Recombinant humanized anti-HCV NS3 monoclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses a recombinant humanized anti-HCV NS3 monoclonal antibody, which comprises a light chain and a heavy chain, the monoclonal antibody is named as R-Anti-HCV-NS3-Ab, the amino acid sequence of the light chain is shown as SEQ ID NO.6, the amino acid sequence of the heavy chain is shown as SEQ ID NO.7, and the amino acid sequence of the heavy chain is shown as SEQ ID NO.8. And the amino acid sequence of the heavy chain is as shown in SEQ ID NO.7. The recombinant humanized anti-HCV NS3 monoclonal antibody developed by the patent is designed based on an NS3 conserved epitope region, high affinity to main epidemic subtypes such as 1a, 1b, 2a, 3a, 4a and the like is ensured, and the broad-spectrum detection capability is ensured; through humanized transformation, anti-mouse antibody interference is reduced, false positive is avoided, and reagent stability is improved; multiple platforms such as chemiluminescence and ELISA are adapted; standardized production: a mammalian cell expression system is adopted, and the batch-to-batch consistency is obviously superior to that of a traditional polyclonal antibody; the quality control product disclosed by the invention can be used for accurately evaluating the sensitivity of an HCV antibody detection reagent, and is particularly suitable for regions with complicated genotypes.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering, antibody engineering, and immunodiagnostic technology, and in particular to a recombinant humanized anti-HCV NS3 monoclonal antibody, its preparation method, and its application. Background Technology

[0002] Hepatitis C virus (HCV) infection is a major global public health challenge, with approximately 58% of chronically infected individuals developing cirrhosis or hepatocellular carcinoma (HCC). Early symptoms of HCV infection are often subtle (such as fatigue and loss of appetite), and 70%-80% of cases progress to chronic infection, leading to long-term liver damage. According to the World Health Organization, approximately 58 million people worldwide carry HCV, and about 290,000 die annually from related complications. Its high degree of concealment and chronicity underscores the urgent need for early and accurate diagnosis. HCV exhibits high genetic diversity, with seven genotypes (1-7) and over 67 subtypes (such as 1a, 1b, and 3a), showing significant geographical differences: genotype 1 (Europe and North America, East Asia), genotype 3 (South Asia, Australia), genotype 4 (North Africa), and genotype 6 (Southeast Asia) are the dominant strains in each region. This diversity poses a significant challenge to the broad spectrum of diagnostic reagents and the compatibility of antibody quality control products.

[0003] Hepatitis C virus (HCV) belongs to the genus Hepatitis Virus in the family Flaviviridae. It is a single-stranded positive-sense RNA virus with a genome length of approximately 9.5 kb, encoding structural proteins (Core, E1 / E2) and non-structural proteins (NS2-NS5). Among them, the HCV NS3 protein, due to its crucial function, abundant conserved epitopes, and strong immunogenicity, has become a core target for diagnosis, treatment, and vaccine development. Pan-genotypic antibodies for NS3 can be designed. NS3 antibodies can be detected 2-4 weeks after infection, earlier than Core antibodies, significantly shortening the window period and possessing early diagnostic value. Its T / B cell epitopes can induce a broad-spectrum immune response, improving the detection sensitivity of reagents for multiple subtypes.

[0004] Existing HCV testing quality control products are mostly prepared from animal-derived polyclonal antibodies or mixed human serum, or clinical strongly positive serum / plasma samples, which have drawbacks such as large batch-to-batch variability, potential biosafety risks, and ethical issues.

