Single-chain antibody recognizing extracellular region of PPRV H protein and application thereof

Abstract

This invention discloses a single-chain antibody that recognizes the extracellular region of the PPRV H protein and its application, belonging to the field of genetic engineering technology. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9. This invention provides a single-chain antibody that recognizes the extracellular region (185-609aa) of the PPRV H protein. Mice are immunized with the PPRV H protein extracellular region protein, and the VH and VL genes of the immunized monoclonal antibody are tandemly linked using monoclonal antibody preparation technology and DNA molecular technology to prepare the single-chain antibody. Neutralization tests have verified that this single-chain antibody can neutralize PPRV virus and can be used for the clinical detection of peste des petits ruminants (PPR).

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a single-chain antibody that recognizes the extracellular region of PPRV H protein and its application. Background Technology

[0002] Peste des petits ruminants (PPR) is an acute infectious disease caused by the peste des petits ruminants virus (PPRV). It primarily infects wild ruminants such as goats, sheep, deer, and antelopes, causing extremely high mortality rates in susceptible animal populations and posing a serious threat to global livestock economies and wildlife conservation. In my country, the Ministry of Agriculture and Rural Affairs has included PPR as one of the compulsory immunization diseases in the "National Guidelines for Compulsory Immunization of Animal Diseases (2022-2025)". Currently, the strategy of controlling PPR through compulsory vaccination is still limited by factors such as viral mutation and wild animal hosts. Therefore, developing rapid and effective new therapeutic drugs is crucial for eradicating PPR.

[0003] The H protein of PPRV is a key protein located on the surface of the viral envelope. It possesses hemagglutinin and neuraminidase activity, recognizing and binding to specific receptors on the host cell surface, mediating viral adsorption to the cell, thereby enabling viral invasion. The H protein can synergistically trigger the fusion of the viral envelope with the host cell membrane, releasing viral genetic material into the cell and completing the viral infection process. The H protein is an important antigen that stimulates the production of neutralizing antibodies in the host. Neutralizing antibodies against the H protein can effectively block the binding of the virus to receptors, thereby neutralizing the virus and providing immune protection. Given the crucial role of the H protein in inducing neutralizing antibody production, the development of high-affinity and high-specificity H protein antibodies is of great value for the diagnosis of PPRV.

[0004] Traditional monoclonal antibodies (mAbs) are widely used in disease diagnosis and treatment, but as large protein molecules, they have limitations such as high production costs, limited stability, immunogenicity, and difficulty in genetic engineering. Single-chain variable fragments (scFvs), on the other hand, are small-molecule genetically engineered antibodies composed of a heavy chain variable region (VH) and a light chain variable region (VL) linked together. Their smaller molecular weight compared to monoclonal antibodies allows them to potentially penetrate tissue barriers and exhibits lower immunogenicity. Furthermore, scFvs can be expressed in large quantities using protein expression systems, enabling low-cost, large-scale production.

[0005] In summary, this invention aims to develop a neutralizing scFv targeting the PPRV-H protein. Its development provides a powerful technical tool and product for the rapid diagnosis of PPR, and has broad application prospects and market value. Summary of the Invention

[0006] The purpose of this invention is to provide a single-chain antibody that recognizes the extracellular region of the PPRV H protein and its application, thereby solving the problems existing in the prior art. This invention provides a single-chain antibody that recognizes the extracellular region (185-609aa) of the PPRV H protein. Mice are immunized with the PPRV H protein extracellular region protein, and the VH and VL genes of the immunized monoclonal antibody are tandemly linked using monoclonal antibody preparation technology and DNA molecular technology to prepare the single-chain antibody. Neutralization tests have verified that this single-chain antibody can neutralize PPRV virus and can be used for the clinical detection, treatment, and research of peste des petits ruminants (PPRV).

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] The present invention provides a single-chain antibody that recognizes the extracellular region of PPRV H protein. The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9.

[0009] Furthermore, the single-chain antibody recognizes amino acid region 185-609 of the PPRV H protein.

[0010] The present invention also provides a gene encoding the above-mentioned single-chain antibody, wherein the nucleotide sequence of the gene is obtained by linking the heavy chain variable region gene sequence and the light chain variable region gene sequence of the above-mentioned single-chain antibody together via a linker.

[0011] The heavy chain variable region gene sequence is shown in SEQ ID NO.3; the light chain variable region gene sequence is shown in SEQ ID NO.2.

[0012] The present invention also provides a recombinant vector expressing the above-mentioned single-chain antibody, wherein the recombinant vector contains the above-mentioned gene.

[0013] The present invention also provides a recombinant microorganism expressing the above-mentioned single-chain antibody, wherein the recombinant microorganism contains the above-mentioned gene or the above-mentioned recombinant vector.

