A varicella-zoster virus gL protein monoclonal antibody and application thereof

Abstract

The application discloses a varicella-zoster virus gL protein monoclonal antibody and application thereof, and relates to the technical field of biological immunity. The varicella-zoster virus gL protein monoclonal antibody comprises CDR-H1 with an amino acid sequence as shown in SEQ ID NO. 9, CDR-H2 with an amino acid sequence as shown in SEQ ID NO. 11, CDR-H3 with an amino acid sequence as shown in SEQ ID NO. 13, CDR-L1 with an amino acid sequence as shown in SEQ ID NO. 16, CDR-L2 with an amino acid sequence of LVS and CDR-L3 with an amino acid sequence as shown in SEQ ID NO. 19. The monoclonal antibody provided by the application can be specifically combined with the varicella-zoster virus gL protein, can be applied to the preparation of products for detecting the varicella-zoster virus gL protein or the varicella-zoster virus gL antibody, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of bioimmunotechnology, and in particular to a monoclonal antibody against the varicella-zoster virus gL protein and its application. Background Technology

[0002] Varicella-zoster virus (VZV) is a widespread pathogen. Primary infection causes chickenpox, and viral reactivation leads to shingles. With an aging population and a growing number of immunocompromised individuals, VZV-related diseases have become a significant public health burden. Accurately assessing an individual's VZV immune status is crucial for identifying susceptible populations, guiding vaccination strategies, and aiding in clinical diagnosis.

[0003] Currently, VZV serological testing mainly employs methods such as enzyme-linked immunosorbent assay (ELISA). While these methods offer good reliability, they are complex to perform, time-consuming (typically 2-4 hours), and rely on specialized equipment like ELISA readers, making them unsuitable for the needs of primary healthcare institutions and rapid on-site screening. Although point-of-care testing (POCT) technology has developed rapidly, existing colloidal gold immunochromatographic test strips still have limitations in detection sensitivity, and the interpretation of weak positive results is easily influenced by subjective factors.

[0004] Quantum dots, as a novel fluorescent nanomaterial, possess excellent optical properties such as high fluorescence intensity, strong resistance to photobleaching, and narrow and tunable emission spectrum. Combining them with immunochromatography holds promise for achieving rapid detection with high sensitivity and stability. However, there are currently no quantum dot-labeled VZV gL antibody rapid detection products available.

[0005] Therefore, developing a rapid VZV antibody detection product that is easy to operate, highly sensitive, highly specific, and suitable for on-site screening has significant clinical application value and market prospects. Summary of the Invention

[0006] The purpose of this invention is to provide a monoclonal antibody against varicella-zoster virus gL protein and its applications, thereby addressing the problems existing in the prior art. The monoclonal antibody against varicella-zoster virus gL protein provided by this invention can specifically bind to the varicella-zoster virus gL protein and can be used to prepare products for detecting varicella-zoster virus gL protein or varicella-zoster virus gL antibodies. It has significant application value in the rapid diagnosis, immune level assessment, and epidemiological investigation of varicella-zoster virus infection.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a monoclonal antibody against varicella-zoster virus gL protein, comprising CDR-H1 with the amino acid sequence shown in SEQ ID NO. 9, CDR-H2 with the amino acid sequence shown in SEQ ID NO. 11, CDR-H3 with the amino acid sequence shown in SEQ ID NO. 13, CDR-L1 with the amino acid sequence shown in SEQ ID NO. 16, CDR-L2 with the amino acid sequence of LVS, and CDR-L3 with the amino acid sequence shown in SEQ ID NO. 19.

[0008] Furthermore, the amino acid sequence of the heavy chain variable region of the varicella-zoster virus gL protein monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0009] The present invention also provides the encoding gene of the above-mentioned varicella-zoster virus gL protein monoclonal antibody.

[0010] Furthermore, the coding gene includes a light chain variable region coding gene and a heavy chain variable region coding gene; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.6.

[0011] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0012] The present invention also provides a recombinant non-plant host cell, comprising the above-described recombinant expression vector.

[0013] The present invention also provides the use of the above-mentioned encoding gene, recombinant expression vector or recombinant non-plant host cell in the preparation of the above-mentioned varicella-zoster virus gL protein monoclonal antibody.

