Specific nano antibody of measles virus nucleoprotein as well as preparation method and application of specific nano antibody

Abstract

The invention provides a measles virus nucleoprotein specific nano antibody as well as a preparation method and application thereof. The amino acid sequence of the nano antibody is any one of A1-A18; the rash virus nucleoprotein can be specifically recognized and combined. And the nano antibody can be massively expressed and purified in escherichia coli cells, so that a potential nano antibody drug with a great application prospect is provided for clinical prevention, treatment and detection of measles viruses.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular biology and immunology, and particularly relates to a nanobody specific to measles virus nucleoprotein and a preparation method and application thereof. BACKGROUND

[0002] Measles virus (MV) is a highly contagious pathogen belonging to the Paramyxoviridae family. It is primarily transmitted through airborne droplets and infects the upper respiratory tract and lymphatic system of the host, leading to cellular infection and damage, and subsequently triggering systemic reactions. The incubation period of infection is typically 10-14 days, and common symptoms include high fever, cough, flu-like symptoms, and the characteristic measles rash. Measles virus can occur worldwide, and recurrent infections are common in areas with low vaccination rates.

[0003] As research on measles virus continues to deepen, scientists have gained a more comprehensive understanding of its structure. The structure of measles virus mainly includes viral genome composed of single-stranded negative-sense RNA, envelope, and nucleoprotein (NP) and other important components. Nucleoprotein NP plays a crucial role in the replication and transcription of the virus. Its main functions include: binding to the virus's negative-sense single-stranded RNA to form a ribonucleoprotein complex (RNP) to protect the RNA from degradation. NP helps the viral RNA polymerase recognize and bind to the RNA template during viral transcription and replication, thereby promoting the transcription and replication of viral genes. In addition, during viral assembly, NP participates in the formation of viral particles together with other structural proteins, ensuring the integrity of the virus and its infectivity. Finally, as a strong immunogenic protein, NP can activate the host's immune system and induce the production of specific antibodies. Due to its conservation, NP is relatively consistent in different types of measles virus. Therefore, the development of antibodies against measles virus nucleoprotein has become an important research direction in the field of public health, and is expected to provide new ideas for the prevention and detection of viral infection and its related complications. In summary, the nucleoprotein of measles virus plays an important role in the life cycle of the virus, and the antibodies produced by it are the key to detecting and resisting viral infection. A deeper understanding of the functions of nucleoprotein and the immune response induced by it will help to develop more effective vaccines and treatment strategies to address the challenges of measles virus infection.

[0004] Antibodies are a crucial tool in the study of measles virus, particularly for the prevention of measles. Developing antibodies against the nucleoprotein of measles virus will provide researchers with powerful tools to study the function and regulation of measles virus in various biological processes such as infection, immune evasion, and pathological mechanisms. This will help better understand the infection mechanism of measles virus, provide new directions and targets for prevention and treatment strategies, and provide rich materials for basic biological research. Through the study of antibodies against measles virus, scientists can identify and verify the antigenic properties of the virus, evaluate the neutralizing ability of the antibodies, and explore their potential applications in vaccine development. In addition, antibodies against measles virus can also be used for clinical diagnosis to help doctors quickly identify infected cases and develop more effective treatment plans. In summary, the development and application of antibodies against the nucleoprotein of measles virus not only promote the progress of measles virus research, but also provide important support for prevention and control measures in the field of public health. SUMMARY