[0014] The present invention also provides the use of the above-mentioned gene, the above-mentioned recombinant vector, or the above-mentioned recombinant microorganism in the production of single-chain antibodies against PPRV H protein.

[0015] The present invention also provides the application of the above-mentioned single-chain antibody in the preparation of a drug for preventing and treating peste des petits ruminants virus infection.

[0016] The present invention also provides a drug for preventing and treating peste des petits ruminants virus infection, wherein the drug uses the above-mentioned single-chain antibody as the active ingredient.

[0017] The present invention also provides the application of the above-mentioned single-chain antibody in the preparation of products for detecting peste des petits ruminants virus, said products including reagents or kits.

[0018] The present invention also provides a kit for detecting peste des petits ruminants virus, the kit containing the above-mentioned single-chain antibody.

[0019] The present invention discloses the following technical effects:

[0020] This invention provides a single-chain antibody that recognizes the extracellular region (185-609aa) of the PPRV H protein. First, mice were immunized with the PPRV H extracellular region protein (185-609aa). A hybridoma cell line secreting the specific monoclonal antibody was obtained using monoclonal antibody preparation technology. The gene sequences of the antibody VH and VL regions were amplified from the cDNA of this cell line. The VH and VL genes were tandemly inserted into the plasmid pCMV using DNA molecular techniques to construct a eukaryotic expression vector for the single-chain antibody. After cell transfection, expression, and purification, the PPRV-H-Scfv single-chain antibody was obtained. Neutralization assays confirmed that this single-chain antibody can neutralize PPRV virus and can be used for the clinical detection of peste des petits ruminants (PPR). The single-chain antibody provided by this invention can neutralize PPRV, providing a new material for the development of PPRV diagnostic kits and offering technical support for reducing the production cost of PPR antibodies. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results of the PPRV-H-Scfv protein elution process were validated; where M: protein relative molecular mass standard; 1: flow-through buffer; 2: 2% Buffer B elution buffer; 3: 10% Buffer B elution buffer; 4: 20% Buffer B elution buffer; 5: 40% Buffer B elution buffer; 6: 60% Buffer B elution buffer;

[0023] Figure 2 The results validate the purification process of PPRV-H-Scfv protein; where M: protein relative molecular mass standard; 1-4: samples purified by Superdex 200 gel filtration chromatography in 4 tubes;

[0024] Figure 3 Cytopathic effects in the PPRV-H-Scfv neutralization assay;

[0025] Figure 4 CPE statistics for the PPRV-H-Scfv neutralization test;

[0026] Figure 5 The results of the Western blot analysis for the PPRV-H-Scfv neutralization assay. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0033] Example 1: Preparation of single-chain antibody against peste des petits ruminants (PPR) H protein

[0034] 1. Preparation of positive hybridoma cells

[0035] (1) Experimental materials: BALB / c mice, myeloma cells.

[0036] (2) Experimental method: BALB / c mice were immunized four times with recombinant PPRV-H (185-609 aa, SEQ ID NO.1) protein. Splenic lymphocytes of mice with high levels of anti-PPRV-H (185-609 aa) protein antibody in serum after immunization were fused with myeloma cells. Cells in wells with high levels of anti-PPRV-H (185-609 aa) protein antibody in the supernatant after fusion were subcloned. Hybridoma cells that could still stably secrete positive monoclonal antibodies after three subclonings could be expanded for culture and cryopreserved. This hybridoma cell line was named 3F6.

[0037] SEQ ID NO.1:

[0038] GTGCLGRTVTRAQFSELTLTLMDLDLEMKHNVSSVFTVVEEGLFGRTYTVWRSDTGKPSTSPGIGHFLRVFEIGLVRDLELGAPIFHMTNYLTVNMSDDYRSCLLAVGELKLTALCTPSETVTLSESGVPKREPLVVVILNLAGPTLGGELYSVLPTTDPTVEKLYLSSHRGIIKDNEANWVVPSTDVRDLQNKGECLVEACKTRPPSFCNGT GIGPWSEGRIPAYGVIRVSLDLASDPGVVITSVFGPLIPHLSGMDLYNNPFSRAAWLAVPPYEQSFLGMINTIGFPDRAEVMPHILTTEIRGPRGRCHVPIELSSR IDDDIKIGSNMVVLPTKDLRYITATYDVSRSEHAIVYYIYDTGRSSSYFYPVRLNFRGNPLSRIECFPWYHKVWCYHDCLIYNTITNEEVHTRGLTGIEVTCNPV.

[0039] 2. Constructing recombinant expression vectors for single-chain antibodies

[0040] (1) Experimental materials: Trizol, chloroform.

