A method for preparing IgA dimers

Abstract

The application discloses a method for preparing IgA dimer. The application provides a method for preparing IgA dimer, comprising the following steps: (1) preparing a heavy chain expression plasmid and a light chain expression plasmid; the heavy chain expression plasmid is used for expressing a heavy chain fusion protein; the heavy chain fusion protein comprises the following elements in sequence: J chain, self-cleavage peptide, secretory signal peptide, IgA antibody heavy chain; the light chain expression plasmid is used for expressing a light chain fusion protein; the light chain fusion protein comprises the following elements in sequence: secretory signal peptide, IgA antibody light chain; (2) co-transfecting the heavy chain expression plasmid and the light chain expression plasmid into mammalian cells, and then culturing to obtain IgA dimer. The method can produce a large amount of high-purity dimer IgA. The application has important theoretical guiding value and wide application prospect in the expression production of dimer IgA, research, clinical popularization and industrial production of dimer IgA antibody.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a method for preparing IgA dimers. Background Technology

[0002] The novel coronavirus is mainly transmitted through the respiratory tract. The antigen on the surface of the virus is mainly its spike protein, and the antigen receptor is angiotensin-converting enzyme 2 (ACE2). The virus invades the human body mainly through infection mediated by the contact between the spike protein and ACE2.

[0003] The main antibody types in the human body are IgG, IgA, and IgM. IgA antibodies are second only to IgG in blood concentration. IgA exists primarily in two forms: IgA monomer (mIgA) and IgA dimer (dIgA) formed by J-chain linkages. In the upper respiratory tract, such as the nasal cavity, IgA dimers are secreted along with secretory components (SCs) into the upper respiratory tract, playing a crucial role in the body's defense against invading pathogens.

[0004] Research on IgA antibodies is of great significance for preventing and treating novel coronavirus infection or other respiratory pathogen invasion. However, what worries everyone is that IgA antibodies have diverse components and complex expression, and existing expression methods suffer from difficulties such as low yield and unstable batch expression, which hinder the advancement of IgA antibody research towards clinical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing IgA dimers.

[0006] This invention provides a method for preparing IgA dimers, comprising the following steps:

[0007] (1) Prepare heavy chain expression plasmid and light chain expression plasmid;

[0008] The heavy chain expression plasmid is used to express the heavy chain fusion protein; the heavy chain fusion protein includes the following components in sequence: J chain, self-cleaving peptide, secretory signal peptide, and IgA antibody heavy chain;

[0009] The light chain expression plasmid is used to express a light chain fusion protein; the light chain fusion protein includes the following components in sequence: a secreted signal peptide and an IgA antibody light chain;

[0010] (2) Heavy chain expression plasmid and light chain expression plasmid were co-transfected into mammalian cells and then cultured to obtain IgA dimers.

[0011] Specifically, the heavy chain fusion protein is composed of the following components in sequence: J chain, self-cleaving peptide, secretory signal peptide, and IgA antibody heavy chain.

[0012] Specifically, the light chain fusion protein is composed of the following components in sequence: a secretory signal peptide and an IgA antibody light chain;

[0013] The self-cleaving peptide is a 2A self-cleaving peptide.

[0014] The 2A self-cleaving peptide refers to a polypeptide containing "VEENPGP".

[0015] “VEENPGP” is the active site of the 2A self-cleaving peptide.

[0016] The 2A self-cleaving peptide may specifically be a T2A self-cleaving peptide or a P2A self-cleaving peptide, etc.

[0017] The cleavage site of the self-cleaving peptide is located between amino acid residue G and amino acid residue P of “VEENPGP”.

[0018] Specifically, the self-cleaving peptide is shown in positions 160-177 of SEQ ID NO: 1.

[0019] Specifically, in the heavy chain fusion protein, the secretory signal peptide is shown as positions 178-197 in SEQ ID NO: 1;

[0020] Specifically, in the light chain fusion protein, the secretory signal peptide is shown as positions 1-19 in SEQ ID NO: 3.

[0021] Specifically, the J chain is shown in bits 1-159 of SEQ ID NO: 1.

[0022] The IgA antibody heavy chain consists of a variable region and a constant region from upstream to downstream.