[0005] The embodiments of the present application provide a measles virus nucleoprotein specific nanobody and its preparation method and application to solve the problems existing in the related art. The technical solutions are as follows: In a first aspect, the embodiments of the present application provide a measles virus nucleoprotein specific nanobody, which is any one of the amino acid sequences of the following complementarity determining regions (CDRs) 1, 2, and 3: C1, CDR1 is GRIFSRYTM; CDR2 is TWGGGRTS; and CDR3 is AAGYYCAGYGCYDARQYP; C2, CDR1 is GFRFSRYAM; CDR2 is SWSGGSIY; and CDR3 is AAGSTFIRGLAPSEPTQYA; C3, CDR1 is GRTFSRSRM; CDR2 is GWSGASTY; and CDR3 is AADRIFEPSVWARYS; C4, CDR1 is GRTFSRPTM; CDR2 is SWSGGSTV; and CDR3 is AADYDLDYELGFGDPKYD; C5, CDR1 is GRTLSRYYM; CDR2 is SWTGYSTY; and CDR3 is AKIISSGWAYGMD; C6, CDR1 is GRTFSSYNM; CDR2 is RWSGGIRY; and CDR3 is AADLTSYSDYGDAE; C7, CDR1 is GRTFMPYTM; CDR2 is TWSGGNSY; and CDR3 is NAGLGTLTM; C8, CDR1 is GRTFRSYAM; CDR2 is NWSGGSTY; CDR3 is NADVRSIGTGYLRN; C9, CDR1 is GRTFSSYAM; CDR2 is IYSGGSTY; CDR3 is AAADRKAILTPMGYK; C10, CDR1 is GGTFTTFSKYGV; CDR2 is SWNGGSTY; CDR3 is YADAASYSGRRRGR; C11, CDR1 is GFTFSRNAI; CDR2 is STSGSTN; CDR3 is HAEILDFGLGYGYD; C12, CDR1 is GSAFSINFI; CDR2 is SSGGSTN; CDR3 is NLNSGIAYRRD; C13, CDR1 is GRTFSTYVM; CDR2 is SQRGTVTA; CDR3 is AARLHYDLGAVLQERQSYD; C14, CDR1 is GGTFSRYAM; CDR2 is SRSGLRTY; CDR3 is AAGTMTFSTPSWH; C15, CDR1 is GRTFSIYTM; CDR2 is TSGGSTY; CDR3 is AAAFSSYYSPNTYFTRPSQYN; C16, CDR1 is RRTSSSIAM; CDR2 is TWIGSTW; CDR3 is ASSKYYRGSYPGGPNPYEYD; C17, CDR1 is RRTFSTYAM; CDR2 is RGSAGSTR; CDR3 is AARTRGSYWDTRYTVPNGYE; C18, CDR1 is GRTFTNYVM; CDR2 is TWSGLRKY; CDR3 is GADTPRHVFFYVPVRENEYD.

[0006] In one embodiment, the amino acid sequence of the Nanobody is any one of: A1, the amino acid sequence shown in SEQ ID NO. 1; A2, the amino acid sequence shown in SEQ ID NO. 2; A3, the amino acid sequence shown in SEQ ID NO. 3; A4, the amino acid sequence shown in SEQ ID NO. 4; A5, the amino acid sequence shown in SEQ ID NO. 5; A6, the amino acid sequence shown in SEQ ID NO. 6; A7, the amino acid sequence shown in SEQ ID NO. 7; A8, the amino acid sequence shown in SEQ ID NO. 8; A9, the amino acid sequence shown in SEQ ID NO. 9; A10, the amino acid sequence shown in SEQ ID NO. 10; A11, the amino acid sequence shown in SEQ ID NO. 11; A12, the amino acid sequence shown in SEQ ID NO. 12; A13, the amino acid sequence shown in SEQ ID NO. 13; A14, the amino acid sequence shown in SEQ ID NO. 14; A15, the amino acid sequence shown in SEQ ID NO. 15; A16, the amino acid sequence shown in SEQ ID NO. 16; A17, the amino acid sequence shown in SEQ ID NO. 17; A18, the amino acid sequence shown in SEQ ID NO. 18; or the amino acid sequence shown in A1-A17 is substituted and / or deleted and / or added by one or several amino acid residues and encodes the same active protein.

[0007] In a second aspect, the embodiments of the present application provide a nucleic acid molecule encoding the nanobody described above, and the nucleotide sequence of the nucleic acid molecule is any one of the following: B1, the nucleotide sequence shown in SEQ ID NO. 19; B2, the nucleotide sequence shown in SEQ ID NO. 20; B3, the nucleotide sequence shown in SEQ ID NO. 21; B4, the nucleotide sequence shown in SEQ ID NO. 22; B5, the nucleotide sequence shown in SEQ ID NO. 23; B6, the nucleotide sequence shown in SEQ ID NO. 24; B7, the nucleotide sequence shown in SEQ ID NO. 25; B8, the nucleotide sequence shown in SEQ ID NO. 26; B9, the nucleotide sequence shown in SEQ ID NO. 27; B10, the nucleotide sequence shown in SEQ ID NO. 28; B11, the nucleotide sequence shown in SEQ ID NO. 29; B12, the nucleotide sequence shown in SEQ ID NO. 30; B13, the nucleotide sequence shown in SEQ ID NO. 31; B14, the nucleotide sequence shown in SEQ ID NO. 32; B15, the nucleotide sequence shown in SEQ ID NO. 33; B16, the nucleotide sequence shown in SEQ ID NO. 34; B17, the nucleotide sequence shown in SEQ ID NO. 35; B18, the nucleotide sequence shown in SEQ ID NO. 36; or a nucleotide sequence represented by B1-B18 is substituted and / or deleted and / or added by one or several nucleotides and encodes a nucleotide sequence of the same active protein.

