B-cell epitope of <i>Trichinella spiralis </i>cysteine protease inhibitor, hybridoma cell line, monoclonal antibody and uses thereof
11634481 · 2023-04-25
Assignee
Inventors
- Xiaolei Liu (Changchun, CN)
- Mingyuan Liu (Changchun, CN)
- Yong Yang (Changchun, CN)
- Yan Liu (Changchun, CN)
Cpc classification
C07K2317/34
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
G01N33/5308
PHYSICS
C07K16/38
CHEMISTRY; METALLURGY
G01N2333/8139
PHYSICS
International classification
Abstract
The present disclosure relates to the field of immunology, in particular to a B-cell epitope of Trichinella spiralis cysteine protease inhibitor, a hybridoma cell line, a monoclonal antibody and uses thereof. The present disclosure provides a hybridoma cell line that can generate anti-WN10 antibody, and identifies the specific B-cell epitope of WN10 protein recognized by the monoclonal antibody. These are of great significance for the diagnosis of trichinellosis, for the establishment of competitive ELISA for detecting antibodies and sandwich ELSIA for detecting circulating antigens, for the detection of Trichinella spiralis in different hosts and for the development of subunit vaccines.
Claims
1. An antibody or antigen-binding fragment thereof capable of specifically binding to Trichinella spiralis WN10 protein, comprising a CDR-L1, a CDR-L2, and a CDR-L3 in a light chain variable region, and a CHR-H1, a CDR-H2, and a CDR-H3 in a heavy chain variable region, wherein the CDR-L1, CDR-L2 and CDR-L3 have an amino acid sequence set forth in SEQ ID NOs: 17, 18 and 19, respectively, and wherein the CDR-H1, CDR-H2 and CDR-H3 have an amino acid sequence set forth in SEQ ID NOs: 20, 21 and 22, respectively.
2. The antibody or antigen-binding fragment thereof according to claim 1 comprising a light chain variable region with a sequence set forth in SEQ ID NO: 15 and a heavy chain variable region with a sequence set forth in SEQ ID NO: 16.
3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises a constant region sequence of mouse IgG1.
4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment comprises one or more selected from the group consisting of F(ab′).sub.2, Fab′, Fab, Fv and scFv.
5. A kit for detecting Trichinella spiralis comprising the antibody or antigen-binding fragment thereof according to claim 1 and an instruction for use.
6. A hybridoma cell line deposited at China General Microbiological Culture Collection Center (CGMCC) with an accession number of 18316.
Description
BRIEF DESCRIPTION OF DRAWINGS
(1) In order to explain the embodiments of the present disclosure or the technical solutions in the prior art more clearly, the followings are briefly introduction of the drawings used in the description of the embodiments or the prior art.
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DETAILED DESCRIPTION
(11) The present disclosure provides a B-cell epitope of Trichinella spiralis cysteine protease inhibitor, a hybridoma cell line, a monoclonal antibody and uses thereof. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize the present invention. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve the present invention.
(12) One object of the present disclosure is to provide a hybridoma cell line that secretes monoclonal antibody against Ts-WN10 protein.
(13) Another object of the present disclosure is to provide a monoclonal antibody secreted by the aforementioned hybridoma cell line. The monoclonal antibody can specifically react with the Ts-WN10 protein, and can compete with the positive sera of pig infected with Trichinella spiralis to recognize the recombinant Ts-WN10 antigen.
