Mouse anti-human CD16 recombinant antibody as well as preparation method and application thereof

Abstract

The invention discloses a mouse anti-human CD16 recombinant antibody as well as a preparation method and application thereof, and belongs to the technical field of monoclonal antibody preparation. In a light chain variable region of the mouse anti-human CD16 recombinant antibody, the amino acid sequence of CDR-L1 is RASENIYRYLA, the amino acid sequence of CDR-L2 is NAKTLAE, and the amino acid sequence of CDR-L3 is QHHYRTPYT; the amino acid sequence of the CDR-H1 in the heavy chain variable region is TYWMH, the amino acid sequence of the CDR-H2 in the heavy chain variable region is EINPSNGRTNYNEKFKT, and the amino acid sequence of the CDR-H3 in the heavy chain variable region is NYYGPFPY. According to the preparation method, light and heavy chain variable region sequences are connected through Linker and inserted into an eukaryotic expression vector containing an Fc gene, then CHO-S cells are transfected, the disulfide bond mismatch probability is reduced, and the obtained antibody is high in correct conformation proportion and good in homogeneity and can be used for detecting human CD16 natural molecules through flow cytometry, IF, WB and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of monoclonal antibody preparation, and particularly relates to a murine anti-human CD16 recombinant antibody and a preparation method and application thereof. BACKGROUND

[0002] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal hematopoietic stem cell disease, and its clinical manifestations include varying degrees of paroxysmal intravascular hemolysis, paroxysmal hemoglobinuria, bone marrow failure and formation of venous thrombosis. The pathogenesis of PNH is that the glycosylphosphatidylinositol-A (PIG-A) gene located on the X chromosome (Xp22.1) undergoes somatic mutation at the level of hematopoietic stem cells, resulting in partial or complete glycosylphosphatidylinositol (GPI) anchor synthesis and expression disorders, which causes the lack of cell surface anchor proteins that are normally combined with GPI anchors. So far, more than 20 GPI anchor proteins such as CD55, CD59, CD14 and CD16 have been found, among which CD16 is expressed in natural killer (NK) cells, granulocytes, mononuclear cells and macrophages.

[0003] Flow cytometry (FCM) can detect the loss of GPI anchor proteins and anchors in different blood cell populations, accurately quantify the size and characteristics of PNH clones, and has high sensitivity for detecting small PNH clones. Therefore, it has become the current gold standard for PNH detection (for PNH clone reporting, international standards have been proposed: 1. PNH cells > 1% are called PNH clones; 2. PNH cells are 0.1%-1% are called low PNH clones; 3. PNH cells < 0.1% are called a small number of GPI-deficient cells or a small number of PNH phenotype cells). With the improvement of understanding of PNH, the international community has overdone the detection of PNH to a high sensitivity level. However, the frequency of PNH detection and the detection rate of positive patients in many domestic institutions are relatively low, the antibody combination of PNH-FCM detection is not standardized, the quality control of the instrument is not standardized, and the detection personnel lack clinical practice experience, which leads to large differences in detection results in different units. Among them, the sensitivity and stability of FCM detection antibodies are particularly important. At present, part of the antibodies for clinical FCM detection are prepared from mouse ascites, and part of them are conventional recombinant antibodies. It is found in clinical detection that the sensitivity and stability of the antibodies have further room for improvement.

[0004] The method of conventional recombinant antibody is to construct light chain and heavy chain into corresponding vectors respectively, co-transfect into the same cell, and then combine into complete antibody after the light chain and the heavy chain are respectively transcribed into peptide chains. The light chain vector and the heavy chain vector need to be in a proper proportion, the internal disulfide bond of the light chain, the internal disulfide bond of the heavy chain and the disulfide bond between the light chain and the heavy chain need to be connected at correct positions, so as to form an antibody with correct conformation. The wrong conformation will affect the affinity of the antibody, thereby reducing the sensitivity. The wrong conformation, especially the disulfide bond mismatch, is easy to form a precipitate, thereby affecting the secretion of the antibody and reducing the supernatant expression amount of the antibody. The proportion of the antibody with wrong conformation is uncontrollable, which will affect the uniformity of the antibody and further affect the detection stability. SUMMARY

[0005] The purpose of the present application is to solve the problems of high disulfide bond mismatch rate, low proportion of correct conformation antibody and poor uniformity of conventional recombinant antibody in the prior art, and provide a mouse anti-human CD16 recombinant antibody, and further provide a preparation method and application of the antibody.

[0006] The technical scheme of the present application is described in detail as follows: In a first aspect, the present application provides a mouse anti-human CD16 recombinant antibody, comprising a light chain variable region and a heavy chain variable region, the light chain variable region comprising a complementarity determining region CDR-L1, CDR-L2 and CDR-L3, wherein: the CDR-L1 amino acid sequence is RASENIYRYLA (SEQ ID NO: 1), the CDR-L2 amino acid sequence is NAKTLAE (SEQ ID NO: 2), the CDR-L3 amino acid sequence is QHHYRTPYT (SEQ ID NO: 3); the heavy chain variable region comprises a complementarity determining region CDR-H1, CDR-H2 and CDR-H3, wherein: the CDR-H1 amino acid sequence is TYWMH (SEQ ID NO: 4), the CDR-H2 amino acid sequence is EINPSNGRTNYNEKFKT (SEQ ID NO: 5), the CDR-H3 amino acid sequence is NYYGPFPY (SEQ ID NO: 6).

[0007] Optionally or preferably, the light chain variable region amino acid sequence of the above mouse anti-human CD16 recombinant antibody is connected with the N-terminal of the heavy chain variable region through a Linker.

[0008] Optionally or preferably, the amino acid sequence of the above mouse anti-human CD16 recombinant antibody is shown as SEQ ID NO: 7.

[0009] Optionally or preferably, the above-mentioned mouse anti-human CD16 recombinant antibody further comprises an Fc fragment, which is in the same polypeptide sequence with the light chain variable region and the heavy chain variable region.

[0010] Optionally or preferably, the above-mentioned mouse anti-human CD16 recombinant antibody, the Fc fragment is connected to the C-terminal of the heavy chain variable region through an enzyme cutting site sequence, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 8, which is Uniprot, P01868, https: / / www.uniprot.org / uniprotkb / P01868, amino acids 103-324.

[0011] Optionally or preferably, the above-mentioned mouse anti-human CD16 recombinant antibody, the amino acid sequence is shown in SEQ ID NO: 9. It is a polypeptide comprising a light chain variable region, a linker, a heavy chain variable region and an Fc fragment. The antibody hinge region in the sequence, specifically Uniprot, P01868, https: / / www.uniprot.org / uniprotkb / P01868, amino acids 103-110 (GCKPCICT) can form a dimer structure. This structure can automatically form a dimer inside the cell when the recombinant antibody is expressed.

[0012] In a second aspect, the present application provides a preparation method of a mouse anti-human CD16 recombinant antibody, comprising the following steps: (1) inserting the coding gene of the amino acid sequence shown in SEQ ID NO: 7 into a eukaryotic expression vector containing the Fc fragment gene of mouse IgG1 to construct a recombinant plasmid; (2) transfecting the recombinant plasmid into CHO-S cells and culturing the cells to express the antibody; (3) collecting the cell culture supernatant, purifying the antibody, and obtaining the mouse anti-human CD16 recombinant antibody.

