Hybridoma cell strain and monoclonal antibody produced therefrom against serine protease of Trichinella spiralis in intestinal stage and application thereof

1. Primer design: According to the ZH68 gene sequence registered in Genbank (accession number: EU263332), PCR amplification primers are designed, and the sequences are as follows:

The template used for Ts-ZH68 gene amplification is AD3-stage adult worm cDNA, and the amplification primers are as follows:

The underlined parts are the introduced EcoRI and XhoI enzymatic digestion sites.

2. Extraction and reverse transcription of Trichinella spiralis T1 ( T. spiralis ) AD3-stage adult worm RNA

Rats infected with T. spiralis muscle larvae for 3 days are dissected, the small intestines are dissected, washed with sterile physiological saline and placed on separating sieve filter cloth, the physiological saline is preheated to 37° C., the small intestines are immersed and incubated at 37° C. for 4 hours and washed with sterile physiological saline containing 2% double antibodies, the supernatant is discarded, and AD3-stage adult worm are harvested. The extracted total RNA is used for reverse transcription to synthesize cDNA, and the system is as follows:

RNAase-free water is added to 25 μl,

the substances are uniformly mixed for a reaction at 42° C. for 1 hour. Products are stored at −20° C.

3. Construction of an expression vector: 3-day-old adult worm cDNA obtained after reverse transcription is used as a template for amplifying Ts-ZH68.

A PCR reaction system (50 μL) is as follows:

The reaction conditions are circulation of pre-denaturation at 95° C. for 5 minutes, at 95° C. for 45 seconds, at 55° C. for 45 seconds and at 72° C. for 45 seconds 30 times and final extension at 72° C. for 10 minutes. PCR products is recovered. The target gene Ts-ZH68 obtained after gel recovery and the prokaryotic expression vector pET28a are subjected to double enzymatic digestion separately, and an enzymatic digestion system is as follows:

At the same time, the prokaryotic expression vector pET28a is subjected to double enzymatic digestion, and an enzymatic digestion system is as follows:

The enzymatic digestion reaction systems are placed in a 37° C. water bath for standing still for 2 hours, and then gel recovery is carried out. The target gene after double enzymatic digestion is ligated with the pET28a vector, and the system is: 10*T 4 DNA Ligase Buffer 1 target gene obtained after enzymatic digestion 4 pET28a obtained after enzymatic digestion 1.5 μL, T 4 DNA ligase 1 μL and ddH 2 O 2.5 μL. The substances are ligated overnight at 16° C. All ligation products are transformed into E coli DH5a competent cells, and a single colony is picked for PCR identification and sequencing. Positive recombinant plasmids are transformed into BL21 (DE3) competent cells.

4. BL21(DE3)-pET28a-Ts-ZH68 induced expression and gel cut purification

When 1 ml solution contained recombinant bacteria is added into 100 ml of an LB culture medium for shaking culture at 37° C. until OD600 nm is about 0.5-0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) is added to a final concentration of 1 mmol/L, and the solution is induced at 37° C. for 6-8 hours. An expression product is subjected to SDS-PAGE electrophoresis and purified by a gel cut method. A cut gel block is added into an appropriate amount of PBS and ground into crushed particles, which can be used to immunize mice.

