Antigenic epitopes of parainfluenza virus type 5 f protein, monoclonal antibodies and uses thereof

Abstract

This invention relates to the field of biotechnology and provides an antigenic epitope of the parainfluenza virus type 5 F protein, a monoclonal antibody, and its applications. 135 AAILNLKNAIQKTNAAVADVVQA 157 Furthermore, a monoclonal antibody that specifically recognizes this antigenic epitope is provided. The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2. The discovery of this epitope provides a new target for the development of PIV5 diagnostic reagents. Peptide antigens can be designed and synthesized based on this epitope to develop highly specific ELISA diagnostic kits. Simultaneously, this epitope can be used as a candidate epitope for the design of multi-epitope vaccines, providing a new strategy for the prevention and control of PIV5.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antigenic epitope, monoclonal antibody, and application of parainfluenza virus type 5 F protein. Background Technology

[0002] Parainfluenza virus 5 (PIV5) belongs to the family Paramyxoviridae, subfamily Paramyxovirinae, and genus Rubulavirus. It is an enveloped, non-segmented, single-stranded, negative-sense RNA virus. First isolated from simian cells, it was named Simian virus (SV5). PIV5 can naturally infect various hosts, including dogs, cattle, minks, and humans. It is the core pathogen of kennel cough syndrome, and some strains can cause encephalitis in dogs and cattle, and digestive system diseases in pigs, causing losses to the livestock industry. Its potential tick-borne transmission characteristics and broad host tropism suggest that the virus can spread through tick bites, increasing the complexity of disease control.

[0003] PIV5 belongs to the Paramyxoviridae family, which is one of the leading pathogens causing respiratory diseases in humans and animals. Its genome is approximately 15,246 nucleotides long, following the "six-base rule," and encodes at least eight major proteins: nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), hemagglutinin-neuraminidase protein (HN), large protein (L), and small hydrophobic protein (SH). The F protein is responsible for the fusion of the viral envelope with the host cell membrane and is a key determinant of viral pathogenicity, as well as a major target for eliciting neutralizing antibodies in the host. While the PIV5 genome is relatively conserved, strains from different host sources exhibit certain genetic variations in key genes (especially the F and HN genes). These variations may be related to differences in viral host adaptability, tissue tropism, and pathogenicity. However, there are currently limited reports on the biological functions of the F protein, and more research is needed to clarify the exact function of the F protein in PIV5 infection. Summary of the Invention

[0004] To address the aforementioned issues, this invention utilizes the expressed F protein as an antigen to prepare monoclonal antibodies and identify their antigenic epitopes. These epitopes exhibit variations among different PIV5 strains, enabling the differentiation of some strains and providing an important basis for subsequent vaccine design and the establishment of serological diagnostic methods based on these epitopes.

[0005] Specifically, the present invention provides an antigenic epitope of parainfluenza virus type 5 F protein, wherein the antigenic epitope is... 135 AAILNLKNAIQKTNAAVADVVQA 157 .

[0006] Furthermore, the present invention provides a nucleic acid capable of encoding the above-mentioned antigenic epitope, wherein the nucleic acid is a gene sequence capable of encoding the antigenic epitope of the F protein as described in claim 1.

[0007] Furthermore, the present invention provides a recombinant expression vector carrying the aforementioned nucleic acid.

[0008] Furthermore, the present invention provides a recombinant cell containing the above-mentioned recombinant expression vector.

[0009] This invention provides a monoclonal antibody that specifically recognizes the antigenic epitope of the F protein described above.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0011] This invention provides the application of the above-mentioned antigenic epitope or the above-mentioned monoclonal antibody in the preparation of a kit for detecting parainfluenza virus type 5 infection.

[0012] This invention provides the use of the above-mentioned antigenic epitope or the above-mentioned monoclonal antibody in the preparation of drugs or vaccines for preventing and treating parainfluenza virus type 5 infection.