[0023] The light chain of an IgA antibody consists of a variable region and a constant region from upstream to downstream.

[0024] Specifically, the heavy chain constant region of the IgA antibody heavy chain is shown in positions 325-677 of SEQ ID NO: 1;

[0025] Specifically, the light chain constant region of the IgA antibody light chain is shown in positions 127-233 of SEQ ID NO: 3.

[0026] Specifically, the variable region of the heavy chain of the IgA antibody heavy chain is shown in positions 198-324 of SEQ ID NO: 1.

[0027] Specifically, the variable region of the light chain of the IgA antibody light chain is shown in positions 20-126 of SEQ ID NO: 3.

[0028] Specifically, the heavy chain variable region of the IgA antibody heavy chain is shown in SEQ ID NO: 8.

[0029] Specifically, the variable region of the light chain of the IgA antibody light chain is shown in SEQ ID NO: 10.

[0030] Specifically, the heavy chain variable region of the IgA antibody heavy chain is shown in SEQ ID NO: 12.

[0031] Specifically, the variable region of the light chain of the IgA antibody light chain is shown in SEQ ID NO: 14.

[0032] The heavy chain expression plasmid contains a DNA molecule encoding the heavy chain fusion protein.

[0033] The light chain expression plasmid contains a DNA molecule encoding the light chain fusion protein.

[0034] The DNA molecule encoding the heavy chain fusion protein comprises, in sequence, the following elements: a DNA segment encoding the J chain, a DNA segment encoding a self-cleaving peptide, a DNA segment encoding a secretory signal peptide, and a DNA segment encoding the IgA antibody heavy chain. The DNA molecule encoding the heavy chain fusion protein is composed of, in sequence, the following elements: a DNA segment encoding the J chain, a DNA segment encoding a self-cleaving peptide, a DNA segment encoding a secretory signal peptide, and a DNA segment encoding the IgA antibody heavy chain.

[0035] The DNA molecule encoding the light chain fusion protein comprises, in sequence, the following elements: a DNA segment encoding a secretory signal peptide and a DNA segment encoding an IgA antibody light chain. The DNA molecule encoding the light chain fusion protein is composed of, in sequence, the following elements: a DNA segment encoding a secretory signal peptide and a DNA segment encoding an IgA antibody light chain.

[0036] Specifically, the DNA segment encoding the J chain is shown in positions 1-477 of SEQ ID NO: 2.

[0037] Specifically, the DNA segment encoding the self-cleaving peptide is shown in positions 478-531 of SEQ ID NO: 2.

[0038] Specifically, the DNA segment encoding the secretory signal peptide is shown in positions 532-591 of SEQ ID NO: 2.

[0039] Specifically, the DNA segment encoding the secretory signal peptide is shown in positions 1-57 of SEQ ID NO: 4.

[0040] Specifically, the DNA segment encoding the heavy chain constant region of the IgA antibody heavy chain is shown in positions 973-2034 of SEQ ID NO: 2.

[0041] Specifically, the DNA segment encoding the light chain constant region of the IgA antibody light chain is shown in positions 379-702 of SEQ ID NO: 4.

[0042] Specifically, the DNA segment encoding the variable region of the heavy chain of the IgA antibody heavy chain is shown in positions 592-972 of SEQ ID NO: 2.

[0043] Specifically, the DNA segment encoding the light chain variable region of the IgA antibody light chain is shown in positions 58-378 of SEQ ID NO: 4.

[0044] Specifically, the DNA segment encoding the heavy chain variable region of the IgA antibody heavy chain is shown in SEQ ID NO: 9.

[0045] Specifically, the DNA segment encoding the light chain variable region of the IgA antibody light chain is shown in SEQ ID NO: 11.

[0046] Specifically, the DNA segment encoding the variable region of the heavy chain of the IgA antibody heavy chain is shown in SEQ ID NO: 13.

[0047] Specifically, the DNA segment encoding the light chain variable region of the IgA antibody light chain is shown in SEQ ID NO: 15.

[0048] The heavy chain expression plasmid can specifically be a recombinant plasmid obtained by inserting a DNA molecule encoding the heavy chain fusion protein into the multiple cloning site of the pCDNA3.4 vector.

