A monkeypox virus-specific recombinant protein antigen and its preparation and application

Abstract

This application discloses a monkeypox virus-specific recombinant protein antigen and its preparation and application, belonging to the field of biotechnology. This monkeypox virus-specific recombinant protein antigen possesses two dominant linear antigens: the monkeypox virus B17R protein and the D9L protein, exhibiting good immunogenicity and specificity, and can accurately distinguish monkeypox virus from other orthopoxviruses. Simultaneously, the nucleotide sequence encoding the monkeypox virus-specific recombinant protein antigen is optimized using E. coli-preferred codons to obtain the nucleic acid encoding the monkeypox virus-specific recombinant protein antigen, thereby improving the expression level of the monkeypox virus-specific recombinant protein antigen in E. coli. This method is simple, low-cost, and easily scaled up in a short time, improving detection timeliness. This monkeypox virus-specific recombinant protein antigen can be used as an immunogen for preparing monkeypox virus monoclonal antibodies that specifically recognize monkeypox virus.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to a recombinant monkeypox virus protein antigen and its preparation and application. Background Technology

[0002] Monkeypox (MPX) is a viral zoonotic disease caused by the monkeypox virus (MPXV). It is transspecies, transmitted from animals to humans, and can also spread from person to person. MPXV belongs to the orthopoxvirus family and is closely related to the well-known smallpox virus. Both have similar clinical symptoms, but MPXV infection is less severe, less contagious, and less likely to cause death than smallpox. In 1980, the World Health Organization (WHO) declared smallpox eradicated and subsequently stopped smallpox immunization, leading to a decline or lack of immunity in unvaccinated populations, making them susceptible. MPX is the most severe orthopoxvirus infection in humans since the eradication of smallpox, and it may fill the epidemiological niche vacated by smallpox. The decreased immunity to monkeypox in unvaccinated populations increases human susceptibility to outbreaks. Furthermore, socio-political and ecological changes in endemic areas, the wildlife trade, and convenient international travel all increase human exposure to and infection with MPXV.

[0003] Although monkeypox virus is self-limiting and has a lower mortality rate than smallpox virus, rodents and some mammals serve as natural reservoirs for the virus, resulting in a wide range of host organisms and the risk of genetic modification into more virulent strains. Therefore, developing a monkeypox virus monoclonal antibody with high affinity and specificity is essential to provide a rapid detection method for early monkeypox virus infection.

[0004] The orthopoxvirus genus exhibits a high degree of similarity in viral morphology, life cycle, and structure, with over 90% homology among their genome sequences. Therefore, finding a specific antigen that can effectively distinguish between vaccinia virus (VACV), ectromelia virus (ECTV), and monkeypox virus is crucial, as this is a significant factor affecting the accuracy of monkeypox detection.

[0005] Currently, rapid and specific detection of monkeypox virus in clinical and research settings mainly relies on nucleic acid detection kits and clinical diagnostic methods. However, nucleic acid detection kits are time-consuming, and clinical diagnosis struggles to differentiate them from other poxvirus infections, both of which hinder rapid diagnosis. Summary of the Invention

[0006] 1. Purpose of the invention

[0007] One of the objectives of this application is to provide a monkeypox virus-specific recombinant protein antigen that can effectively distinguish monkeypox virus from other orthopoxviruses.

[0008] The second objective of this application is to provide a monkeypox virus monoclonal antibody, which is prepared using monkeypox virus-specific recombinant protein antigen as an immunogen and can specifically recognize monkeypox virus, thereby improving the accuracy of monkeypox virus detection.

[0009] 2. Technical Solution

[0010] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0011] This application provides a monkeypox virus-specific recombinant protein antigen with the amino acid sequence shown in SEQ ID NO:3. This monkeypox virus-specific recombinant protein antigen contains only the specific dominant antigenic epitopes of the monkeypox virus D9L protein and B17R protein, which can effectively distinguish monkeypox virus from other orthopoxviruses. This ensures that the monkeypox virus monoclonal antibody prepared using it as an immunogen can only specifically recognize monkeypox virus, thus improving the accuracy of monkeypox virus detection.

[0012] This application also provides a nucleic acid that encodes the aforementioned monkeypox virus-specific recombinant protein antigen.

[0013] Furthermore, the nucleic acid sequence encoding the monkeypox virus-specific recombinant protein antigen is shown in SEQ ID NO:4. This nucleic acid sequence is obtained by analyzing the E. coli rare codons in the nucleic acid encoding the monkeypox virus-specific recombinant protein antigen using rare codon online software, while keeping the amino acid sequence unchanged. The rare codons are replaced with E. coli preferred codons encoding the same amino acid, so that the nucleic acid encoding the monkeypox virus-specific recombinant protein antigen does not contain E. coli rare codons, thereby increasing the expression level of the monkeypox virus-specific recombinant protein antigen in E. coli.

[0014] This application also provides a recombinant expression vector containing the above-mentioned nucleic acid encoding monkeypox virus-specific recombinant protein antigen, which can express monkeypox virus-specific recombinant protein antigen.

[0015] Furthermore, the recombinant expression vector includes a pET 28a(+) plasmid containing the nucleic acid encoding the monkeypox virus-specific recombinant protein antigen.

