CN114324878A - Fluorescence-labeled varicella-zoster virus immunochromatography detection test paper and application thereof - Google Patents

Abstract

The invention provides immunochromatography detection test paper for a varicella-zoster virus with a fluorescent marker and application thereof. The test paper comprises a supporting base plate and an adsorption layer fixed on the supporting base plate, wherein the adsorption layer comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad in sequence from a testing end; the nitrocellulose membrane contains a detection line T and a quality control line print; the monoclonal antibody 7E12 marked on the conjugate pad is quantum dot marked; the detection line T is marked by monoclonal antibody 5B 10; the control line marks the SPA. Wherein monoclonal antibody 7E12 and monoclonal antibody 5B10 were prepared using gE protein as antigen. The glycoprotein gE monoclonal antibody can specifically recognize anti-varicella-zoster virus glycoprotein gE protein and varicella-zoster virus, and can be used for detecting varicella-zoster virus glycoprotein gE and varicella-zoster virus.

Description

Fluorescence-labeled varicella-zoster virus immunochromatography detection test paper and application thereof

Technical Field

The invention relates to fluorescence-labeled chicken pox-herpes zoster virus immunochromatography test paper and application thereof, belonging to the technical field of biological detection.

Background

Varicella-zoster virus (VZV) is called varicella-zoster virus, which is a virus that causes varicella in children after primary infection, remains latent in the body after recovery, and recurs after adults in a small number of patients. Varicella is a common disease with high infectivity for children, which is better at 2-6 years old, the infection source is mainly patients, and the varicella content and respiratory tract secretion in the acute stage of the patients contain viruses.

In recent years, researches at home and abroad find that varicella clinical complications are increased obviously more than ever, and the varicella becomes a disease of the leading case of the incidence rate of children infectious diseases in China increasingly and becomes one of infectious diseases with more prominent problems in relevant emergent public health incidents of schools. Therefore, schools and young holding institutions also become sites for varicella group epidemic diseases, and viruses are easy to spread rapidly through air and contact. And the detection result is accurately provided in time, and the method has great significance for quickly controlling the epidemic situation. In addition, the main component of the existing vaccines for preventing varicella and herpes zoster is the varicella attenuated live virus OKA strain, and in the research and development and production processes of the vaccines, the virus titer is taken as a main quality control project and needs to be detected for many times in multiple steps.

The conventional method (pharmacopoeia method) for detecting the VZV virus is a plaque method at present, but the method has long detection period, high detection requirement and complicated operation, and is not suitable for being developed in a basic laboratory; the method for detecting the VZV virus titer by adopting a PCR amplification method can also be adopted, but the method has higher requirements on detection equipment. Therefore, the development of the VZV antigen detection method which can be fast, accurate, sensitive, specific, low in cost and low in requirements for operator operation experience has great significance for fast diagnosis of VZV virus infection and quality control in vaccine development and production processes.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an immunochromatography test paper for detecting varicella-zoster virus by fluorescence labeling and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a fluorescence-labeled varicella-zoster virus immunochromatography test paper comprises a supporting base plate and an adsorption layer fixed on the supporting base plate, wherein the adsorption layer comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad in sequence from a testing end; the nitrocellulose membrane contains a detection line T and a quality control line print; the monoclonal antibody 7E12 marked on the conjugate pad is quantum dot marked; the detection line T is marked by monoclonal antibody 5B 10; the control line marks the SPA.

The amino acid sequence of the B cell epitope recognized by the monoclonal antibody 7E12 is shown in SEQ ID NO. 7; the amino acid sequence of the B cell epitope recognized by the monoclonal antibody 5B10 is shown in SEQ ID NO. 9.

The monoclonal antibody 7E12 and monoclonal antibody 5B10 were prepared using the gE protein as an antigen.

The preparation method of the gE protein comprises the following steps:

(1) optimizing gE protein signal peptide and gE protein extracellular region sequence into CHO preferred codon, and synthesizing optimized gE gene sequence; the nucleotide sequence of the optimized gE gene is shown as SEQ ID NO: 13 is shown in the figure;

(2) constructing a eukaryotic recombinant lentivirus expression vector pLVX-gE-IRES-ZsGreen1, which is called pLVX-gE for short, wherein the nucleotide sequence of the pLVX-gE is shown in SEQ ID NO. 14;

(3) co-transfecting 293T cells with pLVX-gE, a lentivirus packaging plasmid PSPAX2 and an envelope plasmid PMD2.G, collecting lentivirus suspension, transducing CHO cells, and screening CHO-gE positive cells;

(4) performing enlarged culture on CHO-gE positive cells, adding an SMS CHO-SUPI culture medium additive solution, and inducing and expressing gE protein of varicella-zoster virus;

(5) the culture supernatant of the gE protein was collected and purified.

The immunochromatography test paper is applied to antigen detection of varicella-zoster virus.

The invention has the beneficial effects that:

the anti-varicella-zoster virus glycoprotein gE monoclonal antibody provided by the invention utilizes a lentivirus expression system to express gE protein in CHO cells, and VZV positive serum can specifically recognize the gE protein. The gE monoclonal antibody is prepared by taking the purified gE protein as immunogen and immunizing BALB/c mice by an immunological method, and can specifically recognize and combine with the gE protein.

The gE7E12mAb was identified by the overlapping polypeptide method to recognize B cell epitope P1, while the gE5B10mAb recognized B cell epitope P3. According to the principle of double-antibody sandwich antigen detection, the quantum dot labeled 7E12mAb and 5B10mAb are used to prepare the fluorescence immunochromatographic test paper for detecting VZV antigen, and the test paper can be used for quickly, accurately, sensitively and specifically detecting the VZV antigen, thereby providing guarantee for quick diagnosis of VZV virus infection.

The monoclonal antibody against varicella-zoster virus glycoprotein gE can specifically recognize varicella-zoster virus glycoprotein gE protein and varicella-zoster virus, and can be used for detecting varicella-zoster virus glycoprotein gE and varicella-zoster virus. In addition, in the process of developing VZV vaccine, the test paper provides the most effective means for accurately controlling the content of virus antigen, guiding vaccine development, controlling vaccine production and guaranteeing vaccine release.