[0013] The beneficial effects of this invention are:

[0014] This invention focuses on amino acid positions 20–300 of the PIV5 F protein, which contains the fusion peptide region of the F protein. Using overlapping peptide scanning technology, a series of overlapping peptides covering the positive truncated fragment were designed to ultimately determine the core epitope sequence as 135–157 aa. Further analysis of whether this site is conserved across all strains revealed six variations by comparing sequences from 57 strains submitted to GenBank. Site-directed mutagenesis and Western blot identification showed that Ala alanine at position 143 of this core epitope is a key residue determining the binding specificity of the monoclonal antibody. This residue may bind to the antibody through hydrogen bonding, hydrophobic interactions, etc. Mutations in this residue can disrupt the epitope structure, resulting in loss of antigenic activity. The discovery of this epitope provides a new target for the development of PIV5 diagnostic reagents. Peptide antigens can be designed and synthesized based on this epitope to develop highly specific ELISA diagnostic kits. Simultaneously, this epitope can serve as a candidate epitope for the design of multi-epitope vaccines, providing a new strategy for PIV5 prevention and control.

[0015] Simultaneously, this invention successfully obtained the complete variable region nucleotide sequences of the heavy chain (VH) and light chain (VL) of cells secreting this monoclonal antibody through antibody gene sequencing analysis. The variable region, as the core structure for the specific binding of the antibody to the antigen, provides a foundation for further in-depth analysis of the binding mechanism of this monoclonal antibody to the 135-157 aa epitopes and for conducting structural biology research on antigen-antibody interactions. It also provides a key gene template for the heterologous expression and modification of recombinant antibodies. Genetic engineering techniques can be used to optimize the affinity, stability, and expression level of monoclonal antibodies, overcoming the limitations of high cost and large batch-to-batch variability in traditional hybridoma antibody preparation. Furthermore, after transfecting cells with recombinant plasmids containing the variable region sequence, the supernatant collected and detected by indirect immunofluorescence assay (IFA) and Western blot (WB) accurately identified the target antigen. This result not only verifies the functional integrity of the obtained variable region sequence and confirms its ability to mediate specific antigen binding activity, but also provides a feasible path for the subsequent large-scale preparation of recombinant functional antibodies, providing technical support for promoting the practical application of this type of monoclonal antibody in clinical testing and targeted prevention and control. Attached Figure Description

[0016] Figure 1SDS-PAGE was used to identify the expression of recombinant pCold TF-CPIV5-F (20-200 aa) protein; where M: protein molecular weight standard; 1: uninduced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3); 2: induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3); 3: supernatant of induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3) after sonication; 4: precipitate of induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3) after sonication.

[0017] Figure 2 SDS-PAGE identification of recombinant pCold TF-CPIV5-F (20-200 aa) protein purification; where M: protein molecular weight standard; 1: supernatant of induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3) after sonication; 2: flow-through solution of induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3); 3-7: purification of induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3);

[0018] Figure 3 Western blot analysis results of purified pCold TF-CPIV5-F (20-200 aa) protein; where 1: uninduced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3) bacterial culture; 2: induced pCold-TF-CPIV5-F (20-200 aa) / BL21(DE3);

[0019] Figure 4 The images show the IFA results of hybridoma cell lines; the first row shows the IFA results of hybridoma cell line 2E8 in MDCK cells infected with CPIV5 / Dog / 2015; the second row shows the IFA results of hybridoma cell line 2E8 in uninfected MDCK cells.

[0020] Figure 5 Western blot identification of MAb 2E8 antigenic epitopes; (A) fragment truncation display, (B) Western blot detection of MAb 2E8 on truncation fragments, (C) final identification of linear epitope amino acid display;

[0021] Figure 6The results of the MAb 2E8 antigenic epitope sequence alignment analysis included (a) amino acid alignment results of 57 PIV5 strains (6 amino acid changes in total); and (b) Western blotting analysis of MAb 2E8 against 6 mutant strains.