[0014] The present invention also provides the use of the above-mentioned varicella-zoster virus gL protein monoclonal antibody in the preparation of products for detecting varicella-zoster virus gL protein or varicella-zoster virus gL antibody.

[0015] The present invention also provides a product for detecting varicella-zoster virus gL protein or varicella-zoster virus gL antibody, comprising the above-mentioned varicella-zoster virus gL protein monoclonal antibody.

[0016] Furthermore, the product for detecting varicella-zoster virus gL antibodies is a quantum dot test strip; The quantum dot test strip includes a base plate, a conjugate pad, a sample pad, a nitrocellulose membrane, and an absorbent pad; The nitrocellulose membrane is attached to the base plate, the absorbent pad and the binding pad are respectively tightly pressed against both ends of the nitrocellulose membrane, and the sample pad is tightly pressed against the end of the binding pad away from the nitrocellulose membrane; from the end near the binding pad to the end away from the binding pad, the nitrocellulose membrane is provided with detection lines and quality control lines in sequence; The detection line is coated with Staphylococcus aureus protein A; the control line is coated with the above-mentioned varicella-zoster virus gL protein monoclonal antibody; and the binding pad is coated with quantum dot-labeled varicella-zoster virus gL protein.

[0017] The present invention discloses the following technical effects: 1. This invention is the first to select VZV gL protein as the antibody detection target. Anti-gL antibodies have high specificity and can serve as a reliable serological indicator for assessing immune status after natural infection and vaccination. Unlike existing technologies that mostly focus on gE antigens, this invention fills the gap in the application of the gL target in the field of immunochromatography.

[0018] 2. This invention employs a prokaryotic expression system and a co-expression strategy with the molecular chaperone TF16 to successfully achieve soluble expression of VZV gL protein, avoiding the formation of inclusion bodies. The purified protein has a purity of over 90% and exhibits good immunoreactivity, laying a material foundation for subsequent monoclonal antibody preparation and detection system construction.

[0019] 3. This invention uses hybridoma technology to screen and obtain monoclonal antibody 1B7, which is characterized by high purity and strong specificity. This antibody, used as a quality control line capture antibody, ensures the stability and reliability of the detection system.

[0020] 4. The VZV antibody quantum dot test strip prepared in this invention has the technical advantages of high specificity, high sensitivity, and rapid detection. It can accurately identify VZV antibodies in the test sample, with a concordance rate of 95.5% with commercial ELISA kits, effectively reducing the false negative rate and improving detection accuracy. This test strip can be directly applied to VZV susceptible population screening, post-vaccination immunization efficacy evaluation, and epidemiological surveys, providing a convenient and reliable technical means for the early prevention and control of VZV-related diseases, and has significant promotional value and social benefits. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the PCR electrophoresis results of the strain transfected with the pET-28a-gL recombinant plasmid; lane M is a 2000bp DNA marker, and lanes 1, 2, 3, and 4 are the PCR amplification results. Figure 2 Figure 1 shows the SDS-PAGE and Western Blot results of the VZV gL recombinant plasmid expression product. In Figure 2, A represents the SDS-PAGE results; M: Marker; 1: pET-28a empty vector; 2: pET-28a-gL supernatant; 3: pET-28a-gL precipitate; 4: pET-28a-gL + pTF16 supernatant; 5: pET-28a-gL + pTF16 precipitate. Figure 3 shows the Western blot results; M: Marker; 1: pET-28a empty vector; 2: pET-28a-gL + pTF16. Figure 3 This is a graph showing the SDS-PAGE results of purified gL protein; where M: 180kDa protein marker; 1: supernatant before protein purification; 2: target protein; Figure 4 The figures show the Western Blot results of VZV gL recombinant protein with anti-His monoclonal antibody and VZV positive serum, respectively; where A represents the reaction result of VZV gL recombinant protein with anti-His-tag monoclonal antibody; and B represents the reaction result of VZV gL recombinant protein with VZV positive serum; in A and B, M: Marker; 1: pET-28a empty vector supernatant; 2: gL protein; Figure 5 The image shows the SDS-PAGE results of ascites fluid purification using monoclonal antibody 1B7; where M: 180kDa protein marker; 1 is the ascites fluid before purification; 2 is the ascites fluid after purification. Figure 6 The double reciprocal curves represent the affinity constant of monoclonal antibody 1B7; Figure 7 This is a schematic diagram of the structure of a quantum dot test strip; Figure 8 The image shows the sensitivity test results for the quantum dot test strips. Figure 9 This is a diagram showing the specific detection results of the quantum dot test strip. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0027] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0028] Example 1: Preparation of VZV gL recombinant protein 1. Amplification of the VZV gL encoding gene: Based on the gL protein gene sequence of the VZV standard laboratory reference strain Dumas in the NCBI database (GenBank: NC_001664.4), specific amplification primers were designed, and primers were introduced at the 5' ends of the upstream and downstream primers, respectively. EcoR I and Hind III. Enzyme cleavage sites and corresponding protective bases.