Drawings

FIG. 1 SDS-PAGE identification of the purified gE protein of example 1 of the present invention;

wherein, M: a protein Marker; 1-2: purified gE protein;

FIG. 2 shows the Western-blot identification result of the gE protein purified in example 1 of the present invention;

wherein, M: a protein Marker; 1-2: purified gE protein;

FIG. 3IFA identification result of example 3 of the present invention;

wherein, PC, positive control; NC, negative control; BC, blank control; the original image shows fluorescence signals, and the effect is not obvious after the original image is changed into a black and white image.

FIG. 4 is a schematic view of a test strip of example 5 of the present invention;

wherein, 1, a sample pad; 2, a bonding pad; 3, a nitrocellulose membrane; 4, a water absorption pad; 5, detecting a line; 6, quality control line; and 7, supporting the bottom plate.

Detailed Description

The following examples are given to illustrate specific embodiments of the present invention in further detail, but the scope of the present invention is not limited thereto; the instruments and equipment involved in the following examples are conventional instruments and equipment unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the test methods involved are conventional methods unless otherwise specified. Example 1 expression and purification of VZV gE protein

VZV glycoprotein gE is the most abundant in the viral envelope, is the main antigen of the virus, and is also the main candidate antigen for preparing virus subunit vaccine and detection reagent. The expression of VZV gE protein and the preparation of the monoclonal antibody thereof have important significance for the research and development of subunit vaccines of herpes zoster and detection reagents thereof. Mammalian cells have the ability to facilitate proper folding and post-translational modification of proteins, including glycosylation patterns found in human proteins, and are the most widespread host for the manufacture of complex biopharmaceuticals. Thus, the present invention selects for expression of gE glycoprotein in CHO cells. Specifically, the preparation of gE glycoprotein comprises the following steps:

1. screening of CHO-gE cells:

referring to sequence information of Dumas standard strains published on GenBank, firstly, a gE protein signal peptide and a gE protein extracellular region sequence are optimized into CHO preferred codons, a target gene sequence after optimization of the gE protein is shown as SEQ ID NO.13, then, the optimized target gene is cloned into a pLVX-IRES-ZsGreen1 lentiviral vector, and a eukaryotic recombinant lentiviral expression vector pLVX-gE-IRES-ZsGreen1, which is called as pLVX-gE for short, and a gene sequence of the pLVX-gE is shown as SEQ ID NO. 14. Co-transfecting the pLVX-gE, the PSPAX2 plasmid and the PMD2.G plasmid into 293T cells to package lentiviruses, collecting lentivirus suspension after transfection for 48 hours, transducing CHO cells, and screening CHO-gE positive cells with high green fluorescence expression quantity through BD FACS Aria III.

2. Expression and purification of gE protein

(1) Mass culture of cells: CHO-gE positive cells were inoculated into 50mL shake flasks, cultured at 120rpm, and the growth density of the cells was determined by cell counting. Expanding cells in a 50mL shake flask into a 500mL shake flask for high-density culture when the cell culture density is kept unchanged, counting the cells every day, starting to add an SMS CHO-SUPI culture medium additive solution when the cell density is more than 1.5 times of the initial density, adding 1.5% (v/v%) of the SMS CHO-SUPI culture medium additive solution every day, stopping adding the SMS CHO-SUPI culture medium additive solution when the cell density is kept unchanged or the death rate of the cells starts to increase, collecting cell suspension, and centrifuging to collect supernatant liquid;

(2) the culture supernatant expressing the gE protein was collected and centrifuged at 12000r/min for 10 min. The centrifuged supernatant was filtered through a 0.45 μm filter head and then placed in an ice box for further use.

(3) Sucking 2mL of Q-SepgEroseFF anion exchange column filler by a liquid shifter, adding the filler to the installed protein purification column, and carefully adding an upper gasket after the filler is settled; open the control valve to allow 20% (v/v%) ethanol to flow out and continue to rinse the column with 10mL of deionized water;

(4) adjusting a control valve of a protein purification device to stabilize the flow rate, and flushing the column with 10mL or more of balance buffer solution at a speed of 1mL/min after the flow rate is adjusted;

(5) and (3) dropwise adding the cell culture supernatant filtered by the filter membrane into the well-balanced column in batches, 5mL each time, adjusting the control valve to ensure that the flow rate does not exceed 1mL/min, quickly collecting the filtrate and repeatedly loading the filtrate for 2-3 times, collecting the filtrate of the last time, and standing at-20 ℃ for later use.

(6) Washing the column with washing buffer solutions containing NaCl of different concentrations to remove foreign proteins;

(7) finally, 10mL of elution buffer solution containing 500mM NaCl is added into the column to elute the target protein; adjusting the control valve to reduce the speed of liquid flowing through as much as possible; collecting the eluted liquid into 1.5mL of EP tubes, wherein each tube contains 1mL of EP tubes, measuring the concentration of protein in each tube by using an enzyme-labeling instrument, marking the protein to obtain purified gE protein, and placing the gE protein at the temperature of-20 ℃ for later use;

(8) after the purification was completed, the column was washed with 20mL of ddw, then washed again with NaOH solution (1mol/L), washed again with ddw, and finally stored in 20% ethanol at 4 ℃ in a refrigerator.

(9) 5 XLoading Buffer was added to the eluate collected after purification, boiled for 10min, and the purified result was analyzed by 12% SDS-PAGE (shown in FIG. 1), and then Western-blot identification was performed on the gE protein after purification using VZV positive serum (shown in FIG. 2).

EXAMPLE 2 preparation of monoclonal antibodies

1. Animal immunization

(1) Adding Freund's complete adjuvant into immunogen gE protein (gE protein purified in example 1), and emulsifying for first immunization;

(2) immunizing 2 female BALB/c mice of 4-8 weeks old by a back subcutaneous multipoint injection method, wherein the immunization dose is 10 mu g/mouse;

(3) BALB/c mice were boosted with Freund's incomplete adjuvant emulsified with the immunizing antigen (gE protein purified in example 1) every 2 weeks for 4 total immunizations in the same manner and dose;

(4) after 4-immunization, tail vein blood collection is carried out to determine the titer of a specific antibody aiming at the gE protein, a mouse with higher titer is selected, the BALB/c mouse is subjected to super-strong immunity by using immunogen without adjuvant through a tail vein injection method 3-4 days before cell fusion, and the immunization dose is 20 mu g/mouse.