[0008] In a third aspect, the embodiments of the present application provide a recombinant vector; the vector is pET 28a.

[0009] In a fourth aspect, the embodiments of the present application provide a recombinant cell comprising the nucleic acid molecule or the recombinant vector or the recombinant cell.

[0010] In a fifth aspect, the embodiments of the present application provide a method for constructing the recombinant cell, comprising introducing the recombinant vector into a host cell.

[0011] In an embodiment, the host cell comprises a prokaryotic host cell or a eukaryotic host cell.

[0012] In an embodiment, the host cell is Escherichia coli.

[0013] In a fifth aspect, the embodiments of the present application provide a method for constructing the measles virus nucleoprotein specific nanobody, comprising the following steps: Step 1: constructing a recombinant expression plasmid using a DNA sequence comprising human Measles virus-NP, transfecting a recombinant cell to construct a recombinant cell and recombinantly expressing a recombinant Measles virus-NP protein; Step 2: immunizing a camelid with the recombinant Measles virus-NP protein to promote the camelid to produce an antibody; Step 3: screening a specific nanobody using a helper phage surface display; sequencing and expressing the target antibody to obtain a measles virus nucleoprotein specific nanobody.

[0014] In an embodiment, the amino acid sequence of the recombinant Measles virus-NP protein is shown as SEQ ID NO. 37; the corresponding nucleotide sequence is shown as SEQ ID NO. 38.

[0015] In a sixth aspect, the embodiments of the present application provide an application of the nanobody, the nucleic acid molecule, the recombinant vector, the recombinant cell or the recombinant cell constructed by the method in the preparation of a measles virus detection reagent.

[0016] In a seventh aspect, the embodiments of the present application provide a measles virus detection reagent, comprising the nanobody, the nucleic acid molecule, the recombinant vector, the recombinant cell or the recombinant cell constructed by the method.

[0017] In an eighth aspect, the embodiments of the present application provide a kit for detecting a measles virus, the kit comprising the nanobody described above.

[0018] The advantages or beneficial effects of the above technical solutions at least include: The present application provides a measles virus nucleoprotein specific nanobody, which has high affinity for measles virus nucleoprotein NP, and can be expressed and purified in large quantities in E. coli cells, with a batch capacity of about several milligrams. The present application provides a potential and highly promising nanobody drug for the clinical prevention, treatment and detection of measles virus.

[0019] The above summary is merely intended to illustrate the present application and is not intended to limit in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present application will be readily apparent to those skilled in the art by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0020] In the drawings, like reference numerals refer to like elements throughout the various drawings. The drawings are not necessarily to scale, the emphasis instead being placed upon illustrating certain principles of the application. It should be understood that the drawings are merely depictions of some embodiments of the application and should not be construed as limiting the scope of the application.

[0021] Figure 1 Figure 9A10 (A1) and MV-NP specific affinity results chart; Figure 2 Figure 2G7 (A2) and MV-NP specific affinity results chart; Figure 3 Figure 5H8 (A3) and MV-NP specific affinity results chart; Figure 4 Figure 1B4 (A4) and MV-NP specific affinity results chart; Figure 5 Figure 9B6 (A5) and MV-NP specific affinity results chart; Figure 6 Figure 8G6 (A6) and MV-NP specific affinity results chart; Figure 7 Figure 11H3 (A7) and MV-NP specific affinity results chart; Figure 8 Figure 9A11 (A8) and MV-NP specific affinity results chart; Figure 9 Figure 9F10 (A9) and MV-NP specific affinity results chart; Figure 10Figure for 10H2 (A10) and MV-NP specific affinity results; Figure 11 Figure for 5B3 (A11) and MV-NP specific affinity results; Figure 12 Figure for 8E9 (A12) and MV-NP specific affinity results; Figure 13 Figure for 10A8 (A13) and MV-NP specific affinity results; Figure 14 Figure for 11G8 (A14) and MV-NP specific affinity results; Figure 15 Figure for 3C11 (A15) and MV-NP specific affinity results; Figure 16 Figure for 2H8 (A16) and MV-NP specific affinity results; Figure 17 Figure for 8H1 (A17) and MV-NP specific affinity results; Figure 18 Figure for 2B3 (A18) and MV-NP specific affinity results. DETAILED DESCRIPTION

[0022] Hereinafter, only certain exemplary embodiments are simply described. As can be appreciated by one skilled in the art, the described embodiments can be modified in various different manners without departing from the spirit or scope of the present application. Therefore, the drawings and descriptions are to be regarded as illustrative in nature rather than restrictive.