[0013] Optionally or preferably, in the above-mentioned preparation method, the coding gene of the amino acid sequence shown in SEQ ID NO: 7 is connected to the 5' end of the Fc fragment gene through an enzyme cutting site gene at the 3' end, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO: 8.

[0014] The eukaryotic expression vector can be modified using a commercial vector, and the commercial vector can use pcDNA3.4.

[0015] In a third aspect, the present application provides the use of any of the above-mentioned mouse anti-human CD16 recombinant antibodies in flow cytometry, IF and WB detection reagents.

[0016] Optionally or preferably, in the above applications, the detection reagent is used for flow cytometry or ELISA, and the detection reagent used for flow cytometry contains a mouse anti-human CD16 recombinant antibody labeled with a fluorescent protein.

[0017] Fourthly, the present invention provides a flow cytometry, IF, and WB detection reagent, including any of the mouse anti-human CD16 recombinant antibodies described above.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes the extracellular domain of human CD16 as an immunogen to prepare hybridoma cells, and extracts antibody genes from high-quality hybridoma cells. The light chain variable region and the heavy chain variable region can bind to PBMCs and accurately recognize CD16.

[0019] The recombinant antibody preparation method of this invention optimizes the recombinant antibody design scheme. The light chain variable region and heavy chain variable region genes are linked using a linker while retaining the Fc fragment. This is constructed into a vector, transcribed into a polypeptide (i.e., VL-Linker-VH-Fc, as shown in SEQ ID NO:9), which then forms a dimer through the antibody hinge region. This process is completed directly during transcriptional expression, reducing the steps involved in intracellular antibody assembly, lowering the probability of disulfide bond mismatch, thereby increasing the proportion of correctly conformated antibodies, improving antibody uniformity, and ultimately enhancing detection stability. Correctly conformated antibodies have higher affinity, which improves sensitivity. Correct conformation also facilitates antibody secretion, increases antibody yield, reduces antibody batches, and further contributes to improved antibody detection stability. Attached Figure Description

[0020] Figure 1 The following is a process for preparing mouse anti-human CD16 recombinant antibody in the examples. Figure 1 .

[0021] Figure 2 The following is a process for preparing mouse anti-human CD16 recombinant antibody in the examples. Figure 2 .

[0022] Figure 3 This is a flow cytometry analysis of the results of the purified monoclonal antibody and the control antibody mouse IgG1κ binding to human PBMCs prepared from monoclonal hybridoma cells E419-C1 in the example.

[0023] Figure 4 The image shows an electrophoresis diagram of RNA extracted from monoclonal hybridoma cells E419-C1 in this example.

[0024] Figure 5 The image shown is an electrophoresis diagram of cDNA synthesized by reverse transcription of RNA extracted from monoclonal hybridoma cells E419-C1 in this example.

[0025] Figure 6 The image shown is an electrophoresis diagram of the product obtained by PCR amplification using primer number 40201 in the example. UPM is a single primer control, which helps to distinguish non-specific amplification background products.

[0026] Figure 7 The image shown is an electrophoresis diagram of the PCR amplification product obtained using primer number 39649 in the example. UPM is a single primer control, which helps to distinguish non-specific amplification background products.

[0027] Figure 8 This is a diagram showing the results of homology comparison of light chain variable region genes using IMGT / V-QUEST software in the examples.

[0028] Figure 9 This is a diagram showing the results of amino acid sequence analysis of the CDR variable region of the light chain using NovoPro software in the examples.

[0029] Figure 10 The sequencing diagram for nucleotides 1-321 of the light chain variable region gene in the example is shown.

[0030] Figure 11 This is a diagram showing the results of homology comparison of heavy chain variable region genes using IMGT / V-QUEST software in the examples.

[0031] Figure 12 This is a diagram showing the results of the NovoPro software analysis of the amino acid sequence of the heavy chain variable region CDR in the examples.

[0032] Figure 13 The sequencing diagram for nucleotides 1-351 of the heavy chain variable region gene in the example is shown.

[0033] Figure 14 This is an electrophoresis image of the purified antibody obtained from the supernatant of a six-well plate culture after co-transfection of CHO-S cells with ea4241 and ea4242 plasmids in the example.

[0034] Figure 15 The image shows an electrophoresis diagram of the purified antibody obtained from the supernatant after co-transfecting CHO-S cells with ea4241 and ea4242 plasmids in an Erlenmeyer flask culture, as shown in the example.

[0035] Figure 16 The image shows the p3.4-IL2-Chis vector obtained after modification in the example.

[0036] Figure 17 This is an electrophoresis image of the purified antibody obtained from the supernatant of a six-well plate culture after transfecting CHO-S cells with the ea6051 plasmid in the example.

[0037] Figure 18The image shows the pMuFc-IL2 vector obtained after modification in the example.

[0038] Figure 19 This is an electrophoresis image of the purified antibody obtained from the supernatant of a six-well plate culture after transfecting CHO-S cells with the ea6103 plasmid in the example.

[0039] Figure 20 The pie chart shows the percentage of light and heavy chains co-transfected into the same cell using different vectors, as shown in the example.

[0040] Figure 21 The image shown is an electrophoresis diagram of the purified antibody obtained from the supernatant after CHO-S cells were transfected with the ea6103 plasmid and cultured in four flasks, as described in the example.

[0041] Figure 22 This figure shows the percentage of expression of 69 conventional recombinant antibodies with high expression levels cultured in six-well plates and expanded using Erlenmeyer flasks, as illustrated in the example.

[0042] Figure 23 This figure shows the expression percentage of 22 out of 69 conventional recombinant antibodies cultured in Erlenmeyer flasks and transfected with CHO-S cells in the example.

[0043] Figure 24 The image shown is an undenatured SDS-PAGE electrophoresis image of the mouse anti-human CD16 recombinant antibody A72 in the example.

[0044] Figure 25 This is a statistical chart showing the ELISA detection results of different concentrations of mouse anti-human CD16 recombinant antibody A72 binding to recombinant antigen E419 in the examples.

[0045] Figure 26 The image shows the results of flow cytometry detection of the binding of AF488-labeled mouse anti-human recombinant antibody A72 to human PBMCs in the examples. The top row is the isotype control mouse IgG-FITC, the middle row is the positive control (BD-FITC), the bottom row is the mouse anti-human recombinant antibody A72-AF488, the left column is monocytes, and the right column is lymphocytes.

[0046] Figure 27 The images shown are fluorescence micrographs of the binding of AF488-labeled mouse anti-human recombinant antibody A72 to human PBMCs detected by IF in the examples. The top row shows mouse IgG1 from the control group, and the bottom row shows mouse anti-human recombinant antibody A72 from the experimental group.

[0047] Figure 28 The images shown are fluorescence micrographs of the binding of AF488-labeled mouse anti-human recombinant antibody A72 to pig spleen tissue detected by IHC in the examples. The top row shows mouse IgG1 from the control group, and the bottom row shows mouse anti-human recombinant antibody A72 from the experimental group.

