Preparation method and application of anti-influenza b virus monoclonal antibody

Abstract

The present application relates to the technical field of antibodies, in particular to preparation and application of anti-influenza B virus monoclonal antibody, and provides the antibody and application thereof, nucleic acid molecule, expression vector, genetic engineering biological material, quality control product and preparation method, reagent and kit thereof.The anti-FluB monoclonal antibody can be used as quality control product and calibration product, can significantly cause enzyme-linked immuno reaction with Victoria and Yamagata system recombinant antigens of FluB virus HA gene, and the 37 DEG C acceleration stability experiment result shows that it meets the kit use requirement, the preparation method is simple, and is suitable for mass production.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to the preparation and application of anti-influenza B (FluB) monoclonal antibodies. Background Art

[0002] Influenza viruses are classified into three types: A (A), B (B), and C (C), based on the antigenic differences between their nucleoprotein (NP) and matrix protein (MP). Type A causes the most severe symptoms, while type B is less severe but can still cause outbreaks. Type C typically causes milder symptoms, and type D is a newly discovered virus, with much research still underway. Types A, B, and C are very similar in overall structure. Virus particles are 80 to 120 nanometers in diameter and are typically roughly spherical, but may also occur in filamentous forms.

[0003] Influenza virus B belongs to the Orthomyxoviridae family and the genus Influenza B. It consists of approximately 14,648 nucleotides, divided into eight distinct segments, encoding approximately 11 proteins. The HA protein on the surface of the FluB virus is a trimer, with its gene segments consisting of two parts, HA1 and HA2. HA1, located at the head of the trimer, is a spherical structure containing receptor binding sites and is the primary antigenic region of the influenza virus. Amino acid mutations in this region can alter the antigenicity of the influenza virus, leading to outbreaks and epidemics of influenza B. The HA2 site, located at the base of the trimer, has a handle-like structure and contains a fusion peptide that mediates the fusion of the viral envelope with the host envelope. Some monoclonal antibodies targeting HA2 can neutralize the virus by inhibiting membrane fusion of the influenza virus.

[0004] Based on differences in antigenicity and genetic characteristics, influenza B is divided into two major lineages: Victoria and Yamagata. The HA1 genes of the two lineages encode different amino acids (340 AAs in the V lineage and 338-339 AAs in the Y lineage. The Y lineage lacks AAs at position 163, and some strains lack AAs at position 166). It is also speculated that over the past decade, four antigenic determinants on HA1 (region A: 116-137; region B: 141-150; region C: 162-167; and region D: 194-202), totaling 17 AAs, have changed. The average difference between the V and Y lineages is 5.4%-10.2%, and the amino acid difference and genetic distance between the two lineages have tended to increase over time.

[0005] Compared to influenza A virus, there are relatively few reports of monoclonal antibodies targeting influenza B virus, and most of them are mouse antibody sequences obtained through hybridoma technology. The quality control and calibrator products in most kits need to be bound to anti-human IgM secondary antibodies when used, so they require a human Fc to bind to them. Therefore, natural human antibodies screened from phage-derived fully human antibody libraries have inherent advantages and are more efficient than later chimeric expression construction of mouse antibodies.

[0006] Influenza B virus can cause influenza virus pneumonia. Patients with weakened immune systems, advanced age, or those with serious underlying medical conditions are more likely to contract pneumonia. Therefore, influenza B virus detection is of great clinical significance. However, in vitro diagnostics (IVDs) primarily utilize plasma from positive patients, which presents numerous challenges, such as difficulty collecting and scaling up. Therefore, the development of in vitro methods to generate anti-FluB monoclonal antibodies for use as quality controls and calibrators for use in test kits is highly urgent. Summary of the Invention

[0007] In view of this, the present invention provides the preparation and application of a fully human monoclonal antibody against FluB. This monoclonal antibody, as a quality control and calibrator, can undergo a significant enzyme-linked immunosorbent assay (ELISA) reaction with recombinant antigens of the Victoria and Yamagata lines of the FluB virus HA gene. Its accelerated stability at 37°C meets the requirements for use in the kit, and the preparation method is simple, making it suitable for mass production.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides antibodies, including single-chain antibodies, whose heavy chain variable regions have:

[0010] (1) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or

[0011] (2) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0012] (3) an amino acid sequence that is at least 95% homologous to the amino acid sequence shown in (1) or (2);

[0013] Its light chain variable region has:

[0014] (4) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6; or

[0015] (5) An amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or

[0016] (6) an amino acid sequence having at least 95% homology to the amino acid sequence shown in (4) or (5);

[0017] The plurality is 2 to 5.

[0018] In some specific embodiments of the present invention, the above-mentioned antibody includes Antibody 1 or Antibody 2;

[0019] The heavy chain variable region of the antibody 1 has the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 2;

[0020] The heavy chain variable region of antibody 2 has the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region has the amino acid sequence shown in SEQ ID NO: 6.

[0021] In some specific embodiments of the present invention, the above-mentioned antibody is a single-chain antibody.

[0022] The present invention also provides a nucleic acid molecule encoding the above antibody.

[0023] In some specific embodiments of the present invention, the nucleic acid molecule has:

[0024] (7), the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8; or

[0025] (8) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as (7); or

[0026] (9) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (7) or (8);

[0027] The plurality is 2 to 30.

[0028] In some specific embodiments of the present invention, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0029] In some specific embodiments of the present invention, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8.

[0030] The present invention also provides an expression vector comprising the above nucleic acid molecule.

[0031] In some specific embodiments of the present invention, the backbone of the expression vector includes a phage vector backbone, a lentiviral vector backbone, a prokaryotic vector backbone or a eukaryotic vector backbone.

[0032] The present invention also provides genetically engineered biological materials, including bacteriophages, bacteria, fungi, plant cells or animal cells, which have the above expression vectors.

[0033] The present invention also provides the use of any of the following in preparing a standard substance:

[0034] i) the above-mentioned antibodies;

[0035] ii), the above-mentioned nucleic acid molecule;

[0036] iii) the above-mentioned expression vector;

[0037] iv) the above-mentioned genetically engineered biological materials.

[0038] In some specific embodiments of the present invention, the standard substance used is an influenza detection standard substance.

[0039] The present invention also provides a standard substance, including the above-mentioned antibody, and acceptable adjuvants or auxiliary agents.

[0040] In some specific embodiments of the present invention, the above-mentioned standard substance is an influenza detection standard substance.

[0041] The present invention also provides a reagent or kit comprising any of the following:

[0042] i) the above-mentioned antibodies;

[0043] ii), the above-mentioned nucleic acid molecule;

[0044] iii) the above-mentioned expression vector;

[0045] iv) the above-mentioned genetically engineered biological materials;

[0046] v) The above-mentioned standard substances.

[0047] In some specific embodiments of the present invention, the above reagent or kit is an influenza detection reagent or kit.

[0048] The present invention also provides a method for preparing a standard substance, which is prepared based on any of the following:

[0049] a), the above-mentioned nucleic acid molecule;

[0050] b) the above-mentioned expression vector;

[0051] c) The above-mentioned genetically engineered biological materials.

[0052] In some specific embodiments of the present invention, the preparation method comprises:

[0053] A), expressing the nucleic acid molecule to obtain the antibody, and mixing it with an acceptable excipient or auxiliary agent to obtain a standard substance;

[0054] B) expressing the above expression vector to obtain the antibody, and mixing it with an acceptable excipient or auxiliary agent to obtain a standard substance;

[0055] C) Cultivate the above genetically engineered biological materials to obtain antibodies, and mix them with acceptable excipients or auxiliary agents to obtain standard substances.

