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

Abstract

The invention relates to the technical field of antibodies, in particular to preparation and application of an anti-influenza B virus monoclonal antibody. The invention provides an antibody and application thereof, a nucleic acid molecule, an expression vector, a genetic engineering biological material, a quality control product and a preparation method thereof, a reagent and a kit. The anti-FluB monoclonal antibody is used as a quality control material and a calibration material, and can generate a remarkable enzyme-linked immunosorbent assay reaction with Victoria and Yamagata series recombinant antigens of a FluB virus HA gene, and a 37 DEG C accelerated stability experiment result shows that the anti-FluB monoclonal antibody meets the use requirements of a kit, and the preparation method is simple and 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 can be divided into three types: type A (A), type B (B), and type C (C) based on the antigenic differences between the internal nucleoprotein (NP) and the matrix protein (MP). Type A influenza virus causes the most severe symptoms, type B influenza virus is not serious but can still cause outbreaks, and type C influenza virus usually causes mild symptoms. Type D influenza virus is newly discovered and many questions are still being studied. Type A, B, and C influenza viruses are very similar in overall structure. The virus particles are 80 to 120 nanometers in diameter and are usually roughly spherical, but may appear in filamentous form.

[0003] Influenza virus B belongs to the family Orthomyxoviridae and the genus Influenza virus B. It consists of approximately 14,648 nucleotides, divided into 8 different segments, encoding approximately 11 proteins. The HA protein on the surface of the FluB virus is a trimer structure, and its gene segments include two parts, HA1 and HA2. HA1 is located at the head of the trimer, has a spherical structure, contains receptor binding sites, and is the main antigenic region of the influenza virus. Amino acid mutations in this region will lead to changes in the antigenicity of the influenza virus, thereby causing the outbreak and spread of influenza B. The base of the HA2 site trimer is a handle-like structure containing a fusion peptide that can mediate the fusion of the viral envelope with the host envelope. Some monoclonal antibodies against HA2 can inhibit the membrane fusion of the influenza virus, thereby neutralizing the virus.

[0004] According to the difference in antigenicity and genetic characteristics, influenza B is divided into two major lineages, Victoria and Yamagata. The amino acid differences encoded by the HA1 gene of the two lineages are different (340 AA in the V lineage and 338-339 AA in the Y lineage. The Y lineage lacks 163 AA and 166 AA in some strains. It is also speculated that a total of 17 AA in the four antigenic determinant clusters on HA1 (A region: 116-137; B region 141-150; C region: 162-167; D region: 194-202) have changed in the past 10 years. The average difference rate between the V and Y lineages is 5.4%-10.2%, and the amino acid difference and genetic distance between the two lineages have a tendency to gradually increase over time.

[0005] Compared with influenza A virus, there are relatively few reports on monoclonal antibodies against influenza B virus, and most of them are mouse antibody sequences obtained through hybridoma technology. The quality control and calibration products in most test kits need to be combined with anti-human IgM secondary antibodies when used, so human Fc is required to combine with them. It can be seen that natural human antibodies screened by phage fully human antibody libraries have inherent advantages and are more efficient than mouse antibodies in the later stage of chimeric expression construction.

[0006] Influenza B virus may cause influenza virus pneumonia. Patients with low immunity, older age or serious underlying diseases are more likely to be infected with pneumonia. Therefore, influenza B virus detection has important clinical significance. In the field of in vitro diagnosis, most of the plasma used is positive patient plasma, which has many problems such as difficult collection and amplification. It is very urgent to prepare anti-FluB monoclonal antibodies by in vitro methods as quality control products and calibrators for kit testing. Summary of the invention

[0007] In view of this, the present invention provides the preparation and application of anti-FluB fully human monoclonal antibody, which can react significantly with Victoria and Yamagata recombinant antigens of FluB virus HA gene as quality control and calibrator, and its 37°C accelerated stability meets the requirements of kit use, and the preparation method is simple and suitable for mass production.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides antibodies, including single-chain antibodies, whose heavy chain variable regions have: (1) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or (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 (3) an amino acid sequence having at least 95% homology to the amino acid sequence shown in (1) or (2); Its light chain variable region has: (4) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6; or (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 (6) an amino acid sequence having at least 95% homology to the amino acid sequence shown in (4) or (5); The plurality is 2 to 5.