[0048] Figure 29 The images shown are fluorescence micrographs of the binding of AF488-labeled mouse anti-human recombinant antibody A72 to porcine lymph node tissue detected by IHC in the examples. The top row shows mouse IgG1 from the control group, and the bottom row shows mouse anti-human recombinant antibody A72 from the experimental group.

[0049] Figure 30 The results show the amino acid sequence alignment of the extracellular segment of recombinant antigen E419 and porcine CD16.

[0050] Figure 31 The results show the amino acid sequence alignment of the extracellular segment of recombinant antigen E419 and mouse CD16.

[0051] Figure 32 The electrophoresis results shown in the example are those obtained by Western blot (WB) detection of the binding of mouse anti-human recombinant antibody A72 to recombinant antigen E419.

[0052] Figure 33 This is an SDS-PAGE electrophoresis image of the recombinant antigen E419 in the example.

[0053] Figure 34 The electrophoresis results shown in the example are those obtained by Western blot (WB) detection of the binding of mouse anti-human recombinant antibody A72 to human PBMCs. Detailed Implementation

[0054] To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial channels.

[0055] Example 1: Preparation of mouse anti-human CD16 recombinant antibody refer to Figure 1 and Figure 2 This is a schematic diagram of the overall process in this embodiment.

[0056] 1. Fishing for genes: (1) Six-week-old BALB / c mice were selected, and the extracellular segment of human CD16 molecule expressed in eukaryotes was used as the antigen. The extracellular segment of human CD16 molecule was a self-prepared recombinant antigen, numbered E419, with the sequence Uniprot, O75015, amino acids 24-203, https: / / www.uniprot.org / uniprotkb / O75015, named recombinant antigen E419, and expressed in 293F cells (Thermo Biotech).

[0057] The amino acid sequence of the extracellular segment of human CD16 (recombinant antigen E419): PKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNENLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFS (SEQ ID NO: 10).

[0058] Immunization was performed on days 1, 15, 29, and 43. The dose of recombinant antigen E419 was 50 μg / time / mouse. Freund's adjuvant was used to enhance the immune response. Blood was collected on day 48 for serum titer testing (2 μg / ml of recombinant antigen E419 coated on a plate). Mice with the highest serum titer were selected and immunized with the number E419-2. The specific results of the mouse serum titer are shown in Table 1 below.

[0059] Table 1. Serum dilution factors and corresponding antibody titers of mouse E419-2 mice. The serum titer of mouse E419-2 was qualified and can be used for subsequent antigen shock immunization.

[0060] On day 50, mice E419-2 were immunized with a shock antigen of 50 μg / mouse. On day 55, the spleens of mice E419-2 were harvested, and a single-cell suspension was prepared. These spleen cells were then fused with SP2 / 0 mouse myeloma cells using polyethylene glycol (PEG) fusion medium to form hybridoma cells. Hybridoma cells capable of unlimited proliferation and secreting specific antibodies against CD16 were obtained through screening in HAT and HT selective media (Sigma) and flow cytometry analysis of the supernatant. Using limiting dilution and flow cytometry analysis of the culture supernatant, positive clones with clear flow cytometry clustering and high fluorescence intensity were selected. These positive hybridomas were then subjected to subclonal culture to obtain monoclonal hybridoma clones E419-C1 and E419-C2. The E419-C1 and E419-C2 hybridoma cells were injected into the peritoneal cavity of mice to produce ascites, and the monoclonal antibodies in the ascites were then purified. The purified antibody was tested for titer (using 2 μg / ml recombinant antigen E419 coated on a plate), and the results are as follows: Table 2. Results of monoclonal antibody titers obtained from different monoclonal hybridoma cells. The results showed that the E419-C1 clonal monoclonal antibody had a higher titer than the E419-C2 clonal monoclonal antibody, and it is proposed to use the E419-C1 clonal monoclonal antibody hybridoma cells to extract the antibody gene.

[0061] Flow cytometry was used to detect the binding of E419-C1 clonal monoclonal antibody to human PBMCs (peripheral blood mononuclear cells) to further verify the efficacy of E419-C1 clonal monoclonal antibody: Fresh human peripheral blood was collected from voluntary donors, PBMCs were extracted and blocked, and then the cells were resuspended at 10⁻⁶ mcg / mL. 6 Incubate 100 μl of primary antibody E419-C1 clonal monoclonal antibody (10 μg / ml) and 1:2000 diluted goat anti-mouse secondary antibody (BioLegend). (The incubation process is as follows:) Figure 3 The results showed that the E419-C1 clonal monoclonal antibody can specifically bind to the natural CD16 molecule on the surface of PBMC cells, which can be used for the next step of gene fishing.

[0062] (2) Detection of E419-C1 clonal monoclonal antibody subtypes. The subtypes of E419-C1 clonal monoclonal antibody were detected using a mouse monoclonal antibody subtype identification kit (Southern Biotech). The results are shown in Table 3 below: Table 3. Subtype detection results of E419-C1 clonal monoclonal antibody The results showed that the OD value of the antibody heavy chain subtype IgG1 was higher than that of IgG2a, IgG2b, IgG3, and IgM, while the OD value of the light chain subtype Kappa was higher than that of Lambda. IgG1 and Kappa showed significant color development, while the others showed almost no color development. These results suggest that the heavy chain subtype of the E419-C1 clonal monoclonal antibody is IgG1, and the light chain subtype is Kappa.

[0063] (3) E419-C1 clonal hybridoma cells were cultured, RNA was extracted using the Trizol method, and the results of agarose gel electrophoresis were as follows: Figure 4 The RNA was displayed as three distinct bands: 28S, 18S, and 5S. The band brightness was 28S > 18S > 5S, indicating that the RNA was intact.

[0064] (4) Using the RNA obtained in step (3) as a template, the first-strand cDNA was synthesized by reverse transcription using a RACE kit (Vazyme). The amplified product was obtained by agarose gel electrophoresis as shown in the figure. Figure 5 The molecular weight of the product amplified in lane E419-C1 was approximately 4500 bp, which was similar to the molecular weight of the product from the positive control HEK 293 Control, indicating that reverse transcription was successful.

[0065] (5) Amplify the heavy and light chain variable region genes of the E419-C1 clone monoclonal antibody.

[0066] Using the cDNA obtained in step (4) as a template, upstream primers from the RACE kit (Vazyme) were used. Based on the IgG1 heavy chain constant region sequence and the Kappa light chain constant region sequence of the mouse antibody, downstream amplification primers for the heavy chain and light chain were designed as shown in Table 4. PCR amplification was performed using upstream and downstream primers. UPM single primers were used as controls to help distinguish non-specific amplification background products.

[0067] Table 4. Downstream primers for E419-C1 cloning monoclonal antibody amplification of the variable region gene Agarose gel electrophoresis results are as follows Figure 6 The results showed: The E419-C1 cloned monoclonal antibody heavy chain variable region gene was successfully amplified using Mu-IgG1-CONST-GSP primers (number 40201). Figure 6 The IgG1 amplification product was constructed into a T vector using a kit (TransGen Biotech), named E419-C1-IgG1-T5 heavy chain variable region gene T vector.