[0056] The preparation and application of the anti-FluB fully human monoclonal antibody of the present invention have the following effects:

[0057] The anti-FluB fully human IgM monoclonal antibodies FH105 and FH128 provided by the present invention have a purity of >90%, transient expression levels of 200 mg / L and 150 mg / L, respectively, and an IgM multimer ratio of >90%. After identification, FH105 has a higher potency. This monoclonal antibody can be used as a positive quality control product. Its potency and stability meet the requirements of use, providing a reliable basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0059] Figure 1 Shows the agarose gel electrophoresis of RNA extracted from peripheral blood lymphocytes;

[0060] Figure 2 Shows the agarose gel electrophoresis of VL and VH gene PCR products;

[0061] Figure 3 Shows the agarose gel electrophoresis of the scFv gene PCR product;

[0062] Figure 4 Figure 2 shows SDS-PAGE and HPLC analysis charts; 2 in A shows non-reduced FH105 IgM, and 1 in A shows reduced band (treated with reducing agent / DTT); 3 in B shows non-reduced FH128 IgM, and 4 in B shows reduced band (treated with reducing agent / DTT); C shows SEC-HPLC chart, in which the blue peak is the marker, and the sizes from left to right are 1340 KDa, 670 KDa, 300 KDa, 150 KDa, 45 KDa, 17 KDa, and 1 KDa respectively; the light green peak and red peak correspond to the HPLC peak charts of FH105 IgM and FH128 IgM, respectively. DETAILED DESCRIPTION

[0063] The present invention discloses the preparation and application of fully human anti-FluB monoclonal antibodies. Those skilled in the art can refer to the contents herein and appropriately improve the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications obvious to those skilled in the art are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0064] The amino acid sequences of the heavy chain variable regions of the anti-FluB fully human IgM monoclonal antibodies FH105 and FH128 are shown in SEQ ID NO: 1 and SEQ ID NO: 5, and the amino acid sequences of the light chain variable regions are shown in SEQ ID NO: 2 and SEQ ID NO: 6.

[0065] The anti-FluB fully human monoclonal antibody of the present invention further comprises a heavy chain constant region and a light chain constant region. The heavy chain constant regions are all human IgM subtype, and the light chain constant regions are all Kappa type.

[0066] The present invention also provides a DNA molecule encoding the anti-FluB fully human monoclonal antibody.

[0067] Wherein, the DNA molecule includes a sequence encoding the heavy chain variable region as shown in SEQ ID NO: 3, SEQ ID NO: 7, or a nucleotide sequence complementary to SEQ ID NO: 3, SEQ ID NO: 7; a nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 4, SEQ ID NO: 8, or a nucleotide sequence complementary to SEQ ID NO: 4, SEQ ID NO: 8.

[0068] The present invention also provides an expression vector containing a DNA molecule encoding the anti-FluB fully human IgM monoclonal antibody.

[0069] In some specific embodiments, the backbone vector of the expression vector of the present invention is pCMV3.

[0070] The present invention also provides a recombinant host comprising the expression vector. In some specific embodiments, the host cell is an Expi CHO cell.

[0071] The present invention also provides a method for preparing a fully human anti-FluB IgM monoclonal antibody, comprising:

[0072] (1) Constructing an expression vector containing the above-mentioned DNA molecule;

[0073] (2) Transforming the expression vector described in step (1) into a host cell;

[0074] (3) culturing the host cells obtained in step (2);

[0075] (4) Isolate and purify to obtain the monoclonal antibody.

[0076] The present invention also provides the use of the anti-FluB fully human IgM monoclonal antibody in in vitro diagnostic reagents.

[0077] The present invention also provides a blocking agent comprising the anti-FluB fully human IgM monoclonal antibody and an acceptable adjuvant.