(14) Another object of the present disclosure is to identify a specific B-cell epitope of Ts-WN10 protein. The Ts-WN10 amino acid sequence (without signal peptide) used in the present disclosure is set forth in SEQ IN NO: 13 as
(15) TABLE-US-00001 QILGETTHYGRNDPVMLRNAHEALFSSDLKQESGVFHKLLELEESSTMGI LTTMKVVMQDTDCPVSFALLSYYDVLVNCQGEGRRKHCTMEYTHRNPSKA TVSKCFEEVEEPLIIPQRVKMIGGRAVYIDSNADVEEQMQMLGETTHYGR NDPVMLPKAREALFSSDSKEQSGVLHKLVELEESSTMGILTTMKVVIQDT ECRVSSAYSSYYDVLHYCHGKGPRKHCTLEYRHRTPSTATVSECFEEVEE PLIVPQRVQRVNGRTIYLDSSDDVEEQVVSQRSQMLGGTTKYTDSNVHIK EEVKQAIFESDKKKSSGTYLLLDKIVEGFNMGISSRFQVLVKETECGIKE KAFNSYEDVYKNCSGSGDSK VCSVEYKYFDPTKSTVEC.
(16) In the present disclosure, the total RNA of Trichinella spiralis was extracted using TRIZOL, and then the WN10 cDNA fragment (SEQ IN NO: 14) was cloned by reverse transcription. The cDNA was inserted into the prokaryotic expression vector pET28a, and Ts-WN10 was expressed using the pET28a prokaryotic expression vector. The expressed Ts-WN10 protein in the form of inclusion bodies was isolated and purified, and used as an immunogen to immunize BALB/c mice, and then spleen cells were fused with SP2/0 myeloma cells. In addition, the Ts-WN10 protein was purified by one-step on-column purification using Ni column and AKTA protein purification system. The purified Ts-WN10 was used as the antigen for detection, and also in the indirect ELISA detection to screen positive hybridoma cells. In addition, the positive hybridoma cells were screened again according to the indirect ELISA method recommended in the standard OIE Terrestrial Manual 2017, Chapter 2.1.20-Trichinellosis. Finally, a hybridoma cell line that stably generates monoclonal antibody against Ts-WN10 was obtained.
(17) The present disclosure also provides a monoclonal antibody generated by the aforementioned hybridoma cell line WN10-1H9, which was named WN10-1H9-Ab (1H9 for short). Competitive ELISA test results showed that the monoclonal antibody WN10-1H9-Ab can compete with Trichinella spiralis-infected pig serum to bind Ts-WN10. Western blot results showed that the monoclonal antibody prepared by the present disclosure can specifically bind to the soluble antigen of Trichinella spiralis (T. spiralis) muscle larvae.
(18) The present disclosure uses peptide scanning assay to identify the B-cell epitope recognized by WN10-1H9, and determines that the epitope recognized by WN10-1H9-Ab is VNCQGEGRRKHCTME (as shown in SEQ ID No: 1).
(19) The corresponding nucleotide sequence is
(20) TABLE-US-00002 (SEQ ID No: 2) GTTAATTGTCAAGGAGAAGGCCGACGAAAGCATTGTACAATGGAA.
(21) The present disclosure provides uses of the hybridoma cell line in the detection of Trichinella spiralis infection, and the uses of B-cell epitope of Ts-WN10 in the detection of Trichinella spiralis infection.
(22) In summary, the present disclosure has prepared and identified a monoclonal antibody against Ts-WN10 protein. The monoclonal antibody and the B-cell epitope of Ts-WN10 recognized by the monoclonal antibody can be used to prepare diagnostic reagent for Trichinella spiralis infection, which has laid the foundation for the establishment of a serological diagnosis method for Trichinella spiralis.
(23) Materials
(24) 1. Proteins, Cells, Worms
(25) The Ts-WN10 protein was expressed in prokaryotic cells and purified by gel separation. The Ts-WN10 protein expressed in prokaryotic cells was further purified by one-step on-column purification and refolding. SP2/0 cells was obtained from National Collection of Authenticated Cell Cultures (Cat. TCM18) and Trichinella spiralis (strain ISS534) species is preserved in the lab oratory.