2. Cell fusion and monoclonal antibody preparation

The method of polyethylene glycol is adopted, and the spleen cells of the immunized mice and the mouse myeloma cells SP2/0 are mixed according to the cell number of 8: 1, and screening the fused cells by using HAT selective medium; 12 days after the fusion, gE protein is used as a coating antigen, and positive hybridoma cells are primarily screened by an indirect ELISA method;

the indirect ELISA method comprises the following specific steps:

(1) diluting the purified gE protein into a coating solution with the concentration of 3 mu g/mL by using CBS solution to coat the ELISA plate, incubating for 2h at 37 ℃ in a 100 mu L/hole manner;

(2) discarding the coating solution, washing the plate with PBST, drying, sealing the ELISA plate with 5% (w/v%) skimmed milk, incubating at 37 deg.C for 2h at 200 μ L/well;

(3) discarding the blocking solution, washing the plate with PBST, drying, diluting hybridoma supernatant (primary antibody) with 5% (w/v%) skimmed milk 2 times, sequentially adding into enzyme-labeled plate at 100 μ L/hole with VZV positive serum as positive control, and incubating at 37 deg.C for 30 min;

(4) discarding the primary antibody, washing the plate by PBST, cleaning and drying;

(5) adding diluted goat anti-mouse IgG (secondary antibody) marked by HRP into a reaction hole, incubating for 30min at 37 ℃ at a concentration of 100 mu L/hole;

(6) discarding the secondary antibody, washing with PBST, and patting dry;

(7) adding 100 mu L of TMB color developing solution prepared in situ into each hole, and reacting for 5min in a dark room;

(8) add 100. mu.L of 2M H to each well₂SO₄Terminating the reaction;

(9) microplate reader for reading OD of each well₄₅₀The value is obtained.

3. Subcloning of hybridoma cells by limiting dilution method

The positive hybridoma cells were diluted with 1640/10 complete medium to approximately 10cells/mL per wellmu.L of the suspension was added to a 96-well plate pre-plated with 100. mu.L of feeder cells, and the mixture was incubated at 37 ℃ with 5% CO₂Culturing for 6-8 days in an incubator; further screening positive hybridoma cells by an indirect ELISA method; carrying out subcloning for 2-3 times until hybridoma cell strains which stably secrete anti-gE protein monoclonal antibodies are obtained, carrying out expanded culture on the screened positive monoclonals, wherein the cell number is 1-2 multiplied by 10⁶Freezing and storing in a tube.

4. Stability identification of monoclonal hybridoma cell strain

Continuously culturing the established monoclonal hybridoma cell strain for 3 months and repeatedly freezing and storing by liquid nitrogen for resuscitation so as to identify the stability of the hybridoma cell; the results show that the monoclonal hybridoma cell strain has good stability.

5. In vivo induced ascites method for preparing monoclonal antibody

Female BALB/c parturient mice were selected, intraperitoneally injected with 500. mu.L of sterilized paraffin, and one week later, intraperitoneally injected again with the obtained monoclonal hybridoma cells (7E12 and 5B10) in an amount of 2X 10⁵After one week, ascites is extracted after the abdomen of the mouse is enlarged, the supernatant is centrifuged, and the ascites is purified by ammonium caprylate method.

EXAMPLE 3 purification and characterization of antibodies

1. The saturated ammonium sulfate precipitation method is used for purifying the antibody and the operation method is as follows:

(1) 5mL of the monoclonal antibody ascites (obtained in example 2) was added with 5mL of PBS buffer, and 2.5mL of saturated ammonium sulfate solution was added dropwise thereto so that the mixture became a 20% (wt%) final ammonium sulfate solution, and the mixture was stirred while adding, and then allowed to stand for 30 min.

(2)8000r/min, centrifuging for 20min, and discarding the precipitate to remove fibrin.

(3) Adding 12.5mL saturated ammonium sulfate solution into the supernatant, mixing well, standing for 30 min.

(4)8000r/min, centrifuging for 20min, and discarding the supernatant.

(5) The precipitate was dissolved by adding 10mL of PBS buffer, and then 5mL of saturated ammonium sulfate solution was added to make it a 33% (wt%) ammonium sulfate solution, and after mixing well, it was left to stand for 30 min.

(6)8000r/min, centrifuging for 20min, and discarding supernatant to remove albumin.

(7) And (5) repeating the step (5) for 2-3 times.

(8) Dissolving the precipitate with 5ml PBS buffer solution, placing into dialysis bag, dialyzing with PBS buffer solution at 4 deg.C, and changing the solution for 4 times.

(9)8000r/min, centrifuging for 20min, discarding the precipitate to obtain the supernatant, which is purified monoclonal antibody 7E12, measuring the antibody concentration, packaging, and storing at-20 deg.C. The purified monoclonal antibody 5B10 was obtained in the same manner and stored at-20 ℃.

2. Monoclonal antibody potency assay

The indirect ELISA assay was performed with reference to example 2, with a slight difference in primary antibody: diluting the purified monoclonal antibody by PBS from 1:1000 in a multiple ratio, sequentially adding the diluted monoclonal antibody into an enzyme label plate at 100 mu L/hole, taking VZV positive serum as a positive control, and incubating for 30min at 37 ℃; other steps are carried out according to example 2, and ELISA detection results show that the titers of the monoclonal antibodies 7E12 and 5B10 can reach 1: 2.56X 10⁵。

3. Results of IFA identification of reactivity of monoclonal antibody with gE protein

The gE gene is connected to a pTrip-IRES-puro vector in full length to construct a pTrip-gE-IRES-puro recombinant vector, HEK293T cells are transduced, the recombinant vector is respectively incubated with monoclonal antibody 7E12 and monoclonal antibody 5B10, and a goat anti-mouse IgG antibody combined with FITC (fluorescein isothiocyanate) is added. All nuclei were stained blue with DAPI staining solution. Wherein, fluorescence signals were detected in all wells incubated with mab 7E12 and mab 5B10, and no fluorescence signals were detected in the Negative Control (NC) and Blank Control (BC) wells, as shown in fig. 3. IFA results prove that the monoclonal antibody 7E12, the monoclonal antibody 5B10 screened by the experiment have better reactivity with the VZV gE protein transiently expressed by 293T cells.