[0029] The nucleotide sequence of the VZV gL encoding gene is shown in SEQ ID NO.1; the nucleotide sequence of the forward primer for amplifying the VZV gL encoding gene is shown in SEQ ID NO.2, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.3.

[0030] SEQ ID NO.1: ATGCTGCATCTGCATCTGCAAGACGATACACCTCTGTTCTTCGGAGCCAAGCCTCTGAGCGATGTCTCCTCATCATTACCGAGCCTTGCGTCAGCAGCGTCTACGAAGCCTGGGATTACGCCGCTCCACCTGTGTCCAACCTGAGCGAAGCTCTGAGCGGCATCGTGGTCAAGACCAAGTGTCCAGTGCCAGAAGTCATCTTGTGGTTCAAAGACAAGCAGATGGC CTACTGGACCAATCCATACGTCACTCTGAAGGGCCTGGCACAGAGCGTCGGTGAAGAACACAAGTCTGGCGACATCAGGGATGCTCTGCTGGACGCACTGTCTGGCGTGGGTGGACAGCACACCTTCTAGCACTAACATACCTGAGAACGGATGCGTCTGGGGTGCAGACCGGCTGTTCCAGAGAGTGTGTCAGGGCGGATCTCACCACCACCATCATCACCACCATTAA.

[0031] SEQ ID NO.2: 5'-GGAATTCATGCTGCATCTGCATCTGCAAGACGATACACCTCT-3'; SEQ ID NO. 3: 5'-CCCAAGCTTTAATGGTGGTGATGATGGTGGTGGTGAG-3'.

[0032] The amplification reaction system consisted of: 1 μL of target gene template, 1 μL each of upstream and downstream primers, 0.5 μL of PrimeSTAR HSDNA Polymerase, 4 μL of dNTPs, 10 μL of 5×Primer STAR Buffer, 32.5 μL of ddH2O, and a total volume of 50 μL. The PCR amplification program was as follows: pre-denaturation at 95℃ for 3 min; followed by 34 cycles with the following parameters: denaturation at 95℃ for 30 s, annealing at 65℃ for 30 s, extension at 72℃ for 31 s, and extension at 72℃ for 5 min.

[0033] 2. Purification and recovery of PCR products: After PCR amplification, the products were analyzed by 1% (w / v) agarose gel electrophoresis. The remaining PCR products were recovered using a universal DNA recovery kit. The concentration and purity of the recovered DNA were determined using NanoDrop 2000 and stored at -20°C for later use.

[0034] 3. Construction of pET-28a-gL recombinant plasmid: The VZV gL encoding gene and pET-28a vector were double-digested at 37℃. The digestion reaction mixture contained 2 μg each of pET-28a vector and VZV gL encoding gene. EcoR I and Hind 1 μL of enzyme III, 5 μL of 10×CutSmart Buffer, and ddH2O were added to bring the total volume to 50 μL. After enzyme digestion, the VZV gL encoding gene and the linearized vector were excised from the gel and ligated. The ligation reaction system was as follows: 2 μL of T4 DNA Ligase, 2 μL of 10×T4 DNA ligase Buffer, and the VZV gL encoding gene and vector were added at a molar ratio of 3:1. The total volume was brought to 20 μL with ddH2O. The ligation was carried out at 16℃ for 12 h to obtain the recombinant plasmid pET-28a-gL, which was stored at -20℃ for later use.