[0009] In some specific embodiments of the present invention, the above-mentioned antibody includes Antibody 1 or Antibody 2; The heavy chain variable region of 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; 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.

[0010] In some specific embodiments of the present invention, the above-mentioned antibody is a single-chain antibody.

[0011] The present invention also provides a nucleic acid molecule encoding the above antibody.

[0012] In some specific embodiments of the present invention, the above-mentioned nucleic acid molecule has: (7), the nucleotide sequence shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8; or (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 (9) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (7) or (8); The plurality is 2 to 30.

[0013] In some specific embodiments of the present invention, the above-mentioned nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] In some specific embodiments of the present invention, the above-mentioned nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8.

[0015] The present invention also provides an expression vector, comprising the above nucleic acid molecule.

[0016] In some specific embodiments of the present invention, the backbone of the above-mentioned expression vector includes a phage vector backbone, a lentiviral vector backbone, a prokaryotic vector backbone or a eukaryotic vector backbone.

[0017] The present invention also provides genetic engineering biological materials, including bacteriophages, bacteria, fungi, plant cells or animal cells, having the above expression vector.

[0018] The present invention also provides the use of any of the following in preparing a standard substance: i) the above antibodies; ii), the above-mentioned nucleic acid molecule; iii) the above-mentioned expression vector; iv) the above-mentioned genetically engineered biological materials.

[0019] In some specific embodiments of the present invention, the standard substance used above is an influenza detection standard substance.

[0020] The present invention also provides a standard substance, including the above-mentioned antibody, and acceptable auxiliary materials or adjuvants.

[0021] In some specific embodiments of the present invention, the above-mentioned standard substance is an influenza detection standard substance.

[0022] The present invention also provides a reagent or a kit, which comprises any of the following: i) the above antibodies; ii), the above-mentioned nucleic acid molecule; iii) the above-mentioned expression vector; iv) the above-mentioned genetically engineered biological materials; v) The above-mentioned standard substances.

[0023] In some specific embodiments of the present invention, the above reagent or kit is an influenza detection reagent or kit.

[0024] The present invention also provides a method for preparing a standard substance, which is prepared based on any of the following: a), the above-mentioned nucleic acid molecule; b) the above-mentioned expression vector; c) the above-mentioned genetically engineered biological materials.

[0025] In some specific embodiments of the present invention, the above preparation method comprises: A), expressing the above nucleic acid molecules to obtain antibodies, and mixing with acceptable excipients or adjuvants to obtain standard substances; B), expressing the above expression vector to obtain antibodies, and mixing with acceptable excipients or adjuvants to obtain standard substances; C) Cultivate the above genetically engineered biological materials to obtain antibodies, and mix them with acceptable excipients or adjuvants to obtain standard substances.

[0026] The preparation and application of the anti-FluB fully human monoclonal antibody of the present invention have the following effects: The anti-FluB fully human IgM monoclonal antibodies FH105 and FH128 provided by the present invention have a purity of >90%, an instantaneous expression level of 200 mg / L and 150 mg / L, respectively, and an IgM multimer ratio of >90%. It has been identified that FH105 has a higher potency. The monoclonal antibody can be used as a positive quality control product, and its potency and stability meet the use requirements, providing a reliable basis for clinical diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0028] Figure 1 Shown is the agarose gel electrophoresis of RNA extracted from peripheral blood lymphocytes; Figure 2 Shows the agarose gel electrophoresis of VL and VH gene PCR products; Figure 3 Shows the agarose gel electrophoresis of scFv gene PCR products; Figure 4 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 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 the red peak correspond to the HPLC peak charts of FH105 IgM and FH128 IgM, respectively. DETAILED DESCRIPTION

[0029] The present invention discloses the preparation and application of anti-FluB fully human monoclonal antibodies. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously 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.