[0023] The present application aims to develop nanobodies based on the immune system of a llama, design and implement effective and feasible nanobody screening and preparation technical solutions, and obtain nanobodies that can specifically recognize and bind MV-NP and are capable of batch production and preparation. The present application constructs a DNA sequence encoding human Measles virus-NP into a vector to form a Measles virus-NP recombinant expression plasmid; recombinant expression obtains a recombinant Measles virus-NP protein; the recombinant Measles virus-NP protein is used to immunize a camelid to promote the camelid to produce antibodies; a helper phage surface display is used to screen specific nanobodies; the target antibodies are sequenced and expressed to obtain measles virus nucleoprotein specific nanobodies.

[0024] Therefore, the present application provides a measles virus nucleoprotein specific nanobody, and the amino acid sequence of the nanobody is any one of the following: A1, the amino acid sequence shown in SEQ ID NO. 1; A2, the amino acid sequence shown in SEQ ID NO. 2; A3, the amino acid sequence shown in SEQ ID NO. 3; A4, the amino acid sequence shown in SEQ ID NO. 4; A5, the amino acid sequence shown in SEQ ID NO. 5; A6, the amino acid sequence shown in SEQ ID NO. 6; A7, the amino acid sequence shown in SEQ ID NO. 7; A8, the amino acid sequence shown in SEQ ID NO. 8; A9, the amino acid sequence shown in SEQ ID NO. 9; A10, the amino acid sequence shown in SEQ ID NO. 10; A11, the amino acid sequence shown in SEQ ID NO. 11; A12, the amino acid sequence shown in SEQ ID NO. 12; A13, the amino acid sequence shown in SEQ ID NO. 13; A14, the amino acid sequence shown in SEQ ID NO. 14; A15, the amino acid sequence shown in SEQ ID NO. 15; A16, the amino acid sequence shown in SEQ ID NO. 16; A17, the amino acid sequence shown in SEQ ID NO. 17; A18, the amino acid sequence shown in SEQ ID NO. 18; or the amino acid sequence shown in A1-A17 is substituted and / or deleted and / or added by one or several amino acid residues and the amino acid sequence encoding the same active protein.

[0025] wherein the amino acid sequence of A1-A17 all comprises the complementarity determining region CDR1, CDR2 and CDR3, and the conservative framework region FR1, FR2 and FR3; the FR1, FR2, FR3 and FR4 are staggered with CDR1, CDR2 and CDR3 in order.

[0026] Specifically, the complementarity determining region sequence and the conservative framework region sequence of the amino acid sequence of A1-A17 are shown in Table 1.

[0027] Table 1

[0028]

[0029] The nanobody has high affinity to the nucleoprotein of measles virus and inhibits the infection of measles virus. The present application provides a potential and promising nanobody drug for the clinical prevention, treatment and detection of measles virus.

[0030] Correspondingly, the nucleic acid molecules encoding the above-mentioned nanobodies are as follows: Nucleotide sequences B1, SEQ ID NO. 19; B2, SEQ ID NO. 20; B3, SEQ ID NO. 21; B4, SEQ ID NO. 22; B5, SEQ ID NO. 23; B6, SEQ ID NO. 24; B7, SEQ ID NO. 25; B8, SEQ ID NO. 26; B9, SEQ ID NO. 27; B10, SEQ ID NO. 28; B11, SEQ ID NO. 29; B12, SEQ ID NO. 30; B13, SEQ ID NO. 31; B14, SEQ ID NO. 32; B15, SEQ ID NO. 33; B16, SEQ ID NO. 34; B17, SEQ ID NO. 35; B18, SEQ ID NO. 36. Or, nucleotide sequences shown in B1-B18 that have been substituted and / or deleted and / or added with one or more nucleotides and encode the same active protein.

[0031] The nucleotide sequences of B1-B17 correspond to the amino acid sequences of A1 and A17, respectively. The nucleotide sequences of B1 and B17 correspond to the complementarity-determining regions CDR1, CDR2, and CDR3 of A1-A17, as well as the conserved framework regions FR1, FR2, and FR3, which will not be listed in detail here.

[0032] This application provides a recombinant vector. A vector refers to a medium capable of delivering exogenous DNA or a target gene into a host cell for amplification and / or expression. The vector can be a cloning vector or an expression vector. As one embodiment, the vector in this application is a pET vector. 28a.

[0033] This application provides recombinant cells comprising the aforementioned nucleic acid molecules, recombinant vectors, or nanobodies. Exogenous target genes or recombinant vectors are introduced into host cells, or endogenous genes in the host cells are directly edited. This manipulates and modifies the genes of the host cells, altering their function.