4. Blocking ELISA for identifying reactivity of monoclonal antibody and gE protein

Coating the purified gE protein in an ELISA plate, sealing according to an indirect ELISA step, and washing the plate; in blocking ELISA, a VZV positive serum sample is diluted by PBS buffer solution, added into the ELISA plate, and incubated for 30min at 37 ℃; then 2 purified monoclonal antibodies were usedDiluting the bodies (7E12 and 5B10) with PBS buffer solution, adding the diluted bodies into an enzyme label plate, and incubating for 30min at 37 ℃; finally adding goat anti-mouse IgG/HRP secondary antibody diluted by 1:5000, and incubating for 30min at 37 ℃; adding a color developing solution, developing for 5-10min, and adding 2mol/L H₂SO₄Stopping the reaction; while the negative sera were processed, the absorbance values at 450nm were read, the data recorded and analyzed.

And (4) judging a result: blocking rate (percent inhibition/PI) (negative serum OD)₄₅₀Value-serum OD to be detected₄₅₀Value)/negative serum OD₄₅₀Value X100%. The results showed that the mean OD values of all monoclonal antibodies obtained from the negative and positive sera were significantly different, with the PI values of monoclonal antibodies 7E12 and 5B10 both being greater than 80%, indicating that VZV positive sera were able to block the binding of monoclonal antibodies to gE protein.

Example 4 identification of epitope recognized by monoclonal antibody

1. Design of overlapping polypeptides

The overlapping polypeptide method is adopted to pronucleus express 6 segments of polypeptide, covering the whole extracellular region, and the adjacent polypeptide overlaps 50 amino acids. Firstly, dividing the gene full length of gE protein into six segments, wherein the gene sequences are shown as SEQ ID NO.1-6, respectively constructing prokaryotic expression vectors PET28a-P1, PET28a-P2, PET28a-P3, PET28a-P4, PET28a-P5 and PET28a-P6, transforming DH5 alpha competent cells, respectively selecting positive clone bacteria, extracting recombinant plasmids, then transforming BL21 competent cells, respectively selecting positive expression bacteria, respectively expressing overlapping polypeptides P1-P6 under the induction of IPTG, and the amino acid sequences are shown as SEQ ID NO. 7-12.

(1)SEQ ID NO.1

GGAACAGTGAATAAGCCTGTGGTGGGCGTGCTGATGGGCTTTGGCATCATCACAGGCACACTGAGAATCACAAACCCTGTGAGAGCCTCTGTGCTGAGATATGATGATTTTCACATCGATGAGGATAAGCTGGACACAAACTCTGTGTACGAGCCTTACTACCACTCTGATCACGCTGAGTCTAGCTGGGTGAATAGAGGAGAATCTTCTAGAAAGGCTTATGATCATAACTCTCCTTACATCTGGCCTAGAAACGATTACGATGGATTCCTGGAGAACGCCCACGAGCACCACGGC

(2)SEQ ID NO.2

TACGAGCCTTACTACCACTCTGATCACGCTGAGTCTAGCTGGGTGAATAGAGGAGAATCTTCTAGAAAGGCTTATGATCATAACTCTCCTTACATCTGGCCTAGAAACGATTACGATGGATTCCTGGAGAACGCCCACGAGCACCACGGCGTGTACAACCAGGGTAGAGGCATCGACAGCGGCGAGAGACTGATGCAGCCTACACAGATGTCTGCTCAGGAGGATCTGGGCGATGACACCGGCATTCACGTGATCCCAACACTGAACGGCGATGATAGACACAAGATCGTGAACGTGGACCAGAGACAGTACGGCGATGTGTTTAAGGGAGACCTGAACCCTAAGCCTCAGGGACAGAGACTGATTGAGGTGAGCGTGGAGGAGAACCACCCATTCACACTGAGAGCCCCTATCCAGAGGATCTACGGCGTGAGATACACTGAGACCTGG

(3)SEQ ID NO.3

CAGAGACAGTACGGCGATGTGTTTAAGGGAGACCTGAACCCTAAGCCTCAGGGACAGAGACTGATTGAGGTGAGCGTGGAGGAGAACCACCCATTCACACTGAGAGCCCCTATCCAGAGGATCTACGGCGTGAGATACACTGAGACCTGGTCTTTCCTGCCTTCTCTGACCTGTACAGGCGACGCTGCTCCTGCTATCCAGCACATCTGTCTGAAGCACACAACATGTTTTCAGGATGTGGTGGTGGATGTGGATTGTGCCGAGAACACCAAGGAAGATCAGCTGGCTGAGATCTCTTACAGATTTCAGGGAAAGAAGGAGGCCGATCAGCCTTGGATCGTGGTGAACACCTCTACCCTGTTCGATGAGCTGGAGCTGGATCCTCCTGAAATCGAGCCAGGCGTGCTGAAGGTGCTGAGAACTGAGAAGCAGTATCTGGGCGTGTACATC

(4)SEQ ID NO.4

AGATTTCAGGGAAAGAAGGAGGCCGATCAGCCTTGGATCGTGGTGAACACCTCTACCCTGTTCGATGAGCTGGAGCTGGATCCTCCTGAAATCGAGCCAGGCGTGCTGAAGGTGCTGAGAACTGAGAAGCAGTATCTGGGCGTGTACATCTGGAACATGAGAGGTTCTGACGGCACTAGCACATACGCCACCTTTCTGGTGACCTGGAAGGGCGATGAAAAGACCAGGAATCCTACACCAGCTGTGACACCTCAGCCTAGAGGCGCCGAGTTTCACATGTGGAACTATCACTCTCACGTGTTCTCTGTGGGCGATACCTTCTCTCTGGCCATGCACCTGCAGTATAAGATCCACGAGGCCCCTTTCGACCTGCTGCTGGAGTGGCTGTACGTGCCTATCGACCCTACCTGTCAGCCTATGAGACTGTACAGCACCTGTCTGTACCACCCT