[0016] This application also provides a recombinant expression bacterium, which includes the above-mentioned recombinant expression vector or the above-mentioned nucleic acid encoding monkeypox virus-specific recombinant protein antigen, and can express monkeypox virus-specific recombinant protein antigen.

[0017] Furthermore, the recombinant expression bacteria mentioned above include Escherichia coli BL21(DE3) with the above recombinant expression vector.

[0018] This application also provides the application of the above-mentioned nucleic acid, recombinant expression vector, and recombinant expression bacteria in the preparation of monkeypox virus-specific recombinant protein antigen.

[0019] This application also provides a method for preparing the above-mentioned monkeypox virus-specific recombinant protein antigen, which specifically includes the following steps:

[0020] (1) Construction of recombinant expression vector: The nucleic acid encoding the monkeypox virus-specific recombinant protein antigen was cloned into the expression vector to obtain the recombinant expression vector:

[0021] (2) Construction of recombinant expression bacteria: The obtained recombinant expression vector was transferred into the host bacteria to obtain the recombinant expression bacteria:

[0022] (3) Culture of recombinant expression bacteria: Recombinant expression bacteria are used to induce the expression of monkeypox virus-specific recombinant protein antigen.

[0023] Furthermore, the above-mentioned method for preparing a monkeypox virus-specific recombinant protein antigen further includes:

[0024] (4) Isolation and purification of monkeypox virus-specific recombinant protein antigen: The monkeypox virus-specific recombinant protein antigen was obtained by isolation and purification step (3).

[0025] This application also provides the application of the above-mentioned monkeypox virus-specific recombinant protein antigen, nucleic acid, recombinant expression vector and / or recombinant expression bacteria in the preparation of monkeypox virus monoclonal antibodies.

[0026] Furthermore, the above applications include: preparing and purifying monkeypox virus monoclonal antibodies by immunizing New Zealand white rabbits with the above-mentioned monkeypox virus-specific recombinant protein antigen and Freund's adjuvant.

[0027] This application also provides a monkeypox virus monoclonal antibody that specifically binds to monkeypox virus, including a heavy chain variable region and a light chain variable region, including any one of the following:

[0028] (1) Monoclonal antibody H15L114, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:6;

[0029] (2) Monoclonal antibody H31L137, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0030] (3) Monoclonal antibody H51L152, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12;

[0031] (4) Monoclonal antibody H61L161, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:14;

[0032] (5) Monoclonal antibody H71L172, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

[0033] Furthermore, the amino acid sequence of the heavy chain constant region of the above-mentioned monkeypox virus monoclonal antibody is shown in SEQ ID NO:17, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:18.

[0034] This application also provides a nucleic acid that encodes the heavy chain variable region, light chain variable region, heavy chain constant region, or light chain constant region of the aforementioned monkeypox virus monoclonal antibody.

[0035] Furthermore, the above-mentioned nucleic acid encodes monkeypox virus monoclonal antibody H15L114, the nucleic acid sequence encoding the heavy chain variable region is shown in SEQ ID NO:7, and the nucleic acid sequence encoding the light chain variable region is shown in SEQ ID NO:8.

[0036] Furthermore, the nucleic acid sequence encoding the heavy chain constant region of the monkeypox virus monoclonal antibody is shown in SEQ ID NO:19, and the nucleic acid sequence encoding the light chain constant region is shown in SEQ ID NO:20.

[0037] This application also provides the application of the aforementioned nucleic acid in the preparation of monkeypox virus monoclonal antibodies.

[0038] This application also provides the use of one or more of the above-mentioned monkeypox virus monoclonal antibodies in the detection of monkeypox virus.

[0039] This application also provides the use of one or more of the above-mentioned monkeypox virus monoclonal antibodies in the preparation of monkeypox virus detection products.

[0040] Furthermore, the above applications include using one monoclonal antibody as the coating monoclonal antibody and another monoclonal antibody as the colloidal gold particle-labeled monoclonal antibody, with the two monoclonal antibodies being different.

[0041] This application also provides a monkeypox virus kit, which includes one or more of the above-mentioned monkeypox virus monoclonal antibodies.

[0042] Furthermore, the aforementioned monkeypox virus kit includes any two of the aforementioned monkeypox virus monoclonal antibodies, one of which is a coated monoclonal antibody, and the other is a colloidal gold particle-labeled monoclonal antibody, and the two monoclonal antibodies are different.

[0043] Furthermore, the aforementioned monkeypox virus kit includes monoclonal antibodies H15L114 and H61L161, with monoclonal antibody H15L114 as the coating monoclonal antibody and monoclonal antibody H61L161 as the colloidal gold particle-labeled monoclonal antibody.

[0044] 3. Beneficial effects

[0045] Compared with the prior art, the advantages of this application are as follows:

[0046] (1) The monkeypox virus-specific recombinant protein antigen provided in this application contains only the specific dominant antigenic epitopes of the monkeypox virus D9L protein and B17R protein, which can effectively distinguish monkeypox virus from other orthopoxviruses, ensuring that the monkeypox virus monoclonal antibody prepared with it as an immunogen can only specifically recognize monkeypox virus, thus improving the accuracy of monkeypox virus detection.