[0068] The E419-C1 cloned monoclonal antibody light chain variable region gene was successfully amplified using Mu-Vk-CONST-s primers (number 39649). See [link to primer]. Figure 7 No nonspecific amplification was observed. The E419-C1 light chain variable region gene could be amplified using three PCR programs: A, B, and C. The annealing temperatures of the three PCR programs were different: 62℃ for program A, 65℃ for program B, and 68℃ for program C. The amplification product of program A had the brightest band, indicating that the amplified gene copy number was the highest. The amplification product of program A was selected, and the amplification product of program A was constructed into a T vector using a kit (TransGen Biotech). The vector was named E419-C1-kappa-T3 light chain variable region gene T vector.

[0069] (6) Sequencing and analysis of the variable region gene of the E419-C1 cloned monoclonal antibody: The E419-C1-IgG1-T5 heavy chain variable region gene T-load obtained in step (4) and the E419-C1-kappa-T3 light chain variable region gene T-load obtained in step (5) were sequenced.

[0070] ① The light chain variable region gene sequencing results were analyzed using the IMGT / V-QUEST online analysis software to perform homology comparisons on the light chain variable region genes. The results are as follows: Figure 8(https: / / www.imgt.org / IMGT_vquest / input). The green region gene is related to Musmus IGKV12-44. The 01 F family gene showed a 97.13% match with Musmus IGKJ2. The 01 F family gene, with a matching rate of 97.37%, can be identified as a valid light chain variable region gene (322 nucleotides in total, not a multiple of 3, so one nucleotide needs to be removed).

[0071] ② The amino acid sequence of nucleotides 1-321 is as follows (a total of 107 amino acids): DIQMTQSPASSLSASVGETVTITCRASENIYRYLAWYQQRQGKSPHLLVYNAKTLAEGVPSRFSGSGSDTQFSLKIDNLQPEDFGSYYCQHHYRTPYTFGGGTKLEIK (SEQ ID NO: 13).

[0072] ③ The amino acid sequence of the CDR in the light chain variable region was analyzed using the online software NovoPro (https: / / www.novopro.cn / tools / cdr.html). See Table 5 and [other tables]. Figure 9 .

[0073] Table 5. Results of amino acid sequence analysis of the CDR region of the light chain. ④ Sequencing results corresponding to nucleotides 1-321 of the light chain variable region gene are as follows: Figure 10 This indicates that the sequencing results are reliable.

[0074] ⑤ The heavy chain variable region gene sequencing results were analyzed using the IMGT / V-QUEST online analysis software to perform homology comparisons on the heavy chain variable region genes. The results are as follows: Figure 11 (https: / / www.imgt.org / IMGT_vquest / input). The green region showing the inverse complementary sequence is related to Musmus IGHV1-69-5. The 01 F family gene matching rate is 97.57%, and it is compatible with MusmusIGHJ3. The 01 F family gene matching rate is 87.50%, which can be identified as a valid heavy chain variable region gene (352 nucleotides in total, not a multiple of 3, so one nucleotide needs to be removed).

[0075] ⑥ The amino acid sequence of nucleotides 1-351 is as follows (a total of 117 amino acids): QVQLQQPGAELVKPGASLKLSCKASGYTFTTYWMHWLKQRPGQGPEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARNYYGPFPYWGQGTLVTVSA (SEQ ID NO: 18).

[0076] ⑦ The amino acid sequence of the CDR region of the heavy chain was analyzed using the online software NovoPro (https: / / www.novopro.cn / tools / cdr.html). See Table 6 and [other tables]. Figure 12 .

[0077] Table 6. Results of amino acid sequence analysis of the CDR region of the heavy chain. ⑧ Sequencing results corresponding to the reverse complementary sequence of nucleotides 1-351 of the heavy chain variable region gene are as follows: Figure 13 This indicates that the sequencing results are reliable.

[0078] 2. The mouse anti-human CD16 recombinant antibody A50 (without a linker linking the light and heavy chain variable regions, containing the complete constant regions of the light and heavy chains, and therefore containing the Fc fragment) was constructed using the conventional recombinant antibody construction protocol. The primers are designed as shown in Table 7 below.

[0079] Table 7. Primer sequences for conventional recombinant antibodies Building rechains: Using primers 40269 and 36432, PCR was performed with the E419-C1-IgG1-T5 heavy chain variable region gene T vector (containing nucleotides 1-351 of the above heavy chain variable region gene) as a template to obtain the heavy chain PCR product. After treatment with restriction endonucleases, the PCR product was ligated into the pFUSEss-CHIg-mG2b vector (InvivoGen, containing the complete constant region of the heavy chain) and named ea4241 plasmid.

[0080] Building Light Chains: Using primers 36433 and 39160, PCR was performed with the E419-C1-kappa-T3 light chain variable region gene T vector (containing nucleotides 1-321 of the light chain variable region gene) as a template to obtain the light chain PCR product. After treatment with restriction endonucleases, the PCR product was ligated into the pFUSE2ss-CLIg-mk vector (InvivoGen, containing the complete constant region of the light chain) and named ea4242 plasmid.

[0081] CHO-S cells were co-transfected with ea4241 and ea4242 plasmids, cultured in six-well plates, and the supernatant was collected for purification to obtain recombinant antibodies. SDS-PAGE electrophoresis analysis was then performed. Figure 14 The expression level was moderate. The ea4241 / ea4242 mediator lanes contained purified antibodies. The heavy chain molecular weights expressed by the ea4241 plasmid were approximately 46 kDa and 48 kDa, while the light chain molecular weights expressed by the ea4242 plasmid were approximately 24 kDa and 25 kDa. The recombinant antibody obtained by co-transfection of the ea4241 and ea4242 plasmids was designated A50 and named recombinant antibody A50.

[0082] CHO-S cells were co-transfected with ea4241 and ea4242 plasmids and cultured in Erlenmeyer flasks. A total of four flasks were cultured, and the supernatants were collected and purified to obtain recombinant antibody A50. SDS-PAGE electrophoresis analysis was performed. Figure 15 Based on the total mass of the recombinant antibody A50 obtained from the four vials and the total culture volume, the expression level was calculated to be 11.25 μg / ml.

[0083] 3. To increase the yield of recombinant antibodies, a linker was designed to connect the variable region genes of the light chain and heavy chain, forming VL-Linker-VH, which is a construction scheme for single-chain antibodies.

[0084] The amino acid sequence of the VL-Linker-VH recombinant antibody is as follows (239 amino acids in total): DIQMTQSPASLSSASVGETVTITCRASENIYRYLAWYQQRQGKSPHLLVYNAKTLAEGVPSRFSGSGSDTQFSLKIDNLQPEDFGSYYCQHHYRTPYTFGGGTKLEIK GGGGSGGGGSGGGGS QVQLQQPGAELVKPGASLKLSCKASGYTFTTYWMHWLKQRPGQGPEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARNYYGPFPYWGQGTLVTVSA (SEQ ID NO:7, where the underlined part is the Linker).