(26) 2. Reagents
(27) Purification column HisTrap™ HP (packed with Ni Sepharose High Performance (HP) affinity resin) was purchased from GE, USA; fetal bovine serum and 1640 medium were purchased from Biological Industries; HAT medium (50×), HT medium (50×) and antibody subclass identification kit were purchased from sigma; soluble TMB substrate solution was purchased from TIANGEN; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody was purchased from Beijing Bioss; pre-stained protein marker was purchased from Fermentas; restriction endonucleases EcoRI and XhoI, reverse transcriptase, Ex Taq DNA polymerase, and T4 DNA ligase were purchased from TaKaRa (Dalian) Co., Ltd; ECL luminescent substrate was purchased from Solarbio Life Sciences (Beijing).
(28) 3. Experimental Animals
(29) BALB/c mice aged 6 weeks were provided by Changchun Yisi Experimental Animal Technology Co., Ltd.
(30) In the following, the present disclosure will be further explained in conjunction with the examples.
EXAMPLES
Example 1 Prokaryotic Expression and Purification of Ts-WN10 Protein
(31) 1. Primer Design
(32) According to the Ts-WN10 gene sequence registered in Genbank (accession number: EU263325), PCR amplification primers were designed. The sequences are as follows:
(33) TABLE-US-00003 TsWN10-EcoRI-atg: 5′-TAACGAATTCATGCAGATACTTGGTGA-3′ (as shown in SEQ ID No: 3); TsWN10-XhoI-tta: 5′-GACGCTCGAGTTAACATTCAACA-3′ (as shown in SEQ ID No: 4).
(34) The underlined parts are the introduced EcoRI and XhoI restriction sites, and the length of the amplified product is expected to be 1187 bp.
(35) 2. RNA Extraction from Trichinella spiralis T1 and Reverse Transcription
(36) The mouse carrying Trichinella spiralis (strain ISS534) was killed by cervical dislocation, and the skin, tail, internal organs and claws were removed. The body was washed and minced, placed in 300 ml of digestion solution containing 1% HCl and 1% pepsin, and stirred and digested in a 37° C. incubator for 2 hours. The digestion solution was filtered with 80-mesh filter to remove the residues, and the filtrate was collected after 2 hours of precipitation in a separatory funnel, about 500 ml. After the filtrate was precipitated for 30 minutes, the upper layer liquid was gently sucked off with a 20 ml syringe, and physiological saline was added for precipitation again, the supernatant was discarded, and the washing was repeated until there was no impurity. The worms were moved to EP tube and centrifuged at 1,000 rpm for 3 minutes to remove the excess liquid. 1 ml Trizol was added to the harvested worms and mixed well, and allowed to stand at room temperature for 5 minutes. 0.2 ml chloroform was added, the mixture was shaken vigorously for 15 s, incubated at room temperature for 2-3 min, and centrifuged at 12,000×g at 4° C. for 15 min. The upper layer was carefully collected, an equal volume of pre-cooled isopropanol was added, mixed, and incubated at room temperature for 10 min. The mixture was centrifuged at 12,000×g at 4° C. for 10 min, and the supernatant was discarded. 1 ml 75% ethanol (prepared with DEPC-treated water) was added to the pellet, gently shaken for 15 seconds, centrifuged at 7,500×g at 4° C. for 5 minutes, and the supernatant was carefully discard. The precipitate was placed at room temperature and dried for 3-5 minutes, 20-30 μl DEPC-treated water was added to dissolve the precipitate, and then stored at −20° C.
(37) The extracted total RNA was used for reverse transcription to synthesize cDNA. The reaction system was as follows:
(38) TABLE-US-00004 M-MLV 5 × Reaction Buffer 5.0 μl dNTP Mixture 1.0 μl M-MLV RT 1.0 μl RNAase inhibitor 0.5 μl oligo dT.sub.18 1.0 μl RNAase-free H.sub.2O Up to 25 μl
(39) The reaction system was mixed well in reacted at 42° C. for 1 hour, and then stored at −20° C.