[0034] This application provides a method for constructing the above-described recombinant cells, including introducing the above-described recombinant vector into host cells.

[0035] In one embodiment, the host cell includes a prokaryotic host cell or a eukaryotic host cell.

[0036] In an embodiment, the host cell is E. coli.

[0037] In a fifth aspect, the embodiments of the present application provide a method for constructing the measles virus nucleoprotein specific nanobody as described above, comprising the following steps: Step 1: constructing a recombinant expression plasmid using a DNA sequence encoding a human-derived Measles virus-NP, transfecting a recombinant cell to construct the recombinant cell, and recombinantly expressing a recombinant Measles virus-NP protein; Step 2: immunizing a camelid with the recombinant Measles virus-NP protein to induce the camelid to produce antibodies; Step 3: screening specific nanobodies using helper phage surface display; sequencing and expressing the target antibodies to obtain a measles virus nucleoprotein specific nanobody.

[0038] In an embodiment, the amino acid sequence of the recombinant Measles virus-NP protein is shown as SEQ ID NO. 37; and the corresponding nucleotide sequence is shown as SEQ ID NO. 38.

[0039] The embodiments of the present application provide an application of the nanobody as described above, the nucleic acid molecule as described above, the recombinant vector as described above, the recombinant cell as described above, or the recombinant cell constructed by the method as described above, in the preparation of a measles virus detection reagent.

[0040] The embodiments of the present application provide a measles virus detection reagent, which comprises the nanobody as described above, the nucleic acid molecule as described above, the recombinant vector as described above, the recombinant cell as described above, or the recombinant cell constructed by the method as described above.

[0041] The embodiments of the present application provide a measles virus detection kit, which comprises the nanobody as described above.

[0042] The present application is further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods, or directly purchased from the market.

[0043] Example 1 1. Preparation of antigen 1.1 Constructing a DNA sequence encoding a human-derived Measles virus-NP into a pet-28a E. coli expression vector to form a Measles virus-NP recombinant expression plasmid; the amino acid sequence of the completely expressed recombinant Measles virus-NP protein is shown as SEQ ID NO. 37.

[0044] The corresponding DNA sequence is shown as SEQ ID NO. 38.

[0045] 1.2 Transfect the Measles virus-NP recombinant expression plasmid into BL21(DE3) competent cells, and culture to obtain a monoclonal strain expressing Measles virus-NP protein; 1.3 After culturing the monoclonal strain at 37°C, induce the expression of Measles virus-NP protein at 16°C by adding 0.4mM inducer (IPTG, isopropyl-β-D-thiogalactoside); 1.4 Collect all bacteria, and after processes such as lysis, centrifugation, affinity chromatography, and gel filtration chromatography, obtain recombinantly expressed Measles virus-NP protein.

[0046] 2, Immunization of alpaca (1) Immunize the alpaca a total of 4 times, each time injecting 0.2 mg of antigen (recombinantly expressed Measles virus-NP protein) into the animal subcutaneously. The first immunization is recorded as day 1, and the subsequent immunizations are on day 11, day 21, and day 31, respectively; (2) On day 30, before the fourth immunization injection, collect about 200 mL of alpaca venous peripheral blood; (3) On day 45, i.e. 14 days after the fourth immunization, collect about 200 mL of alpaca venous peripheral blood.

[0047] Compared with traditional mouse, rabbit, and other animal antibody immunization technical solutions, the present technology has the advantage of collecting a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening of highly diverse nanobodies.

[0048] 3. Construction of alpaca nanobody library The 2 batches of alpaca venous peripheral blood collected in the above stages are used as raw materials to construct a high-diversity nanobody library.

[0049] The processing methods of the 2 batches of alpaca venous peripheral blood are the same: (1) Use density gradient centrifugation and other methods to separate lymphocytes from the alpaca venous peripheral blood; (2) Extract total mRNA from the lymphocytes and reverse transcribe it into cDNA; (3) Use appropriate DNA primers (Table 2) to amplify the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e. the DNA fragments of nanobodies, from the above cDNA as templates by polymerase chain reaction (PCR); Table 2

[0050] (4) Link the DNA of VHH to the phage surface display selection vector (Phen1) to form a VHH-pIII fusion protein expression vector plasmid library; where pIII is a protein present on the flagella of the phage surface.

[0051] (5) The DNA ligation product was transformed into TG1 competent bacteria by electroporation. After appropriate culture, all colonies were collected to form the alpaca nanobody library.

[0052] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this technical solution can preserve all nanobody fragments (i.e., libraries) of alpacas for a long time, which can continuously support the subsequent screening and development of nanobodies.