[0085] The corresponding nucleotide sequence is (717 nucleotides in total): GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGGTATTTAGCATGGTATCAGCAGAGACAGGGAAAATCTCCTCACCTCCTGGTCTATAATGCAAAAACCT TAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGACACACAGTTTTCCTGAAGATCGACAACCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCACTATCGTACTCCGTACACGTTCGGAGGGGGGACCAAGTTGGAAAATAAAA GGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCG CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCACTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCACCTACTGGATGCACTGGCTGAAGCAGAGGCCTGGACAAGGCCCTGAGTGGATTGGAGAGATTAATCCTAGTAACGGTCGTACTAAC TACAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTGGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAATTACTACGGGCCCTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO:27, the underlined part is Linker).

[0086] 4. Construction and expression of VL-Linker-VH recombinant antibody (without Fc fragment): (1) Based on the design scheme of light chain and heavy chain variable region genes and above recombinant antibodies, and combined with the enzyme restriction sites and reading frames of the expression vector, primers were designed as shown in Table 8.

[0087] Table 8 Recombinant Antibody Primer Sequences (2) Construction of VL-Linker-VH: Using primers 40270 and 40272, PCR was performed with the E419-C1-kappa-T3 light chain variable region gene T vector (containing nucleotides 1-321 of the light chain variable region gene) as a template to obtain the light chain PCR product.

[0088] Using primers 40271 and 40273, PCR was performed with the E419-C1-IgG1-T5 heavy chain variable region gene T vector (containing nucleotides 1-351 of the above heavy chain variable region gene) as a template to obtain the heavy chain PCR product.

[0089] Using primers 40270 and 40271, and with the light chain PCR product and heavy chain PCR product as templates, PCR was performed to tandem the light chain and heavy chain to form the VL-Linker-VH recombinant antibody sequence, named GEA E419-C1-1 plasmid.

[0090] Using primers 41342 and 40271, and with GEA E419-C1-1 plasmid as a template, the VL-Linker-VH sequence was constructed into the P3.4-IL2-Chis vector (modified from the commercial vector pcDNA3.4, without the Fc fragment, containing a C-terminal 6×His tag; see diagram). Figure 16 The plasmid was named ea6051.

[0091] (3) Expression of VL-Linker-VH: The ea6051 plasmid was transfected into CHO-S cells, and antibody expression levels were detected after culturing in six-well plates. Electrophoresis results are shown below. Figure 17 The expression level was close to zero, and the predicted molecular weight of the antibody expressed by the ea6051 plasmid was approximately 27.6 kDa. Figure 17 The ea6051 medium lane contained the purified antibody. No obvious bands were observed in the 22kD-44kD range indicated by the marker, so further expansion culture in Erlenmeyer flasks was not performed.

[0092] Experimental results show that the VL-Linker-VH recombinant single-chain antibody constructed by linking the light and heavy chains using a Linker alone cannot be expressed normally for this sequence.

[0093] 5. To increase the yield of recombinant antibodies, further optimize the recombinant antibody design scheme: Considering that the reason why recombinant antibodies with the VL-Linker-VH structure cannot be expressed normally may be that the Fc fragment is conducive to maintaining the recombinant antibody structure close to the natural antibody structure, which in turn is conducive to antibody expression, folding and secretion, and increases antibody yield, when further optimizing the recombinant design scheme, the light chain variable region gene and the heavy chain variable region gene are linked with a Linker, while retaining the Fc fragment to form VL-Linker-VH-Fc, which is then constructed into a vector.

[0094] Because a segment of amino acid sequence in the Fc fragment can serve as a hinge region, recombinant antibodies with a VL-Linker-VH-Fc structure can form a dimer through the antibody hinge region after being transcribed into a polypeptide. Compared to conventional recombinant antibodies without linker linkage, this reduces the steps involved in intracellular antibody assembly, lowers the probability of disulfide bond mismatch, thereby increasing the proportion of antibodies with the correct conformation, improving antibody uniformity, and ultimately enhancing detection stability.

[0095] In addition, adding the Fc fragment can increase the number of fluorescent labeling sites, thereby increasing the number of fluorescent molecules labeled by each antibody, which in turn enhances the detection signal and further improves the detection sensitivity.

[0096] For some experimental tests that do not require direct antibody labeling of fluorescence, retaining the mouse Fc fragment makes it easier to use commercially available anti-mouse IgG secondary antibodies for detection, such as Western blot (WB) tests.

[0097] Select the Fc fragment sequence and design the recombinant antibody sequence: Because the variable region gene of the heavy chain of this mouse anti-human CD16 antibody originates from IgG1 (see...) Figure 6 (Middle IgG1), therefore the Fc fragment of mouse IgG1 was selected, with the gene and amino acid sequence referenced from the constant region sequence of secretory mouse IgG1: Uniprot, P01868, Ig gamma-1 chain C region secreted form, mouse ( https: / / www.uniprot.org / uniprotkb / P01868The total length is 324 amino acids, of which amino acids 1-97 are the CH1 region, amino acids 98-110 are the hinge region, amino acids 111-217 are the CH2 region, and amino acids 218-324 are the CH3 region. The recombinant antibody selects the fragment of amino acids 103-324 as the Fc fragment of mouse IgG1. The specific amino acid sequence is: GCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID) NO:8 contains a portion of the hinge region (amino acids 103-110, GCKPCICT), the CH2 region, and the CH3 region, totaling 222 amino acids, of which 2 amino acids are natural mutations (N276D and N278D).

[0098] The amino acid sequence of the mouse anti-human CD16 recombinant antibody VL-Linker-VH-Fc is designed as shown in SEQ ID NO:9, containing the VL-Linker-VH sequence (239 amino acids) of SEQ ID NO:7, two amino acids (LE) translated from the XhoI (CTCGAG) restriction site, and the aforementioned mouse IgG1-Fc fragment (222 amino acids), totaling 463 amino acids. The specific amino acid sequence is as follows: DIQMTQSPASLSASVGETVTITCRASENIYRYLAWYQQRQGKSPHLLVYNAKTLAEGVPSRFSGSGSD TQFSLKIDNLQPEDFGSYYCQHHYRTPYTFGGGTKLEIK GGGGSGGGGSGGGGS QVQLQQPGAELVKPGASLKLSC KASGYTFTTYWMHWLKQRPGQGPEWIGEINPSNGRTNYNEKFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCAR NYYGPFPYWGQGTLVTVSALEGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:9), in the sequence, the underlined part is the linker, the italicized text before and after the linker indicates the variable regions of the light chain and heavy chain, respectively, the bolded LE after the heavy chain is the restriction enzyme site, the restriction enzyme site is XhoI, the corresponding nucleotide sequence is CTCGAG; after LE is the mouse IgG1-Fc fragment.