(40) 3. Construction of pET28a-Ts-WN10 Expression Vector
(41) The Ts-WN10 gene was amplified using the cDNA obtained by reverse transcription as a template. The PCR reaction system (50μl) was as follows:
(42) TABLE-US-00005 10 × Ex Taq Buffer 5.0 μl 10 mM dNTPs 1 μl Ex Taq 0.5 μl 10 pM Forward Primer 2 μl 10 pM Reverse Primer 2 μl cDNA Template 3 μl Sterile ddH.sub.2O 36.5 μl
(43) The reaction conditions: 95° C. pre-denaturation for 5 minutes; 95° C. for 45 s, 53° C. for 45 s, 72° C. for 45 s, 30 cycles; 72° C. final extension for 10 minutes. The amplified product showed a band around 1161 bp in the 1% agarose gel electrophoresis (
(44) The result of cloning experiment for Ts-WN10 gene (
(45) The Ts-WN10 fragment recovered from the gel was subjected to double digestion, and the digestion system was as follows:
(46) TABLE-US-00006 10 × H Buffer 2 μl EcoRI 2 μl XhoI 2 μl WN10 Fragment 10 μl Sterile ddH.sub.2O 4 μl
(47) The prokaryotic expression vector pET28a was subjected to double digestion, and the digestion system was as follows:
(48) TABLE-US-00007 10 × H Buffer 2 μl EcoRI 2 μl XhoI 2 μl pET28a Vector 10 μl Sterile ddH.sub.2O 4 μl
(49) The digestion reaction system was put into a 37° C. water bath for 2 hours, and then subjected to gel purification. The digested WN10 fragment was ligated with pET28a vector, and the reaction system was:
(50) TABLE-US-00008 10 × T.sub.4DNA Ligase Buffer 1 μl Digested WN10 fragment 4 μl Digested pET28a 1.5 μl T.sub.4DNA Ligase 1 μl ddH.sub.2O 2.5 μl
(51) The ligation was carried out at 16° C. overnight. All ligation products were transformed into E. coli DH5α competent cells, and single colonies were picked for double enzyme digestion identification. The positive recombinant plasmid was transformed into BL21 (DE3) competent cells.
(52) 4. Expression and Purification of Ts-WN10
(53) 1 ml of broth containing recombinant bacteria was added to 100 ml of LB medium and cultured at 37° C. with shaking until O.D. 600 nm value was about 0.5-1. IPTG was added to a final concentration of 1 mmol/L, and the culture was induced at 37° C. for 6-8 h. The expressed products were subjected to SDS-PAGE electrophoresis and purified by cutting the gel. An appropriate amount of PBS was added to the gel, and the gel was ground into crushed particles, which can be used to immunize mice.
(54) 5. Induced Expression of Ts-WN10 and One-Step On-Column Purification and Refolding
(55) The broth after induction was centrifuged and resuspended in 30 mL resuspension buffer (20 mM Tris-HCl PH 8.0). The mixture was placed in an ice bath for 10 minutes, and sonicated on ice (3 seconds sonication and 3 seconds interval for a total of 30 minutes). The mixture was centrifuged at 8,000 rpm for 10 min to collect the precipitate, and the precipitate was resuspended in 30 ml of pre-cooled inclusion body washing solution (2M urea, 20 mM Tris-HCl, 0.3M NaCl pH 8.0), put on ice for 10 min, and sonicated on ice (3 seconds sonication and 3 seconds interval for a total of 30 minutes). This step was repeated 3 times. The mixture was centrifuged at 8,000 rpm for 10 minutes to collect the precipitate, and the precipitate was resuspended in 30 ml of pre-cooled PBS washing solution (0.01M PBS containing 4M urea), put on ice for 10 min, and sonicated on ice (3 seconds sonication and 3 seconds interval for a total of 30 minutes). This step was repeated 2 times. The mixture was centrifuged at 8,000 rpm for 10 min to collect the precipitate, and the precipitate was resuspended in 5 ml binding buffer (8M urea, 20 mM Tris-HCl, 0.3M NaCl, 5 mM imidazole pH 8.0) and dissolved overnight at 4° C. The mixture was centrifuged at 8,000 rpm for 30 min to collect the supernatant, and the supernatant was filtered and used for sample loading. Refer to the “Purifying Challenging Proteins” pages 77-79 of manual (GE Healthcare) for the refolding procedure on the AKTA protein purification system. Purification column has 1 ml HisTrap′ HP. After loading the sample, the refolding time on the column was 2 h, and the solution was 100% replaced with Refolding Buffer (20 mM Tris-HCl, 0.3M NaCl, 5 mM imidazole, 1 mM 2-mercaptoethanol pH 8.0) solution. The solution was changed to Elution Buffer (20 mM Tris-HCl, 0.3M NaCl, 500 mM imidazole pH 8.0) to elute the target protein. The above purification experiment was repeated on 4 batches of culture.