[0005] Therefore, existing technologies need further improvement. Summary of the Invention

[0006] This invention provides a recombinant humanized monoclonal antibody against the non-structural protein NS3 of hepatitis C virus (HCV). This recombinant monoclonal antibody is secreted and expressed in HEK293 suspension cells. The invention also provides a method for obtaining the nucleotide sequence of this recombinant monoclonal antibody. The variable region sequence of the recombinant monoclonal antibody is obtained by immunizing mice with recombinant NS3 subtype antigens, screening and preparing hybridoma cells, and sequencing the sequences using hybridoma gene sequencing technology. The recombinant monoclonal antibody is constructed by humanizing the light and heavy chain constant regions, optimizing the codons, and then constructing it into the cell expression vector pCDNA3.4. This vector is then transfected into HEK293 suspension cells for recombinant expression. The expression product is purified by affinity chromatography using a Protein A column to obtain the recombinant humanized anti-HCV non-structural protein NS3 monoclonal antibody, named R-Anti-HCV-NS3-Ab. This recombinant monoclonal antibody, expressed in mammalian cells using gene recombination technology, features high yield, high purity, and small batch-to-batch variation. The present invention also provides the application of the recombinant monoclonal antibody as a quality control in HCV antibody detection reagents; the recombinant monoclonal antibody as a quality control has the advantages of high sensitivity, excellent specificity, good interoperability, multi-platform compatibility, high safety, and batch-to-batch stability.

[0007] Specifically, the technical solution provided in this application is as follows: In a first aspect, this application provides a recombinant humanized anti-HCV NS3 monoclonal antibody, comprising a light chain and a heavy chain, wherein the anticlonal antibody is named R-Anti-HCV-NS3-Ab, the amino acid sequence of its light chain is shown in SEQ ID NO.6, and the amino acid sequence of its heavy chain is shown in SEQ ID NO.7.

[0008] Furthermore, the amino acid sequence of the light chain from the N-terminus to the C-terminus is Signal-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-LH (hIgκC); the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.1. The amino acid sequence of the heavy chain from the N-terminus to the C-terminus is Signal-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-CH (HIgG1); the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.2. Analysis of the light chain complementarity-determining regions CDR1, CDR2, and CDR3 sequences shows the following: CDR1: SSSVSY, CDR2: DTS, CDR3: QQWSTYPLT; the heavy chain complementarity-determining regions CDR1, CDR2, and CDR3 are shown as follows: CDR1: GFTFTNSY, CDR2: TRNKANGYTL, CDR3: VRSHRYDNFALDY.

[0009] The light and heavy chain signal peptide sequences are shown in SEQ ID NO.3; their humanized constant region sequences are shown in SEQ ID NO.4 and 5, respectively.

[0010] Furthermore, the nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO. 8. The nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO. 9. The above sequences are preferred nucleotide sequences after codon optimization using the online codon optimization website Jcat for mammalian cell expression.

[0011] Secondly, this application also provides a recombinant expression vector, which is obtained by inserting the nucleotide sequence of the codons shown in SEQ ID NO. 8 and 9 into the pCDNA3.4 vector, and successfully obtaining light chain recombinant plasmid and heavy chain recombinant plasmid; the gene synthesis and recombinant vector construction work were completed by Qingke Biotechnology.

[0012] Thirdly, this application also provides a host cell comprising a recombinant expression vector as described in the second aspect, wherein the host cell is HEK293 suspension cells.

[0013] Fourthly, this application also provides a method for preparing the monoclonal antibody as described in the first aspect, comprising the following steps: S1: Mice were immunized with HCV NS3 recombinant antigen, and hybridoma cells were obtained by screening. S2: Extract mRNA from hybridoma cells, reverse transcribe it into cDNA, amplify the variable regions of the light and heavy chains, and sequence it. S3: Ligate the light and heavy chain variable region sequences obtained from sequencing with the human constant region sequences to construct a recombinant expression vector; S4: Transfect HEK293 suspension cells with the light and heavy chain recombinant plasmids for culture and secretion expression; S5: Purify to obtain the monoclonal antibody.