(5)SEQ ID NO.5

TTCTCTGTGGGCGATACCTTCTCTCTGGCCATGCACCTGCAGTATAAGATCCACGAGGCCCCTTTCGACCTGCTGCTGGAGTGGCTGTACGTGCCTATCGACCCTACCTGTCAGCCTATGAGACTGTACAGCACCTGTCTGTACCACCCTAACGCCCCCCAGTGTCTGAGCCACATGAACAGCGGCTGTACTTTCACAAGCCCTCATCTGGCCCAGAGAGTGGCCAGCACCGTGTACCAGAACTGTGAGCACGCCGATAATTACACAGCCTATTGCCTGGGCATCAGCCACATGGAACCTAGCTTTGGCCTGATCCTGCATGACGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGTCTCTGTCTGGACTGTATGTGTTCGTGGTGTACTTTAACGGACACGTGGAGGCTGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTC

(6)SEQ ID NO.6

AGCTTTGGCCTGATCCTGCATGACGGCGGCACAACCCTGAAGTTCGTGGATACCCCTGAGTCTCTGTCTGGACTGTATGTGTTCGTGGTGTACTTTAACGGACACGTGGAGGCTGTGGCCTACACCGTGGTGTCTACCGTGGACCACTTCGTGAACGCCATTGAGGAGAGAGGATTCCCTCCTACAGCTGGCCAGCCTCCTGCTACCACCAAGCCAAAGGAGATCACCCCTGTGAACCCTGGCACCTCTCCTCTGCTGAGATACGCCGCTTGGACCGGCGGCCTGGCC

(7)SEQ ID NO.7

MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHG

(8)SEQ ID NO.8

YEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETW

(9)SEQ ID NO.9

QRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYI

(10)SEQ ID NO.10

RFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHP

(11)SEQ ID NO.11

FSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHF

(12)SEQ ID NO.12

SFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLA

2. Identification of monoclonal antibody recognition epitope

6 sections of polypeptides expressed by pronucleus are used for coating an enzyme label plate, indirect ELISA detection is carried out on the gE monoclonal antibody and the polypeptides, the reactivity of the monoclonal antibody and the polypeptides is identified, and the result shows that the polypeptide P1(SEQ ID NO.7) can be identified by the monoclonal antibody 7E 12; the polypeptide P3(SEQ ID NO.9) is recognized by monoclonal antibody 5B 10.

Example 5 application of monoclonal antibody in preparation of varicella-zoster virus antigen test paper

This embodiment provides a varicella-zoster virus antigen detection reagent, which comprises the anti-varicella-zoster virus gE protein monoclonal antibody provided by the invention.

1. Quantum dot labeled monoclonal antibody 7E12

Activating carboxyl on Quantum Dots (QDs) by adopting an EDC method, and coupling with amino of monoclonal antibody 7E12 to obtain quantum dot-labeled QDs-7E12 mAb.

2. Development of test paper

Spraying QDs-7E12 mAb onto the pretreated conjugate pad; after the NC membrane (the nitrocellulose membrane) is pretreated, 5B10 monoclonal antibody is sprayed on a detection line (T line) of the NC membrane, and SPA (staphylococcal protein A) is sprayed on a quality control line (C line) of the NC membrane; the materials are assembled into a test paper board by using an LM5000 test paper assembler or manual assembly. Firstly, sticking the NC membrane to the center of a supporting base plate, then sticking the bonding pad and the sample pad which are sprayed with QDs-7E12 mAb to the sample end of the NC membrane in sequence, overlapping each layer by 1-2mm, sticking the water absorption pad to the other end of the NC membrane, and overlapping the water absorption pad and the NC membrane by 1-2 mm. Finally, the test paper strips are cut into test paper strips with the width of 4mm by using a strip cutting instrument (as shown in figure 4), dried, sealed and stored.

And (4) interpretation of results: dripping a sample to be detected on the sample pad, irradiating the test paper by using a handheld ultraviolet lamp after 5-10min, and if the C line and the T line are developed, determining that the result is positive; if the C line is colored, the T line is not colored, and the result is negative; if the C line is not colored, the interpretation is invalid.

3. Detecting actual samples

The VZV attenuated live vaccine is used as a detection sample, and the detection result shows that the varicella-zoster virus antigen detection fluorescence immunochromatographic test paper has high sensitivity and specificity, and the detection limit is 2.725 multiplied by 10³TCID₅₀mL, no cross-reaction with other viruses of the Herpesviridae family (HSV-1, HSV-2, CMV, EBV and KSHV).

While the present invention has been described in detail with reference to the drawings and the embodiments, those skilled in the art will understand that various specific parameters in the above embodiments can be changed without departing from the spirit of the present invention, and a plurality of specific embodiments are formed, which are common variation ranges of the present invention, and will not be described in detail herein.

Sequence listing

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tacgagcctt actaccactc tgatcacgct gagtctagct gggtgaatag aggagaatct 60

tctagaaagg cttatgatca taactctcct tacatctggc ctagaaacga ttacgatgga 120

ttcctggaga acgcccacga gcaccacggc gtgtacaacc agggtagagg catcgacagc 180

ggcgagagac tgatgcagcc tacacagatg tctgctcagg aggatctggg cgatgacacc 240

ggcattcacg tgatcccaac actgaacggc gatgatagac acaagatcgt gaacgtggac 300

cagagacagt acggcgatgt gtttaaggga gacctgaacc ctaagcctca gggacagaga 360

ctgattgagg tgagcgtgga ggagaaccac ccattcacac tgagagcccc tatccagagg 420

atctacggcg tgagatacac tgagacctgg 450

<210> 3

<211> 450

<212> DNA

<213> Artificial sequence ()

<400> 3

cagagacagt acggcgatgt gtttaaggga gacctgaacc ctaagcctca gggacagaga 60

ctgattgagg tgagcgtgga ggagaaccac ccattcacac tgagagcccc tatccagagg 120

atctacggcg tgagatacac tgagacctgg tctttcctgc cttctctgac ctgtacaggc 180

gacgctgctc ctgctatcca gcacatctgt ctgaagcaca caacatgttt tcaggatgtg 240

gtggtggatg tggattgtgc cgagaacacc aaggaagatc agctggctga gatctcttac 300

agatttcagg gaaagaagga ggccgatcag ccttggatcg tggtgaacac ctctaccctg 360

ttcgatgagc tggagctgga tcctcctgaa atcgagccag gcgtgctgaa ggtgctgaga 420

actgagaagc agtatctggg cgtgtacatc 450

<210> 4

<211> 450

<212> DNA

<213> Artificial sequence ()