[0047] (2) The nucleic acid encoding monkeypox virus-specific recombinant protein antigen provided in this application is optimized by using Escherichia coli preferred codons to optimize the nucleotide sequence corresponding to the encoding monkeypox virus-specific recombinant protein antigen, thereby improving the expression level of monkeypox virus-specific recombinant protein antigen in Escherichia coli. This method is simple to prepare, low in cost, easy to scale up production in a short time, and improves detection timeliness.

[0048] (3) The monkeypox virus monoclonal antibody provided in this application can specifically recognize monkeypox virus and can be used to detect monkeypox virus. In particular, combining any two of them and using them in pairs improves the detection sensitivity and accuracy of monkeypox virus. Attached Figure Description

[0049] Figure 1 This is the result of amino acid sequence analysis of the monkeypox virus D9L protein.

[0050] Figure 2 These are the amino acid sequence analysis results of the monkeypox virus B17R protein.

[0051] Figure 3This is an electrophoresis image of a monkeypox virus-specific recombinant protein antigen expression vector digested with double enzymes on a 1% agarose gel. Lane 1 is the DNA marker band, and lane 2 is the band of the recombinant expression vector after double digestion with BamHI and HindIII.

[0052] Figure 4 This is an SDS-PAGE result of positive recombinant expression bacteria induced expression. Lane 1 is the protein molecule marker, lanes 2 to 4 are positive induced expression bacteria, and lane 5 is the uninduced control group.

[0053] Figure 5 This refers to the immunogenicity and specificity of ELISA detection of monkeypox virus-specific recombinant protein antigens.

[0054] Figure 6 It is a flow cytometer for sorting monkeypox virus-specific B lymphocytes.

[0055] Figure 7 This is an electrophoresis image of the variable regions of the heavy and light chains of monkeypox virus antibodies on a 1% agarose gel. The middle band is the DNA marker band, lanes 1-8 are the heavy chain bands, and lanes 9-16 are the light chain bands.

[0056] Figure 8 This is an ELISA test showing the binding of monoclonal antibodies to monkeypox virus-specific recombinant protein antigens.

[0057] Figure 9 This is an immunoblot diagram showing the binding of monkeypox virus monoclonal antibodies to monkeypox virus-specific recombinant protein antigens.

[0058] Figure 10 This is an SDS-PAGE result of monkeypox virus monoclonal antibody. Lanes 1-5 show the purified monoclonal antibody. Detailed Implementation

[0059] The present application will be further described below with reference to specific embodiments.

[0060] 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 pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0061] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0062] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0063] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0064] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as “less than about 4.5,” which should be interpreted to include all the aforementioned values ​​and ranges. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0065] Example 1

[0066] This embodiment provides the selection of dominant antigenic epitopes for monkeypox virus.

[0067] To ensure the specificity of monkeypox virus antigens, the inventors compared, repeatedly verified, and conducted numerous experiments on the genomes of multiple viruses in the orthopoxvirus genus. They ultimately determined that amino acids 154 to 309 of the monkeypox virus D9L protein and amino acids 555 to 674 of the B17R protein are missing amino acids from vaccinia virus and murine pox virus, and can be used as candidate proteins for monkeypox virus-specific antigens.

[0068] In this embodiment, monkeypox virus D9L and B17R proteins were used as target antigens. Referring to the amino acid sequences of monkeypox virus D9L protein (AAV84851.1) and B17R protein (AGR37974.1) published on NCBI, the secondary and tertiary domains of the D9L and B17R proteins were analyzed using analytical tools such as DNAstar V7.10 and UniProt. The analysis results are as follows: Figure 1 and 2As shown, online software such as TMHMM-2.0, BepiPred-2.0, Discotope-2.0, and Epitome were used to analyze transmembrane structure, linear site prediction, and antigen-antibody interaction residues. Based on this, specific dominant antigenic epitopes of D9L and B17R proteins were comprehensively analyzed and screened. Specifically, amino acids 154 to 309 of the monkeypox virus D9L protein have the sequence: NWDDEFDYLDYDYTTDYDNRMGKTVLYYYIITRSQDGYVTSLDV INYLISHENEMCHYTYRERTILYYYVDKCDIKREIFDVLFDSNYSGNELMHILSIYLRKQYRK KNHKIDNYIVDKLLSAHDTFYILELCNSLRNNVIISSILKRYTDSIQDL (SEQ ID NO: 1); and amino acids 555 to 674 of the monkeypox virus B17R protein have the sequence: SLCSYIPLKWTSFLI SRLPPKSVICSLTNHIIDYVLTNNRRIIWQSQMINKYVLLLDPSFYYRFRNAIENKLDQYNNR YNMFEHDRDVNEKYGKVLHDLDTYIKDVQVLKSTSITNNITL (SEQ ID NO: 2).

[0069] Example 2

[0070] This embodiment provides a monkeypox virus-specific recombinant protein antigen.