[0030] 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.

[0031] The anti-FluB fully human monoclonal antibody of the present invention further comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant regions are all human IgM subtype, and the light chain constant regions are all Kappa type.

[0032] The present invention also provides a DNA molecule encoding the anti-FluB fully human monoclonal antibody.

[0033] 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.

[0034] The present invention also provides an expression vector containing a DNA molecule encoding the anti-FluB fully human IgM monoclonal antibody.

[0035] In some specific embodiments, the backbone vector of the expression vector of the present invention is pCMV3.

[0036] The present invention also provides a recombinant host containing the expression vector. In some specific embodiments, the host cell is an Expi CHO cell.

[0037] The present invention also provides a method for preparing an anti-FluB fully human IgM monoclonal antibody, comprising: (1) constructing an expression vector containing the above-mentioned DNA molecule; (2) transforming the expression vector described in step (1) into a host cell; (3) culturing the host cells obtained in step (2); (4) Isolating and purifying to obtain the monoclonal antibody.

[0038] The present invention also provides the use of the anti-FluB fully human IgM monoclonal antibody in an in vitro diagnostic reagent.

[0039] The present invention also provides a blocking agent comprising the anti-FluB fully human IgM monoclonal antibody and an acceptable adjuvant.

[0040] The sequence involved in the present invention is as follows: FH105 heavy chain variable region amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASRGTSSSNAISWVRQTPGHGLEWMGRIIPVLAIENYAQKFEGRITITADKSTSTAYMELTSLTSEDTAVYYCARERGTHLSKADAFDLWGQGTKVTVSS (SEQ ID NO: 1) FH105 light chain variable region amino acid sequence: NIQMTQSPSSSLSASVGDRVTVTCRASQTISNYLNWYQQKPGKAPKLLIYGASSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYNTPLTFGPGTKVDIK (SEQ ID NO: 2) FH105 heavy chain variable region nucleotide sequence: CAGGTTCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCTAGAGGCACCTCCAGTAGCAATGCTATCAGCTGGGTGCGCCAGACCCCTGGCCATGGGCTTGAGTGGATGGGAAGGATCATCCCTGTCCTTGCCATAGAAAACTACGCACAGA AGTTCGAGGGCAGAATCACCATCACCGCGGACAAATCCACGAGCACAGCCTACATGGAGTTGACCAGCCTGACATCTGAGGACACGGCCGTGTATTATTGTGCGAGAGAGCGTGGGACTCACTTAAGTAAGGCTGATGCTTTTGATCTATGGGGCCAAGGGACAAAGGTCACCGTCTCCTCA (SEQID NO: 3) FH105 light chain variable region nucleotide sequence: AACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCGTCACTTGCCGGGCAAGTCAGACCATTAGCAACTATTTAAATTGGTATCAGCAGAAACCTGGGAAAGCCCCTAAACTCCTGATCTATGGTGCATCCAGTTTG CACAGTGGCGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCGACTTACTACTGTCAGCAGAGTTACAATACCCCCCTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA (SEQ ID NO: 4) FH128 heavy chain variable region amino acid sequence: QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGNFYNSGITNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYNCARVGQWPGDHWFDPWGQGTLVTVSS (SEQ ID NO: 5) FH128 light chain variable region amino acid sequence: NIQLTQSPSTLSASVGDRVTITCRASQSIRSWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSPITFGQGTRLEIK (SEQ ID NO: 6) FH128 heavy chain variable region nucleotide sequence: CAGGGTGCAGCTACAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGGAATTTCTATAACAGTGGGATCACCAACTACAACCC CTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTCAGCTCTGTGACCGCTGCGGACACGGGCCGTGTATAACTGTGCGAGAGTAGGGCAGTGGCCGGGAGACCACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQ ID NO:7) FH128 light chain variable region nucleotide sequence: AACATCCAGTTGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGAAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCATCTACTTTAGA AAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGTAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCACAGTATAATAGTTATTCTCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA (SEQ ID NO: 8) 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.