(56) The UV absorption spectrum for the Ts-WN10 protein refolding on column showed that after the solution was 100% replaced with the Refolding Buffer, and washed the column with 5 column volumes of the Refolding Buffer, an obvious elution peak appeared. After the solution was exchanged to Elution Buffer, obvious elution peaks also appeared. SDS-PAGE results showed that both of the two peaks were the elution peak of the target protein. In the 4 repeat purification experiments, the SDS-PAGE results showed that a highly uniform soluble target protein was obtained in each experiment.
(57) It can be seen from the results of SDS-PAGE that the supernatant of the purified protein showed only one clear band, and the size was consistent with the theoretical value (
Example 2 Preparation of Anti-Ts-WN10 Monoclonal Antibody
(58) 1. Immunization of Mice
(59) The purified rTs-WN10 protein was used to immunize five 6-week-old female BALB/c mice. The immunization was carried out 5 times. The immunization interval was two weeks. The immunization dose was 50μg/mouse. The immunization route was intraperitoneal immunization.
(60) Blood samples were collected from the tail of the mice one week after the fourth and the fifth immunizations, and the serum was separated (4° C., 3,000 rpm centrifugation for 30 min). Antibody levels were detected by Ts-WN10 and indirect ELISA method. The indirect ELISA was performed as follows: the purified and refolding Ts-WN10 protein was diluted with a coating solution (0.01M NaOH, pH 12), and the coating amount was 0.125μg/well, 100 μl per well. Coating was carried out at 37° C. for 2 h or 4° C. overnight. The plate was washed 3 times with PBST (containing 0.05% Tween 20), and then blocked with a blocking buffer (5% non-fat milk) at 37° C. for 2 hours. The plate was washed 3 times with PBST. The serum to be tested (or hybridoma supernatant in other experiments) was diluted (at a 2-fold ratio) and added to the plate with 100 μl per well. After incubating at 37° C. for 1 hour, the plate was washed 3 times with PB ST. The goat anti-mouse secondary antibody was diluted 1:1000 and incubated with the plate at 37° C. for 30 min. The plate was washed 4 times with PBST. The plate was developed at 37° C. for 10 minutes, and the reaction was terminated with 2M H2504. The absorbance value at O.D. 450 nm was recorded.
(61) Three days before the cell fusion, BALB/c mice with high anti-Ts-WN10 level were boosted, and each mouse was injected with 50 μg immunogen into the abdominal cavity.
(62) 2. Cell Fusion
(63) Feeder cells were prepared one day before fusion. Macrophages from BALB/c mouse were collected according to conventional methods and plated in 96-well cell culture plates for later use. The mice were sacrificed by cervical dislocation, the spleen was taken out under aseptic conditions, and the spleen cells were isolated. 1×10.sup.8 spleen cells were mixed with 2.5×10.sup.7 SP2/0 myeloma cells at a ratio of 4:1. 1 ml PEG1000 was added to the plate for cell fusion, and the dripping was over within 1 min. Then, within 1 min, 1 ml of 1640 basal medium preheated to 37° C. was added dropwise to the cell suspension with stirring. Furthermore, within 3 minutes, 1 ml of 1640 basal medium preheated to 37° C. was added dropwise to the cell suspension with stirring. Finally, 10 ml of 1640 basal medium at 37° C. was slowly added to the cell suspension. The whole process was operated in a 37° C. water bath. The cell suspension was centrifuged at 1,000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in HAT medium. The cells were evenly spread into a 96-well plate pre-plated with feeder cells, and placed in a 37° C., 5% CO2 incubator for culture.