[0071] The specific dominant antigenic epitopes screened in Example 1 were tandemly arranged in a certain order to construct and synthesize a multi-epitope recombinant protein antigen, thus obtaining a monkeypox virus-specific recombinant protein antigen. In this example, to enhance the activation effect of the selected specific dominant antigenic epitopes on the immune system of New Zealand rabbits and shorten the preparation time of monoclonal antibodies, two specific dominant antigenic epitope sequences of monkeypox virus D9L and B17R proteins were linked by a flexible fragment (Gly)4 to obtain a monkeypox virus-specific recombinant protein antigen with the following amino acid sequence:

[0072] NWDDEFDYLDYDYTTDYDNRMGKTVLYYYIITRSQDGYVTSLDVINYLISHENEMCHYTYRERTILYYYVDKCDIKREIFDVLFDSNYSGNELMHILSIYLRKQYRKKNHKIDNYIVDKLLSAHDTFYILELCNSLRNNVII SSILKRYTDSIQDLGGGGSLCSYIPLKWTSFLISRLPPKSVICSLTNHIIDYVLTNNRRIIWQSQMINKYVLLLDPSFYYRFRNAIENKLDQYNNRYNMFEHDRDVNEKYGKVLHDLDTYIKDVQVLKSTSITNNITL(SEQ ID NO: 3).

[0073] Example 3

[0074] This embodiment provides a nucleic acid encoding the monkeypox virus-specific recombinant protein antigen in Example 2. This nucleic acid is a nucleic acid encoding the monkeypox virus-specific recombinant protein, optimized by E. coli preferred codons. Specifically:

[0075] To improve the expression level of the monkeypox virus-specific recombinant protein antigen in *E. coli* while maintaining the amino acid sequence unchanged, rare codons from *E. coli* in the nucleic acid encoding the monkeypox virus-specific recombinant protein antigen were analyzed using online rare codon software. These rare codons were then replaced with *E. coli*-preferred codons encoding the same amino acids, ensuring that the nucleic acid encoding the monkeypox virus-specific recombinant protein antigen did not contain any rare codons from *E. coli*. This resulted in the final nucleic acid encoding the monkeypox virus-specific recombinant protein antigen.Its sequence is: AACTGGGA TGATGAATTTGATTATCTGGATTATGATTATACCACCGATTATGATAACCGCATGGGCAAAACCGTGCTGTATTATTATATTATTACCCGCAGCCAGGATGGCTATGTGACCAGCCTGGATGTGATTAACTATCTGATTAGCCATGAAAACGAAATGTGCCATTATACCTATCGCGAACGCACCATTCTGTATTATTATGTGGATAAATGCGATATTAAACGCGAAATTTTTGATGTGCTGTTTGATAGCAACTATAGCGGCAACGAACTGATGCATATTCTGAGCATTTATCTGCGCAAACAGTATCGCAAAAAAAACCATAAAATTGATAACTATATTGTGGATAAACTGCTGAGCGCGCATGATACCTTTTATATTCTGGAACTGTGCAACAGCCTGCGCAACAACGTGATTATTAGCAGCATTCTGAAACGCTATACCGATAGCATTCAGGATCTGGGCGGCGGCGGCAGCCTGTGCAGCTATATTCCGCTGAAATGGACCAGCTTTCTGATTAGCCGCCTGCCGCCGAAAAGCGTGATTTGCAGCCTGACCAACCATATTATTGATTATGTGCTGACCAACAACCGCCGCATTATTTGGCAGAGCCAGATGATTAACAAATATGTGCTGCTGCTGGATCCGAGCTTTTATTATCGCTTTCGCAACGCGATTGAAAACAAACTGGATCAGTATAACAACCGCTATAACATGTTTGAACATGATCGCGATGTGAACGAAAAATATGGCAAAGTGCTGCATGATCTGGATACCTATATTAAAGATGTGCAGGTGCTGAAAAGCACCAGCATTACCAACAACATTACCCTG (SEQ ID NO: 4).

[0076] Example 4<0000,173>

[0077] This example provides an expression vector for a monkeypox virus-specific recombinant protein antigen and its construction.

[0078] It should be noted that there seems to be a formatting issue in the original text where " " might be incorrect as " <0000,173>". This has been translated as is but might need to be corrected in the original source.The sequence was synthesized by Beijing Qingke Biotechnology Co., Ltd. after adding the nucleotide sequences corresponding to the restriction endonuclease sites BamHⅠ and HindⅢ upstream and downstream of the nucleic acid of the sequence shown in SEQ ID NO: 4.

[0079] The pET 28a(+) plasmid (Novagen) was extracted using a kit. The plasmid was double-digested with BamHI and HindIII (NEB). After electrophoresis on a 1% agarose gel, the single vector fragment was recovered using a gel recovery kit (Tiangen Biotech (Beijing) Co., Ltd.). At the same time, the nucleic acid fragment encoding the monkeypox virus-specific recombinant protein antigen was double-digested with BamHI and HindIII. After electrophoresis, the fragment was recovered by gel recovery and stored at -20℃ for later use.

[0080] The double-digested pET 28a(+) plasmid (Novagen) and the double-digested nucleic acid fragment encoding monkeypox virus-specific recombinant protein antigen were mixed at a molar ratio of 1:3 and ligated with T4 DNA ligase (NEB) at 16℃ for 2 hours. The resulting expression vector for monkeypox virus-specific recombinant protein antigen was named pET 28a(+)-SDT151.

[0081] The ligation product was transformed into BL21(DE3) competent cells (Shanghai Weidi Biotechnology Co., Ltd.) and plated on LB agar plates containing Kanab+ resistant (50 μg / mL) and incubated at 37°C for 12 h. Single clones were picked from the plates and transferred to LB liquid medium containing Kanab+ resistant (50 μg / mL) and incubated at 37°C with a shaker for 4 h. Plasmids were extracted using a plasmid purification kit (Tiangen Biotech (Beijing) Co., Ltd.), and identified by BamHI and HindIII double digestion to obtain the correct monkeypox virus-specific recombinant protein antigen expression vector, such as... Figure 3 As shown.