[0022] Figure 7 To validate the function of the MAb 2E8 expression plasmid; (A) IFA identification of 293T cell supernatant expressing antibody sequence in MDCK cells inoculated with CPIV5 / Dog / 2015; (B) Western blot analysis of CPIV5 / Dog / 2015 virus particles and recombinant F (20-200aa) protein in 293T cell supernatant expressing antibody sequence. Detailed Implementation

[0023] The embodiments of the present invention will be described in detail below. The following embodiments are implemented based on the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0024] As a pathogen that infects a wide range of hosts and causes certain economic losses, the development of prevention and control technologies for PIV5 has always been a focus of attention. The F protein is the main immunogenic protein of PIV5, and its antigenic epitopes are the core regions that induce specific immune responses in the body. Therefore, identifying the functional antigenic epitopes of the F protein is of great value for the diagnosis and vaccine development of PIV5.

[0025] This invention focuses on the 20-200 amino acid fragment of the PIV5 F protein, which contains the fusion peptide region of the F protein. Previous studies have shown that this region plays a key role in viral membrane fusion and may contain important immunoepitaxes, but there have been no previous reports on systematic epitope identification.

[0026] Example 1: Construction, expression and identification of recombinant plasmids

[0027] RNA was extracted from the CPIV5 (Dog / HLJ / 2015) strain using the FineQuick Viral DNA / RNA Extraction Kit. First-strand cDNA of CPIV5 (Dog / HLJ / 2015) was synthesized using PrimeScript™ II Reverse Transcriptase. The obtained cDNA was used as a template for PCR amplification of the CPIV5 (Dog / HLJ / 2015) F gene fragment. The primers used for PCR are as follows (underlined are the introduced KpnⅠ and EcoRI restriction sites):

[0028] Upstream primer pCold-TF-CPIV5-F (58-600bp):

[0029] 5'- GGTAGGCATATGGAGCTC GGTACC ATGGGTACTATAATTC -3'

[0030] Downstream primer pCold-TF-CPIV5-R (58-600bp):

[0031] 5'- TGCAGGTCGACAAGCTTTTA GAATTC TTACAAATAGAGATTGAGG -3'

[0032] The CPIV5 (Dog / HLJ / 2015) strain mentioned therein has been published in the article “Preparation of canine oligodendrocyte precursor cells and establishment of a neurotropic virus infection model” (Chinese Journal of Preventive Veterinary Medicine, April 2025, Vol. 47, No. 4).

[0033] PCR products were purified and recovered using a DNA gel extraction kit. KpnⅠ and EcoRI were selected as the double restriction enzyme sites for vector construction. The purified PCR products were recombined with the double-digested pCold TF linearized cloning vector. The mixture was incubated in a 50°C metal bath for 30 min and then immediately cooled on ice. The resulting product was then transformed into DH5α competent cells and plated on LB agar plates containing ampicillin. Single colonies were picked, cultured, and plasmids were extracted. DNA sequencing confirmed the recombinant plasmid was named pCold TF-CPIV5-F (58-600 bp).

[0034] The recombinant plasmid pCold TF-CPIV5-F (58-600bp) was transformed into E. coli BL21(DE3). After culturing until the OD600nm value reached 0.6-0.8, IPTG was added to one tube to a final concentration of 1 mmol / L, while the other tube was left untreated. Expression was induced at 37 ℃ for 4 h, and the expression of the recombinant protein was detected by SDS-PAGE. His-Tag-tagged antibody (1:8000) was used as the primary antibody, and IRDye 800CW-labeled goat anti-mouse IgG (1:8000) was used as the secondary antibody. Western blot was used to identify the reactivity of the protein.