[0035] 4. Transformation with pET-28a-gL recombinant plasmid: Add 10 μL of the recombinant plasmid pET-28a-gL to 100 μL of DH5α competent cells thawed on ice, mix gently, incubate on ice for 15-30 min, heat shock at 42℃ for 90 s, and quickly transfer to ice to cool for 5 min; add 900 μL of LB liquid medium, incubate at 37℃ on a shaker at 220 rpm for 1 h; remove the bacterial culture, centrifuge at 3000 rpm for 10 min, discard 900 μL of supernatant, resuspend the precipitate with the remaining liquid, take 60 μL of the bacterial culture for spreading, and then incubate inverted at 37℃ overnight; pick a single colony and transfer it to LB liquid medium containing kanamycin resistance, incubate at 37℃ on a shaker at 220 rpm for 4-5 h; take 1 μL of the bacterial culture as template DNA, select T7 universal primers for bacterial PCR identification, the amplified product is about 600 bp, and the detection results are as follows. Figure 1 As shown, positive monoclonal strains were screened and sequenced. The correctly sequenced strains were then expanded and cultured, followed by plasmid extraction using a plasmid extraction kit. Plasmid concentration was determined using NanoDrop 2000, and the concentration and time were recorded. The samples were then stored at -20°C.

[0036] 5. Expression, purification, and identification of VZV gL recombinant protein: The recombinant plasmid pET-28a-gL was transformed into BL21(DE3) competent cells containing the molecular chaperone pTF16 to obtain pET-28a-gL+pTF16 recombinant expression bacteria. The recombinant expression bacteria were inoculated at a ratio of 1:100 into LB liquid medium containing kanamycin and ampicillin resistance and cultured at 37°C until OD500. 600When the value is 0.6, add L-arabinose solution with a final concentration of 2 mg / mL, and incubate at 37℃ in a shaker for 20 min. Then add 0.2 mM IPTG solution for induction and incubate at 16℃ in a shaker at 220 rpm for 14 h.

[0037] The expression of recombinant gL protein was identified using SDS-PAGE and Western Blot, and the results are as follows: Figure 2 As shown, the pET-28a-gL+pTF16 recombinant expression bacteria showed a clear band of approximately 20 kDa in the supernatant after induction, while the target protein of the pET-28a-gL recombinant expression bacteria (without molecular chaperone) was mainly present in the precipitate after induction. This confirms that the molecular chaperone co-expression strategy achieved soluble expression of the gL protein.

[0038] The induced bacterial culture was collected by centrifugation at 8000 rpm for 10 min at 4℃. After sonication, the culture was centrifuged at 12000 rpm for 10 min at 4℃ to separate the supernatant and precipitate. The supernatant was filtered through a 0.45 μm filter membrane. The recombinant protein was purified using a Ni-NTA affinity chromatography column. The target protein was eluted with 500 mM imidazole elution buffer, and the eluent was collected. The purified protein was analyzed by SDS-PAGE, and the results are shown below. Figure 3 As shown, the purity is higher than 90%, and the molecular weight is approximately 20 kDa. Western blotting was used to identify the purified protein, and the results are as follows. Figure 4 As shown, the protein specifically reacts with anti-His-tag monoclonal antibody and can be recognized by VZV antibody-positive serum, indicating that soluble VZV gL recombinant protein with good immunoreactivity was obtained from the supernatant. The protein concentration was determined to be 0.42 mg / mL by the BCA method.

[0039] Example 2: Preparation of VZV gL protein monoclonal antibody hybridoma cell line 1. Immunization and serum titer determination in BALB / c mice (1) Immunogen: VZV gL recombinant protein was emulsified with Freund's complete adjuvant and Freund's incomplete adjuvant at a volume ratio of 1:1 to prepare Freund's complete adjuvant immunogen and Freund's incomplete adjuvant immunogen.

[0040] (2) Immunization regimen: Two 6-8 week old female BALB / c mice were immunized with Freund's complete adjuvant immunogen via subcutaneous injection at multiple sites on the back, approximately 100 μL / mouse, of which 40 μg / mouse contained VZV gL recombinant protein. BALB / c mice were boosted with Freund's incomplete adjuvant immunogen at the same method and dosage 14 days and 28 days after the initial immunization. (3) Serum titer determination: Blood was collected from the tail of the mice 14 days after booster immunization, and the serum titer of the mice was determined by ELISA. The results showed that mouse No. 1 had the highest ELISA titer, which was 1:102400. Therefore, mouse No. 1 was selected for cell fusion to prepare monoclonal antibody hybridoma cell lines.