[0082] Example 5

[0083] This embodiment provides a monkeypox virus-specific recombinant protein antigen expression strain and its construction.

[0084] The correctly identified pET 28a(+)-SDT151 recombinant expression vector bacterial suspension was streaked onto LB agar plates containing Kana+ resistance (50 μg / mL) and incubated overnight at 37°C. Single clones from the plates were picked and transferred to 2×YT liquid medium containing Kana+ resistance (50 μg / mL) and incubated at 37°C with a shaker for 12 h. The overnight bacterial suspension was then inoculated at a ratio of 1% into 2×YT liquid medium containing Kana+ resistance (50 μg / mL) and incubated at 37°C until OD (outcome limit). 600The concentration was between 0.5 and 0.8, and expression was induced overnight at 20°C with IPTG (final concentration 0.5 mmol / L). The bacterial cells were collected by centrifugation and analyzed by SDS-PAGE. The results are as follows: Figure 4 As shown, lane 1 is the Maker, lane 2 is the uninduced expression control, and lane 3 is the induced expression strain. The size of the specific recombinant protein antigen expressed by pET 28a(+)-SDT151 is between 25kD and 45kD, and around 35kD, which is consistent with the prediction results of the online software. Moreover, there is no target protein band in the control strain, indicating that the expression strain expressing the monkeypox virus specific recombinant protein antigen was successfully constructed.

[0085] Example 6

[0086] This embodiment provides the purification of monkeypox virus-specific recombinant protein antigen.

[0087] Inoculate monkeypox virus-specific recombinant protein antigen-expressing strains into 2×YT liquid medium, add Kana to a final concentration of 50 μg / mL, and incubate at 37°C in a shaker until OD50. 600 The concentration was between 0.5 and 0.8. IPTG (final concentration 0.5 mmol / L) was added as an inducer, and the mixture was induced overnight at 20°C. After centrifugation to collect the bacterial cells, the cells were lysed by low-temperature sonication for 30 min. After centrifugation at low temperature (4°C), the supernatant was collected and subjected to nickel agarose affinity chromatography. After washing and elution, the purified monkeypox virus-specific recombinant protein antigen was finally obtained.

[0088] Example 7

[0089] This embodiment provides a monkeypox virus monoclonal antibody and its preparation. The monkeypox virus monoclonal antibody was prepared and purified by immunizing New Zealand white rabbits with the purified monkeypox virus-specific recombinant protein antigen and Freund's adjuvant as described in Example 6.

[0090] The monkeypox virus-specific recombinant protein antigen purified in Example 6 was used as an immunogen to immunize three New Zealand rabbits. Each rabbit was immunized with 600 μg of monkeypox virus-specific recombinant protein antigen via a multi-site dorsal immunization. Specifically: for the first immunization, 1 mL of complete Freund's adjuvant was mixed with 600 μg of monkeypox virus-specific recombinant protein antigen and emulsified. Three weeks later, for the second immunization, 1 mL of incomplete Freund's adjuvant was mixed with 600 μg of monkeypox virus-specific recombinant protein antigen and emulsified, again via a multi-site dorsal immunization. Five weeks later, for the third immunization, 300 μg of monkeypox virus-specific recombinant protein antigen was mixed with an equal volume of 0.01 M PBS (pH 7.4), again via a multi-site dorsal immunization. Seven weeks later, the same immunization procedure (i.e., 300 μg of monkeypox virus-specific recombinant protein antigen mixed with an equal volume of 0.01 M PBS (pH 7.4)) was repeated. Seven days after the last immunization, blood was collected from the marginal ear vein of the rabbits, and rabbit antiserum was obtained by centrifugation at 4000 rpm for 20 min.

[0091] Monkeypox virus-specific recombinant protein antigen was diluted with carbonate buffer (50 mM, pH 9.6), and 100 μL / well was coated onto an ELISA plate and incubated overnight at 4°C. The next day, the plate was blocked with 1% BSA at 37°C for 1 hour. Rabbit antiserum was serially diluted as primary antibody and incubated at 37°C for 1 hour. Then, 1:5000 secondary antibody (goat anti-rabbit) was added and incubated at 37°C for 1 hour. Finally, chromogenic reagent was added for color development. The results showed that the antibody titer against the monkeypox virus-specific recombinant protein antigen was not lower than 1:50000, indicating that the monkeypox virus-specific recombinant protein antigen possessed good antigenicity. Figure 5 As shown.

[0092] To obtain specific B lymphocytes, the spleen of New Zealand white rabbits was ground to obtain lymphocytes, which were then resuspended at 10⁻⁶ ppm. 5 At a density of / mL, 1mL of resuspended cells was directly incubated with an antibody for screening specific B cells, followed by flow cytometry screening. The results are as follows: Figure 6 As shown.