[0041] The present invention will be further described below in conjunction with the embodiments.

[0042] Example 1: Primer synthesis and gene acquisition 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 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 ® First Strand cDNA Synthesis Kit. Cat No. E6300S) was used to reverse transcribe cDNA, which was used as a template to amplify the scFv heavy chain and light chain gene fragments respectively. The primer library sequences used are as follows: The heavy chain upstream primer is as follows: VHF1: CAGGTBCAGCTGGTRCAGTC (SEQ ID NO: 9); VHF2: CAGGTCAACTTAAGGGAGTCTGG (SEQ ID NO: 10); VHF3: CAGGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 11); VHF4: CAGGGTGCAGCTGCAGGAGTCGGG (SEQ ID NO: 12); VHF5: CAGGTGCAGCTGTTGCAGTCTGC (SEQ ID NO: 13); VHF6: CAGGTACAGCTGCAGCAGTCAGG (SEQ ID NO: 14); VHF7: CAGGTCCAGCTKGTGCARTC (SEQ ID NO: 15); VHF8: CAGGTCCAGCTGGTGCAGTC (SEQ ID NO: 16); VHF9: CAGGTACAGCTGGTGGAGTC (SEQ ID NO: 17); Heavy chain downstream primer: VHR1: TGAGGAGACGGTGACCTTTG (SEQ ID NO: 18); VHR2: TGAGGAGACGGTGACCAGGG (SEQ ID NO: 19); VHR3: TGAAGAGACGGTGACCATTG (SEQ ID NO: 20); VHR4: TGAGGAGACGGTGACCAGGG (SEQ ID NO: 21); VHR5: TGAGGAGACGGTGACCGTGG (SEQ ID NO: 22); Light chain kappa subtype upstream primer: Kappa1F:GACATCCAGATGACCCAGTCTCC (SEQ ID NO: 23); Kappa2F:GATGTTGTGATGACTCAGTCTCC (SEQ ID NO: 24); Kappa3F:GAAATTGTGTTGACGCAGTCTCC (SEQ ID NO: 25); Kappa4F:GACATCGTGATGACCCAGTCTCC (SEQ ID NO: 26); Kappa5F: GAAACGACACTCACGCAGTCTCC (SEQ ID NO: 27); Kappa6F:GAAATTGTGCTGACTCAGTCTCC (SEQ ID NO: 28); Light chain kappa subtype downstream primers: KappaR1:TTTGATATCCACTTTGGTCC (SEQ ID NO: 29); KappaR2:TTTGATCTCCACCTTGGTCC (SEQ ID NO: 30); KappaR3:TTTGATTTCCAGCTTGGTCC (SEQ ID NO: 31); KappaR4:TTTAATCTCCAGTCGTGTCC (SEQ ID NO: 32); KappaR5: TAGGACGGTCAGCTTGGTCC (SEQ ID NO: 33).

[0043] 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, extension at 72°C for 30 s, for a total of 30 cycles; and extension at 72°C for another 10 min. The variable regions of the light and heavy chains were connected to the T vector after gel excision and purification, and then sent for testing to obtain the sequences of the variable regions of the light and heavy chains. 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 variable regions of the heavy and light chains was about 350 bp, and the size of the scFv fragment after splicing was about 750 bp (such as Figure 2 and Figure 3 as shown).