[0041] 2. Cell fusion and culture (1) Enhanced immunization: 3-5 days before cell fusion, mice No. 1 were given a super-strong immunization by intraperitoneal injection of VZV gL recombinant protein without adjuvant. The immunization dose was 50 μg / mouse. (2) Preparation of spleen cells: BALB / c mice that had undergone hyperimmunization for 5 days were euthanized by cervical induction. The spleen was disinfected with 75% ethanol and removed under aseptic conditions. The spleen was washed twice with GNK solution preheated to 37°C. A small amount of GNK solution was added to the spleen and it was cut into pieces with scissors on sterile 120-mesh nylon gauze. The spleen cells were then thoroughly ground to obtain a spleen cell suspension. The spleen cells were filtered into a sterile beaker and transferred to a sterile cell centrifuge tube. The cells were centrifuged at 1000 r / min for 10 min and washed with GNK solution 1-2 times for later use.

[0042] (3) Cell fusion and culture of fused cells: Cell fusion was performed using the polyethylene glycol method. Spleen cells from immunized mice were fused with mouse myeloma cells SP2 / 0 at a ratio of 8:1. The fused cells were gently suspended in HAT selective culture medium and dispersed into 96-well cell culture plates at 200 μL / well. The plates were then cultured at 37°C in a 5% CO2 incubator.

[0043] 3. Screening, identification, and subcloning of hybridoma cells (1) ELISA screening: After culturing the fused cells for 3-4 days, small cell clusters can be observed under a microscope. On day 10, 50 μL of cell culture supernatant was collected and screened using the ELISA method.

[0044] First round of screening: VZV gL recombinant protein (diluted to 1 μg / mL with CBS) was used as the coating agent to coat 10 ELISA plates for initial screening of hybridoma cell culture supernatant; Second round of screening: VZV gL recombinant protein (diluted to 1 μg / mL with CBS) and pET-28a empty vector supernatant (diluted 1:1000 with CBS) were selected as coating agents, and positive hybridoma cell lines were screened out after eliminating false positives. Third round of screening: The same method as the second round of screening was used to screen the above positive hybridoma cells in the third round of ELISA. The results showed that 6 positive hybridoma cell lines were screened out (named 8C4, 7F3, 5C9, 1F6, 1B7 and 3D10, respectively).

[0045] (2) Subcloning of positive hybridoma cells: positive hybridoma cells were subcloned by limiting dilution: the above positive hybridoma cells were diluted with 1640 complete medium to about 8 cells / mL, and 100 μL of each cell was added to a 96-well plate pre-coated with 100 μL of feeder cells. After culturing in a 37℃, 5% CO2 incubator for 6-8 days, the cells were screened and identified by ELISA. The positive monoclonal cell lines were then transferred to 48-well cell culture plates for expansion culture and subcloning was performed 2-3 times to obtain hybridoma cell lines that stably secrete monoclonal antibodies against VZV gL recombinant protein.

[0046] 4. Determination of the variable region sequence of monoclonal antibodies RNA was extracted from the monoclonal positive hybridoma cell line 1B7, reverse transcribed into cDNA, and then amplified by PCR to obtain the heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody 1B7, which was then sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0047] The sequencing results of monoclonal antibody 1B7 are as follows: Nucleotide sequence of the gene encoding the heavy chain variable region (SEQ ID NO.4): ATGCAGCAGTCAGGGGCTGAACTGGTGAAGCCTGGGACTTCAATGAAGCTGTCCTGTAAGGCTTCTGGCTACACCTTCACCGACTACTGGATACATTGGCTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGAGATTTATCCTAGGAACGGT CGTACAAACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTATATACAACTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAGGGGCTCTTGATGCTTACTGGGGCCAAGGGACCTGA.

[0048] Amino acid sequence of the heavy chain variable region (SEQ ID NO.5): MQQSGAELVKPGTSMKLSCKASGYTFTDYWIHWLKQRPGQGLEWIGEIYPRNGRTNYNEKFKNKATLTVDKSSSTAYIQLNSLTSEDSAVYFCAGALDAYWGQGT.