[0041] (2) Experimental methods: Total RNA was extracted from hybridoma cells 3F6 using Trizol and chloroform. cDNA was obtained using a reverse transcription kit. The antibody light chain variable region (SEQ ID NO.2) and heavy chain variable region (SEQ ID NO.3) were amplified by PCR. The heavy chain variable region and the light chain variable region were tandemly linked by a linker to obtain a recombinant DNA fragment. A mouse IgK signal peptide was inserted at the front end of this recombinant DNA fragment, and a 6×HIS tag and a FLAG tag were inserted at the end. The two tags were linked by a linker. The resulting recombinant sequence was named PPRV-H-Scfv (SEQ ID NO.4). The PPRV-H-Scfv sequence was inserted into the pCMV plasmid to construct the recombinant expression vector pCMV-PPRV-H-Scfv for single-chain antibodies.

[0042] Light chain variable region (VL), SEQ ID NO.2:

[0043] GACATTTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAATTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAGCTACTTGGTCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGC ATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTTATTACTGTCAACAATATTATAGTTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA.

[0044] Heavy chain variable region (VH), SEQ ID NO.3:

[0045] CAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTATTTTATAGAGTGGATAAAACAGAGGCCTGGACAGGGCCTTGAGTGGATTGGACTGATTAATCCTGGAAGTGGCGGTACTAACTACAATGCGAAGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAACTCTGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTACAAGAGTGGATTACGACGTAGATTATTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCTTCA。

[0046] PPRV-H-Scfv sequence, SEQ ID NO. 4:

[0047] GCCACCATGGAAACAGACACCCTGCTGCTGTGGGTCCTGCTGCTCTGGGTGCCTGGCAGCACCGGCCAGGTCCAGCTGCAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGGTGTCCTGCAAGGCTTCTGGATACGCCTTCACTAATTATTTTATAGAGTGGATAAAACAGAGGCCTGGACAGGGCCTTGAGTGGATTGGACTGATTAATCCTGGAAGTGGCGGTACTAACTACAATGCGAAGTTCAAGGGCAAGGCAACACTGACTGCAGACAAATCCTCCAACTCTGCCTACATGCAGCTCAGCAGCCTGACATCTGATGACTCTGCGGTCTATTTCTGTACAAGAGTGGATTACGACGTAGATTATTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCTTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAATTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAGCTACTTGGTCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAACAATATTATAGTTATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACATCACCACCATCATCACGGCGGTGGCAGCGGTGGCGGTAGCGGCGGTAGCGATTACAAGGATGACGACGATAAGTAA。

[0048] 3. Expression and purification of PPRV-H-Scfv protein

[0049] (1) Experimental materials: PEI transfection reagent, HisTrap™ excel, Superdex 200, 293F cells, SMM293-TII medium, SMS 293-SMPI cell growth medium.

[0050] (2) Experimental method:

[0051] 400 μg of the transfection plasmid and 1200 μg of PEI transfection reagent were introduced into 293F cells. The first addition of cell growth medium (SMS 293-SMPI) was given within 24 h of transfection, followed by additional additions on days 3 and 5. On day 7 of expression, the cell suspension was collected, and the supernatant was collected by centrifugation and filtered through a 0.45 μm filter once and a 0.22 μm filter once to obtain the cell supernatant containing the target protein.

[0052] After pretreatment of the HisTrap™ excel (5ml) affinity chromatography column, the cell supernatant was loaded onto the column. After loading the Ni affinity chromatography column, the sample solution was introduced. When the λ (280nm) (mAu) peak value remained constant, the protein sample was eluted using a gradient of different concentrations of elution buffer B (Buffer B: 1.2114g Tris, 14.61g NaCl, 17.02g imidazole dissolved in 400mL distilled water, brought to a final volume of 500mL, and pH adjusted to 7.4). After sample collection, SDS-PAGE staining was performed to analyze protein purification. Results are as follows: Figure 1 As shown, the target protein can be eluted when 10%-60% of Buffer B is used.

[0053] Based on the above validation results, the target protein sample was collected and concentrated. Simultaneously, the Superdex 200 gel filtration chromatography column was pretreated. During this process, the concentrated protein sample, after concentration determination, underwent pretreatment before loading by centrifugation at 12000 rpm for 10 min, and the supernatant was collected. The protein sample was then loaded, and quantitative collection began after peak elution. SDS-PAGE staining analysis was performed to assess the purification status of the target protein. After loading with the Superdex 200 molecular sieve, an absorption peak was observed at 16 mL. Sample collection began at this point, and four tubes of samples were ultimately collected. The staining results are as follows: Figure 2 As shown, the collected samples are purified target protein samples.