[0053] 4. Displaying and screening specific nanobodies on the surface of bacteriophages Using the nanobody library obtained in the above stages as the source, antigen-specific nanobodies were obtained through phage surface display screening. The specific technical solution is as follows: (1) Take an appropriate amount of frozen nanobody library, inoculate it into bacterial culture medium, and after appropriate culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture under appropriate conditions. (2) Phages amplified in bacterial culture supernatant were extracted using the PEG-NaC method; (3) Incubate the phage with the antigen appropriately, and fix the antigen in the immunoassay tube (Maxisorp immunoassay tube, ThermoFisher Scientific); (4) Washing: Discard the phages, then wash the antigen with PBS buffer an appropriate number of times (3-5 times) to wash away the phages that are not specifically bound to the antigen, and retain the phages that are specifically bound to the antigen. (5) Elution: The phages were eluted with an acidic glycine solution to dissociate them from the antigen and retain them. This yielded phages expressing specific nanobodies, which can then be subjected to the following technical operations: (6) Transformation into a specific nanobody library. The phage is re-infected with E. coli cultured to a suitable state, but no helper phage is added. After complete phage infection, the specific nanobody exists in the E. coli in the form of DNA plasmids. Collecting all these E. coli results in an antigen-specific nanobody library. This library can be used as raw material to return to step (1) for the next round of phage surface display screening. (7) Transform into single clone nanobody colony. Take a small amount of phage obtained in step (5) (e.g. 0.5%), dilute it and then infect E. coli which has been cultured to an appropriate state again, but do not add helper phage again. After the phage infection is complete, evenly spread the E. coli on a bacterial culture dish and culture it under appropriate conditions to obtain single clone colonies containing nanobody DNA plasmids.

[0054] 5. Identify positive single clone nanobodies.

[0055] The bacterial culture dish with single clone colonies obtained through the above stage 4 step (7) can be used to identify positive single clone nanobodies. The specific technical scheme is as follows: (1) Pick single clone colonies and culture them in a microplate; (2) Add IPTG (isopropyl-β-D-thiogalactoside) to induce VHH-pII (i.e. a fusion protein containing nanobodies) expression; (3) Collect the bacterial culture supernatant containing nanobodies, incubate it with antigens, and fix the antigens in advance in a 96-well microplate (Maxisorp transparent microplate, ThermoFisher Scientific); (4) Use enzyme-linked immunosorbent assay (ELISA) to detect whether the single clone nanobodies bind to antigens, and the steps are as follows: a. Coating: Dilute the antigens with PBS to 5 μg / mL, 50 μL / well, and shake and incubate for coating overnight at 4°C; b. Blocking: The next day, discard the antigens, add 100 μL / well of PBS-2% BSA, and shake and incubate for 1 hour at room temperature; c. Washing: 3 times with PBST and 3 times with PBS, 150 μL / well; d. Add the culture supernatant, 50 μL / well, and shake and incubate for 1-2 hours at room temperature; e. Washing: 3 times with PBST and 3 times with PBS, 150 μL / well; f. Add diluted anti-myc HRP, and incubate for 1 hour at room temperature; g. Washing: 3 times with PBST and 3 times with PBS, 150 μL / well; h. Add ELISA color developing substrate, and incubate for 30 min at room temperature in the dark; i. Read OD405nm.

[0056] (5) For single clone nanobody microbial colonies that can bind to antigens, extract the DNA plasmid after appropriate culture again and perform DNA sequencing to obtain the nanobody nucleic acid sequence. After translation, the complete amino acid sequence of the nanobody can be obtained, as shown in Table 1.

[0057] 6. Small batch recombinant expression and purification of monoclonal nanobodies (1) The monoclonal nanobodies obtained in the previous stage can specifically recognize and bind to antigens. The DNA plasmid of the nanobodies is transformed into BL21(DE3) competent cells, and the monoclonal nanobodies can be expressed and purified in small batches by means of the E. coli expression system, with a batch production capacity of about several milligrams.

[0058] (2) Using the ELISA method, incubate nanobodies of different concentrations, and measure the affinity of the nanobodies to the antigen according to the binding ability of the nanobodies to the antigen. The results are shown in Figures 1-18 .

