expression, which offers advanced post-translational modifications, improves the solubility of the recombinant protein, resolves the problem of inclusion bodies in E. coli expression, and further improves the stability of the antigen.

[0058] In some embodiments, the cell comprises Escherichia coli BL21. It is understood that the cell includes but is not limited to Escherichia coli BL21 and its derivative strains.

[0059] On the other hand, the present application provides a method for preparing the above-mentioned cells, which comprises the step of introducing the above-mentioned nucleic acid molecule or the above-mentioned vector into the target cell.

[0060] On the other hand, the present application provides a method for preparing the above-mentioned hepatitis D virus recombinant antigen, comprising: culturing the cells as described above, and isolating the hepatitis D virus recombinant antigen from the obtained culture.

[0061] In some embodiments, the culture conditions include: a temperature of 14°C to 18°C, shaking at 180 rpm to 220 rpm, and a time of 22 to 26 hours. For example, the temperature is 14°C, 15°C, 16°C, 17°C, or 18°C, and any values ​​therebetween. For example, the time is 22 hours, 23 hours, 24 hours, 25 hours, or 26 hours, and any values ​​therebetween.

[0062] Wherein, oscillation is cultured on a shaker with a rotation speed of 180 rpm, 181 rpm, 182 rpm, 183 rpm, 184 rpm, 185 rpm, 186 rpm, 187 rpm, 188 rpm, 189 rpm, 190 rpm, 191 rpm, 192 rpm, 193 rpm, 194 rpm, 195 rpm, 196 rpm, 197 rpm, 198 rpm, 199 rpm, 200 rpm, 201 rpm, 202 rpm, 203 rpm, 204 rpm, 205 rpm, 206 rpm, 207 rpm, 208 rpm, 209 rpm, 210 rpm, 211 rpm, 212 rpm, 213 rpm, 214 rpm, 215 rpm, 216 rpm, 217 rpm, 218 rpm, 219 rpm or 220 rpm and any value in between.

[0063] This application utilizes 16°C shaking culture to induce protein expression in BL21 cells, which can significantly maintain protein structural stability, increase the proportion of soluble recombinant antigen, and reduce the proportion of inclusion bodies. Prokaryotically expressed HDV recombinant antigens are highly hydrophobic. Furthermore, the addition of Triton X-405 during the purification process can significantly improve antigen solubility. The mass percentage of Triton X-405 is 1% to 3%, for example, 1%, 2%, or 3%, or any other value in between.

[0064] Optionally, the mass percentage of TritonX-405 is 1.5%.

[0065] On the other hand, the present application provides an anti-HDV detection product, which uses the above-mentioned HDV recombinant antigen as a detection antigen.

[0066] Another aspect of the present application provides a method for detecting hepatitis D virus. The method uses the aforementioned hepatitis D virus recombinant antigen to prepare a detection antibody, and determines the presence of hepatitis D virus in a test sample through an immune binding reaction. Another aspect of the present application provides the use of the aforementioned hepatitis D virus recombinant antigen, nucleic acid molecule, vector, or cell in the preparation of a hepatitis D virus detection product.

[0067] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0068] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0069] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean 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 the present application.

[0070] Example 1

[0071] This embodiment provides a method for preparing a recombinant antigen HDV-SE for detecting human HDV antibodies in a biological sample, comprising:

[0072] 1. Plasmid construction: Construct the pET28a-S-HDAg-P1-P2-P3-Avi-tag expression clone, whose amino terminal sequence composition is shown above.

[0073] S-HDAg utilizes the full-length protein sequence, ensuring detection of HDV antibodies in human blood. P1, P2, and P3 represent three linear epitopes within HDAg (aa59-84, aa92-114, and aa149-172, respectively), expressed in duplicate to enhance 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, facilitating the development of highly stable diagnostic reagents.

[0074] 2. Construct the specific amino acid sequence of the expression clone, such as Figure 1 As shown:

[0075] His-tag: HHHHHHHHHH (SEQ ID NO. 1).

[0076] S-HDAg (NCBI Reference Sequence:NP_077804.1):

[0077] MSRPEGRKNRGGREEVLEQWVSGRKKLEELERDLRKVKKKIKKLEDEHPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPRKRPSRGGFTDKERQDHRRR KALENKRKQLSAGGKNLSKEEEEELRRLTEEDERRERRIAGPQVGGVNPLEGGTRGAPGGGFVPSMQGVPESPFTRTGEGLDIRGSQGFP; amino acid sequence 1-195; (SEQ IDNO.2).