[0049] The heavy chain expression plasmid can specifically be a recombinant plasmid obtained by replacing a small fragment between two multiple cloning sites (e.g., XBaI and AgeI restriction sites) of the pCDNA3.4 vector with a DNA molecule encoding the heavy chain fusion protein.

[0050] The light chain expression plasmid can specifically be a recombinant plasmid obtained by inserting a DNA molecule encoding the light chain fusion protein into the multiple cloning site of the pCDNA3.1 vector.

[0051] The light chain expression plasmid can specifically be a recombinant plasmid obtained by replacing a small fragment between the two multiple cloning sites (BamHI and EcoRI restriction sites) of the pCDNA3.1 vector with a DNA molecule encoding the light chain fusion protein.

[0052] The present invention also includes a kit for preparing IgA dimers, comprising any of the heavy chain expression plasmids and any of the light chain expression plasmids described above.

[0053] The kit also includes mammalian cells.

[0054] The IgA consists of an IgA antibody heavy chain and an IgA antibody light chain.

[0055] The IgA antibody heavy chain is any one of those described above.

[0056] The IgA antibody light chain is any one of those described above.

[0057] Any of the mammalian cells mentioned above may be 293 cells or their derivatives.

[0058] The mammalian cells mentioned above can specifically be 293F cells.

[0059] This invention also protects the use of the kit in the preparation of IgA dimers.

[0060] The method of this invention can produce large quantities of high-purity dimer IgA.

[0061] This invention is used for the expression and production of dimer IgA, and has important theoretical guiding value and broad application prospects for the research, clinical promotion and industrial production of dimer IgA antibodies. Attached Figure Description

[0062] Figure 1 This is a schematic diagram of the expression strategies for Example 1 and Comparative Example 2.

[0063] Figure 2 This is a chromatogram of a small-scale SEC chromatography column used in Example 1.

[0064] Figure 3 Electrophoresis images of reducing SDS-PAGE and non-reducing SDS-PAGE in Example 1.

[0065] Figure 4 The tomography diagram is shown in Comparative Example 1, which uses a small-scale SEC chromatography column.

[0066] Figure 5 This is a graph showing the neutralization activity results of Example 2.

[0067] Figure 6 This is a graph showing the neutralization activity results of Example 4. Detailed Implementation

[0068] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are performed in triplicate, and the average value is taken. In the examples, the recombinant plasmids have all been sequenced for verification. SMM 293-TII medium: Sinocare Biotechnology Co., Ltd., catalog number M293TII. PEI transfection reagent: PEI Max, Polysciences Biotechnology Co., Ltd., catalog number 24765. The pCDNA3.4 vector is a circular double-stranded DNA molecule, the full sequence of which is shown in SEQ ID NO: 16. The pCDNA3.1 vector is a circular double-stranded DNA molecule, the full sequence of which is shown in SEQ ID NO: 17.

[0070] hACE2-hela cells (ie "HeLa cell lines stably expressing theACE2molecules"), recorded in the following literature: Wang, R., Zhang, Q., Ge, J., Ren, W., Zhang, R., Lan, J., Ju, B., Su, B., Yu, F., Ch. en,P.,Liao,H.,Feng,Y.,Li,X.,Shi,X.,Zhang,Z.,Zhang,F.,Ding,Q.,Zhang,T.,Wang,X.&Zhang,L.Analysis of SARS-CoV-2 variant mutations reveal neutralization escape mechanisms and the ability to use ACE2receptors from additional species. Immunity 54, 1611-1621.e1615, doi:10.1016 / j.immuni.2021.06.003(2021).

[0071] Example 1: Preparation of antibody 311 using the expression strategy of the present invention.

[0072] I. Construction of recombinant plasmids

[0073] See the diagram of the expression strategy. Figure 1 A.

[0074] 1. Prepare a heavy chain expression plasmid, named 311-2AHC plasmid.

[0075] The 311-2AHC plasmid is a recombinant plasmid obtained by replacing the small fragment between the XBaI and AgeI restriction sites in the pCDNA3.4 vector with the double-stranded DNA molecule shown in SEQ ID NO: 2. The double-stranded DNA molecule shown in SEQ ID NO: 2 encodes the protein shown in SEQ ID NO: 1.