(64) 3. Screening and Cloning of Positive Hybridoma Cell Lines
(65) The Ts-WN10 and indirect ELISA were used to screen positive hybridoma cells, and the positive hybridoma cells were expanded and cultured. The first subcloning of the positive hybridoma cells was performed by the limiting dilution method. Refer to the ES indirect ELISA method described in Chapter 2.1.20-Trichinellosis of OIE Terrestrial Manual 2017, to screen the positive hybridoma cell lines again, to expand the culture of the positive hybridoma cells, and to subclone the positive hybridoma cells by the limiting dilution method. The subcloning was performed at least 3 times, and the subcloned positive hybridoma cell lines were frozen. Finally, a hybridoma cell line that can stably generate anti-Ts-WN10 monoclonal antibody was obtained, which was named WN10-1H9, with a deposit number of CGMCC No. 18316. The antibody generated by WN10-1H9 cell line was named WN10-1H9-Ab (1H9 for short).
(66) WN10-1H9 was subjected to sequencing followed the standard protocol in the art. The amino acid sequence of the light chain variable region of the antibody is
(67) TABLE-US-00009 (SEQ ID NO: 15) DVVVTQSPASLAVSLGQRATISCRASKSVDNYGISFMNWFQQKPGQPPKL LIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQIKEVPY TFGGGTKLEIK;
(68) and the amino acid sequence of the heavy chain variable region of the antibody is
(69) TABLE-US-00010 (SEQ ID NO: 16) QVQLQQPGSELVRPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGN ISPGSGNTNYDEKFKTKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTRHG TVDYWGQGTTVTVSS.
(70) Complementarity-determining regions (CDRs) are shown below:
(71) TABLE-US-00011 CDR-L1: (SEQ ID NO: 17) KSVDNYGISF; CDR-L2: (SEQ ID NO: 18) AAS; CDR-L3: (SEQ ID NO: 19) QQIKEVPYT; CDR-H1: (SEQ ID NO: 20) GYTFTSYW; CDR-H2: (SEQ ID NO: 21) ISPGSGNT; CDR-H3: (SEQ ID NO: 22) TRHGTVDY.
4. Preparation of Ascites Containing Antibodies
(72) Healthy BALB/c mice about 12 weeks of age were intraperitoneally injected with paraffin oil, 0.5 ml/mouse. One week later, the mice were intraperitoneally injected with 1×10.sup.6 hybridoma cells. After 7 to 10 days, when the mouse's belly was extremely big, the ascites was collected every 2 days. The extracted ascites was centrifuged at 10,000 rpm for 10 min to remove the upper layer of grease and sediment, and the supernatant was aliquoted and stored at −20° C.
Example 3 Identification of Monoclonal Antibody
(73) 1. Subclass Identification of Monoclonal Antibody
(74) The monoclonal antibodies obtained in Example 2 was subjected to subclass identification according to the instructions of the antibody subclass identification kit. The results show that the heavy chains of the monoclonal antibodies of the present disclosure are all IgG1, and the light chains are kappa chains. See Table 1 for details.
(75) TABLE-US-00012 TABLE 1 Subclass identification of monoclonal antibody Subclass lgG1 lgG2a lgG2b lgG3 lgM lgA κ chain λ chain OD 450 0.837 0.062 0.045 0.048 0.031 0.046 0.757 0.039 nm
2. Competitive ELISA
(76) Coating and blocking were performed the same as indirect ELISA. Sera of pig infected with or without Trichinella spiralis were diluted at a 2-fold ratio starting from 1:1. After dilution, 50μl of each serum was mixed with 50μl of monoclonal antibody supernatant in equal volume. After mixing, 100 μl of the solution was added to the plate and incubated at 37° C. for 1 h. The following steps were the same as indirect ELISA. The analysis was performed to see whether the monoclonal antibody could compete with the positive serum of pig infected with Trichinella spiralis to bind to the Ts-WN10 protein.