[0044] Example 2: Construction of scFv phage library The scFv gene fragment was connected to the phage vector pComb3XSS via the SfiⅠ restriction site, and the ligation product was electroporated into E. coli TG1 competent cells. 2×YT-AG medium (containing 2% glucose, 100 μg / mL AMP) was added and cultured at 37°C for 1 h with shaking. 10 μL of bacterial solution was taken and inoculated into 2×YT-AG medium for overnight culture. The plaque formation was observed and the antibody library titer was calculated: Antibody library titer (PFU / mL) = dilution × number of plaques (PFU) / volume of bacterial solution used for plating (mL).

[0045] Randomly pick 10-20 positive colonies and send them to Sangon Technology Co., Ltd. for sequencing, perform colony PCR identification, and analyze the diversity of the antibody library. Add helper phage-M13K07 to the remaining bacterial solution, incubate at 37°C for 30 min, collect the bacteria by centrifugation, add 100 mL 2×YT medium (containing 1 μM IPTG, 100 μg / mL AMP) to resuspend, and culture overnight at 30°C in a shaking incubator; collect the supernatant by centrifugation, add a quarter volume of 20% PEG / 2.5 M NaCl, ice bath for 50 minutes to precipitate the phage, collect the precipitate by centrifugation, and resuspend the precipitate with PBS to obtain the phage antibody library.

[0046] Example 3: Enrichment and screening of human anti-FluB virus phage antibody library FluB Victoria and Yamagata recombinant antigens were mixed in a 1:1 ratio and coated on an immunotube at a concentration of 10 μg / mL, incubated at 4°C overnight, and blocked with 5% skim milk at 37°C for 2 h; washed twice with PBST, added with 1 mL of the scFv phage library prepared in Example 2, incubated at room temperature for 2 h, washed 10 times with PBST, added with 1 mL of triethylamine dissociation buffer (100 mM), incubated at room temperature for 10 min, neutralized with 1 M Tris-HCl (pH = 6.4), and added with 50 mL of TG1 bacterial solution in the logarithmic growth phase. After incubation at 37°C for 30 min, 10 μL was taken and coated with 2×YT / GA, and the plate was incubated at 37°C overnight to detect the output. The remaining bacterial solution was expanded and cultured until the bacterial solution A600 was about 0.5, and the helper phage M13K07 was added. The subsequent process was consistent with Example 2, and the above screening process was repeated for 4 rounds to enrich the phages that bind to the FluB antigen.

[0047] Example 4: Induced expression of positive phage clones Single colonies after four rounds of enrichment were randomly selected in 96-well plates, and a total of 6 plates were selected. 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 overnight cultured bacterial solution was transferred to a 96-well plate containing 100 μL of culture medium for activation, cultured at 37°C for 2-3 h. When the OD value was about 2, 20 μL of 2×YT+helper phage (MOI=5) mixture was added to each well and incubated at 37°C for 30 min. After shaking culture for 1.5 h, 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 h. The next day, the supernatant was centrifuged for detection.

[0048] Example 5: Phage monoclonal ELISA detection V and Y antigens were coated on ELISA plates at 4°C overnight. The plates were blocked with Casein blocking solution at 37°C for 2h, and then dried for later use. Monoclonal phage was added, and the plates were incubated at 37°C for 30 min. After washing with PBST solution for 6 times, the plates were dried and HRP-M13 was added. The plates were incubated at 37°C for 30 min, and then washed with PBST solution for 5 times. After drying and color development for 15 min, 2 MH 2 SO 4 The reaction was terminated and the absorbance at 450 nm was measured by an ELISA reader. The two clones with strong reactivity were sent for sequencing.

[0049] Example 6: Nucleic acid sequence analysis of variable region genes and construction of full-length IgM antibodies 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.

[0050] 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.

[0051] Using the two selected positive monoclonal clones as templates, the variable region gene sequences were amplified by PCR technology and constructed into the laboratory vectors IgM-PCMV3 and LC-PCMV3 (including constant region sequences) through HindIII / XbaI. After the recombinant plasmids were transferred into competent DH5α cells, positive clones were selected for sequencing and plasmid extraction was performed.