[0076] In SEQ ID NO: 1, positions 1-159 form the J chain, positions 160-177 form the self-cleaving peptide, positions 178-197 form the IL2 signal peptide, positions 198-324 form the variable region of the heavy chain of antibody 311, and positions 325-677 form the constant region of the heavy chain of antibody 311. The self-cleaving peptide has a self-cleaving function, and the signal peptide is cleaved, ultimately producing the following two proteins: the protein shown in positions 1-176 of SEQ ID NO: 1 (which contains the J chain), and the protein shown in positions 198-677 of SEQ ID NO: 1 (i.e., the heavy chain of antibody 311).

[0077] 2. Prepare a light chain expression plasmid, named 311-LC plasmid.

[0078] The 311-LC plasmid is a recombinant plasmid obtained by replacing the small fragment between the BamHI and EcoRI restriction sites in the pCDNA3.1 vector with the double-stranded DNA molecule shown in SEQ ID NO: 4. The double-stranded DNA molecule shown in SEQ ID NO: 4 encodes the protein shown in SEQ ID NO: 3.

[0079] In SEQ ID NO: 3, positions 1-19 constitute the secretory signal peptide, positions 20-126 constitute the variable region of the light chain of antibody 311, and positions 127-233 constitute the constant region of the light chain of antibody 311. The signal peptide is cleaved, ultimately producing the protein shown in positions 20-233 of SEQ ID NO: 3 (i.e., the light chain of antibody 311).

[0080] II. Preparation of Antibody 311

[0081] 1. Add 5 ml of PEI transfection reagent to 50 ml of SMM 293-TII medium, then add 1 mg of 311-2AHC plasmid and 1 mg of 311-LC plasmid. Mix thoroughly and let stand for 15-20 min. Then add to 1 L of 293F cell suspension and culture for 96 hours. 293F cell suspension: 293F cells are suspended in SMM 293-TII medium at a cell density of (2-3) × 10⁻⁶ cells / mL. 6 Cells / ml

[0082] 2. After completing step 1, collect the entire culture system, centrifuge at 4000 rpm and discard the cell pellet, and collect the supernatant (approximately 1 L of supernatant was obtained).

[0083] 3. Take all the supernatant obtained in step 2 and use CaptureSelect. TM IgA affinity matrix (Thermofisher, Cat. 194288005) was used to purify the antibody according to its instructions. Specific steps: 2 ml of Capture Select was added to the supernatant. TM The IgA affinity matrix was incubated overnight at 4°C. The entire system was then transferred to an empty chromatography column, washed with 10-20 ml of PBS buffer (pH 7.2, 10 mM), eluted with 10 ml of glycine-HCl buffer (pH 3.0, 0.1 M), and the eluent was collected.

[0084] 4. Take all the eluent collected in step 3, adjust the pH to 7, then filter it through a 0.45 μm membrane for sterilization and collect the filtrate. Concentrate the filtrate using a Merck 30KD Amicon Ultra-15 centrifugal filter to obtain 3 ml of concentrate. The antibody protein concentration in the concentrate was measured using a Thermo nanodrop and found to be 3 mg / ml. Therefore, the total protein yield was 9 mg.

[0085] 5. Take 1 ml of the concentrate obtained in step 4 and load it onto Superose. TM 6. Increase the number of 10 / 300GL small-scale SEC chromatography columns (Cytiva, Cat. 29091596) and then elute with PBS buffer (pH 7.2, 10 mM). See the chromatogram below. Figure 2 The chromatography spectrum showed characteristic peaks of the IgA dimer (based on the column and expected molecular weight, the theoretical retention volume corresponding to the characteristic peaks is in the range of 11-13 ml), with peak intensities exceeding 800 mAU. The chromatogram indicated that high-purity IgA dimers were obtained in high yield. The post-column solution corresponding to the characteristic peaks of the IgA dimer was collected, which is the dIgA-311 antibody solution.

[0086] 6. Take the dIgA-311 antibody solution prepared in step 5 and perform reducing SDS-PAGE and non-reducing SDS-PAGE respectively. The results are shown in the figure. Figure 3 . Figure 3 The antibody bands were observed to be concentrated and uniform, indicating that the antibody solution contained high-purity IgA dimers.