[0093] The selected specific B lymphocytes were lysed, and total RNA was obtained using the TurboCapture 96mRNA Plate (QIAGEN) kit. cDNA was obtained using a reverse transcription kit (Shanghai Tongke Biotechnology Co., Ltd.). The reverse transcription product was used as a template for PCR. Corresponding primers were designed to amplify the antibody-encoded heavy chain variable region (VH) and light chain variable region (VL) sequences. The reaction program was as follows: VH: 95℃ 5 min, 95℃ 30 s, 70℃ 30 s, 72℃ 1 min, 72℃ 10 min for 35 cycles; VL: 95℃ 5 min, 95℃ 30 s, 55℃ 30 s, 72℃ 1 min, 72℃ 10 min for 35 cycles. The amplification products were detected by 1% agarose gel electrophoresis. Figure 7 Then, a single target band was selected for gel recovery.

[0094] The target band recovered from the gel was ligated to the corresponding vector via homologous recombination and transformed into TOP10 competent cells (Shanghai Weidi Biotechnology Co., Ltd.). The cells were cultured at 37°C for 12 hours. Single clones were picked and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The sequencing results were analyzed using software to screen for sequences encoding the monoclonal antibodies VH and VL.

[0095] Example 8

[0096] This embodiment provides performance testing of monkeypox virus monoclonal antibodies.

[0097] To determine whether the screened monoclonal antibodies specifically bind to the monkeypox virus-specific recombinant protein antigen and their function, verification was performed using ELISA and Western blot assays.

[0098] Nucleic acids encoding the VH and VL variable regions were selected and ligated into vectors containing the heavy chain constant region and light chain constant region, respectively, via homologous recombination. Plasmids containing complete sequences encoding monoclonal antibody nucleic acids were transfected into healthy Expi 293F cells plated in six-well plates to express monkeypox virus monoclonal antibodies at low levels. After 5 days of culture, the supernatant was collected for antibody ELISA and Western blot analysis.

[0099] (1) H15L114 monoclonal antibody:

[0100] The amino acid sequence of the heavy chain variable region (H15L114VH) of the H15L114 monoclonal antibody is: GVQCQSLEESGGRLVTPGTPLTLTCTASGFDISSYSVGWVRQAPGEGLEYIGWISVYGNIYYTTWAKGRFTISRTST TVDLKILGPTTEDTATYFCARGDGHTGFGGFWGPGTLVTVSS (SEQ ID NO:5);

[0101] The amino acid composition of the light chain variable region (H15L114VL) of the H15L114 monoclonal antibody is: GATFAQVLTQTPSPVSVAVGGTVTINCQASQSVYSNNNLAWFQQKPGQPPKRLIYFASTLASGVSSRFKGSGSGTQFTLT ISDVQCDDAATYYCLGEFSCSSADCSAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVT (SEQ ID NO:6);

[0102] The nucleic acid sequence encoding the H15L114 monoclonal antibody heavy chain variable region (H15L114VH-DNA) is: GGCGTGC AGTGCCAGTCCCTGGAGGAGTCCGGCGGCCGCTGGTGACCCCCGGCACCCCCCTGACCCTGACCTGCACCGCCTCCGGCTTCGACATCTCCTCCTACTCCGTGGGCTGGGTGCGCCAGGCCCCCGGCGAGGGCCTGGAGTACATCGGCTGGATCTCCGTGTACGGCAACATCTACT ACACCACCTGGGCCAAGGGCCGCTTCACCATCTCCCGCACCTCCACCACCGTGGACCTGAAGATCCTGGGCCCCACCACCGAGGACACCGCCACCTACTTCTGCGCCCGCGGCGACGGCCACACCGGCTTCGGCGGCTTCTGGGGCCCCGGCACCCTGGTGACCGTGTCCTCC(SEQ ID NO:7);

[0103] The nucleic acid sequence encoding the light chain variable region (H15L114VL-DNA) of the H15L114 monoclonal antibody is as follows: GGCGCCACCTTCGCCCAGGTGCTGACCCAGACCCCCTCCCCCGTGTCCGTGGCCGTGGGCGGCACCGTGACCATCAACTGCCAGGCCTCCCAGTCCGTGTACTCCAACAACAACCTGGCCTGGTTCCAGCAGAAGCCCGGCCAGCCCCCCAAGCGCCTGATCTACTTCGCCTCCACCCTGGCCTCCGGCGTGT CCTCCCGCTTCAAGGGCTCCGGCTCCGGCACCCAGTTCACCCTGACCATCTCCGACGTGCAGTGCGACGACGCCGCCACCTACTACTGCCTGGGCGAGTTCTCCTGCTCCTCCGCCGACTGCTCCGCCTTCGGCGGCGGCACCGAGGTGGTGGTGAAGGGCGACCCCGTGGCCCCCACCGTGCTGATCTTCCCCCCCGCCGCCGACCAGGTGGCCACCGGCACCGTGACC(SEQ ID NO:8).