[0099] The nucleotide sequence corresponding to VL-Linker-VH-Fc is (1392 nucleotides in total, including the final stop codon TGA): GACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATG TCGAGCAAGTGAGAATATTTACAGGTATTTAGCATGGTATCAGCAGAGACAGGGAAAATCTCCTCACCTCCTGGTC TATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGACACACAGTTTTCTCTGA AGATCGACAACCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCACTATCGTACTCCGTACACGTTCGG AGGGGGGACCAAGTTGGAAATAAAAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTC CAACTGCAGCAGCCTGGGGCTGAACTGGTGAAGCCTGGGGCTTCACTGAAGCTGTCCTGCAAGGCTTCTGGCTACA CCTTCACCACCTACTGGATGCACTGGCTGAAGCAGAGGCCTGGACAAGGCCCTGAGTGGATTGGAGAGATTAATCC TAGTAACGGTCGTACTAACTACAATGAGAAGTTCAAGACCAAGGCCACACTGACTGTGGACAAATCCTCCAGCACA GCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAAATTACTACGGGCCCT TTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA CTCGAG(SEQ ID NO:33). The italicized text represents the nucleotide sequence corresponding to VL-Linker-VH, the underlined part is the restriction enzyme site XhoI, the sequence following the restriction enzyme site is the Fc fragment of mouse IgG1, and the last three TGAs are the stop codons.

[0100] 6. The construction process of VL-Linker-VH-Fc: Using primers 41342 and 40271 as a template, PCR was performed to obtain the VL-Linker-VH sequence. The PCR product was then treated with restriction endonucleases and ligated into the modified pMuFc-IL2 vector (derived from the commercial vector pcDNA3.4; see diagram). Figure 18The plasmid, containing the mouse IgG1-Fc fragment gene, was recombined. After being transformed into *E. coli* DH5α, positive clones were selected, and the corresponding plasmid was designated ea6103. The ea6103 plasmid was sent to a sequencing company for sequencing. The sequencing results showed that the nucleotide sequence matched the design scheme described above.

[0101] 7. Expression and purification of the ea6103 plasmid: (1) CHO-S cells were transfected with the ea6103 plasmid and cultured in six-well plates: E. coli transfected with the ea6103 plasmid were cultured extensively, and the ea6103 plasmid was extracted. The ea6103 plasmid was then transfected into CHO-S cells, and the cells were cultured in six-well plates for 4-5 days. The culture supernatant was collected for purification and analyzed by SDS-PAGE electrophoresis. (See [link to documentation]). Figure 19 The results showed that the ea6103 plasmid could express the recombinant antibody VL-Linker-VH-Fc at a high level, which was consistent with the predicted molecular weight of about 51kD.

[0102] Compared with the expression levels in six-well plates, the recombinant antibody expressed by the ea6103 plasmid (see...) Figure 19 The recombinant antibody A50 expressed by co-transfection with ea4241 and ea4242 plasmids (see [link]). Figure 14 That is, for this mouse anti-human CD16 antibody gene sequence, the expression level of VL-Linker-VH-Fc is higher than that of conventional recombinant antibodies. In addition, the VL-Linker-VH single-chain antibody does not contain Fc, and its expression level is close to zero, which is considered as no expression (see [link to relevant documentation]). Figure 17 The cells transfected and cultured in the six-well plates had the same cell density and a culture volume of 2 ml. All supernatants were collected for purification, and the purified antibody volumes were the same. The same volume of antibody was used for pre-electrophoresis sample processing, with the same processing reagents and volumes. The loading volume for SDS-PAGE was also the same. Therefore, the grayscale value of the antibody bands shown in the SDS-PAGE results can directly indicate the expression level.

[0103] Previously, 515 recombinant antibodies were constructed using conventional recombinant antibody methods. This involved constructing the light and heavy chains into separate vectors (the light and heavy chains were not linked by a linker and contained complete constant regions). The two vectors were then co-transfected into the same cell for expression. The expression of these 515 recombinant antibodies in six-well plates is shown below. Figure 20As shown: 219 cells did not express the protein, accounting for 43%; 69 cells expressed the protein weakly, accounting for 13%; 186 cells expressed the protein moderately, accounting for 36%; and 41 cells expressed the protein highly, accounting for 8%. CHO-S cells transfected with the ea6103 plasmid and cultured in six-well plates showed high expression levels, ranking in the top 8%. Only recombinant antibodies expressed with the ea6103 plasmid were included in this statistical analysis.

[0104] (2) CHO-S cells were transfected with the ea6103 plasmid and cultured in Erlenmeyer flasks: The ea6103 plasmid was transfected into CHO-S cells (Thermo Scientific). Cells were cultured in Erlenmeyer flasks for 4-5 days, and a total of four flasks were cultured. The culture supernatant was collected and purified, and analyzed by SDS-PAGE electrophoresis. Results are shown below. Figure 21 Based on the total mass of the recombinant antibodies obtained from the four vials and the total culture volume, the expression level was calculated to be 30.99 μg / ml. The recombinant antibody expressed by the ea6103 plasmid was named recombinant antibody A72.

[0105] Compared with the expression levels in Erlenmeyer flask culture, the recombinant antibody A72 (30.99 μg / ml) was higher than that of recombinant antibody A50 (11.25 μg / ml). That is, for the antibody gene sequence of this mouse anti-human CD16, the expression level of VL-Linker-VH-Fc is higher than that of conventional recombinant antibody, which is consistent with the expression level in six-well plate culture.

[0106] Of the 515 conventional recombinant antibodies previously cultured, 69 showed relatively high expression levels after further expansion culture in Erlenmeyer flasks. The expression levels after Erlenmeyer flask expansion culture are shown below. Figure 22 As shown: 18 antibodies had expression levels < 5 μg / ml, accounting for 26.09%; 29 antibodies had expression levels between 5 μg / ml and 20 μg / ml, accounting for 42.03%; and 22 antibodies had expression levels ≥ 20 μg / ml, accounting for 31.88%. The expression level of recombinant antibody A72, cultured in Erlenmeyer flasks, was 30.99 μg / ml, ranking in the top 31.88%. Only conventional recombinant antibodies are included in this statistical analysis; recombinant antibody A72 is not included.

[0107] Of the 69 cells cultured in Erlenmeyer flasks, 22 were transfected with CHO-S cells for expression, and the expression results are as follows: Figure 23As shown: 11 antibodies had expression levels < 5 μg / ml, accounting for 50%; 8 antibodies had expression levels 5 μg / ml ≤ < 20 μg / ml, accounting for 36.36%; and 3 antibodies had expression levels ≥ 20 μg / ml, accounting for 13.64%. Recombinant antibody A72 transfected into CHO-S cells and cultured in Erlenmeyer flasks had an expression level of 30.99 μg / ml, ranking in the top 13.64%. Only conventional recombinant antibodies are included in this analysis; recombinant antibody A72 is not included. (Because CHO-S cells have relatively fewer host proteins than 293F cells, the purity of antibodies expressed and purified from CHO-S cells is higher. As one factor, current antibody drugs are all expressed using CHO-S cells, so the expression level in CHO-S cells is more meaningful. The expression level in CHO-S cells is counted separately here; the rest were expressed by transfecting 293F cells.)

[0108] After screening through six-well plate culture and Erlenmeyer flask expansion culture, antibodies with relatively high expression levels will be further increased in yield through measures including, but not limited to, transfection with further improved CHO-S cells, use of improved culture media, use of anti-apoptotic genes to improve the survival rate of CHO-S cells, and use of fermenter culture.