[0049] Nucleotide sequence of the gene encoding the light chain variable region (SEQ ID NO.6): ATGGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTA TCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCATGA.

[0050] Amino acid sequence of the light chain variable region (SEQ ID NO.7): MDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGP.

[0051] The arrangement of amino acids in the heavy chain variable region is shown in Table 1, and the arrangement of amino acids in the light chain variable region is shown in Table 2.

[0052] Table 1. Arrangement of amino acids in the variable region of the heavy chain. Table 2. Arrangement of amino acids in the variable region of the light chain Example 3: Preparation and purification of VZV gL protein monoclonal antibody (1B7) The monoclonal hybridoma cell line 1B7 from Example 2 was expanded and cultured. The titer of the culture supernatant was measured by ELISA to ensure the stability of the monoclonal cell line. The hybridoma cells 1B7 were collected for the large-scale preparation of monoclonal antibodies.

[0053] Multiparous female BALB / c mice were selected and injected intraperitoneally with 500 μL of sterile paraffin. 7–10 days later, approximately 1 × 10⁻⁶ mmol / L was administered per mouse. 7A sample of [number] cells was injected into pre-prepared monoclonal positive hybridoma cells; 10 days after injection, ascites fluid was aspirated, centrifuged at 8000 rpm and 4℃ for 20 min to remove grease, and the middle layer of ascites fluid was collected and stored at -80℃ for later use; mouse IgG was purified using a protein A column to obtain high-purity monoclonal antibody 1B7, and the purification results were detected by SDS-PAGE. The results are as follows: Figure 5 As shown, the purified band is single and correctly positioned, indicating that a high-purity monoclonal antibody 1B7 has been obtained.

[0054] Affinity assay: The affinity of monoclonal antibodies is usually expressed using the affinity constant "Ka" and is determined by ELISA. The procedure is as follows: Antigens at different dilutions are coated onto the plate, i.e., one plate each at concentrations of 1 μg / mL and 2 μg / mL. The purified monoclonal antibody is used as the primary antibody, serially diluted starting at 1:1000. The secondary antibody is HRP-labeled goat anti-mouse antibody (1:5000 dilution). OD values ​​are read at different coating concentrations. 450 value.

[0055] The horizontal axis represents antibody concentration, and the vertical axis represents OD. 450 The OD value at which the reaction curve flattens is taken as the 100% reading; then the reciprocal of the monoclonal antibody concentration and the OD value are used as the 100% OD value. 450 Plot a double reciprocal curve with the reciprocals of the values ​​as the x and y coordinates, and calculate the OD using the formula. 450 The numerical values ​​correspond to the concentrations of monoclonal antibodies, and their units are converted to (mol / mL). The affinity constant is then calculated using the formula.

[0056] Kaff=(n-1) / 2(n[Ab']t-[Ab]t); n=[Ag]t / [Ag']t [Ag]t corresponds to a high coating concentration, and [Ag']t corresponds to a low coating concentration; [Ab']t and [Ab]t represent 50% OD. 450 Plot a double reciprocal curve based on the corresponding molar concentration of the monoclonal antibody. Figure 6 The affinity constant of monoclonal antibody 1B7 was found to be 2.9 × 10⁻⁶. 9 L / mol.

[0057] Example 4: Preparation of varicella-zoster virus gL antibody quantum dot test strip 1. Preparation of quantum dot-labeled VZV gL recombinant protein (1) Mix 1.25 μL of carboxylated water-soluble quantum dots with a concentration of 8 μmol / L with 4.8 μL of EDC solution with a concentration of 1.0 mg / mL, vortex mix, and then react in a shaker at 220 rpm for 30 min in the dark at 25℃.

[0058] (2) Take 5 μL of VZV gL recombinant protein (protein concentration of 0.4 mg / mL) and add it to a centrifuge tube, then add 8.95 μL of borate buffer (0.01 mol / L, pH 8.0), and continue to react in the dark at 220 rpm in a shaker at 25℃ for 2.5 h.

[0059] (3) After centrifuging at 4000 rpm for 5 min, take the supernatant to obtain the conjugated product (quantum dot labeled VZV gL recombinant protein) and store it at 4℃ in the dark for later use.