[0035] The recombinant plasmid pCold TF-CPIV5-F (58-600 bp) was transformed into *E. coli* BL21(DE3). Single colonies were selected and cultured on LB medium containing ampicillin. When the OD600 value reached 0.4–0.6, 0.5 mM IPTG was added as an inducer, and the culture was incubated at 16°C with shaking at 180 rpm for 18 h to induce protein expression. After induction, the bacterial cells were collected, resuspended in PBS, and sonicated until the bacterial solution was clear. The solution was then centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was discarded, and the bacterial pellet was stored at -20°C or -80°C. The pCold TF-CPIV5-F (20-200 aa) protein was purified according to the HisSep Ni-NTA Agarose Resin (His-tagged protein agarose purification resin) instructions.

[0036] Results of expression and identification of recombinant plasmids:

[0037] The constructed recombinant plasmid pCold TF-CPIV5-F (58-600 bp) was transformed into E. coli BL21(DE3). SDA-PAGE analysis showed that the expressed protein molecular weight was consistent with expectations, approximately 71.7 kDa (see [link to SDA-PAGE]). Figure 1 The recombinant protein was identified by sonication as being expressed in a soluble form in the supernatant. The expressed product was purified by nickel column affinity chromatography to obtain the purified protein, which was then identified by SDS-PAGE (see [link to SDS-PAGE]). Figure 2 The results showed that a high-purity recombinant protein was obtained. Western blot was used to validate the purified protein, and detection with an anti-His monoclonal antibody confirmed correct protein expression (see...). Figure 3 ).

[0038] Example 2: Preparation of F (20-200 aa) protein MAb

[0039] 50 µg of purified F (20-200 aa) protein was emulsified with an equal volume of Freund's complete adjuvant and administered as a primary immunization to mice via intraperitoneal injection. Second and third booster immunizations were performed using the same method on days 15 and 30 post-primary immunization, respectively. One week after the third immunization, tail blood was collected from immunized mice. PIV5-infected cells were fixed and used as a detection plate, and serum antibody titers were measured using indirect immunofluorescence (IFA). Mice with the highest serum titers were selected for a booster immunization 3 days before cell fusion, via intramuscular injection of 50 µg of adjuvant-free F (20-200 aa) protein into the hind leg. Three days later, spleen cells from these mice were fused with mouse myeloma cells SP2 / 0. Using indirect immunofluorescence, hybridoma cell culture supernatant was used as the primary antibody, and HRP-labeled goat anti-mouse IgG (dilution ratio 1:5000) was used as the secondary antibody to screen for positive hybridoma cell lines capable of secreting F (20-200 aa) protein-specific monoclonal antibodies (MAb). The selected positive hybridoma cell lines were subjected to three subcloning processes and then expanded into larger cultures. Subsequently, the expanded positive monoclonal cell lines were injected into the peritoneal cavity of mice pretreated with Freund's incomplete adjuvant to induce the production of mouse monoclonal antibody ascites.

[0040] After fusing immunized mouse spleen cells with SP / 20 cells, an IFA assay was performed, ultimately identifying a hybridoma cell line capable of stably secreting MAb (anti-F (20-200 aa) protein), named 2E8. The MAb subtype was identified as IgG1, and the light chain as κ. The IFA results of hybridoma cell line 2E8 are as follows: Figure 4 As shown, the result was positive, proving that the monoclonal antibody is effective against CPIV5 and is a monoclonal antibody targeting CPIV5.

[0041] Example 3: Identification of MAb

[0042] Identification of MAb subclasses and potency: The potency of MAbs was determined using an ELISA method. Subclasses of differentially identified MAbs were determined according to the Pierce® RapidELISA Mouse MAb Isotyping Kit instructions.

[0043] Western blot identification of MAb reactivity: The expressed PIV5 F (20-200 aa) protein and ultracentrifuged purified CPIV5 (Dog / HLJ / 2015) virus particles were transferred to a nitrocellulose membrane (NC membrane) after SDS-PAGE electrophoresis. MAb (1:100) was used as the primary antibody and IRDye 800CW-labeled goat anti-mouse IgG (1:8000) was used as the secondary antibody. The reactivity of MAb with the virus and recombinant F (20-200 aa) protein was detected by Western blot.