[0014] Specifically, the preparation method is as follows: S1: Hybridoma preparation: BALB / C mice were immunized with NS3 recombinant antigen prepared from HCV NS3 subtype 1b. Hybridoma cells were prepared by screening HCV NS3 recombinant proteins from subtypes 1b, 1a, 2a, 3a, and 4a. The HCV NS3 recombinant antigen was obtained by cloning selected fragments of NS3 from different subtypes into the pET-28a(+) vector, expressing and purifying it in BL21(DE3) after induction with isopropyl thiogalactoside.

[0015] S2: Hybridoma cell sequencing: After the prepared hybridoma cells are cultured, their mRNA is extracted and cDNA is prepared by reverse transcription. The prepared cDNA is used as a template and amplified using primers for the light and heavy chain variable regions. The amplified products are ligated into a vector using a TA ligation kit, and the constructed recombinant vector is sent for sequencing.

[0016] S3: Sequencing sequence analysis and light / heavy chain recombinant plasmid construction: The sequenced light and heavy chain variable regions are analyzed. The successfully sequenced light and heavy chain variable regions are ligated with the human light and heavy chain constant regions, codon optimization is performed, and the plasmids are constructed into the pCDNA3.4 vector. Light and heavy chain recombinant vectors are prepared.

[0017] S4: HEK293 suspension cells were transfected with the light and heavy chain recombinant plasmid for culture and secretion expression.

[0018] S5: Collect the supernatant of HEK293 suspension cell culture, purify it using a protein A affinity chromatography column, and finally obtain the purified recombinant monoclonal antibody.

[0019] Fifthly, based on the same invention, this application also provides an anti-HCV quality control product, characterized in that the quality control product comprises the monoclonal antibody described in the first aspect.

[0020] Sixthly, this application also provides the use of the monoclonal antibody as described in the first aspect or the anti-HCV quality control product as described in the fifth aspect in the preparation of an HCV antibody detection kit.

[0021] Furthermore, the detection kit is a double-antigen sandwich method, indirect method, or capture method ELISA kit.

[0022] The present invention has the following beneficial effects: The recombinant humanized anti-HCV NS3 monoclonal antibody developed in this patent overcomes technical bottlenecks through the following innovative points: Designed based on the conserved epitope region of NS3, it ensures high affinity for major prevalent subtypes such as 1a, 1b, 2a, 3a, and 4a, guaranteeing broad-spectrum detection capabilities; through humanization modification, it reduces interference from anti-mouse antibodies (HAMA), avoids false positives, and improves reagent stability; it is compatible with multiple platforms such as chemiluminescence and ELISA; and it is produced in a standardized manner using a mammalian cell expression system, achieving antibody purity >95% and batch-to-batch consistency (CV <3%) significantly superior to traditional polyclonal antibodies. The quality control material of this patent can accurately assess the sensitivity of HCV antibody test kits (detection limit down to 1 ng / mL), making it particularly suitable for regions with complex genotypes. Its standardized production system provides a reliable tool for quality control of HCV diagnostic reagents, contributing to the implementation of the global strategy to eliminate hepatitis C. In the future, this antibody can also be extended to the development of therapeutic drugs (such as blocking NS3 / 4A protease activity), showing multiple application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0024] Figure 1 In Example 2, the variable regions of the light and heavy chains were amplified by PCR, where M: DL2000; 1, 2: amplification products of the light chain variable region; 3, 4: amplification products of the heavy chain variable region. Figure 2 Results of SDS-PAGE and SEC-HPLC after purification. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0026] Example 1: Method for preparing hybridoma cells resistant to the non-structural protein NS3 of hepatitis C virus (HCV) (1) Preparation of immunogen NS3: The hepatitis C virus NS3 protein sequence was analyzed using the uniprot database (subtypes 1a, 1b, 2a, 3a, and 4a were identified by referring to P27958, P26663, P26660, Q81258, and O39929, respectively). Different subtype recombinant NS3 antigens were obtained through gene synthesis, plasmid construction (construction into the pET28a vector), transformation and expression in Escherichia coli, and purification.