[0060] 2. Preparation of varicella-zoster virus gL antibody quantum dot test strips The structure of quantum dot test strips is as follows: Figure 7 As shown, the device includes a base plate, a conjugate pad, a sample pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. The NC membrane is attached to the base plate, and the absorbent pad and the conjugate pad are tightly pressed against both ends of the NC membrane. The sample pad is tightly pressed against the end of the conjugate pad furthest from the NC membrane. From the end closest to the conjugate pad to the end furthest from the conjugate pad, a detection line (T line) and a control line (C line) are sequentially arranged on the NC membrane. The detection line is coated with Staphylococcus aureus protein A (SPA), and the control line is coated with anti-VZV gL protein monoclonal antibody 1B7. The conjugate pad is coated with quantum dot-labeled VZV gL recombinant protein.

[0061] The specific assembly process is as follows: (1) Preparation of the conjugate pad Cut glass wool (commercially available glass fiber cotton, 30 cm × 25 cm in size, with a clean, smooth, and foreign-free surface) into strips of 0.75 cm × 30 cm, place them on the XYZ 3060 spray dot apparatus platform, and fix them with pressure strips; spray evenly with conjugate pad buffer, place in a 42℃ drying oven for 50 min, and remove for later use; take 4.5 mL of quantum dot-labeled VZV gL recombinant protein, and use an Airjet Quanti 3060 to spray the quantum dot-labeled gL recombinant protein solution onto the glass wool at a rate of 8 μL / cm; place in a 42℃ drying oven for 50 min; finally, add desiccant to the conjugate pad and store it in a sealed container at room temperature (15~25℃) for later use.

[0062] The binding pad buffer is a 20 mmol / L Na2B4O7 buffer containing 1% BSA, 0.1% Tween 20 (v / v), 3% (w / v) sucrose and 0.1% NaN3.

[0063] (2) Preparation of sample pad Cut the glass cellulose membrane into strips of 1.5cm × 30cm and soak them in the sample pad buffer for 5 min; dry them in a 42℃ drying oven for 4 h; place the sample pad in a plastic bag, add desiccant, and store it in a sealed container at room temperature (15~25℃) for later use.

[0064] The sample pad buffer was a PBS (0.01 mol / L, pH 7.2) solution containing 0.5% Triton-100 (v / v) and 0.1% (w / v) sodium azide.

[0065] (3) Preparation of absorbent pad Cut the absorbent filter paper into strips of 1.9cm × 30cm, add desiccant, and store in a sealed container at room temperature (15~25℃) for later use.

[0066] (4) Preparation of nitrocellulose membrane Purified Staphylococcus aureus protein A (SPA) and anti-VZV gL protein monoclonal antibody 1B7 were sprayed onto nitrocellulose membranes using the BioDot spot spraying platform, serving as the test line (T) and control line (C), respectively. The concentration of the SPA test line was 1.0 mg / mL, and the concentration of the 1B7 control line monoclonal antibody was 1.5 mg / mL. After spraying, the membranes were placed in a drying oven and dried at 37°C for 4 hours.

[0067] (5) Assemble the pretreated conjugate pad, sample pad, absorbent pad and nitrocellulose membrane along the length of the plastic base plate, with each part overlapping by about 2 mm. Finally, cut into test strips with a width of 2.8 mm, put the test strips into a foil bag containing desiccant, and dry and store at room temperature.

[0068] 3. Method for interpreting test strip results Add the diluted sample to the sample pad and determine the results after 10 minutes. Observe under UV light. If no fluorescent band appears on the C line, the test strip is invalid. If a fluorescent band appears on the C line, the test strip is valid. If the test strip is valid, and no fluorescent band appears on the T line, it indicates that there are no varicella-zoster virus antibodies in the sample, and the result is negative. If a fluorescent band appears on the T line, it indicates that there are varicella-zoster virus antibodies in the sample. The immune complex formed by the quantum dot-labeled gL recombinant protein and the antibodies in the sample will be captured by the SPA on the T line, and the result is positive.

[0069] 4. Performance determination of quantum dot test strips (1) Sensitivity detection: VZV antibodies were detected using the prepared quantum dot test strips. VZV antibody-positive serum was diluted 10-fold (1:10). 2 1:10 3、 1:104 1:10 5 1:10 6 1:10 7 The diluted samples were then tested separately. The results are as follows: Figure 8 As shown, when the serum dilution reaches 1:10 6 Even after dilution, the C and T fluorescent bands on the test strip are still clearly visible. This indicates that the limit of detection for this test strip is 1:10 at a serum dilution. 6 It has high sensitivity.