[0044] Indirect immunofluorescence assay (IFA) for MAb reactivity: CPIV5 (Dog / HLJ / 2015) strain was inoculated into MDCK cells. After cytopathic effects appeared at 72 h, the cells were fixed, then permeabilized and blocked. The reactivity of MAb with the viral native F protein in MDCK cells was detected using Mab 2E8 (1:50) as the primary antibody and AlexaFluor 488-labeled goat anti-mouse IgG (H+L) (1:200) as the secondary antibody.

[0045] To determine the epitopes of MAb 2E8, three overlapping truncated fragments, FD1 (20-100 aa), FD2 (55-142 aa), and FD3 (110-200 aa), were first cloned into the pCold-TF vector to construct a recombinant plasmid expressing the truncated F protein, with the full-length F protein (20-200 aa) serving as a control. All fragments were sequenced and confirmed to be correct. The constructed plasmids were then transformed into BL21(DE3) competent cells, and expression was induced by IPTG. SDS-PAGE was used to detect the expression of the recombinant plasmids. MAb2E8 (1:100) was used as the primary antibody, and IRDye 800CW-labeled goat anti-mouse IgG (1:8000) was used as the secondary antibody. Western blotting was used to identify the antigenic region recognized by MAb 2E8. Figure 5 Western blot analysis initially identified the MAb 2E8 antigenic epitope in the F (110-200 aa) region. The F (110-200 aa) region was truncated and expressed. Each truncated gene fragment was fused with the pCold-TF vector and identified using Western blot. The results showed that... 135 AAILNLKNAIQKTNAAVADVVQA 157 It is the smallest linear epitope of MAb 2E8 (see...) Figure 5 ).

[0046] Example 4: Identification and Analysis of MAb Recognition Epitopes

[0047] The recombinant plasmid pCold TF-CPIV5-F (58-600 bp) contains a 543 bp gene fragment encoding a 181-amino acid F (20-200 aa) protein fragment. By expressing a series of overlapping truncated F (20-200 aa) protein fragments, the epitope region was gradually narrowed, thereby precisely locating the smallest epitope recognized by the monoclonal antibody. Specific primers were designed using Oligo 7 software (see Table 1), and EcoRI and KpnI restriction enzyme sites were introduced into the forward and reverse primers, respectively. The primers were synthesized by Beijing Ruibo Biotechnology Co., Ltd. To verify the length of the located epitope, the recombinant plasmids containing each truncated gene fragment were transformed into E. coli for induced expression to obtain the corresponding recombinant proteins. Subsequently, using the prepared monoclonal antibody as the primary antibody, these recombinant proteins were detected by Western blot to further confirm the shortest linear epitope recognized by the monoclonal antibody.

[0048] Table 1

[0049] Note: The underlined areas represent the EcoRI and HindIII restriction sites, respectively.

[0050] The MAb 2E8 antigenic epitope sequence, i.e., the amino acid sequence at positions 135–157, was compared with the F protein sequences of 56 PIV5 strains in the GenBank database. Six amino acid variations were found in this region: A135V (3 / 56), N142H / A149T (2 / 56), A149T (11 / 56), A149S (2 / 56), A143T (5 / 56), and A143S / A149T (1 / 56).

[0051] When the amino acid at position 143 is mutated from alanine (A) to threonine (T) or serine (S), the monoclonal antibody completely loses its ability to recognize the corresponding mutant strain (see [link to relevant documentation]). Figure 6 The sequence number indicates that amino acid position 143 is a key residue determining the binding specificity of this monoclonal antibody. The reported mutant strains at this site are five porcine isolates from China and one porcine isolate from South Korea, with sequence numbers DX 2020 PP711545 (2020), LZ-1 2021PP189887 (2021), ZY2021 PP711548 (2021), LZ-2 2021 PP711547 (2021), and JY 2022 PP711546 (2022). These are five porcine isolates from China and one porcine isolate from South Korea, M197 MK423240 (2017).