(77) The test results show that the monoclonal antibody prepared by the present disclosure can compete with the positive serum of pig infected with Trichinella spiralis to bind to the Ts-WN10 protein, as shown in Table 2 and
(78) TABLE-US-00013 TABLE 2 1H9 monoclonal antibody competes with Trichinella spiralis-infected pig serum for binding to Ts-WN10 antigen Dilutions of Sera 1:1 1:2 1:4 1:8 1:16 1:32 OD 450 nm of 0.3470 0.3065 0.2700 0.3480 0.5680 0.7685 positive serum (P) OD 450 nm of 0.9380 0.8650 0.7525 0.7590 0.9080 0.9775 negative serum (N) P/N Value 0.369936 0.354335 0.358804 0.458498 0.625551 0.786189
3. Western Blot Experiments
(79) Trichinella spiralis muscle larvae were washed 3 times with ddH.sub.2O, and a small amount of ddH.sub.2O was added. In an ice bath, the worms were grinded to pieces by a tissue grinder, and then broken by an ultrasonic cell pulverizer on ice (voltage: 300V; ultrasonication: 5 s; interval: 9 s; operation: 5 times; total 3 cycles). Observing under the light microscope, when the worm bodies were crushed into smaller pieces, it was placed at 4° C. and −20° C. alternately for freeze-thaw lysis, 5 times. After freeze-thaw lysis, the mixture was centrifuged at 4° C. at 1,600 g for 30 minutes, and the supernatant was collected as the soluble antigen. The supernatant was subjected to SDS-PAGE electrophoresis, and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk at 4° C. overnight. Monoclonal antibody was added and incubated at room temperature for 1 hour. The membrane was washed 3 times with PBST, and then incubated with HRP-labeled goat anti-mouse IgG antibody (diluted at 1:3000) for 1 h at room temperature. After the membrane was washed 3 times with PBST, ECL luminescent substrate was added for color development. The cell culture medium was used as a negative control.
(80) The test results show that the monoclonal antibody can bind to the soluble antigen from the worms, and the position of the recognized band is consistent with the position of WN10, indicating that the obtained monoclonal antibody can recognize the natural WN10 antigen. The monoclonal antibody 1H9 prepared by the present disclosure can specifically react with the soluble antigen of Trichinella spiralis muscle larvae (
Example 4 Identification of B-Cell Epitope
(81) 1. Expression of Truncated Ts-WN10 Protein (1.sup.st Round)
(82) Using the sequence of the recombinant plasmid pET28a-WN10 as a template, 2 pairs of primers were designed:
(83) TABLE-US-00014 TsWN10-A-EcoRI-atg: 5′-TAACGAATTCATGCAGATACTTGGTGA-3′ (as shown in SEQ ID No: 5); TsWN10-A-XhoI-tta: 5′-GACGCTCGAGTTAATTCACCCTT-3′ (as shown in SEQ ID No: 6); TsWN10-B-EcoRI-atg: 5′-TAACGAATTCATGCGTGTCCAAAGG-3′ (as shown in SEQ ID No: 7); TsWN10-B-XhoI-tta: 5′-GACGCTCGAGTTAACATTCAACA-3′ (as shown in SEQ ID No: 8).