[0070] (2) Specificity detection: The prepared quantum dot test strip was used to detect serum positive for pseudorabies virus (PRV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) antibodies. At the same time, VZV antibody positive serum was set as a positive control to evaluate the specificity of the quantum dot test strip.

[0071] The results are as follows Figure 9 As shown, from left to right, the antibodies are: VZV, PRV, HSV-1, and HSV-2. Only the VZV group has a fluorescent band on the T line; the other T lines do not have fluorescent bands. This indicates that the test strip has good specificity and no cross-reaction with other common herpesvirus antibodies.

[0072] (3) Stability test: The prepared test strips were stored at room temperature in the dark and dry environment, and the fluorescence signal intensity was measured on day 1, day 7, day 14, day 21 and day 35. The results showed that after 35 days of storage, the fluorescence intensity values ​​of the test line and the control line of the test strip did not decrease significantly compared with the initial measured values, indicating that the test strip has good stability.

[0073] Example 5: Clinical Sample Validation The test strip prepared in Example 4 was used to test 22 samples (5 clinical samples, 7 serially diluted VZV standard references, and 10 physiological saline samples) in parallel with a commercial VZV IgG ELISA kit. The test results are shown in Table 3. The concordance rate between the two methods was 95.5% (21 / 22), indicating that the test strip has good clinical accuracy.

[0074] Table 3. Comparison of detection results between quantum dot antibody test strips and commercial VZV IgG ELISA kits The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody against varicella-zoster virus gL protein, characterized in that, This includes CDR-H1 with the amino acid sequence shown in SEQ ID NO. 9, CDR-H2 with the amino acid sequence shown in SEQ ID NO. 11, CDR-H3 with the amino acid sequence shown in SEQ ID NO. 13, CDR-L1 with the amino acid sequence shown in SEQ ID NO. 16, CDR-L2 with the amino acid sequence LVS, and CDR-L3 with the amino acid sequence shown in SEQ ID NO.

19.

2. The varicella-zoster virus gL protein monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the varicella-zoster virus gL protein monoclonal antibody is shown in SEQ ID NO.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

3. The encoding gene of a monoclonal antibody against varicella-zoster virus gL protein as described in claim 1 or 2.

4. The encoding gene according to claim 3, characterized in that, The coding genes include light chain variable region coding genes and heavy chain variable region coding genes; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.4; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.

6.

5. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 3 or 4.

6. A recombinant non-plant host cell, characterized in that, Includes the recombinant expression vector as described in claim 5.

7. The use of the encoding gene as described in claim 3 or 4, the recombinant expression vector as described in claim 5, or the recombinant non-plant host cell as described in claim 6 in the preparation of the varicella-zoster virus gL protein monoclonal antibody as described in claim 1.

8. The use of a monoclonal antibody against varicella-zoster virus gL protein as described in claim 1 or 2 in the preparation of a product for detecting varicella-zoster virus gL protein or varicella-zoster virus gL antibody.

9. A product for detecting varicella-zoster virus gL protein or varicella-zoster virus gL antibody, characterized in that, Including the varicella-zoster virus gL protein monoclonal antibody as described in claim 1 or 2.

10. The product according to claim 9, characterized in that, The product for detecting varicella-zoster virus gL antibodies is a quantum dot test strip. The quantum dot test strip includes a base plate, a conjugate pad, a sample pad, a nitrocellulose membrane, and an absorbent pad; The nitrocellulose membrane is attached to the base plate, the absorbent pad and the binding pad are respectively tightly pressed against both ends of the nitrocellulose membrane, and the sample pad is tightly pressed against the end of the binding pad away from the nitrocellulose membrane; from the end near the binding pad to the end away from the binding pad, the nitrocellulose membrane is provided with detection lines and quality control lines in sequence; The detection line is coated with Staphylococcus aureus protein A; the control line is coated with the varicella-zoster virus gL protein monoclonal antibody as described in claim 1 or 2; and the binding pad is coated with quantum dot-labeled varicella-zoster virus gL protein.

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