[0059] Figure 1 Figure 9A10 (A1) and MV-NP specific affinity results, with an OD405nm value of 48.71 nM; Figure 2 Figure 2G7 (A2) and MV-NP specific affinity results, with an OD405nm value of 26.39 nM; Figure 3 Figure 5H8 (A3) and MV-NP specific affinity results, with an OD405nm value of 9.555 nM; Figure 4 Figure 1B4 (A4) and MV-NP specific affinity results, with an OD405nm value of 51.72 nM; Figure 5 Figure 9B6 (A5) and MV-NP specific affinity results, with an OD405nm value of 26.03 nM; Figure 6 Figure 8G6 (A6) and MV-NP specific affinity results, with an OD405nm value of 51.72 nM; Figure 7 Figure 11H3 (A7) and MV-NP specific affinity results, with an OD405nm value of 7.561 nM; Figure 8 Figure 9A11 (A8) and MV-NP specific affinity results, with an OD405nm value of 34.07 nM; Figure 9 Figure 9F10 (A9) and MV-NP specific affinity results, with an OD405nm value of 51.33 nM; Figure 10 Figure 10H2 (A10) and MV-NP specific affinity results, with an OD405nm value of 14.30 nM; Figure 11 Figure 5B3 (A11) and MV-NP specific affinity results, with an OD405nm value of 50.59 nM; Figure 12 Figure 8E9 (A12) and MV-NP specific affinity results, with an OD405nm value of 39.21 nM; Figure 13Figure 8 is a graph of the affinity results for 10A8 (A13) and MV-NP specificity, wherein the OD405nm value is 84.63 nM; Figure 14 Figure 9 is a graph of the affinity results for 11G8 (A14) and MV-NP specificity, wherein the OD405nm value is 9.051 nM; Figure 15 Figure 10 is a graph of the affinity results for 3C11 (A15) and MV-NP specificity, wherein the OD405nm value is 8.461 nM; Figure 16 Figure 11 is a graph of the affinity results for 2H8 (A16) and MV-NP specificity, wherein the OD405nm value is 36.31 nM; Figure 17 Figure 12 is a graph of the affinity results for 8H1 (A17) and MV-NP specificity, wherein the OD405nm value is 13.26 nM; Figure 18 Figure 13 is a graph of the affinity results for 2B3 (A18) and MV-NP specificity, wherein the OD405nm value is 58.54 nM.

[0060] Therefore, the measles virus nucleoprotein specific nanobodies of the present application have good affinity with measles virus nucleoprotein, and can realize specific recognition and binding of MV-NP; the nanobodies are obtained based on the immune system of a llama and are prepared in batches; the nanobodies can realize clinical diagnosis of measles virus and help doctors quickly identify infection cases, so as to formulate more effective treatment plans.

[0061] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. In addition, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0062] In addition, the terms "first", "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specifically limited.

[0063] The above merely provides the specific implementation of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of various changes or replacements within the technical range disclosed by the present application, and these should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A measles virus nucleoprotein-specific nanobody, characterized in that, The nanobody is any one of the amino acid sequences including the following complementarity-determining regions CDR1, CDR2 and CDR3; C1 and CDR1 are GRIFSRY™; CDR2 is TWGGGRTS; CDR3 is AAGYYCAGYGCYDARQYP; C2 and CDR1 are GFRFSRYAM; CDR2 is SWSGGSIY; CDR3 is AAGSFIRGLAPSEPTQYA; C3 and CDR1 are GRTFSRSRM; CDR2 is GWSGASTY; CDR3 is AADRIFEEPSVWARYS; C4 and CDR1 are GRTFSRPTM; CDR2 is SWSGGSTV; CDR3 is AADYDLDYELGFGDPKYD; C5 and CDR1 are GRTLSRYYM; CDR2 is SWTGYSTY; CDR3 is AKIISSGWAYGMD; C6 and CDR1 are GRTFSSYNM; CDR2 is RWSGGIRY; CDR3 is AADLTSYSDYGDAE; C7 and CDR1 are GRTMPY™; CDR2 is TWSGGNSY; CDR3 is NAGLGTL™. C8 and CDR1 are GRTFRSYAM; CDR2 is NWSGGSTY; CDR3 is NADVRSIGTGYLRN; C9 and CDR1 are GRTFSSYAM; CDR2 is IYSGGSTY; CDR3 is AAADRKAILTPMGYK; C10 and CDR1 are GGTFTTFSKYGV; CDR2 is SWNGGSTY; CDR3 is YADAASYSGRRRGR; C11 and CDR1 are GFTFSRNAI; CDR2 is STSGSTN; CDR3 is HAEILDFGLGYGYD; C12 and CDR1 are GSAFSINFI; CDR2 is SSGGSTN; CDR3 is NLNSGIAYRRD; C13 and CDR1 are GRTFSTYVM; CDR2 is SQRGTVTA; CDR3 is AARLHYDLGAVLQERQSYD; C14 and CDR1 are GGTFSRYAM; CDR2 is SRSGLRTY; CDR3 is AAGMTFSTPSWH; C15 and CDR1 are GRTFSIYTM; CDR2 is TSGGSTY; CDR3 is AAAFSSYYSPNTYFTRPSQYN; C16 and CDR1 are RRTSSSIAM; CDR2 is TWIGSTW; CDR3 is ASSKYYRGSYPGGPNPYEYD; C17 and CDR1 are RRTFSTYAM; CDR2 is RGSAGSTR; CDR3 is AARTRGSYWDTRYTVPNGYE; C18 and CDR1 are GRTFTNYVM; CDR2 is TWSGLRKY; CDR3 is GADTPRHVFFYVPVRENEYD.