[0078] Peptide 1: amino acid sequence is GKKDKDGEGAPPAKRARTDQMEVDSG: amino acid sequence 221-246; (SEQ ID NO. 3).

[0079] Peptide 2: amino acid sequence is GGFTDKERQDHRRRKALENKRKQ, amino acid sequence 262-284; (SEQ ID NO. 4).

[0080] Peptide 3: amino acid sequence is VGGVNPLEGGTRGAPGGGFVPSMQ, amino acid sequence 300-323; (SEQ ID NO. 5).

[0081] Flexible linker: amino acid sequence is GGGGSGGGGSGGGGS, amino acid sequences are 206-220, 247-261, 285-299, 324-338; (SEQ ID NO. 6).

[0082] Avi-tag: amino acid sequence is GLNDIFEAQKIEWHE, amino acid sequence 339-353, (SEQ ID NO. 7).

[0083] Amino acid sequence of recombinant protein HDV-SE:

[0084] MSRPEGRKNRGGREEVLEQWVSGRKKLEELERDLRKVKKKIKKLEDEHPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPRKRPSRGGFTDKERQDHRRRKALENKRKQLSAGGKNLSKEEEEELRRLTEEDERRERRIAGPQVGGVNPLEGG TRGAPGGGFVPSMQGVPESPFTRTGEGLDIRGSQGFPGGGGSGGGGSGGGGSGKKDKDGEGAPPAKRARTDQMEVDSGGGGGSGGGGSGGGGSGGFTDKERQDHRRRKALENKRKQGGGGSGGGGSGGGGSVGGVNPLEGGTRGAPGGGFVPSMQ(SEQ ID NO.8).

[0085] Amino acid sequence of recombinant protein HDV-SE (including fusion tag):

[0086] MHHHHHHHHHHSRPEGRKNRGGREEVLEQWVSGRKKLEELERDLRKVKKKIKKLEDEHPWLGNIKGILGKKDKDGEGAPPAKRARTDQMEVDSGPRKRPSRGGFTDKERQ DHRRRKALENKRKQLSAGGKNLSKEEEEELRRLTEEDERRERRIAGPQVGGVNPLEGGTRGAPGGGFVPSMQGVPESPFTRTGEGLDIRGSQGFPGGGGSGGGGSGGGGS GKKDKDGEGAPPAKRARTDQMEVDSG GGGGSGGGGSGGGGS GGFTDKERQDHRRRKALENKRKQ GGGGSGGGGSGGGGS VGGVNPLEGGTRGAPGGGFVPSMQ GGGGSGGGGSGGGGSGLNDIFEAQKIEWHE (SEQ ID NO. 9).

[0087] The constructed plasmid map is as follows Figure 1 As shown, the orange arrow represents the HDV recombinant-S-HDAg-P1-P2-P3-protein, which has a His-tag at its N-terminus and an Avi-tag at its C-terminus.

[0088] 3. Plasmid transformation: The obtained vector was transformed into Escherichia coli BL21 containing the encoding ligase BirA. Adding IPTG (0.5 mmol / L) can induce the co-expression of HDV recombinant protein and BirA to achieve biotinylation.

[0089] 4. Inoculation: Inoculate Escherichia coli BL21 containing the recombinant plasmid into LB liquid medium (containing 50 μg / mL kanamycin) and culture at 37°C with shaking overnight.

[0090] 5. Induction: dilute overnight bacteria at 1:100, culture at 37°C with shaking at 200 rpm, and continuously monitor the OD value of the bacterial solution. 600 , when OD 600 =0.4 (Blank: ddH2O), remove the shake flask and add 1M IPTG in a clean bench to a final IPTG concentration of 0.5mM. Set the shaker temperature to 16°C and the speed to 200 rpm and continue incubation for 24 hours. Inducing protein expression in BL21 cells at 16°C with shaking can greatly maintain protein structural stability, increase the proportion of soluble recombinant antigen, and reduce the proportion of inclusion bodies.

[0091] 6. Protein purification: Prepare an NTA chromatography column. Add 5 mL of 1.5% Triton X-405 lysis buffer to the bacterial pellet and resuspend. Ultrasonicate the cells and gently mix the sample using a shaker for 60 minutes. Centrifuge at 12,000 rpm at 4°C for 30 minutes and discard the pellet. Apply the supernatant to a Ni2+-NTA column at a flow rate of 15 mL / h and collect the flow-through. Wash the column with cleaning solution at a flow rate of 15 mL / h. Collect the eluate at OD280 = 0.01 for 3 hours. Wash the column with eluate and collect 1 mL fractions. Take 10 μL of each of the collected liquids for SDS-PAGE verification. Figure 2 It is the purified HDV-SE antigen recombinant protein (molecular weight: 36.38 KDa).