[0027] (2) Mouse immunization: Female balb / c mice aged 6-8 weeks were immunized with the 1b subtype recombinant NS3 antigen; the immunoglobulin was mixed with an equal volume of Freund's complete adjuvant at a dose of 75 μg / mouse, emulsified completely, and administered subcutaneously to multiple sites on the back of the mice; three weeks later, a booster immunization was performed, with the immunoglobulin mixed with an equal volume of incomplete Freund's adjuvant at a dose of 35 μg / mouse, emulsified completely, and administered subcutaneously to multiple sites on the back of the mice; one week later, blood was collected from the tails of the mice to detect serum titers. When the serum titer reached 100,000 or higher, fusion was prepared; three days before fusion, the mice were immunized intraperitoneally without adjuvant at a dose of 100 μg / mouse.

[0028] (3) Cell fusion: During fusion, the spleen of immunized mice was taken, the cells were ground and dispersed, and mouse B lymphocytes were collected after centrifugation. They were mixed at a ratio of 7:1 (B lymphocytes: SP2 / 0), centrifuged, and washed with serum-free DMEM medium to ensure that there was no fetal bovine serum in the mixed cells. Cell fusion was performed with PEG. After full fusion, the fusion was terminated with DMEM medium. Finally, the fused cells were resuspended with HAT medium, placed in a 96-well cell culture plate, and cultured in a carbon dioxide incubator.

[0029] (4) Hybridoma cell screening and subcloning: The medium was completely replaced with HAT medium on the 7th day after fusion, and the culture supernatant was detected by indirect ELISA on the 9th day. The specific testing methods are as follows: Coating: Dilute different subtypes of NS3 recombinant antigen protein to 1 μg / mL with CBS buffer (pH 9.6), coat 100 μL / well into microplates, and incubate overnight at 4°C. Wash the plate 5 times with PBST buffer and blot dry. Block: Block the plate with PBST buffer containing 3% BSA, 200 μL / well, and incubate at 37°C for 2 h. Wash the plate 5 times with PBST buffer and blot dry. Detection: Add the cell supernatant to be tested to the plate, 100 μL / well, with mouse immune serum as a positive control and HAT medium as a blank control, and incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and blot dry. Add enzyme-labeled secondary antibody: Dilute HRP-labeled goat anti-mouse antibody 20,000 times with PBST buffer and add 100 μL / well to the plate, incubate at 37°C for 1 h. Wash the plate 5 times with PBST buffer and blot dry. Color development: Develop the color with TMB chromogenic solution in the dark, 100 μL / well, and incubate at 37°C for 15 min. Terminate with 2M sulfuric acid stop solution, 50 μL / well, and detect at 450 nm. Cells that reacted to different NS3 subtypes and had high titers (OD450 ≥ 2.0) were selected for subcloning. Subcloning was performed using a serial dilution method. 200-500 cells were transferred to well A1 of a new 96-well cell culture plate and diluted 2-fold both longitudinally and laterally. After 7-9 days, the culture supernatant was tested using an indirect ELISA method. Wells with a single positive cell cluster were selected for subcloning until the positive rate of the 96-well plate reached 100%. Subcloning was repeated twice more. When the positive rate was 100% in both cases, subcloning was terminated to ensure the stability of the hybridoma cell line.

[0030] Example 2: Sequencing of NS3 non-structural protein against hepatitis C virus (HCV) in hybridoma cells (1) Hybridoma cell line culture and RNA extraction The prepared hybridoma cells were cultured in a large-scale manner, and the cultured cells were collected. RNA was extracted from the cells according to the instructions of Thermo's RNA extraction kit.

[0031] (2) Reverse transcription Following the instructions of Thermo's kit, the extracted RNA was immediately reverse transcribed to prepare cDNA.

[0032] (3) PCR amplification and recovery Using universal primers for the variable region of the IgG subtype mouse monoclonal antibody, and with the above-mentioned cDNA as a template, PCR amplification was performed on the variable regions of the light and heavy chains. The amplification products were identified by DNA gel extraction, and the PCR products were recovered by gel cutting using a TIANGEN kit. The light and heavy chain PCR products are shown below. Figure 1 .