<400> 4

agatttcagg gaaagaagga ggccgatcag ccttggatcg tggtgaacac ctctaccctg 60

ttcgatgagc tggagctgga tcctcctgaa atcgagccag gcgtgctgaa ggtgctgaga 120

actgagaagc agtatctggg cgtgtacatc tggaacatga gaggttctga cggcactagc 180

acatacgcca cctttctggt gacctggaag ggcgatgaaa agaccaggaa tcctacacca 240

gctgtgacac ctcagcctag aggcgccgag tttcacatgt ggaactatca ctctcacgtg 300

ttctctgtgg gcgatacctt ctctctggcc atgcacctgc agtataagat ccacgaggcc 360

cctttcgacc tgctgctgga gtggctgtac gtgcctatcg accctacctg tcagcctatg 420

agactgtaca gcacctgtct gtaccaccct 450

<210> 5

<211> 450

<212> DNA

<213> Artificial sequence ()

<400> 5

ttctctgtgg gcgatacctt ctctctggcc atgcacctgc agtataagat ccacgaggcc 60

cctttcgacc tgctgctgga gtggctgtac gtgcctatcg accctacctg tcagcctatg 120

agactgtaca gcacctgtct gtaccaccct aacgcccccc agtgtctgag ccacatgaac 180

agcggctgta ctttcacaag ccctcatctg gcccagagag tggccagcac cgtgtaccag 240

aactgtgagc acgccgataa ttacacagcc tattgcctgg gcatcagcca catggaacct 300

agctttggcc tgatcctgca tgacggcggc acaaccctga agttcgtgga tacccctgag 360

tctctgtctg gactgtatgt gttcgtggtg tactttaacg gacacgtgga ggctgtggcc 420

tacaccgtgg tgtctaccgt ggaccacttc 450

<210> 6

<211> 288

<212> DNA

<213> Artificial sequence ()

<400> 6

agctttggcc tgatcctgca tgacggcggc acaaccctga agttcgtgga tacccctgag 60

tctctgtctg gactgtatgt gttcgtggtg tactttaacg gacacgtgga ggctgtggcc 120

tacaccgtgg tgtctaccgt ggaccacttc gtgaacgcca ttgaggagag aggattccct 180

cctacagctg gccagcctcc tgctaccacc aagccaaagg agatcacccc tgtgaaccct 240

ggcacctctc ctctgctgag atacgccgct tggaccggcg gcctggcc 288

<210> 7

<211> 100

<212> PRT

<213> Artificial sequence ()

<400> 7

Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly

1 5 10 15

Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val

20 25 30

Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly

100

<210> 8

<211> 150

<212> PRT

<213> Artificial sequence ()

<400> 8

Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp Val Asn

1 5 10 15

Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro Tyr Ile

20 25 30

Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His Glu His

35 40 45

His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu Arg Leu

50 55 60

Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp Asp Thr

65 70 75 80

Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His Lys Ile

85 90 95

Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly Asp Leu

100 105 110

Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val Glu Glu

115 120 125

Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr Gly Val

130 135 140

Arg Tyr Thr Glu Thr Trp

145 150

<210> 9

<211> 150

<212> PRT

<213> Artificial sequence ()

<400> 9

Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly Asp Leu Asn Pro Lys Pro

1 5 10 15

Gln Gly Gln Arg Leu Ile Glu Val Ser Val Glu Glu Asn His Pro Phe

20 25 30

Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr Gly Val Arg Tyr Thr Glu

35 40 45

Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys Thr Gly Asp Ala Ala Pro

50 55 60

Ala Ile Gln His Ile Cys Leu Lys His Thr Thr Cys Phe Gln Asp Val

65 70 75 80

Val Val Asp Val Asp Cys Ala Glu Asn Thr Lys Glu Asp Gln Leu Ala

85 90 95

Glu Ile Ser Tyr Arg Phe Gln Gly Lys Lys Glu Ala Asp Gln Pro Trp

100 105 110

Ile Val Val Asn Thr Ser Thr Leu Phe Asp Glu Leu Glu Leu Asp Pro

115 120 125

Pro Glu Ile Glu Pro Gly Val Leu Lys Val Leu Arg Thr Glu Lys Gln

130 135 140

Tyr Leu Gly Val Tyr Ile

145 150

<210> 10

<211> 150

<212> PRT

<213> Artificial sequence ()

<400> 10

Arg Phe Gln Gly Lys Lys Glu Ala Asp Gln Pro Trp Ile Val Val Asn

1 5 10 15

Thr Ser Thr Leu Phe Asp Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu

20 25 30

Pro Gly Val Leu Lys Val Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val

35 40 45

Tyr Ile Trp Asn Met Arg Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr

50 55 60

Phe Leu Val Thr Trp Lys Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro

65 70 75 80

Ala Val Thr Pro Gln Pro Arg Gly Ala Glu Phe His Met Trp Asn Tyr

85 90 95

His Ser His Val Phe Ser Val Gly Asp Thr Phe Ser Leu Ala Met His

100 105 110

Leu Gln Tyr Lys Ile His Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp

115 120 125

Leu Tyr Val Pro Ile Asp Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser

130 135 140

Thr Cys Leu Tyr His Pro

145 150

<210> 11

<211> 150

<212> PRT

<213> Artificial sequence ()

<400> 11

Phe Ser Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys

1 5 10 15

Ile His Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro

20 25 30

Ile Asp Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr

35 40 45

His Pro Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr

50 55 60

Phe Thr Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln

65 70 75 80

Asn Cys Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser

85 90 95

His Met Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr

100 105 110

Leu Lys Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe

115 120 125

Val Val Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val

130 135 140

Ser Thr Val Asp His Phe

145 150

<210> 12

<211> 96

<212> PRT

<213> Artificial sequence ()

<400> 12

Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys Phe Val

1 5 10 15

Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val Tyr Phe

20 25 30

Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr Val Asp

35 40 45

His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr Ala Gly

50 55 60

Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val Asn Pro

65 70 75 80

Gly Thr Ser Pro Leu Leu Arg Tyr Ala Ala Trp Thr Gly Gly Leu Ala

85 90 95

<210> 13

<211> 1641

<212> DNA

<213> Artificial sequence ()