[0109] (3) The recombinant antibody A72 was analyzed by non-denaturing SDS-PAGE electrophoresis. The results are shown in [reference needed]. Figure 24 The data showed a band at approximately 100 kD in the non-reduced lane, and the expected molecular weight of the recombinant antibody A72 dimer was 102 kD. This indicates that the gene for recombinant antibody A72 can form a dimer structure after expression in CHO-S cells (the predicted molecular weight of the monomer of the recombinant antibody expressed by the VL-Linker-VH-Fc sequence is 51 kD, so the predicted molecular weight of the dimer is approximately 102 kD).

[0110] 8. Validation of the efficacy of recombinant antibody A72: (1) ELISA (Enzyme-Linked Immunosorbent Assay) detection of the binding of recombinant antibody A72 to recombinant antigen E419: The plate was coated with recombinant antigen E419 (2 μg / ml). The primary antibody was serially diluted recombinant antibody A72, and the secondary antibody was goat anti-mouse IgG polyclonal antibody-HRP (horseradish peroxidase) (a self-prepared antibody, catalog number SAA544Mu19). TMB (tetramethylbenzidine) was used for color development, and the reaction was terminated with a stop solution. OD values ​​were read at 450 nm and 630 nm. Results are shown in [reference needed]. Figure 25 The analysis results showed that recombinant antibody A72 could bind to recombinant antigen E419, with an EC50 of 0.071 μg / ml.

[0111] (2) Flow cytometry detection of the binding of recombinant antibody A72 to human PBMCs: ① Labeling fluorescence: Recombinant antibody A72 was labeled with green fluorescent AF488 using a condensation method to obtain the directly labeled antibody A72-AF488 at a concentration of 2 mg / ml.

[0112] ② Flow cytometry titration of A72-AF488 antibody concentration: Fresh peripheral blood from voluntary donors was collected, PBMCs were extracted and blocked, and then the cells were resuspended at 10⁻⁶. 6 100 μl of samples were incubated with A72-AF488 and then analyzed by flow cytometry. The results are shown in Table 9 below. Table 9 Flow cytometry results Among them, A72-AF488 showed the highest SI value and the strongest fluorescence intensity (Pos Mediam) at a dose of 5 μl / test, and can be used as the recommended dose.

[0113] ③ Detection of CD16 expression in human PBMCs using A72-AF488 antibody: Fresh peripheral blood was collected from voluntary donors, PBMCs were extracted and blocked, and then the cells were resuspended at 10⁻⁶. 6 100 μl of recombinant antibody A72-AF488 (5 μl / test) was incubated and flow cytometry was performed. The isotype control was mouse IgG-FITC, and the positive control was mouse anti-human CD16-FITC (BD Biosciences). Results are shown below. Figure 26 The results showed that the positive rate of CD16 in monocytes detected by recombinant antibody A72-AF488 (12.22%) was close to that detected by the positive control (BD-FITC in the figure) (9.85%); the positive rate of CD16 in lymphocytes detected by recombinant antibody A72-AF488 (13.45%) was close to that detected by the positive control (BD-FITC in the figure) (13.81%). This suggests that recombinant antibody A72 can recognize the naturally expressed CD16 molecule in human PBMCs, and the detection rate is close to that of the positive control BD-FITC.

[0114] (3) IF (immunofluorescence) detection of the binding of the directly labeled recombinant antibody A72-AF488 to human PBMCs: ① Slide preparation: Take a slide that has been soaked in 2% sodium silicate overnight, circle the experimental area with a histochemical pen, add 0.01% poly-lysine (0.1 mg / ml), and incubate for 30 min.

[0115] ② Extraction of PBMCs: Extract PBMCs using human lymphocyte separation medium, and resuspend in PBS (phosphate buffered saline) to a final concentration of 10. 6 per ml.

[0116] ③ Fixation: Remove polylysine, wash 3 times with PBS, add 20 μl of cell suspension, air dry for 30 min, add 4% paraformaldehyde fixative, and fix at room temperature for 20 min.

[0117] ④ Blocking: Remove fixative, wash 3 times with PBS, add 1% BSA (bovine serum albumin) blocking solution, and incubate at 37°C for 30 min.

[0118] ⑤ Incubation of A72-AF488: Remove blocking solution, wash 3 times with PBS, add recombinant antibody A72-AF488 (5µg / ml), incubate overnight at 4℃, and use mouse IgG1-AF488 (5μg / ml) as isotype control.

[0119] ⑥ Nuclear staining: Remove A72-AF488, wash 3 times with PBS, add working solution of nuclear counterstain DAPI (4',6-diamidinyl-2-phenylindole) (blue), and incubate at room temperature in the dark for 5 min.

[0120] ⑦ Mounting: Remove DAPI, wash 3 times with PBS, mount with anti-fluorescence attenuation mounting medium, and observe and acquire images under a fluorescence microscope.

[0121] See results Figure 27 The detection area shown in the figure shows that the green fluorescence of human PBMC samples detected by recombinant antibody A72-AF488 is significantly stronger than that of the Mouse IgG1 isotype control (blue represents cell nuclei). The results indicate that recombinant antibody A72-AF488 can bind to the natural CD16 molecule expressed in human PBMCs, which is consistent with the flow cytometry results.

[0122] (4) IHC (immunohistochemistry) detection of the binding of the directly labeled recombinant antibody A72-AF488 to porcine spleen and lymph node tissues: ① Sample processing: Paraffin sections were dewaxed with xylene and graded alcohols, then antigen retrieval was performed, followed by rinsing with 0.01M PBS for 5 min, and repeated 3 times.

[0123] ② Blocking: Add 5% BSA blocking solution, place in a humidified chamber at room temperature for 30 minutes to block.

[0124] ③ Incubation of A72-AF488: Remove the blocking solution, add A72-AF488 (20µg / ml), incubate overnight in a humidified chamber at 4℃, and use mouse IgG1-AF488 (20μg / ml) as an isotype control.

[0125] ④ Nuclear staining: Wash three times with PBST (phosphate buffer containing 0.05% Tween 20) for 5 minutes each time, shaking occasionally to wash away the free A72-AF488 fluorescent antibody. Add nuclear counterstaining agent DAPI working solution (blue) and incubate at room temperature in the dark for 5 minutes.

[0126] ⑤ Mounting: Remove DAPI, wash 3 times with PBS, mount with anti-fluorescence attenuation mounting medium, and observe and acquire images under a fluorescence microscope.

[0127] See the results for spleen tissue fluorescence. Figure 28 The tissue region shown in the figure reveals that the green fluorescence of the porcine spleen tissue sample detected by recombinant antibody A72-AF488 is significantly stronger than that of the Mouse IgG1 isotype control (blue represents cell nuclei), indicating that recombinant antibody A72-AF488 can bind to the naturally expressed CD16 molecule in porcine spleen tissue. Human spleen tissue highly expresses CD16, suggesting that porcine spleen tissue also highly expresses CD16.