[0033] Example 3: Variable region sequence analysis, humanization modification, and construction of recombinant plasmids Sequencing results were analyzed using the IMGT antibody variable region analysis tool in the database. The amino acid sequence of the light chain variable region (VL) of the anti-hepatitis C virus non-structural protein NS3 monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.2. The sequencing results show that the light and heavy chain variable region sequences include one signal peptide sequence, three complementarity-determining regions (CDR1, CDR2, CDR3), and four backbone regions (FR1-FR4). Analysis revealed the following sequences for the light chain complementarity determination regions (CDR1, CDR2, and CDR3): CDR1: SSSVSY, CDR2: DTS, CDR3: QQWSTYPLT; and the following sequences for the heavy chain complementarity determination regions (CDR1, CDR2, and CDR3): CDR1: GFTFTNSY, CDR2: TRNKANGYTL, CDR3: VRSHRYDNFALDY.

[0034] The same signal peptide sequence was added to the N-terminus of the variable regions of the light and heavy chains, as shown in SEQ ID NO.3; a human light chain constant region (hIgκC) sequence was added to the C-terminus of the light chain variable region: the sequence is referenced from Genbank: P01834.2, as shown in SEQ ID NO.4; a human heavy chain constant region (hIgG1-CH) was added to the C-terminus of the heavy chain variable region, the sequence is referenced from Genbank: AFR78282.1, as shown in SEQ ID NO.5; the full-length amino acid sequences of the humanized light and heavy chains were obtained: the sequences are shown in SEQ ID NO.6 and SEQ ID NO.7, respectively; and their translation codons were optimized using the online codon optimization website Jcat. The optimized full-length base sequence of the light chain is shown in SEQ ID NO.8; the full-length base sequence of the heavy chain is shown in SEQ ID NO.9. The light and heavy chain base sequences were given to a gene synthesis company (Qingke Biotechnology Co., Ltd.) and synthesized into the cell expression vector pCDNA3.4. The light chain recombinant plasmid was named pCDNA3.4-NS3-hL; the heavy chain recombinant plasmid was named pCDNA3.4-NS3-hH.

[0035] Example 4: Expression and purification of recombinant humanized anti-hepatitis C virus non-structural protein NS3 monoclonal antibody (1) Cell resuscitation (a) Remove one HEK293 suspension cell from the liquid nitrogen tank. After removal, quickly place the cryovial into a water bath for rapid thawing, no more than 1 minute. The facility temperature is 37°C, ensuring the temperature remains stable at 37°C during thawing. When placing the cryovial into the water bath, ensure the liquid level does not exceed 2 / 3 of the tube's height to avoid water contact with the tube opening and causing contamination.

[0036] (b) In a clean bench, transfer HEK293 cells to a centrifuge tube containing 10 mL of culture medium, centrifuge at 1000 rpm for 10 min, collect the cell pellet, resuspend in culture medium, count the cells, and adjust the cell density to 0.4-0.6 × 10⁻⁶. 6 cells / mL.

[0037] (c) Transfer the resuspended cells to a shake flask. In principle, the culture volume should not exceed 25% of the total volume of the shake flask. Place the shake flask in a CO2 shaker and culture it under the following conditions: 37°C, 5% CO2, and 110 rpm. Culture for about 3 days, then observe and passage the cells.

[0038] (2) Cell passage (a) After about 3 days of cell resuscitation, the cells are in the logarithmic growth phase. Cells are then harvested for cell density (×10⁻⁶). 6Cell counts (cells / mL) and viability (%) were measured, and cells were passaged after the tests.

[0039] (b) Calculate cell density ≥2×10 6 When the cell count / mL is above 90%, use 0.8-1.2 × 10⁻⁶ cells / mL. 6 The cells were passaged at a density of cells / mL and then transferred to shake flasks for culture.