[0087] Comparative Example 1: Antibody 311 was prepared using the traditional method (three-plasmid expression strategy).

[0088] I. Construction of recombinant plasmids

[0089] 1. Prepare the heavy chain expression plasmid, named 311-HC plasmid.

[0090] The 311-HC plasmid is a recombinant plasmid obtained by replacing the small fragment between the XBaI and AgeI restriction sites in the pCDNA3.4 vector with the double-stranded DNA molecule shown in SEQ ID NO: 5.

[0091] In SEQ ID NO: 5, nucleotides 1-60 encode the IL2 signal peptide, and nucleotides 61-1503 encode the heavy chain of antibody 311.

[0092] 2. Prepare the J-chain expression vector, named Human-J plasmid.

[0093] The Human-J plasmid is a recombinant plasmid obtained by replacing the small fragment between the XBaI and AgeI restriction sites in the pCDNA3.4 vector with the double-stranded DNA molecule shown in SEQ ID NO: 6.

[0094] The DNA molecule shown in SEQ ID NO: 6 encodes the J strand.

[0095] 3. Light chain expression plasmid, namely the 311-LC plasmid prepared in Example 1.

[0096] II. Preparation of Antibody 311

[0097] 1. Add 5 ml of PEI transfection reagent to 50 ml of SMM 293-TII medium, then add 1 mg of 311-HC plasmid, 1 mg of Human-J plasmid, and 1 mg of 311-LC plasmid. Mix thoroughly and let stand for 15-20 min. Then add to 1 L of 293F cell suspension and culture for 96 hours. 293F cell suspension: 293F cells are suspended in SMM 293-TII medium at a cell density of (2-3) × 10⁻⁶ cells / mL. 6 Cells / ml

[0098] 2. Same as step 2 of Example 1.

[0099] 3. Same as step 3 in Example 1.

[0100] 4. Same as step 4 in Example 1. The total protein yield is 0.8 mg.

[0101] 5. Same as step 5 of step two in Example 1.

[0102] See the chromatogram Figure 4 The chromatography showed characteristic peaks for both IgA dimers (the theoretical retention volume for these peaks is 11-13 ml, based on the column and expected molecular weight) and IgA monomers (the theoretical retention volume for these peaks is 13-15 ml, based on the column and expected molecular weight). The intensity of the characteristic peak for the IgA dimer did not exceed 100 mAU. This indicates that the traditional method for preparing IgA dimers results in low yield and low purity.

[0103] Comparative Example 2: Preparation of Antibodies using Other Expression Strategies 311

[0104] I. Construction of recombinant plasmids

[0105] See the diagram of the expression strategy. Figure 1 B.

[0106] 1. Prepare a heavy chain expression plasmid and name it the heavy chain control plasmid.

[0107] The heavy chain control plasmid is a recombinant plasmid obtained by replacing the small fragment between the XBaI and AgeI restriction sites in the pCDNA3.4 vector with the double-stranded DNA molecule shown in SEQ ID NO: 7.

[0108] In SEQ ID NO: 7, nucleotides 1-60 encode the IL2 signal peptide, nucleotides 61-1500 encode the heavy chain of antibody 311, nucleotides 1501-1554 encode the self-cleaving peptide, and nucleotides 1555-2034 encode the J chain.

[0109] 2. Light chain expression plasmid, namely the 311-LC plasmid prepared in Example 1.

[0110] II. Preparation of Antibody 311

[0111] 1. Add 5 ml of PEI transfection reagent to 50 ml of SMM 293-TII medium, add 1 mg of heavy chain control plasmid and 1 mg of 311-LC plasmid, mix thoroughly, and let stand for 15-20 min. Then add to 1 L of 293F cell suspension and culture for 96 hours. 293F cell suspension: 293F cells are suspended in SMM 293-TII medium at a cell density of (2-3) × 10⁻⁶ cells / mL. 6 Cells / ml

[0112] 2. Same as step 2 of Example 1.

[0113] 3. Same as step 3 in Example 1.

[0114] 4. Same as step 4 in Example 1. The total protein yield is 100 μg.