[0052] The above results indicate that the present invention has successfully obtained a monoclonal antibody targeting the linear epitope 135–157 of the CPIV5 F protein. This epitope exhibits a certain degree of variation among different PIV5 strains. This monoclonal antibody can be used to distinguish some porcine strains from other strains, providing an important basis for subsequent vaccine design and the establishment of serological diagnostic methods based on this epitope.

[0053] Example 5: Sequence analysis and functional identification of cell lines secreting monoclonal antibodies

[0054] To characterize the obtained monoclonal antibody at the molecular level, 2E8 hybridoma cells were sent to Jiangsu Baiying Biotechnology Co., Ltd. for antibody sequencing analysis. The complete variable region nucleotide sequences of the heavy chain (VH) and light chain (VL) of the monoclonal antibody were obtained, and the sequences are as follows.

[0055] The amino acid sequence of the heavy chain variable region:

[0056] MGWTYIILFLVATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTDYYMYWVKQRPGQGLEWIGGITPSNGRTNFNEKFKSKATLTVDYSSNTAYMQLSSLTSEDSAVYYCSRGGPIYYYGSTPFDYWGQGTTLTVSS

[0057] The amino acid sequence of the light chain variable region:

[0058] MSVPTQVLGLLLLWLTGARCDIQMTQSPASSLSASVGETVTITCRASENVYSYLAWYQQKQGKSPQLLVYDAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGTYYCQHHYGTPPTFGGGTKLEIK

[0059] The synthetic heavy chain expression plasmid (12 μg) and light chain expression plasmid (8 μg) were co-transfected into 293T cells. After 48 h, the supernatant was collected and used to perform indirect immunofluorescence experiments and Western blotting to verify the results in MDCK cells infected with PIV5.

[0060] To identify the functionality of the obtained sequences, heavy chain expression plasmids (12 μg) and light chain expression plasmids (8 μg) expressing antibody 2E8 were constructed, and their functions were verified by indirect immunofluorescence and Western blot experiments. The antibody expression plasmids were transiently transfected into 293T cells, and the cell culture supernatant was collected as the primary antibody. IFA detection was performed on MDCK cells inoculated with the CPIV5 / Dog / 2015 strain. Figure 7A) The results showed that specific green fluorescence signals appeared in MDCK cells inoculated with the virus, while no fluorescence appeared in the negative control. Simultaneously, Western blot analysis was performed on purified CPIV5 / Dog / 2015 virus particles and recombinant F (20-200 aa) protein obtained by ultracentrifugation. Figure 7 (B) The results showed that the viral particles exhibited a specific band at approximately 70 kDa, and the recombinant F(20-200) protein also showed a clear band at the corresponding position. These results indicate that the obtained heavy and light chain sequences encoding the 2E8 monoclonal antibody are accurate and have good functionality.

Claims

1. The antigenic epitope of parainfluenza virus type 5 F protein, characterized in that, The antigen epitope is 135 AAILNLKNAIQKTNAAVADVVQA 157 .

2. A nucleic acid capable of encoding the above-mentioned antigenic epitope, wherein the nucleic acid is a gene sequence capable of encoding the antigenic epitope of the F protein according to claim 1.

3. A recombinant expression vector carrying the nucleic acid of claim 2.

4. A recombinant cell, said recombinant cell containing the recombinant expression vector of claim 3.

5. A monoclonal antibody that specifically recognizes the antigenic epitope of the F protein of claim 1.

6. The monoclonal antibody as described in claim 5, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

7. The use of the antigenic epitope of claim 1 or the monoclonal antibody of claim 5 in the preparation of a kit for detecting parainfluenza virus type 5 infection.

8. The use of the antigenic epitope of claim 1 in the preparation of a vaccine against parainfluenza virus type 5.

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