[0040] (c) Continue passage 1-2 more times. Before transient transfection, cells need to be passaged stably at least 3 times. If the cell condition is poor or cell acclimatization is required during passage, the cells need to be passaged stably 3 times after the cell condition is stable or acclimatization is complete before transfection.

[0041] (3) Instantaneous transfection (a) Plasmid and PEI pretreatment: On the day of transfection, add an appropriate amount of HEK293 suspension cell culture medium with light chain recombinant plasmid: heavy chain recombinant plasmid: transfection reagent (PEI) in a ratio of 2:1:3. After mixing thoroughly, let stand for 10-15 minutes.

[0042] (b) Transfection: Count the prepared cells and adjust the cell density to 3.0-3.5 × 10⁶ cells / year using HEK293 suspension culture medium (model: 293CD01). 6 cells / mL.

[0043] (c) The transfected cells are loaded into sterile shake flasks (volume ≤30%) and the shake flasks are placed in a carbon dioxide shaker for incubation.

[0044] (4) Feeding 24 hours after transfection, add feed medium (model 293 F01) at 3-5% of the culture volume; add L-glutamine solution at 1% of the culture volume, and continue culturing. On days 3 and 5 post-transfection (day 0 is the day of transfection), cell viability is calculated (3-day cell viability ≥90%; 5-day cell viability ≥75%), and feed medium is added at 3-5% of the culture volume; L-glutamine solution is added at 1% of the culture volume, and culturing continues. On day 7, cell viability is measured (cell viability ≥50%), and the culture supernatant is collected. (Note: HEK293 suspension cell culture medium and feed medium were purchased from Peiding Biotechnology.) (5) Collection and purification of expression supernatant (a) Collection of supernatant: The culture product was placed in a centrifuge bottle, balanced, centrifuged at 8°C and 9000 rpm for 30 min, and the supernatant was collected for purification.

[0045] (b) Protein A-column affinity chromatography purification Equilibration: Take the Protein-A chromatography column and equilibrate it with 10 volumes of loading buffer (20mM PBS, pH 7.4); Sample loading: Add the processed cell expression supernatant to the chromatography column and collect the flow-through using a centrifuge tube; after all the sample has passed through the column, load the flow-through again and collect the flow-through at the same time, and label the flow-through (during the sample loading process, control the flow rate to be slow so that the sample and the purification column can fully combine). Equilibration: After loading, equilibrate the column with 10 column volumes of loading buffer. At the same time, use Coomassie Brilliant Blue reagent to detect the outlet liquid. When the liquid protein concentration is below 1 mg / mL, the equilibration is complete. Elution: Add 30 μl of neutralization buffer (1 M Tris-HCl, pH 9.0) to a 600 μL LEP tube beforehand, and elute with 10 column volumes of elution buffer (0.1 M Gly-HCl, pH 2.5-3.0); Elution: During elution, collect 400 μL of eluent from each tube and mix well immediately; use Coomassie Brilliant Blue reagent to detect the concentration of antibody in the eluent (note the start and end points of collection) and label accordingly; Equilibration: Equilibrate with 10 column volumes of loading buffer. Use pH test paper to check the pH of the outlet liquid. When the pH matches that of the loading buffer, the equilibration is complete. Rinse the column with 20% ethanol and store it in 20% ethanol at 4°C.