(84) WN10 fragments A and B were obtained by PCR amplification respectively and ligated into pET28a vector to construct recombinant plasmids, named pET28a-WN10-A and pET28a-WN10-B, respectively. Recombinant bacteria containing pET28a-WN10-A or pET28a-WN10-B was induced separately to express the recombinant proteins, according to step 4 in Example 1. The expression products were analyzed by SDS-PAGE electrophoresis to confirm that the proteins were successfully expressed. These two proteins were subjected to SDS-PAGE, and the Western blot was performed using the monoclonal antibody according to the method of Example 3. The results showed that the B-cell epitope targeted by the monoclonal antibody 1H9 was located in the WN10-A fragment (
(85) 2. Expression of Truncated Ts-WN10 Protein (2.sup.nd Round)
(86) Using the sequence of the recombinant plasmid pET28a-WN10-A as a template, two pairs of primers were designed, and the PCR amplification products were inserted into the pET28a vector according to the method in Example 1 to construct recombinant plasmids, respectively named pET28a-WN10-A01 and pET28a-WN10-A02. Recombinant proteins were expressed according to the method of Example 1. The expression products were analyzed by SDS-PAGE electrophoresis to confirm that the proteins were successfully expressed. These two proteins were subjected to SDS-PAGE, and the Western blot was performed using the monoclonal antibody according to the method of Example 3. The results showed that the B-cell epitope targeted by the monoclonal antibody 1H9 was located in the WN10-A01 fragment (
(87) Primers are shown below.
(88) TABLE-US-00015 TsWN10-A01-EcoRI-atg: 5′-TAACGAATTCATGCAGATACTTG-3′ (as shown in SEQ ID No: 9); TsWN10-A01-XhoI-tta: 5′-GACGCTCGAGTTAAGCATTTGAA-3′ (as shown in SEQ ID No: 10); TsWN10-A02-EcoRI-atg: 5′-TAACGAATTCATGATTGATTCAAATGC-3′ (as shown in SEQ ID No: 11); TsWN10-A02-XhoI-tta: 5′-GACGCTCGAGTTAATTCACCCTT-3′ (as shown in SEQ ID No: 12).
(89) SDS-PAGE analysis showed that both proteins were successfully expressed. It was identified by Western blot that 1H9 monoclonal antibody reacted specifically with Ts-WN10-A01, and the reactivity was strong.
(90) 3. Identification of Epitope Recognized by the Monoclonal Antibody
(91) The software DNAstar was used to predict the hydrophilicity and antigenicity of WN10, and the epitope prediction programs ABpred and Bepipred were used to predict the epitopes. 14 short peptides were synthesized using PepScan technology. These short peptides were tested by indirect ELISA, and the coating amount was 0.25 μg/well. The results showed that 1H9 reacted specifically with Ts-WN10-W2. Based on the results of Western blot, it is inferred that the B-cell epitope of Ts-WN10 recognized by 1H9 is the amino acid sequence VNCQGEGRRKHCTME (SEQ ID NO: 1).
(92) TABLE-US-00016 TABLE 3 Synthetic Peptides Peptide Sequence SEQ ID NO: W1 ALFSSDLKQESGVFH 23 W2 VNCQGEGRRKHCTME 24 W3 TMEYTHRNPSKATVS 25 W4 TVSKCFEEVEEPLII 26 W5 QILGETTHYGRNDP 27 W6 ALFSSDSKEQSGVLH 28 W7 HKLVELEESSTMGIL 29 W8 CTLEYRHRTPSTATV 30 W9 EEQVVSQRSQMLGGT 31 W10 IFESDKKKSSGTYLL 32 W11 KETECGIKEKAFNSY 33 W12 EKAFNSYEDVYKNCS 34 W13 DVYKNCSGSGDSKVC 35 W14 VEYKYFDPTKSTVEC 36
(93) TABLE-US-00017 TABLE 4 Indirect ELISA results of peptides with WN10-1H9-Ab Peptide No. W1 W2 W3 W4 W5 W6 W7 OD 450 0.0733 1.1533 0.1487 0.1714 0.2860 0.1273 0.1616 nm Peptide No. W8 W9 W10 W11 W12 W13 W14 OD 450 0.1516 0.1441 0.1610 0.1470 0.1600 0.1663 0.1581 nm
(94) The above are only the preferred embodiments of the present disclosure. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications are also It should be regarded as the protection scope of the present invention.