[0078] The sequence involved in the present invention is as follows:

[0079] FH105 heavy chain variable region amino acid sequence:

[0080] QVQLVQSGAEVKKPGSSVKVSCKASRGTSSSNAISWVRQTPGHGLEWMGRIIPVLAIENYAQKFEGRITITADKSTSTAYMELTSLTSEDTAVYYCARERGTHLSKADAFDLWGQGTKVTVSS (SEQ ID NO: 1)

[0081] FH105 light chain variable region amino acid sequence:

[0082] NIQMTQSPSSSLSASVGDRVTVTCRASQTISNYLNWYQQKPGKAPKLLIYGASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLTFGPGTKVDIK (SEQ ID NO: 2)

[0083] FH105 heavy chain variable region nucleotide sequence:

[0084] CAGGTTCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCTAGAGGCACCTCCAGTAGCAATGCTATCAGCTGGGTGCGCCAGACCCCTGGCCATGGGCTTGAGTGGATGGGAAGGATCATCCCTGTCCTTGCCATAGAAAACTACGCACAGAAGTTCGAGGGCAGAATCACCATCACCGCGGACAAATCCACGAGCACAGCCTACATGGAGTTGACCAGCCTGACATCTGAGGACACGGCCGTGTATTATTGTGCGAGAGAGCGTGGGACTCACTTAAGTAAGGCTGATGCTTTTGATCTATGGGGCCAAGGGACAAAGGTCACCGTCTCCTCA(SEQID NO: 3)

[0085] FH105轻链可变区核苷酸序列:

[0086] AACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCGTCACTTGCCGGGCAAGTCAGACCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCTGGGAAAGCCCCTAAACTCCTGATCTATGGTGCATCCAGTTTGCACAGTGGCGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCGACTTACTACTGTCAGCAGAGTTACAATACCCCCCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA(SEQ ID NO: 4)

[0087] FH128重链可变区氨基酸序列:

[0088] QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGNFYNSGITNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYNCARVGQWPGDHWFDPWGQGTLVTVSS(SEQ ID NO: 5)

[0089] FH128 light chain variable region amino acid sequence:

[0090] NIQLTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSPITFGQGTRLEIK (SEQ ID NO: 6)

[0091] FH128 heavy chain variable region nucleotide sequence:

[0092] CAGGGTGCAGCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGAATTTCTATAACAGTGGGATCACCAACTACAACCC CTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTCAGCTCTGTGACCGCTGCGGACACGGGCCGTGTATAACTGTGCGAGAGTAGGGCAGTGGCCGGGAGACCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO:7)

[0093] FH128 light chain variable region nucleotide sequence:

[0094] AACATCCAGTTGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGAAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCTACTTTAGA AAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGTAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCACAGTATAATAGTTATTCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 8)

[0095] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0096] The present invention will be further described below with reference to the embodiments.

[0097] Example 1: Primer synthesis and gene acquisition

[0098] Lymphocytes were isolated from anticoagulated blood of respiratory patients using human peripheral blood lymphocyte separation medium (Solarbio, Beijing), and total cellular RNA was extracted using an RNA extraction kit (Shengang, Shanghai) (electrophoresis results are shown in Figure 1 ), using Oligo-dT primers and the extracted RNA as a template using the NEB first-strand synthesis kit (ProtoScript ® cDNA was generated by reverse transcription using the First Strand cDNA Synthesis Kit (Cat. No. E6300S) and used as template to amplify the scFv heavy and light chain gene fragments. The primer library sequences used are as follows:

[0099] The heavy chain upstream primer is as follows:

[0100] VHF1: CAGGTBCAGCTGGTRCAGTC (SEQ ID NO: 9);

[0101] VHF2: CAGGTCAACTTAAGGGAGTCTGG (SEQ ID NO: 10);

[0102] VHF3: CAGGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 11);

[0103] VHF4:CAGGTGCAGCTGCAGGAGTCGGG(SEQ ID NO: 12);

[0104] VHF5:CAGGTGCAGCTGTTGCAGTCTGC(SEQ ID NO: 13);

[0105] VHF6:CAGGTACAGCTGCAGCAGTCAGG(SEQ ID NO: 14);

[0106] VHF7:CAGGTCCAGCTKGTGCARTC(SEQ ID NO: 15);

[0107] VHF8:CAGGTCCAGCTGGTGCAGTC(SEQ ID NO: 16);