[0104] (2) H31L137 monoclonal antibody:

[0105] The amino acid sequence of the heavy chain variable region (H31L137VH) of the H31L137 monoclonal antibody is: GVQCQSVEESGGR LVTPGTPLTLTYTVSGIDLSSYAMIWVRQAPGKGLEWIGTTGTSGATYYASWAKGRFTISKT STTVDLKITSPTTEDTATYFCVRGVGLWGQGTLVTVSS (SEQ ID NO:9);

[0106] The amino acid composition of the light chain variable region (H31L137VL) of the H31L137 monoclonal antibody is: GATFAQVLTQTPSPVSVAVGGTVTINCQASQSVYSNNNLAWFQQKPGQPPKRLIYFASTLASGVSSRFKGSGSGTQFTLT ISDVQCDDAATYYCLGEFSCSSADCSAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVT (SEQ ID NO:10);

[0107] (3) H51L152 monoclonal antibody:

[0108] The amino acid sequence of the heavy chain variable region (H51L152VH) of the H51L152 monoclonal antibody is: GVQCQEQLEESGG DLVKPEGSLTLTCTVSGFSFSSSYWMCWVRQAPGKGLEWIACIYAGNSGNTDYATWAKGRF TISKTSSTTVTLQMTSLTVADTATYFCARAPYAIYVPYGDPYYFNLWGPGTLVTVSS (SEQ ID NO:11);

[0109] The amino acid composition of the light chain variable region (H51L152VL) of the H51L152 monoclonal antibody is: GATFAQVLTQTPSPVSA VVGGTVTISCQSSESVYSNKNLAWYQQKPGQPPKRLMYSASTLASGVPSRFKGSGSGTQFT LTISDVQCDDAATYYCLGGYDCMLADCYAFGGRTEVVVKGDPVAPTVLIFPPAADQVATGT VT (SEQ ID NO:12);

[0110] (4) H61L161 monoclonal antibody:

[0111] The amino acid sequence of the heavy chain variable region (H61L161VH) of the H61L161 monoclonal antibody is: GVQCQSVEESGGR LVTPGTPLTLTCTVSGFSLSSYYMSWVRQAPGKGLEWIGDIYGSGSIYYANWAKGRFTISKT STTVDLKITSPTTEDTATYFCARGTDNSDYCGFDLWGQGTLVTVSS (SEQ ID NO:13);

[0112] The amino acid composition of the light chain variable region (H61L161VL) of the H61L161 monoclonal antibody is: GATIAQVLTQTPASVSAAVGGTVTINCQSSQSVYNNNWLAWFQQKPGQPPKRLIYFASTLASGVSSRFKGSGSGTHFTL TISGVQCDDAATYFCLGGYSGGIYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVT (SE Q ID NO:14);

[0113] (5) H71L172 monoclonal antibody:

[0114] The amino acid sequence of the heavy chain variable region (H71L172VH) of the H71L172 monoclonal antibody is: GVQCQSVEESGGR LVTPGTPLTLTCTVSGIDLSSNAMIWVRQAPGKGLEWIGTIGGSGVTYCATWAKGRFTISKT STTVYLKITGPTTEDTATYFCARGVTLWGQGTLVTVSS (SEQ ID NO:15);

[0115] The amino acid composition of the light chain variable region (H71L172VL) of the H71L172 monoclonal antibody is: GATFAQVLTQTPSSTSAAVGGTVTINCQSSQSVLDNKWLAWYHQKPGQPPKLLIYAASTLASGVPSRFKGSGSGTHFTL TISDLECDDAATYYCAGTYVSSNWYLDAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTV T (SEQ ID NO:16);

[0116] (6) Constant region

[0117] The amino acid sequence of the heavy chain constant region (CH) of the above monoclonal antibody is: METGLRWLLLVAVLKGVQCQEQ LVESGGGLVQPGASLTLTCKASGFSISSVYGMCWVRQAPGKGLEWIASIYAGVGASTYYANWAKGRFTISRTSSTTVTLQMTSLTAADTATYFCARGFIGDGYAAGAFDPWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTPGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKP MCPPPELPGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKA RGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPTVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK(SEQ ID NO:17);

[0118] The amino acid sequence of the light chain constant region (CL) of the above monoclonal antibody is: MDVVMTQTPASVEAAVGGTVTI KCQASQNIYSNLAWYQQKPGQRPKLLIYKASTLASGVSSRFKGSGSGTEFTLTISDLECADAATYYCQCTWYDNTYVAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:18);

[0119]

[0120] The nucleic acid sequence encoding the light chain constant region (CL-DNA) of the monoclonal antibody is: ATGGATGTCGTGATGACCCA (SEQ ID NO:20).

[0121] ELISA test results as follows Figure 8 As shown, H15L114, H31L137, H51L152, H61L161, and H71L172 are ELISA-positive clones. Among them, the H15L114 monoclonal antibody exhibits the best binding affinity to the monkeypox virus-specific recombinant protein. The immunoblotting results are as follows... Figure 9 As shown, the monoclonal antibodies H15L114, H31L137, H51L152, H61L161, and H71L172 can specifically bind to monkeypox virus.

[0122] Example 9

[0123] This embodiment provides the expression and purification of monkeypox virus monoclonal antibodies.

[0124] The selected nucleic acids encoding the VH and VL variable region sequences were ligated into vectors containing the heavy chain constant region and the light chain constant region, respectively, via homologous recombination. Plasmids containing complete sequences encoding monoclonal antibody nucleic acids were then transfected into cells at a density of 3 × 10⁶ cells / year. 6 In 100 mL of Expi 293F cells, the cell supernatant was collected after 5 days of culture. The antibody was purified by affinity chromatography, and the purification was verified by SDS-PAGE. The results are as follows: Figure 10 As shown, lanes 1-5 are H15L114, H31L137, H51L152, H61L161, and H71L172, respectively, for the purification of antibodies.