[0115] Example 2: Functional verification of dIgA-311 antibody

[0116] I. Preparation of Novel Coronavirus Pseudovirus

[0117] Co-transfection of 293T cells with a plasmid expressing the SARS-CoV-2 membrane protein and a backbone plasmid pNL4-3R-E-luciferase yielded an infectious but non-replicating pseudovirus of SARS-CoV-2, with infectivity similar to that of live SARS-CoV-2. The backbone plasmid pNL4-3R-E-luciferase, i.e., the backbone plasmid pNL4-3R-E containing Luciferase (i.e., vector with the luciferase gene containing backbone pNL4-3R-E in the literature), is described in the following literature: Wang Q, Liu L, Ren W, Gettie A, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang L. Cell Rep. 2019.

[0118] The gene encoding the novel coronavirus membrane protein was inserted between the BamHII and EcoRI restriction sites of the pcDNA3.1(+) vector to obtain a plasmid expressing the novel coronavirus membrane protein. The plasmid expressing the novel coronavirus membrane protein and the backbone plasmid pNL4-3R-E-luciferase were co-transfected into 293T cells and incubated at 37°C (using DMEM medium containing 10% fetal bovine serum). The cell culture supernatant was collected 60 hours after transfection; this was the viral fluid containing the novel coronavirus pseudovirus. The gene encoding the membrane protein of the wild-type (WT) novel coronavirus is shown in SEQ ID NO: 18.

[0119] II. Neutralization Activity Detection

[0120] 1. Take the dIgA-311 antibody solution prepared in Example 1 and dilute it with PBS buffer (pH 7.2, 10mM) to obtain antibody dilution solutions of various concentrations.

[0121] 2. Take a 96-well cell culture plate, add 100 μL of antibody diluent and 50 μL of the virus solution prepared in step one to each well (the virus concentration in the 50 μL virus solution is 1×10⁻⁶). 4Add 100 μL of PBS buffer (pH 7.2, 10 mM) and 50 μL of the virus solution prepared in step one to each virus control well (the virus concentration in the 50 μL virus solution is 1×10⁻⁶). 4 Add 100 μL of PBS buffer (pH 7.2, 10 mM) and 50 μL of DMEM medium containing 10% fetal bovine serum to each cell control well (TCID 50 / ml), and incubate at 37°C for 1 hour.

[0122] 3. After completing step 2, take the cell culture plate and inoculate each well with 100 μL of hACE2-hela cell suspension (the solvent used to prepare the cell suspension is DMEM medium containing 10% fetal bovine serum, and the concentration of hACE2-hela cells in the cell suspension is 2 × 10⁻⁶). 5 (cells / ml), incubated at 37°C for 64 hours.

[0123] 4. After completing step 3, take the cell culture plate, aspirate the supernatant, add 100 μL of lysis buffer (Novizan Biotechnology, catalog number DD1204-03, follow the instructions) to each well, and incubate at 37°C for 5 minutes.

[0124] 5. After completing step 4, take the cell culture plate and detect the luciferase activity.

[0125] Each process is configured with multiple duplicate holes.

[0126] Neutralization activity (%) = [1 - (fluorescence intensity of experimental group - fluorescence intensity of cell control) / (fluorescence intensity of virus control - fluorescence intensity of cell control)] × 100%.

[0127] Neutralization activity results are shown in Figure 5 . Figure 5 The vertical axis represents neutralizing activity (%); the horizontal axis represents the antibody concentration (μg / ml) logarithm to base 10. The antibody concentration refers to the antibody concentration in the mixed system consisting of 100 μL of antibody dilution and 50 μL of test virus solution in step 2.

[0128] The antibody concentration at which the neutralizing activity is 50% was calculated using Prism 5 software, i.e., the IC50 value of the antibody.

[0129] The IC50 value of dIgA-311 antibody against the pseudovirus of the wild-type novel coronavirus was 1.5 ng / ml.

[0130] Example 3: Preparation of three antibodies using the expression strategy of the present invention.

[0131] I. Preparation of antibody dIgA-311

[0132] The method is the same as in Example 1.

[0133] II. Preparation of antibody dIgA-341

[0134] The only difference is between the heavy chain expression plasmid and the light chain expression plasmid.