[0054] The amino acid sequence of the heavy chain variable region (VH) of the PPRV-H-Scfv protein is shown in SEQ ID NO.5:

[0055] QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYFIEWIKQRPGQGLEWIGLINPGSGGTNYNAKFKGKATLTADKSSNSAYMQLSSLTSDDSAVYFCTRVDYDVDYYALDYWGQGTSVTVSS;

[0056] Among them, CDR1 is: GYAFTNYF (SEQ ID NO.6);

[0057] CDR2 is: INPGSGGT (SEQ ID NO.7);

[0058] CDR3 is: TRVDYDVDYYALDY (SEQ ID NO.8).

[0059] The light chain variable region (VL) of the PPRV-H-Scfv protein is shown in SEQ ID NO.9:

[0060] DIVMSQSPSSLAVSIGEKVTMSCKSSQSLLYSSNQKSYLVWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPWTFGGGTKLEIK;

[0061] Among them, CDR1 is: QSLLYSSNQKSY (SEQ ID NO.10);

[0062] CDR2 is: WAS;

[0063] CDR3 is: QQYYSYPWT (SEQ ID NO.11).

[0064] The purified PPRV-H-Scfv protein sample was concentrated using a 10kDa ultrafiltration tube. The final protein sample concentration was determined and aliquoted for storage. After being flash-frozen in liquid nitrogen, the sample was stored at -80°C for later use.

[0065] Example 2: Neutralization efficacy assay of the small ruminant plague H protein single-chain antibody PPRV-H-Scfv

[0066] 1. Experimental materials: Vero cells, DMEM medium, PPRV-vaccine strain Nigeria 75 / 1.

[0067] 2. Experimental methods:

[0068] Mix PPRV-H-Scfv protein with 200 μL of 10 6 TCID 50After thoroughly mixing with PPRV at / mL, the mixture was inverted and reacted at 4℃ for 1 h. The reacted virus was then inoculated into Vero cells. After 2 h of inoculation, the supernatant containing the virus was discarded, and the cells were washed twice with PBS before adding 0.5% DMEM. Cytopathic effects were observed after 48 h. A virus-only inoculation group was set up as a positive control. The results are as follows: Figure 3 and Figure 4 As shown, compared with the PPRV inoculation group, the number of CPEs in the PPRV-H-Scfv (5 μg) + PPRV inoculation group was reduced, indicating that PPRV-H-Scfv has a neutralizing effect on the virus.

[0069] Different amounts of PPRV-H-Scfv (5 μg / 20 μg / 50 μg) were thoroughly mixed with PPRV and reacted at 4℃ for 1 h. The mixture was then inoculated into Vero cells. After 2 h of inoculation, the supernatant containing the virus was discarded, and the cells were washed twice with PBS and then inoculated with 0.5% DMEM. Cell protein samples were collected after 48 h, and the expression level of PPRV-V in each group was detected by Western blotting. A virus-only inoculation group was set up as a positive control. Results are as follows: Figure 5 As shown, compared with the PPRV inoculation group, the addition of a certain dose of PPRV-H-Scfv can relatively reduce the V protein in PPRV-infected cells. This indicates that PPRV-H-Scfv can neutralize PPRV virus, thereby reducing the virus's pathogenicity and replication ability.

[0070] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A single-chain antibody that recognizes the extracellular region of PPRV H protein, characterized in that, The amino acid sequence of the heavy chain variable region of the single-chain antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

9.

2. The single chain antibody according to claim 1, characterized in that, The single-chain antibody recognizes amino acid region 185-609 of the PPRV H protein.

3. A gene encoding the single chain antibody of claim 1 or 2. The nucleotide sequence of the gene is obtained by linking the heavy chain variable region gene sequence and the light chain variable region gene sequence of the single chain antibody according to claim 1 or 2 together with a linker. The heavy chain variable region gene sequence is shown in SEQ ID NO.3; the light chain variable region gene sequence is shown in SEQ ID NO.

2.

4. A recombinant vector expressing the single-chain antibody of claim 1 or 2, characterized in that, The recombinant vector contains the gene described in claim 3.

5. A recombinant microorganism expressing the single chain antibody of claim 1 or 2, wherein, The recombinant microorganism contains the gene of claim 3 or the recombinant vector of claim 4.

6. The use of the gene of claim 3, the recombinant vector of claim 4, or the recombinant microorganism of claim 5 in the production of single-chain antibodies against PPRV H protein.

7. The use of the single-chain antibody according to claim 1 or 2 in the preparation of a drug for preventing and treating peste des petits ruminants virus infection.

8. A medicament for preventing and treating infection of Peste des petits ruminants virus, characterized by, The drug uses the single-chain antibody as the active ingredient as described in claim 1 or 2.

9. Use of a single chain antibody according to claim 1 or 2 for the manufacture of a product for the detection of Peste des petits ruminants virus, characterized in that, The products include reagents or kits.

10. A kit for detecting Peste des petits ruminants virus, characterized in that, The kit contains the single-chain antibody as described in claim 1 or 2.

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