[0125] Example 10

[0126] This embodiment provides a colloidal gold particle-labeled monkeypox virus monoclonal antibody and its preparation method.

[0127] Add colloidal gold solution to potassium carbonate solution, mix thoroughly, add monkeypox virus monoclonal antibody, react at room temperature for 2 hours, add BSA, block for 2 hours, centrifuge, discard supernatant, dissolve precipitate thoroughly with 1 mL reconstitution solution, use a gold spraying membrane instrument (Shanghai Jinbiao Biotechnology Co., Ltd.) to evenly spray the reconstitution solution onto glass fiber, and then place in an electric thermostatic incubator (Shanghai Yiheng Scientific Instruments Co., Ltd.) at 37℃ for 30 minutes.

[0128] The monoclonal antibodies H15L114, H31L137, H51L152, H61L161, and H71L172 were labeled with colloidal gold using the method described above, and colloidal gold particle-labeled monoclonal antibodies were obtained respectively.

[0129] Example 11

[0130] This embodiment provides the application of monkeypox virus monoclonal antibodies in the detection of monkeypox virus.

[0131] Monkeypox virus monoclonal antibodies (H15L114, H31L137, H51L152, H61L161, and H71L172) were diluted with coating buffer and then uniformly coated onto nitrocellulose membranes using a gold-sprayed membrane scrubbing apparatus (Shanghai Jinbiao Biotechnology Co., Ltd.). These were the T-lines. Goat anti-rabbit solution was also uniformly coated onto nitrocellulose membranes using the same apparatus (Shanghai Jinbiao Biotechnology Co., Ltd.). After coating, the nitrocellulose membranes were placed in a thermostatic incubator (Shanghai Yiheng Scientific Instruments Co., Ltd.) at 37°C for 30 minutes.

[0132] Following standard procedures, the sample pad, glass fiber, nitrocellulose membrane, and filter paper are assembled sequentially on a PVC pad, then cut into 4mm wide strips, fitted with reagent card strips, and pressed tightly.

[0133] Clinical serum samples from monkeypox virus patients and normal human serum samples were loaded at 100 μL / well. After being incubated at room temperature for 15 min, the values ​​were read using a chromatography reader (Shanghai Jiehao Scientific Instruments Co., Ltd.) and the P / N value (the ratio of the positive sample detection value to the negative sample detection value) was calculated. See Table 1 for details.

[0134] Table 1. Statistics of P / N values ​​for different paired monkeypox virus monoclonal antibodies

[0135]

[0136] Table 1 shows that when the P / N ratio of the same monoclonal antibody is less than 2.1, a positive result cannot be detected; however, when any two different monoclonal antibodies are used together, a P / N ratio greater than or equal to 2.1 can detect positive samples. Among them, the H15L114 coated monoclonal antibody and the H61L161 colloidal gold particle-labeled monoclonal antibody have the highest affinity for the antigen and are the best combination for detecting monkeypox virus.

[0137] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A monkeypox virus-specific recombinant protein antigen, characterized in that, The amino acid sequence of the monkeypox virus-specific recombinant protein antigen is shown in SEQ ID NO:

3.

2. A nucleic acid, characterized in that, The nucleic acid encodes the monkeypox virus-specific recombinant protein antigen of claim 1.

3. A nucleic acid according to claim 2, characterized in that, The nucleic acid sequence is shown in SEQ ID NO:

4.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the nucleic acid as described in claim 2 or 3.

5. A recombinant expression bacterium, characterized in that, The recombinant expression bacteria includes the recombinant vector described in claim 4.

6. The use of the nucleic acid of claim 2 or 3, the recombinant expression vector of claim 4, or the recombinant bacteria of claim 5 in the preparation of monkeypox virus-specific recombinant protein antigen.

7. The method for preparing a monkeypox virus-specific recombinant protein antigen according to claim 1, characterized in that, The method specifically includes the following steps: (1) Construction of recombinant expression vector: The nucleic acid described in claim 2 or 3 is cloned into the expression vector to obtain the recombinant expression vector; (2) Construction of recombinant expression bacteria: The recombinant expression vector constructed in (1) was transferred into the host bacteria to obtain recombinant expression bacteria; (3) Culture of recombinant expression bacteria: Recombinant expression bacteria are used to induce the expression of monkeypox virus-specific recombinant protein antigen.

8. The method for preparing a monkeypox virus-specific recombinant protein antigen according to claim 7, characterized in that, The method further includes: (4) Isolation and purification of monkeypox virus-specific recombinant protein antigen: The monkeypox virus-specific recombinant protein antigen was obtained by isolation and purification step (3).

9. The application of the method for preparing a monkeypox virus-specific recombinant protein antigen according to claim 1, the nucleic acid according to claim 2 or 3, the recombinant expression vector according to claim 4, the recombinant bacteria according to claim 5, or the method for preparing a monkeypox virus-specific recombinant protein antigen according to any one of claims 6-8 in the preparation of monkeypox virus monoclonal antibodies.

10. The application according to claim 9, characterized in that, The application includes: immunizing New Zealand white rabbits with monkeypox virus-specific recombinant protein antigen and Freund's adjuvant to prepare and purify monkeypox virus monoclonal antibodies.

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