2. The measles virus nucleoprotein-specific nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody is any of the following: A1, the amino acid sequence shown in SEQ ID NO. 1; A2, the amino acid sequence shown in SEQ ID NO. 2; A3, the amino acid sequence shown in SEQ ID NO. 3; A4, the amino acid sequence shown in SEQ ID NO. 4; A5, the amino acid sequence shown in SEQ ID NO. 5; A6, the amino acid sequence shown in SEQ ID NO. 6; A7, the amino acid sequence shown in SEQ ID NO. 7; A8, the amino acid sequence shown in SEQ ID NO. 8; A9, the amino acid sequence shown in SEQ ID NO. 9; A10, the amino acid sequence shown in SEQ ID NO. 10; A11, the amino acid sequence shown in SEQ ID NO. 11; A12, the amino acid sequence shown in SEQ ID NO. 12; A13, the amino acid sequence shown in SEQ ID NO. 13; A14, the amino acid sequence shown in SEQ ID NO. 14; A15, the amino acid sequence shown in SEQ ID NO. 15; A16, the amino acid sequence shown in SEQ ID NO. 16; A17, the amino acid sequence shown in SEQ ID NO. 17; A18, the amino acid sequence shown in SEQ ID NO.

18. Or the amino acid sequence shown in A1-A17, with one or more amino acid residues substituted and / or deleted and / or added, that encodes the same active protein.

3. The nucleic acid molecule encoding the nanobody of claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is any of the following: Nucleotide sequences B1, SEQ ID NO. 19; B2, SEQ ID NO. 20; B3, SEQ ID NO. 21; B4, SEQ ID NO. 22; B5, SEQ ID NO. 23; B6, SEQ ID NO. 24; B7, SEQ ID NO. 25; B8, SEQ ID NO. 26; B9, SEQ ID NO. 27; B10, SEQ ID NO. 28; B11, SEQ ID NO. 29; B12, SEQ ID NO. 30; B13, SEQ ID NO. 31; B14, SEQ ID NO. 32; B15, SEQ ID NO. 33; B16, SEQ ID NO. 34; B17, SEQ ID NO. 35; B18, SEQ ID NO.

36. Or, nucleotide sequences shown in B1-B18 that have been substituted and / or deleted and / or added with one or more nucleotides and encode the same active protein.

4. A recombinant vector containing the nucleic acid molecule of claim 3; characterized in that, The carrier is pET 28a.

5. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid molecule of claim 3 or the recombinant vector of claim 4, or express the nanobody of claim 1 or 2.

6. A method for constructing recombinant cells according to claim 5, characterized in that, This includes introducing the recombinant vector of claim 4 into host cells; Preferably, the host cell includes a prokaryotic host cell or a eukaryotic host cell; more preferably, the host cell is Escherichia coli.

7. A method for constructing the measles virus nucleoprotein-specific nanobody of claim 1, characterized in that, Includes the following steps: Step 1: Construct a recombinant expression plasmid containing the DNA sequence of human Measles virus-NP, transfect the plasmid to construct recombinant cells, and recombinantly express the recombinant Measles virus-NP protein; Step 2: The recombinant Measles virus-NP protein is used to immunize camel animals to induce them to produce antibodies; Step 3: Use helper phage surface display to screen for specific nanobodies; sequence and express the target antibody to obtain measles virus nucleoprotein-specific nanobodies; Preferably, the amino acid sequence of the recombinant Measles virus-NP protein is shown in SEQ ID NO. 37; the corresponding nucleotide sequence is shown in SEQ ID NO.

38.

8. The use of the nanobody of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the recombinant cell of claim 5, or the recombinant cell prepared by the construction method of claim 6 in the preparation of reagents for detecting measles virus.

9. A reagent for detecting measles virus, characterized in that, The reagents include the nanobody of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant vector of claim 4, the recombinant cell of claim 5, or the recombinant cell prepared by the construction method of claim 6.

10. A kit for detecting measles virus, characterized in that, The kit contains the nanobody as described in claim 1 or 2.

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