[0046] Dialysis: The collected protein was placed in a dialysis bag, and dialysis was performed twice daily with 20 mM PBS (pH 7.4) as the dialysate, changing the medium twice a day (morning and evening). Dialysis was performed at 2-8℃ for 3 days (at least 6 times). After dialysis, the protein was removed and placed in a 50 ml centrifuge tube. The protein was centrifuged at 7500-9000 rpm at 4-15℃ for 30 min. The supernatant was collected, and the concentration was measured. The obtained protein sample was the recombinant humanized anti-hepatitis C virus (HCV) non-structural protein NS3 monoclonal antibody (R-Anti-HCV-NS3-Ab). After purification, R-Anti-HCV-NS3-Ab was analyzed by SDS-PAGE gel imaging and SEC-HPLC. The results are shown below. Figure 2 As shown, the SDS-PAGE gel image correctly shows the light and heavy chains; their sizes are consistent with the theoretical values ​​of 25 kDa for the light chain and 50 kDa for the heavy chain. SEC-HPLC detection shows that the recombinant monoclonal antibody can form the correct dimer, and the dimer purity is as high as 97%. Calculation of the total amount after purification shows that the yield of this recombinant monoclonal antibody can reach 500 mg / L, which is a high yield.

[0047] Example 5: ELISA assay to detect the titer affinity of R-Anti-HCV-NS3-Ab for different isotypes of NS3 protein. The R-Anti-HCV-NS3-Ab prepared in this invention can be detected using different subtypes of NS3 antigens. Detection can be performed indirectly using ELISA. (1) Different subtypes of NS3 antigen were diluted at 0.5ug / mL in 0.01M pH9.6 carbonate buffer coating solution, 100ul / well, and coated overnight at 4℃.

[0048] (2) Wash the plate three times with PBST, dry it, add 200ul of blocking buffer per well, and block for 2 hours.

[0049] (3) Wash the plate three times with PBST, dry it, and set aside.

[0050] (4) Sample addition: Add 1 mg / ml R-Anti-HCV-NS3-Ab at a 1:10 ratio. 2 (10μg / ml), 1:10 3 (1μg / ml), 1:10 4 (100ng / ml), 1:10 5 (10ng / ml), 1:10 6 (1ng / ml), 1:10 7 (100pg / ml), 1:10 8 (10 pg / ml). Add 100 μl to each well of a 96-well microplate and allow to react at room temperature for 2 hours.

[0051] (5) Wash the plate three times with PBST and dry it. Add 0.1 mL of mouse anti-human-HRP antibody diluted 1:10000 with PBST to each well and react at room temperature for 1 hour.

[0052] (6) Wash the plate five times with PBST and dry it. Add 100 μL of TMB colorimetric solution and react at room temperature in the dark for 10 minutes.

[0053] (7) Add 50 μL of stop solution to terminate the reaction; measure its OD value at 450 nm, and P / N ≥ 2.1 is considered positive.

[0054] (8) The affinity was calculated by determining the half-maximal effective concentration (EC50) of R-Anti-HCV-NS3-Ab using linear interpolation.

[0055] The results are shown in Table 1: R-Anti-HCV-NS3-Ab showed consistent valence trends across different subtype gradient tests, with valence deviation (CV) ≤ 5% for the same gradient test. EC50 was calculated as EC50 = 1 ng / ml + (50% - 31.02%) / (64.96% - 31.02%) * (10 ng / ml - 1 ng / ml) = 6.0 ng / ml. The calculation shows that EC50 is around 6 ng / ml, indicating high affinity.

[0056] Table 1: ELISA assay for the titer and affinity of recombinant monoclonal antibody R-Anti-HCV-NS3-Ab for different subtypes

[0057] Example 6: Application Testing of Recombinant Humanized Anti-Hepatitis C Virus (HCV) Non-structural Protein NS3 Monoclonal Antibody (R-Anti-HCV-NS3-Ab) as a Quality Control Product (1) Preparation of anti-HCV quality control products Prepare a 20mM PBS buffer (pH 7.4) containing a certain concentration of BSA and Tween-20 to simulate serum matrix; serially dilute the purified R-Anti-HCV-NS3-Ab with the prepared simulated serum matrix to final concentrations of 100 ng / ml, 10 ng / ml, and 1 ng / ml; the concentrations of other components are 0.1% BSA and 0.05% Tween-20; and add 0.1% ProClin 300 or 0.02% NaN3 to prevent microbial contamination.