[0108] VHF9:CAGGTACAGCTGGTGGAGTC(SEQ ID NO: 17);

[0109] 重链下游引物:

[0110] VHR1:TGAGGAGACGGTGACCTTTG(SEQ ID NO: 18);

[0111] VHR2:TGAGGAGACGGTGACCAGGG(SEQ ID NO: 19);

[0112] VHR3:TGAAGAGACGGTGACCATTG(SEQ ID NO: 20);

[0113] VHR4:TGAGGAGACGGTGACCAGGG(SEQ ID NO: 21);

[0114] VHR5:TGAGGAGACGGTGACCGTGG(SEQ ID NO: 22);

[0115] 轻链kappa亚型上游引物:

[0116] Kappa1F:GACATCCAGATGACCCAGTCTCC(SEQ ID NO: 23);

[0117] Kappa2F:GATGTTGTGATGACTCAGTCTCC(SEQ ID NO: 24);

[0118] Kappa3F:GAAATTGTGTTGACGCAGTCTCC (SEQ ID NO: 25);

[0119] Kappa4F:GACATCGTGATGACCCAGTCTCC (SEQ ID NO: 26);

[0120] Kappa5F: GAAACGACACTCACGCAGTCTCC (SEQ ID NO: 27);

[0121] Kappa6F:GAAATTGTGCTGACTCAGTCTCC (SEQ ID NO: 28);

[0122] Light chain kappa subtype downstream primers:

[0123] KappaR1:TTTGATATCCACTTTGGTCC (SEQ ID NO: 29);

[0124] KappaR2:TTTGATCTCCACCTTGGTCC (SEQ ID NO: 30);

[0125] KappaR3:TTTGATTTCCAGCTTGGTCC (SEQ ID NO: 31);

[0126] KappaR4:TTTAATCTCCAGTCGTGTCC (SEQ ID NO: 32);

[0127] KappaR5: TAGGACGGTCAGCTTGGTCC (SEQ ID NO: 33).

[0128] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles; and extension at 72°C for another 10 min. The light and heavy chain variable regions after gel excision and purification were connected to T vectors and sent for testing to obtain the light and heavy chain variable region sequences. Then, the OverLap PCR technology was used to splice the two into a scFv gene fragment through the SfiⅠ restriction site. The PCR products of the light chain, heavy chain and scFv gene fragments were detected by 1% agarose gel electrophoresis. The results showed that the size of the light and heavy chain variable regions was about 350 bp, and the size of the scFv fragment after splicing was about 750 bp (such as Figure 2 and Figure 3 shown).

[0129] Example 2: Construction of scFv phage library

[0130] The scFv gene fragment was ligated to the phage vector pComb3XSS via the SfiⅠ restriction site. The ligation product was electroporated into E. coli TG1 competent cells and incubated with shaking at 37°C for 1 h in 2×YT-AG medium (containing 2% glucose and 100 μg / mL AMP). 10 μL of the bacterial solution was inoculated into 2×YT-AG medium and cultured overnight. Plaque formation was observed and the antibody library titer was calculated:

[0131] Antibody library titer (PFU / mL) = dilution × number of plaques (PFU) / volume of bacterial solution used for plating (mL).

[0132] Randomly select 10-20 positive colonies and send them to Sangon Technology Co., Ltd. for sequencing, colony PCR identification, and analysis of the antibody library diversity. Helper phage M13K07 was added to the remaining bacterial suspension and incubated at 37°C for 30 minutes. After centrifugation, the cells were resuspended in 100 mL of 2×YT medium (containing 1 μM IPTG and 100 μg / mL AMP) and incubated overnight on a shaker at 30°C. The supernatant was collected by centrifugation, and one-quarter volume of 20% PEG / 2.5 M NaCl was added. The phage were sedimented on ice for 50 minutes, the pellet was collected by centrifugation, and resuspended in PBS to obtain the phage antibody library.