[0135] Compared to plasmid 311-2AHC, the heavy chain expression plasmid differs only in that the coding sequence of the heavy chain variable region of antibody 311 (as shown in positions 592-972 of SEQ ID NO: 2) is replaced with the coding sequence of the heavy chain variable region of antibody 341 (as shown in SEQ ID NO: 9). The heavy chain variable region of antibody 341 is shown in SEQ ID NO: 8.

[0136] Compared to the 311-LC plasmid, the light chain expression plasmid differs only in that the coding sequence of the light chain variable region of antibody 311 (as shown in positions 58-378 of SEQ ID NO: 4) is replaced with the coding sequence of the light chain variable region of antibody 341 (as shown in SEQ ID NO: 11). The light chain variable region of antibody 341 is shown in SEQ ID NO: 10.

[0137] Everything else is the same as in Example 1.

[0138] III. Preparation of antibody dIgA-335

[0139] Compared to plasmid 311-2AHC, the heavy chain expression plasmid differs only in that the coding sequence of the heavy chain variable region of antibody 311 (as shown in positions 592-972 of SEQ ID NO: 2) is replaced with the coding sequence of the heavy chain variable region of antibody 335 (as shown in SEQ ID NO: 13). The heavy chain variable region of antibody 335 is shown in SEQ ID NO: 12.

[0140] Compared to the 311-LC plasmid, the light chain expression plasmid differs only in that the coding sequence of the light chain variable region of antibody 311 (as shown in positions 58-378 of SEQ ID NO: 4) is replaced with the coding sequence of the light chain variable region of antibody 335 (as shown in SEQ ID NO: 15). The light chain variable region of antibody 335 is shown in SEQ ID NO: 14.

[0141] Everything else is the same as in Example 1.

[0142] The purity and yield of the antibody in steps one, two, and three are shown in Table 1.

[0143] Table 1. Comparison of expression results of three different IgA antibodies.

[0144] Antibody name Antibody expression volume Training time Final antibody yield antibody purity dIgA-311 800ml 96h 9.2mg >90％ dIgA-341 800ml 96h 10.6mg >90％ dIgA-335 800ml 96h 11.4mg >90％

[0145] Example 4: Functional Verification of the Antibody

[0146] The three antibodies prepared in Example 3 were functionally verified using the same method as in Example 2.

[0147] See results Figure 6 .

[0148] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing IgA dimer, comprising the following steps: (1) preparing a heavy chain expression plasmid and a light chain expression plasmid; the heavy chain expression plasmid is used for expressing a heavy chain fusion protein; the heavy chain fusion protein sequentially comprises the following elements from N-terminal to C-terminal: J chain, self-cleavage peptide, secretory signal peptide, IgA antibody heavy chain; the light chain expression plasmid is used for expressing a light chain fusion protein; the light chain fusion protein sequentially comprises the following elements from N-terminal to C-terminal: secretory signal peptide, IgA antibody light chain; (2) co-transfecting the heavy chain expression plasmid and the light chain expression plasmid into mammalian cells, and then culturing to obtain IgA dimer.

2. The method of claim 1, wherein: The cleavage site of the self-cleavage peptide is between amino acid residue G and amino acid residue P. 3.The method of claim 1, wherein: in the heavy chain fusion protein, the secretory signal peptide is shown as positions 178-197 in SEQ ID NO: 1; in the light chain fusion protein, the secretory signal peptide is shown as positions 1-19 in SEQ ID NO:

3. 4.The method of claim 1, wherein: the J chain is shown as positions 1-159 in SEQ ID NO:

1. 5.The method of claim 1, wherein: the self-cleavage peptide is shown as positions 160-177 in SEQ ID NO:

1. 6.The method of claim 1, wherein: the constant region of the IgA antibody heavy chain is shown as positions 325-677 in SEQ ID NO: 1; the constant region of the IgA antibody light chain is shown as positions 127-233 in SEQ ID NO:

3.

7. The method of any one of claims 1 to 6, wherein: the mammalian cells are 293F cells. 8.A kit for preparing IgA dimer, comprising a heavy chain expression plasmid and a light chain expression plasmid; the heavy chain expression plasmid is as described in any one of claims 1 to 6; the light chain expression plasmid is as described in any one of claims 1 to 6.

9. The kit of claim 8, wherein: the kit further comprises mammalian cells. 10.Use of the kit of claim 8 or 9 in preparing IgA dimer.

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