<400> 13

atgggaacag tgaataagcc tgtggtgggc gtgctgatgg gctttggcat catcacaggc 60

acactgagaa tcacaaaccc tgtgagagcc tctgtgctga gatatgatga ttttcacatc 120

gatgaggata agctggacac aaactctgtg tacgagcctt actaccactc tgatcacgct 180

gagtctagct gggtgaatag aggagaatct tctagaaagg cttatgatca taactctcct 240

tacatctggc ctagaaacga ttacgatgga ttcctggaga acgcccacga gcaccacggc 300

gtgtacaacc agggtagagg catcgacagc ggcgagagac tgatgcagcc tacacagatg 360

tctgctcagg aggatctggg cgatgacacc ggcattcacg tgatcccaac actgaacggc 420

gatgatagac acaagatcgt gaacgtggac cagagacagt acggcgatgt gtttaaggga 480

gacctgaacc ctaagcctca gggacagaga ctgattgagg tgagcgtgga ggagaaccac 540

ccattcacac tgagagcccc tatccagagg atctacggcg tgagatacac tgagacctgg 600

tctttcctgc cttctctgac ctgtacaggc gacgctgctc ctgctatcca gcacatctgt 660

ctgaagcaca caacatgttt tcaggatgtg gtggtggatg tggattgtgc cgagaacacc 720

aaggaagatc agctggctga gatctcttac agatttcagg gaaagaagga ggccgatcag 780

ccttggatcg tggtgaacac ctctaccctg ttcgatgagc tggagctgga tcctcctgaa 840

atcgagccag gcgtgctgaa ggtgctgaga actgagaagc agtatctggg cgtgtacatc 900

tggaacatga gaggttctga cggcactagc acatacgcca cctttctggt gacctggaag 960

ggcgatgaaa agaccaggaa tcctacacca gctgtgacac ctcagcctag aggcgccgag 1020

tttcacatgt ggaactatca ctctcacgtg ttctctgtgg gcgatacctt ctctctggcc 1080

atgcacctgc agtataagat ccacgaggcc cctttcgacc tgctgctgga gtggctgtac 1140

gtgcctatcg accctacctg tcagcctatg agactgtaca gcacctgtct gtaccaccct 1200

aacgcccccc agtgtctgag ccacatgaac agcggctgta ctttcacaag ccctcatctg 1260

gcccagagag tggccagcac cgtgtaccag aactgtgagc acgccgataa ttacacagcc 1320

tattgcctgg gcatcagcca catggaacct agctttggcc tgatcctgca tgacggcggc 1380

acaaccctga agttcgtgga tacccctgag tctctgtctg gactgtatgt gttcgtggtg 1440

tactttaacg gacacgtgga ggctgtggcc tacaccgtgg tgtctaccgt ggaccacttc 1500

gtgaacgcca ttgaggagag aggattccct cctacagctg gccagcctcc tgctaccacc 1560

aagccaaagg agatcacccc tgtgaaccct ggcacctctc ctctgctgag atacgccgct 1620

tggaccggcg gcctggcctg a 1641

<210> 14

<211> 9844

<212> DNA

<213> Artificial sequence ()