[0133] Example 3: Enrichment and screening of human anti-FluB virus phage antibody library

[0134] FluB Victoria and Yamagata recombinant antigens were mixed in a 1:1 ratio and coated onto immunotubes at a concentration of 10 µg / mL. The mixture was incubated overnight at 4°C and blocked with 5% skim milk at 37°C for 2 h. After washing twice with PBST, 1 mL of the scFv phage library prepared in Example 2 was added. The tubes were incubated with shaking at room temperature for 2 h, washed 10 times with PBST, and 1 mL of 100 mM triethylamine dissociation buffer was added. The tubes were incubated at room temperature for 10 min, neutralized with 1 M Tris-HCl (pH 6.4), and 50 mL of TG1 bacteria in the logarithmic growth phase were added. After incubation at 37°C for 30 min, 10 µL of the mixture was plated on 2×YT / GA plates and incubated overnight at 37°C to measure the yield. The remaining culture was expanded until the A600 of the culture was approximately 0.5. Helper phage M13K07 was added. The subsequent process was consistent with Example 2. The above screening process was repeated for four rounds to enrich for phages that bind to the FluB antigen.

[0135] Example 4: Induced expression of positive phage clones

[0136] After four rounds of enrichment, single colonies were randomly selected and plated in six 96-well plates. 400 μL of 2×YT / GA medium (2% glucose, 50 μg / mL Amp) was added to each well and cultured overnight at 37°C. The next day, 5 μL of the overnight culture was transferred to a 96-well plate containing 100 μL of culture medium for activation. The cells were incubated at 37°C for 2-3 hours. When the OD value was approximately 2, 20 μL of a mixture of 2×YT and helper phage (MOI = 5) was added to each well and incubated at 37°C for 30 minutes. After incubation on a shaker for 1.5 hours, 300 μL of 2×YT-AK (100 μg / mL Amp, 50 μg / mL Kan) was added to each well and cultured overnight for 16-18 hours. The next day, the supernatant was centrifuged and collected for analysis.

[0137] Example 5: Phage monoclonal ELISA detection

[0138] V and Y antigens were coated onto ELISA plates separately at 4°C overnight. The plates were then blocked with Casein blocking buffer at 37°C for 2 hours, then dried and set aside. Monoclonal phage was added and the plates were incubated at 37°C for 30 minutes. The plates were then washed six times with PBST and dried. HRP-M13 was added and the plates were incubated at 37°C for 30 minutes. The plates were washed five times with PBST and dried. The plates were then color-developed for 15 minutes and terminated with 2 M H₂SO₄. The absorbance at 450 nm was measured using a microplate reader. Two clones with strong reactivity were sent for sequencing.

[0139] Example 6: Nucleic acid sequence analysis of variable region genes and construction of full-length IgM antibodies

[0140] Antibody 1-FH105: its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 2.

[0141] Antibody 2-FH128: Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 5; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 6.

[0142] Using two selected positive clones as templates, the variable region gene sequences were amplified by PCR and inserted into the laboratory vectors IgM-PCMV3 and LC-PCMV3 (containing the constant region sequences) using HindIII / XbaI. The recombinant plasmids were transformed into competent DH5α cells, and positive clones were selected for sequencing and plasmid extraction.

[0143] Example 7: Expression of full-length IgM antibody

[0144] Adjust the density of Expi CHO cells to 3×10 cells per day before transfection. 6cells / mL to 4 x 10 6 cells / mL, and the cells were diluted to 6 x 10 6 The extracted plasmids were diluted according to the ExpiFectamine CHO transfection reagent instructions, and the plasmid DNA and liposomes were diluted at a ratio of 1 μg: 5 μL to transfect the cells, and the FH105 and FH128 antibody plasmids contained light and heavy chain plasmids and J chain plasmids, wherein HC:LC:J = 1:1:1. After 24 h, 24% CHO feed and 0.6% enhancer were added, and the supernatant was harvested after the cell viability decreased to about 70%-75%.