[0128] See the results for the fluorescence of lymph node tissue. Figure 29 The tissue region shown in the figure reveals that the green fluorescence of the porcine lymph node tissue sample detected by recombinant antibody A72-AF488 is significantly stronger than that of the Mouse IgG1 isotype control (blue represents cell nuclei), indicating that recombinant antibody A72-AF488 can bind to the naturally expressed CD16 molecule in porcine lymph node tissue. Human lymph node tissue highly expresses CD16, suggesting that porcine lymph node tissue also highly expresses CD16.

[0129] The reasons for using porcine spleen and lymph node tissues for IHC testing are as follows: The amino acid sequences of the recombinant antigen E419 (human CD16 extracellular segment) (Uniprot, O75015, amino acids 24-203, https: / / www.uniprot.org / uniprotkb / O75015) and the porcine CD16 extracellular segment (Uniprot, Q28942, https: / / www.uniprot.org / uniprotkb / Q28942, amino acids 20-209) were compared using the sequence alignment software BIOXM 2.6. The alignment results are shown in [link to documentation]. Figure 30 The results showed that the amino acid sequence identity (the number and proportion of completely matching amino acids) was 58%, and the similarity (the number and proportion of amino acids with similar biochemical properties, such as hydrophilicity, hydrophobicity, and charge) was 74%.

[0130] The amino acid sequences of the recombinant antigen E419 (human CD16 extracellular segment) (Uniprot, O75015, https: / / www.uniprot.org / uniprotkb / O75015, amino acids 24-203) and the mouse CD16 extracellular segment (Uniprot, P08508, https: / / www.uniprot.org / uniprotkb / P08508, amino acids 31-215) were compared using the sequence alignment software BIOXM 2.6. The alignment results are shown in [link to documentation]. Figure 31 The results showed that the amino acid sequence identity was 49% and the similarity was 65%.

[0131] The comparison results of the two groups indicated that the consistency and similarity of the extracellular amino acid sequence of CD16 between pigs and humans were higher than those between mice and humans. The human spleen tissue and lymph node tissue highly expressed CD16 molecules, so pig spleen tissue and lymph node tissue were selected for detection.

[0132] (5) Western blot (WB) detection of antibody binding to immunogen or natural molecules ① Western blotting (WB) detects the binding of antibodies to immunogens, and Western blotting detects the binding of recombinant antibody A72 to recombinant antigen E419: The sample to be tested was recombinant antigen E419. The primary antibody was recombinant antibody A72 (0.05 μg / ml), and the secondary antibody was goat anti-mouse IgG-HRP diluted 1:50000 (1.558 mg / ml, self-prepared antibody, catalog number SAA544Mu19). The sample loading volume was 20 ng in lane 1 and 100 ng in lane (3). See results below. Figure 32 This indicates that recombinant antibody A72 can recognize recombinant antigen E419, with a molecular weight of approximately 35-46 kDa. The molecular weight shown by Western blotting is basically consistent with the actual molecular weight of recombinant antigen E419. The molecular weight of recombinant antigen E419 by SDS-PAGE electrophoresis is approximately 35-46 kDa. (See [link to relevant documentation]). Figure 33 .

[0133] ② Western blotting (WB) detection of antibody binding to natural molecules, and Western blotting detection of recombinant antibody A72 binding to human PBMCs: Fresh peripheral blood was collected from voluntary donors, and PBMCs were extracted using human lymphocyte separation medium, counted, and 1×10⁶ cells were extracted. 7Cells were collected by centrifugation, PBMCs were collected, the supernatant was removed, and 80 μl of lysis buffer containing 1% SDS (sodium dodecyl sulfate) and 20 μl of 5× Loading Buffer were added. The mixture was heated in boiling water for 5 min. The primary antibody was recombinant antibody A72 (0.03 μg / ml), and the secondary antibody was goat anti-mouse IgG-HRP diluted 1:50000 (1.558 mg / ml, self-prepared antibody, catalog number SAA544Mu19). The loading volumes for lanes 1, 2 μl, 4 μl, and 8 μl were respectively. See the results below. Figure 34 This indicates that the recombinant antibody A72 can bind to the natural molecules expressed in human PBMCs, with a molecular weight of approximately 40-51 kDa.

[0134] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. A murine anti-human CD 16 recombinant antibody comprising a light chain variable region and a heavy chain variable region, characterized in that, The light chain variable region comprises complementarity determining regions CDR-L1, CDR-L2, CDR-L3, wherein: the CDR-L1 amino acid sequence is RASENIYRYLA (SEQ ID NO: 1), the CDR-L2 amino acid sequence is NAKTLAE (SEQ ID NO: 2), the CDR-L3 amino acid sequence is QHHYRTPYT (SEQ ID NO: 3); The heavy chain variable region comprises complementarity determining regions CDR-H1, CDR-H2, CDR-H3, wherein: the CDR-H1 amino acid sequence is TYWMH (SEQ ID NO: 4), the CDR-H2 amino acid sequence is EINPSNGRTNYNEKFKT (SEQ ID NO: 5), the CDR-H3 amino acid sequence is NYYGPFPY (SEQ ID NO: 6).

2. The murine anti-human CD 16 recombinant antibody according to claim 1, characterized in that, The C-terminal of the light chain variable region amino acid sequence is connected to the N-terminal of the heavy chain variable region through a linker.

3. The murine anti-human CD 16 recombinant antibody according to claim 2, characterized in that, The amino acid sequence of the murine anti-human CD16 recombinant antibody is shown in SEQ ID NO:

7.

4. The murine anti-human CD 16 recombinant antibody according to claim 2, characterized in that, Further comprising an Fc fragment, characterized in that the Fc fragment is on the same polypeptide sequence as the light chain variable region and the heavy chain variable region.

5. The murine anti-human CD 16 recombinant antibody according to claim 4, characterized in that, The Fc fragment is connected to the C-terminal of the heavy chain variable region through an enzyme cutting site sequence, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO:

8.

6. The murine anti-human CD 16 recombinant antibody according to claim 5, characterized by the fact that it is a 158F2 antibody. The amino acid sequence of the murine anti-human CD16 recombinant antibody is shown in SEQ ID NO:

9.

7. A method for preparing a murine anti-human CD 16 recombinant antibody, characterized by, The method comprises the following steps: (1) inserting the coding gene of the amino acid sequence shown in SEQ ID NO: 7 into a eukaryotic expression vector containing the Fc fragment gene of mouse IgG1 to construct a recombinant plasmid; (2) transfecting CHO-S cells with the recombinant plasmid and culturing the cells to express the antibody; (3) collecting the cell culture supernatant, purifying the antibody, and obtaining the murine anti-human CD16 recombinant antibody.

8. The preparation method according to claim 7, characterized in that, The coding gene of the amino acid sequence shown in SEQ ID NO: 7 is connected to the 5' end of the Fc fragment gene through an enzyme cutting site gene at the 3' end, and the amino acid sequence of the Fc fragment is shown in SEQ ID NO:

8.

9. The murine anti-human CD16 recombinant antibody of any one of claims 1-6 for use as a reagent for flow cytometry, IF or WB detection.

10. A flow cytometry, IF or WB detection reagent, characterized in that, The method comprises the murine anti-human CD16 recombinant antibody of any one of claims 1-6.

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