[0092] Example 2

[0093] This embodiment provides a method for preparing a kit for detecting a recombinant antigen HDV-SE for human HDV antibodies in a biological sample, comprising:

[0094] The recombinant protein HDV-SE was prepared using the method of Example 1, and mouse anti-human IgG was labeled with acridinium ester. Commercial SA magnetic beads were used. The biotinylated recombinant protein - S-HDAg-P1-P2-P3-Avi-tag prepared in this example did not need to be labeled and was directly used as the biotin component to prepare a detection kit.

[0095] Verification results:

[0096] 1. Analysis of the test results of the HDV recombinant antigen prepared in this application

[0097] The HDV antibody detection kit (chemiluminescence method) is used, which includes the following steps:

[0098] 1. Prepare magnetic bead diluent with a formula of 50 mM Tris, 0.9 w / v% NaCl, 0.1 w / v% ProClin 300, and a pH of 7.5.

[0099] 2. Prepare acridine diluent with a formula of 50 mM PB, 0.9 w / v% NaCl, 0.1 w / v% ProClin 300, and a pH of 7.5.

[0100] 3. Prepare biotin diluent with the formula of 50 mM PB, 0.9 w / v% NaCl, 0.1 w / v% ProClin 300, and pH 7.5.

[0101] 4. Label the mouse anti-human IgG antibody with acridinium ester to prepare an antibody-labeled conjugate.

[0102] 5. Prepare streptavidin-coupled magnetic beads (SA magnetic beads).

[0103] 6. Dilute the antibody-labeled conjugate with acridine diluent to prepare an acridine working solution; dilute the SA magnetic beads with magnetic bead diluent to prepare a magnetic bead working solution; dilute the HDV recombinant antigen with biotin diluent to prepare a biotin working solution, and assemble the HDV antibody detection kit.

[0104] 2. Comparison of Detection Performance of Different HDV Recombinant Antigens

[0105] 1. Construct pET28a-S-HDAg-Avi-Tag, pET28a-L-HDAg-Avi-Tag and pET28a-S-HDAg-P1-P2-P3-Avi-Tag plasmid vectors, transform and induce host cells to express proteins, and purify the corresponding biotinylated modified proteins.

[0106] 2. The HDV recombinant antigen was used to assemble an HDV antibody detection kit (chemiluminescence method) according to the method of Example 1.

[0107] 3. 31 clinically positive samples and 28 clinically negative samples were tested. The test results of the three HDV recombinant antigen clinically positive samples are shown in Table 1, and the test results of the three HDV recombinant antigen clinically negative samples are shown in Table 2.

[0108] Among them, clinically positive samples came from HBsAg-positive patients, and the presence of HDV infection was confirmed by combined detection of anti-HDV antibodies and HDV RNA; the clinical symptoms were consistent.

[0109] The experimental results showed that: (1) pET28a-L-HDAg-Avi-Tag had critical missed detection and false positives; (2) pET28a-S-HDAg-Avi-Tag reduced false positives compared with pET28a-L-HDAg-Avi-Tag, but the sensitivity needed to be optimized; (3) pET28a-S-HDAg-P1-P2-P3-Avi-Tag had higher detection sensitivity than pET28a-S-HDAg-Avi-Tag and pET28a-L-HDAg-Avi-Tag, with no missed detection and false positives; the recombinant antigen had the highest positive and negative coincidence rate, reaching 100%, and the detection performance was the best.

[0110] Table 1: Detection results of three HDV recombinant antigen clinically positive samples

[0111]

[0112]

[0113] Note: The concentration unit is COI; “+” indicates that both clinical HDV antibody and RNA combined tests are positive.

[0114] Table 2: Detection results of clinical negative samples of 3 HDV recombinant antigens

[0115]

[0116]

[0117] Note: The concentration unit is COI; “-” indicates that the clinical HDV antibody and RNA combined tests are negative.