[0145] Example 8: Purification of human anti-FluB virus full-length IgM antibody

[0146] The supernatant of the harvested FH105 and FH128 antibodies was filtered through a 0.45 μm filter membrane, and the chromatography column filled with Protein L filler was equilibrated with ultrapure water and buffer (0.02 M PBS, pH 7.4) in sequence, and the filtered antibody supernatant was loaded, equilibrated again, and finally eluted with dissociation buffer (0.1 M sodium citrate pH 2.3) to collect the target protein antibody, and the protein content was detected using a spectrophotometer NanoDrop One and the protein expression level was detected by SDS-PAGE and SEC-HPLC.

[0147] The results show that after purification, the expression amounts of the FH105 and FH128 antibodies are 200 mg / L and 150 mg / L, respectively, and the molecular weights of the heavy chains are about 65 KD, and the molecular weights of the light chains are about 25 KD, which are consistent with the expectations. Figure 4 The HPLC detection results show that the purity of the antibodies is more than 98% (about 150 KDa). The HPLC results show that most of the target antibodies exist in the form of aggregates, and a small part exists in the form of monomers (about 200 kDa in size).

[0148] Example 9: ELISA detection of human anti-FluB virus full-length IgM antibody

[0149] The detection was carried out according to the instruction of the IgM kit, and according to the judgment standard of the kit: S / CO value ≥ 10 AU / mL is positive. The detection results of the prepared FH105 IgM and FH128 IgM human antibodies are shown in Table 1, and the average detection concentration of FH105 IgM and FH128 IgM is 27015 and 25227 respectively, which has strong reactivity and meets the reactivity requirement of positive quality control. Then, the antibody titer (S / CO) of FH105 IgM and FH128 IgM was adjusted to low, medium and high levels (target values are 3±1, 7±1 and 13±1 respectively), and after freeze-drying, the titer variation amplitude after 7 days of heat acceleration at 37℃ for 14 days was examined, and the stability data is shown in Table 2. The variation amplitude is within 10%, which meets the requirement of reagent use. Since the reactivity of FH105 IgM antibody is 7% higher than that of FH128 IgM antibody, FH105 is preferred.

[0150] Table 1: FH105 IgM and FH128 IgM antibody reactivity detection results

[0151]

[0152] Table 2: FH105 IgM and FH128 IgM antibody 37℃ acceleration stability evaluation results

[0153]

[0154] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. An antibody, characterized in that: I) its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, and its light chain variable region has the amino acid sequence shown in SEQ ID NO: 2; or II) its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 5, and its light chain variable region has the amino acid sequence shown in SEQ ID NO:

6.

2. The antibody according to claim 1, wherein The antibody is a single-chain antibody.

3. A nucleic acid molecule encoding the antibody according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, wherein: 1) having the nucleotide sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4; or II) having the nucleotide sequence shown in SEQ ID NO: 7 and SEQ ID NO:

8.

5. An expression vector, characterized in that The nucleic acid molecule according to claim 3 or 4.

6. Genetically engineered biological material, including bacteriophage, bacteria, fungi or animal cells, characterized in that: The invention also comprises the expression vector according to claim 5.

7. Application of any of the following in the preparation of reference materials: i), the antibody according to claim 1 or 2; ii), the nucleic acid molecule according to claim 3 or 4; iii) the expression vector according to claim 5; iv) The genetically engineered biological material according to claim 6; The standard substances include quality control products and standard products.

8. Standard substances, including quality control substances or standard substances, characterized in that: The invention comprises the antibody according to claim 1 or 2, and acceptable adjuvants or auxiliary agents.

9. A reagent or a kit, characterized in that Include any of the following: i), the antibody according to claim 1 or 2; ii), the nucleic acid molecule according to claim 3 or 4; iii) the expression vector according to claim 5; iv) The genetically engineered biological material according to claim 6; v) The standard substance according to claim 8.

10. A method for preparing a standard substance, characterized in that: Prepared based on any of the following: a), the nucleic acid molecule according to claim 3 or 4; b) the expression vector according to claim 5; c) The genetically engineered biological material according to claim 6.

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