[0058] (2) Quality control products for sandwich and indirect ELISA kits from different manufacturers The self-made anti-HCV quality control product R-Anti-HCV-NS3-Ab was tested using sandwich and indirect ELISA kits from different manufacturers. The results are shown in Table 2. The results show that the self-made anti-HCV quality control product can be detected by both indirect and sandwich HCV ELISA kits from different manufacturers, indicating that the recombinant monoclonal antibody R-Anti-HCV-NS3-Ab of the present invention has good compatibility, high potency, and good sensitivity.

[0059] Table 2. Results of testing self-made anti-HCV quality control samples with sandwich and indirect ELISA kits from different manufacturers.

[0060] (3) Batch-to-batch differences in self-made anti-HCV quality control products for ELISA testing Following the above method for preparing anti-HCV quality control products, three consecutive batches of purified R-Anti-HCV-NS3-Ab monoclonal antibodies were prepared as anti-HCV quality control products. The batch-to-batch differences were tested using the manufacturer's 3-sandwich HCV ELISA kit. The test results are shown in Table 3. Through deviation calculation, the CV between the three batches was ≤5%, which is small and better than the requirement of CV ≤10% for most products on the market.

[0061] Table 3. Batch-to-batch differences in quality control samples from three consecutive batches of self-made anti-HCV products.

[0062] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A recombinant humanized anti-HCV NS3 monoclonal antibody, comprising a light chain and a heavy chain, characterized in that, The anticlonal antibody is named R-Anti-HCV-NS3-Ab, and its light chain amino acid sequence is shown in SEQ ID NO.6; its heavy chain amino acid sequence is shown in SEQ ID NO.

7.

2. The recombinant humanized anti-HCV NS3 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain from the N-terminus to the C-terminus is Signal-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-LH (hIgκC); the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.1; the amino acid sequence of the heavy chain from the N-terminus to the C-terminus is Signal-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-CH (HIgG1); the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.

2.

3. The recombinant humanized anti-HCV NS3 monoclonal antibody according to claim 1, characterized in that, The nucleotide sequence encoding the light chain of the monoclonal antibody is shown in SEQ ID NO.8; the nucleotide sequence encoding the heavy chain of the monoclonal antibody is shown in SEQ ID NO.

9.

4. A recombinant expression vector, characterized in that, The vector comprises the nucleotide sequence encoding the light or heavy chain of a monoclonal antibody as described in claim 3, wherein the vector is pCDNA3.

4.

5. A host cell, characterized in that, The invention comprises a recombinant expression vector as described in claim 4, wherein the host cell is HEK293 suspension cells.

6. A method for preparing the monoclonal antibody as described in any one of claims 1-3, characterized in that, Includes the following steps: S1: Mice were immunized with HCV NS3 recombinant antigen, and hybridoma cells were obtained by screening. S2: Extract mRNA from hybridoma cells, reverse transcribe it into cDNA, amplify the variable regions of the light and heavy chains, and sequence it. S3: Ligate the light and heavy chain variable region sequences obtained from sequencing with the human constant region sequences to construct a recombinant expression vector; S4: Transfect HEK293 suspension cells with the light and heavy chain recombinant plasmids for culture and secretion expression; S5: Purify to obtain the monoclonal antibody.

7. The preparation method according to claim 6, characterized in that, The HCV NS3 recombinant antigen in step S1 includes at least one of the subtypes 1a, 1b, 2a, 3a, and 4a.

8. An anti-HCV quality control product, characterized in that, The quality control material comprises the monoclonal antibody as described in any one of claims 1-5.

9. The use of the monoclonal antibody as described in any one of claims 1-5 or the anti-HCV quality control product as described in claim 9 in the preparation of an HCV antibody detection kit.

10. The application according to claim 9, characterized in that, The detection kit is a double-antigen sandwich method, indirect method, or capture method ELISA kit.

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