[0118] 3. Inter-batch verification of the recombinant antigen HDV-SE of this application

[0119] 1. Plasmid transformation and amplification: Add 1 μL of the constructed pET28a-His tag-S-HDAg-P1-P2-P3-Avi Tag plasmid vector (≥100 ng / mL) to 100 μL of competent DH5α cells and incubate on ice for 30 minutes. Heat shock the cells at 42°C for 90 seconds, then quickly incubate on ice for 5 minutes. Add 1 mL of antibiotic-free LB liquid medium and shake at 37°C at 200 rpm for 40 minutes. Centrifuge at 5000 rpm for 5 minutes. Remove the supernatant and resuspend the cell suspension in 100 μL of LB medium (containing 50 μg / mL kanamycin). Spread the suspension onto solid LB medium (containing 50 μg / mL kanamycin). Incubate the cells inverted at 37°C overnight. Pick a single colony and add it to 5 mL of LB liquid medium (containing 50 μg / mL kanamycin). Shake the culture at 37°C at 200 rpm for 12 hours.

[0120] 2. Plasmid extraction: Use a plasmid extraction kit to extract the plasmid.

[0121] 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 induced to express the HDV recombinant antigen with a biotin tag.

[0122] 4. Different batches of HDV recombinant antigens were used to assemble HDV antibody detection kits (chemiluminescence method) according to the method of Example 1.

[0123] 5. Test the inter-batch difference samples of HDV antibodies. 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, magnetically separate, add 100 μL of acridine working solution, incubate for 10 minutes, magnetically separate, add excitation solution and pre-excitation solution, and read the results.

[0124] 6. The deviation between different batches is within ±5%, which can better control the batch difference. See Table 3 for specific data.

[0125] Table 3: Verification of inter-batch differences of HDV recombinant antigen HDV-SE

[0126]

[0127]

[0128] The above results demonstrate that the recombinant antigen HDV-SE described in this application demonstrates high sensitivity and excellent batch-to-batch stability. Its adapted detection method can shorten HDV antibody detection time to under 40 minutes, eliminating false positives and missed detections. The recombinant antigen HDV-SE described in this application promotes the development, upgrading, and application of hepatitis D testing products, can be widely used in HDV patient screening, and ultimately provide support for the prevention and treatment of hepatitis D.

[0129] The embodiments described above only express several implementation methods of the present application, which are convenient for understanding the technical solutions of the present application in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of this application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A recombinant hepatitis D virus antigen, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

8.

2. The hepatitis D virus recombinant antigen according to claim 1, characterized in that One or both of the C-terminus and the N-terminus of the recombinant antigen are connected to a tag protein; Optionally, the tag protein is independently selected from one of a His tag, an HA tag, a c-Myc tag, an AviTag tag, a SNAP tag and a Flag tag.

3. A nucleic acid molecule, characterized in that Contains a recombinant antigen for encoding the hepatitis D virus according to claim 1 or 2.

4. A carrier, characterized in that comprising the nucleic acid molecule of claim 3; Optionally, the vector comprises pET28a.

5. A cell, characterized in that Comprising the nucleic acid molecule according to claim 3 or the vector according to claim 4; Optionally, the cell comprises a prokaryotic cell; Optionally, the cell comprises Escherichia coli; Optionally, the Escherichia coli includes Escherichia coli BL21.

6. A method for preparing the cell according to claim 5, comprising: The step of introducing the nucleic acid molecule according to claim 3 or the vector according to claim 4 into the target cell.

7. The method for preparing the recombinant hepatitis D virus antigen according to claim 1 or 2, characterized in that: include: Cultivating the cell according to claim 5, and isolating the hepatitis D virus recombinant antigen from the obtained culture.

8. The method for preparing a recombinant hepatitis D virus antigen according to claim 7, characterized in that: The culture conditions include: temperature of 14°C to 18°C, shaking at a speed of 180 rpm to 220 rpm, and time of 22 h to 26 h; Optionally, the separation step comprises: collecting cells in the culture, adding a lysis solution to disrupt the cells, and performing protein purification on the obtained cell lysis products; Optionally, the lysate comprises Triton X-405 at a final concentration of 1 w / v% to 3 w / v%.

9. An anti-hepatitis D virus detection product, characterized in that: The product uses the hepatitis D virus recombinant antigen described in claim 1 or 2 as a detection antigen.

10. A method for detecting hepatitis D virus, characterized in that: The detection method uses the hepatitis D virus recombinant antigen according to any one of claims 1 to 2 to prepare a detection antibody, and determines whether the sample to be tested contains hepatitis D virus through an immune binding reaction.

11. Use of the hepatitis D virus recombinant antigen according to claim 1 or 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, or the cell according to claim 5 in the preparation of a hepatitis D virus detection product.

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