<400> 14

tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60

cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120

tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180

ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240

agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300

agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360

ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780

aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840

aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900

caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960

gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020

cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080

ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140

aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200

tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260

aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320

gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380

cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440

gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500

ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560

actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620

gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680

aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740

ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800

tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860

actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920

cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980

cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040

gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100

aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160

acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2220

atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2280

ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2340

aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2400

tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2460

ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2520

agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2580

ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2640

acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2700

gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2760

tccatagaag acaccgactc tactagagga tctatttccg gtgaattcat gggaacagtg 2820

aataagcctg tggtgggcgt gctgatgggc tttggcatca tcacaggcac actgagaatc 2880

acaaaccctg tgagagcctc tgtgctgaga tatgatgatt ttcacatcga tgaggataag 2940

ctggacacaa actctgtgta cgagccttac taccactctg atcacgctga gtctagctgg 3000

gtgaatagag gagaatcttc tagaaaggct tatgatcata actctcctta catctggcct 3060

agaaacgatt acgatggatt cctggagaac gcccacgagc accacggcgt gtacaaccag 3120

ggtagaggca tcgacagcgg cgagagactg atgcagccta cacagatgtc tgctcaggag 3180

gatctgggcg atgacaccgg cattcacgtg atcccaacac tgaacggcga tgatagacac 3240

aagatcgtga acgtggacca gagacagtac ggcgatgtgt ttaagggaga cctgaaccct 3300

aagcctcagg gacagagact gattgaggtg agcgtggagg agaaccaccc attcacactg 3360

agagccccta tccagaggat ctacggcgtg agatacactg agacctggtc tttcctgcct 3420

tctctgacct gtacaggcga cgctgctcct gctatccagc acatctgtct gaagcacaca 3480

acatgttttc aggatgtggt ggtggatgtg gattgtgccg agaacaccaa ggaagatcag 3540

ctggctgaga tctcttacag atttcaggga aagaaggagg ccgatcagcc ttggatcgtg 3600

gtgaacacct ctaccctgtt cgatgagctg gagctggatc ctcctgaaat cgagccaggc 3660

gtgctgaagg tgctgagaac tgagaagcag tatctgggcg tgtacatctg gaacatgaga 3720

ggttctgacg gcactagcac atacgccacc tttctggtga cctggaaggg cgatgaaaag 3780

accaggaatc ctacaccagc tgtgacacct cagcctagag gcgccgagtt tcacatgtgg 3840

aactatcact ctcacgtgtt ctctgtgggc gataccttct ctctggccat gcacctgcag 3900

tataagatcc acgaggcccc tttcgacctg ctgctggagt ggctgtacgt gcctatcgac 3960

cctacctgtc agcctatgag actgtacagc acctgtctgt accaccctaa cgccccccag 4020

tgtctgagcc acatgaacag cggctgtact ttcacaagcc ctcatctggc ccagagagtg 4080

gccagcaccg tgtaccagaa ctgtgagcac gccgataatt acacagccta ttgcctgggc 4140

atcagccaca tggaacctag ctttggcctg atcctgcatg acggcggcac aaccctgaag 4200

ttcgtggata cccctgagtc tctgtctgga ctgtatgtgt tcgtggtgta ctttaacgga 4260

cacgtggagg ctgtggccta caccgtggtg tctaccgtgg accacttcgt gaacgccatt 4320

gaggagagag gattccctcc tacagctggc cagcctcctg ctaccaccaa gccaaaggag 4380

atcacccctg tgaaccctgg cacctctcct ctgctgagat acgccgcttg gaccggcggc 4440

ctggcctgac tcgagactag ttctagagcg gccgcggatc ccgcccctct ccctcccccc 4500

cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt 4560

tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct 4620

tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga 4680

atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga 4740

ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac 4800

gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag 4860

ttgtggaaag agtcaaatgg ctcacctcaa gcgtattcaa caaggggctg aaggatgccc 4920

agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg 4980

tttagtcgag gttaaaaaac gtctaggccc cccgaaccac ggggacgtgg ttttcctttg 5040

aaaaacacga tgataatatg gcccagtcca agcacggcct gaccaaggag atgaccatga 5100

agtaccgcat ggagggctgc gtggacggcc acaagttcgt gatcaccggc gagggcatcg 5160

gctacccctt caagggcaag caggccatca acctgtgcgt ggtggagggc ggccccttgc 5220

ccttcgccga ggacatcttg tccgccgcct tcatgtacgg caaccgcgtg ttcaccgagt 5280

acccccagga catcgtcgac tacttcaaga actcctgccc cgccggctac acctgggacc 5340

gctccttcct gttcgaggac ggcgccgtgt gcatctgcaa cgccgacatc accgtgagcg 5400

tggaggagaa ctgcatgtac cacgagtcca agttctacgg cgtgaacttc cccgccgacg 5460

gccccgtgat gaagaagatg accgacaact gggagccctc ctgcgagaag atcatccccg 5520

tgcccaagca gggcatcttg aagggcgacg tgagcatgta cctgctgctg aaggacggtg 5580

gccgcttgcg ctgccagttc gacaccgtgt acaaggccaa gtccgtgccc cgcaagatgc 5640

ccgactggca cttcatccag cacaagctga cccgcgagga ccgcagcgac gccaagaacc 5700

agaagtggca cctgaccgag cacgccatcg cctccggctc cgccttgccc tgaacgcgtc 5760

tggaacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 5820

gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 5880

tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 5940

gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 6000

cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 6060

ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 6120

cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg 6180

ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 6240

gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 6300

cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga 6360

attaattctg cagtcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag 6420

ctaccaatgc tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag 6480

tcacacctca ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact 6540

ttttaaaaga aaagagggga ctggaagggc taattcactc ccaacgaaga caagatatcc 6600

ttgatctgtg gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag 6660

ggccaggggt cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc 6720

cagataaggt agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc 6780

tgcatgggat ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag 6840

catttcatca cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag 6900

cttgctacaa gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg 6960

gggagtggcg agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct 7020

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 7080

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 7140

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt 7200

agttcatgtc atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt 7260

gagaggcctt gacattgcta gcgtttaccg tcgacctcta gctagagctt ggcgtaatca 7320

tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 7380

gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 7440

gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 7500

atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 7560

actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 7620

gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 7680

cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 7740

ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 7800

ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 7860

ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 7920

agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 7980

cacgaacccc ccgttcagcc cgaccgcgcg ccttatccgg taactatcgt cttgagtcca 8040

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 8100

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 8160

gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 8220

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 8280

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 8340

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 8400

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 8460

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 8520

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 8580

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 8640

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 8700

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 8760

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 8820

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 8880

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 8940

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 9000

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 9060

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 9120

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 9180

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 9240

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 9300

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 9360

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 9420

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg 9480

acggatcggg agatcaactt gtttattgca gcttataatg gttacaaata aagcaatagc 9540

atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa 9600

ctcatcaatg tatcttatca tgtctggatc aactggataa ctcaagctaa ccaaaatcat 9660

cccaaacttc ccaccccata ccctattacc actgccaatt acctgtggtt tcatttactc 9720

taaacctgtg attcctctga attattttca ttttaaagaa attgtatttg ttaaatatgt 9780

actacaaact tagtagtttt taaagaaatt gtatttgtta aatatgtact acaaacttag 9840

tagt 9844

Claims (5)

1. The immunochromatography test paper for the fluorescence-labeled varicella-zoster virus is characterized by comprising a supporting base plate and an adsorption layer fixed on the supporting base plate, wherein the adsorption layer sequentially comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad from a testing end; the nitrocellulose membrane contains a detection line T and a quality control line print; the monoclonal antibody 7E12 marked on the conjugate pad is quantum dot marked; the detection line T is marked by monoclonal antibody 5B 10; the control line marks the SPA.

2. The fluorescence-labeled varicella-zoster virus immunochromatographic detection test paper according to claim 1, characterized in that the amino acid sequence of the B cell epitope recognized by the monoclonal antibody 7E12 is shown in SEQ ID No. 7; the amino acid sequence of the B cell epitope recognized by the monoclonal antibody 5B10 is shown in SEQ ID NO. 9.

3. The fluorescence-labeled varicella-zoster virus immunochromatographic test strip according to claim 1, characterized in that the monoclonal antibody 7E12 and monoclonal antibody 5B10 are prepared using the gE protein as an antigen.

4. The fluorescence-labeled varicella-zoster virus immunochromatographic detection strip according to claim 3, characterized in that the gE protein is prepared by a method comprising the steps of:

(1) optimizing gE protein signal peptide and gE protein extracellular region sequence into CHO preferred codon, and synthesizing optimized gE gene sequence; the nucleotide sequence of the optimized gE gene is shown as SEQ ID NO: 13 is shown in the figure;

(2) constructing a eukaryotic recombinant lentivirus expression vector pLVX-gE-IRES-ZsGreen1, which is called pLVX-gE for short, wherein the nucleotide sequence of the pLVX-gE is shown in SEQ ID NO. 14;

(3) co-transfecting 293T cells with pLVX-gE, a lentivirus packaging plasmid PSPAX2 and an envelope plasmid PMD2.G, collecting lentivirus suspension, transducing CHO cells, and screening CHO-gE positive cells;

(4) performing enlarged culture on CHO-gE positive cells, adding an SMS CHO-SUPI culture medium additive solution, and inducing and expressing gE protein of varicella-zoster virus;

(5) the culture supernatant of the gE protein was collected and purified.

5. The immunochromatographic test strip of claim 1 for use in the detection of varicella-zoster virus antigen.

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