[0052] Example 7: Full-length IgM antibody expression Adjust the density of Expi CHO cells to 3×10 cells per day before transfection. 6 cells / mL to 4×106 On the day of transfection, cells were diluted to 6×10 cells / mL using ExpiCHO-S expression medium. 6 The extracted plasmids were diluted with plasmid DNA and liposomes according to the instructions of ExpiFectamine CHO transfection reagent, and the cells were transfected at a ratio of 1 μg: 5 μL. The FH105 and FH128 antibody plasmids contained light and heavy chain plasmids and J chain plasmids, of which HC:LC:J=1:1:1. After 24 h, 24% CHO feed and 0.6% enhancer were added, and the cell viability was reduced to about 70%-75%, and the supernatant was harvested.

[0053] Example 8: Purification of human anti-FluB virus full-length IgM antibody The supernatant of 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 turn. The filtered antibody supernatant was loaded and equilibrated again. Finally, the target protein antibody was eluted with dissociation buffer (0.1 M sodium citrate pH 2.3), and the dissociation peak was collected. The protein content was detected using a spectrophotometer NanoDrop One and the protein expression level was detected by SDS-PAGE and SEC-HPLC.

[0054] The results showed that after purification, the expression levels of FH105 and FH128 antibodies were 200 mg / L and 150 mg / L respectively, the molecular weight of the heavy chain was about 65 KD, and the molecular weight of the light chain was about 25 KD, which was consistent with expectations; Figure 4 The HPLC test results showed that the antibody purity reached more than 98% (about 150 KDa). The HPLC results showed that the target antibody was mostly in the form of aggregates; a small number of monomers (about 200 kDa) existed.

[0055] Example 9: ELISA detection of human anti-FluB virus full-length IgM antibodies The test was performed using the influenza B virus IgM kit according to the instructions. According to the kit judgment standard: S / CO value ≥ 10AU / mL is positive. The test results of the FH105 IgM and FH128 IgM human antibodies prepared in this experiment are shown in Table 1. The average detection concentrations of FH105 IgM and FH128 IgM were 27015 and 25227, respectively, with strong reactivity, which met the reactivity requirements as positive quality control products. Subsequently, the titers (S / CO) of FH105 IgM and FH128 IgM antibodies were adjusted to three levels: low, medium, and high (target values ​​were 3±1, 7±1, and 13±1, respectively). After freeze-drying, the titer variation after reconstitution was assessed by heat acceleration at 37°C for 7 days and 14 days. The stability data is shown in Table 2. The variation is within 10%, which meets the reagent use requirements. Since the reactivity of FH105 IgM antibody is 7% higher than that of FH128 IgM antibody, FH105 is preferred.

[0056] Table 1: FH105 IgM and FH128 IgM antibody reactivity test results

[0057] Table 2: Results of accelerated stability test of FH105 IgM and FH128 IgM antibodies at 37°C

[0058] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Antibodies, including single-chain antibodies, characterized in that Its heavy chain variable region has: (1) the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or (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 (3) an amino acid sequence having at least 95% homology to the amino acid sequence shown in (1) or (2); Its light chain variable region has: (4) the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6; or (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 (6) an amino acid sequence having at least 95% homology to the amino acid sequence shown in (4) or (5); The plurality is 2 to 5.

2. The antibody according to claim 1, characterized in that 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, characterized in that have: (7), the nucleotide sequence as shown in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 7 or SEQ ID NO: 8; or (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 (9) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (7) or (8); The plurality is 2 to 30.

5. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 3 or 4.

6. Genetically engineered biological material, including bacteriophages, bacteria, fungi, plant cells or animal cells, characterized in that: Comprising the expression vector according to claim 5.

7. Application of any of the following in the preparation of standard substances: 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 substances and standard substances.

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 as claimed in 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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