5. BL21(DE3)-pET28a-Ts-ZH68 affinity purification

After induction, the bacterial solution is centrifuged and resuspended with 30 mL of a resuspension buffer (20 mM Tris-HCL, PH 8.0). The bacterial solution is subjected to an ice bath for 10 minutes and ultrasonic treatment on ice for a total of 30 minutes, wherein the ultrasonic treatment is performed for 3 seconds, then stops for 3 seconds, and so on. The bacterial solution is centrifuged at 8000 rpm for 10 minutes, and a precipitate is collected, resuspended with 30 ml of a pre-cooled inclusion body washing solution (2 M urea, 20 mM Tris-HCL, 0.3 M NaCL, PH 8.0) and subjected to an ice bath for 10 minutes and ultrasonic treatment on ice for a total of 10 minutes, wherein the ultrasonic treatment is performed for 3 seconds, then stops for 3 seconds, and so on. This step is repeated 3 times. The bacterial solution is centrifuged at 8000 rpm for 10 minutes, and a precipitate is collected, resuspended with 30 ml of a pre-cooled PBS washing solution (0.01 M PBS containing 4 M urea) and subjected to an ice bath for 10 minutes and ultrasonic treatment on ice for a total of 5 minutes, wherein the ultrasonic treatment is performed for 3 seconds, then stops for 3 seconds, and so on. This step is repeated twice. The bacterial solution is centrifuged at 8000 rpm for 10 minutes, and a precipitate is collected, resuspended with 5 ml of a binding buffer (8 M urea, 20 mM Tris-HCL, 0.3 M NaCL, 5 mM imidazole, pH 8.0) and dissolved overnight at 4° C. The bacterial solution is centrifuged at 8000 rpm for 30 minutes, and a supernatant is collected and filtered for sample loading. Ts-ZH68 with a His tag is purified by using an AKTA pμrifier 100 (GE Healthcare, USA) purification system. Elution Buffers containing 30 mmol/L, 50 mmol/L, 100 mmol/L and 300 mmol/L imidazole are used for elution separately, and purified recombinant ZH68 protein is subjected to SDS-PAGE analysis. It can be seen that the purified protein supernatant has only one clear and obvious band, and the size is consistent with the theoretical value ( FIG. 1 ), indicating that a relatively pure rTs-ZH68 protein is obtained in this study. A BCA protein quantification kit is used for measuring the concentration of the purified protein, and the concentration of rTs-ZH68 protein is 0.85 mg/mL.

1. Mouse Immunization

Five 6-week-old female BALB/c mice are immunized with the recombinant Ts-ZH68 protein obtained after gel cut purification in Example 1 every two weeks for a total of 4 times, the immunization dosage is 50 μg/mouse an equal volume of adjuvant added, Freund's complete adjuvant is used for the first immunization, Freund's incomplete adjuvant is used for the other three times, and the immunation route is intraperitoneal immunization.

One week after the third immunization and the fourth immunization, mice are tail-cleaved to collect blood, the serum is separated (4° C., centrifugation at 3000 rpm for 30 minutes), and the antibody level is detected by an affinity-purified Ts-ZH68-indirect ELISA method. The operation of the Ts-ZH68 indirect ELISA method is as follows: the recombinant Ts-ZH68 protein is diluted with a coating solution (0.1 M carbonate buffer, pH 9.6, Na 2 CO 3 63.6 g, NaHCO 3 33.6 g, constant volume to 1000 mL), and the coating quantity is 0.1 μg/well and 100 μl per well. The recombinant Ts-ZH68 protein is coated at 37° C. for 1 hour and then placed overnight at 4° C. Afterwards, a plate is washed 3 times with PBST (containing 0.05% Tween 20). A blocking solution (5% skim milk) is used for blocking at 37° C. for 2 hours. Afterwards, the plate is washed 3 times with PBST. The serum to be tested is subjected to doubling dilution (or diluted with the hybridoma supernatant at a ratio of 1:2) and then added into a microwell plate with 100 μl per well. The serum is incubated at 37° C. for 1 hour. Afterwards, the plate is washed 3 times with PBST. A goat anti-mouse secondary antibody is diluted at a ratio of 1:1000 and incubated at 37° C. for 30 minutes. Afterwards, the plate is washed 4 times with PBST. Color development is carried out at 37° C. for 10 minutes. A reaction is stopped with 2 M H 2 SO 4 , and the absorbance at OD450 nm is read.

Three days before cell fusion, BALB/c mice with good immune effects are subjected to boosted immunization, and each mouse is injected intravenously with 50 μg of immunogen at the tail vein.

2. Cell Fusion

One day before fusion, mouse feeder layer cells are prepared, and BALB/c mouse peritoneal macrophages are taken according to a conventional method and spread in a 96-well cell culture plate for use. Three days after boosting, mice were sacrificed and the spleens are aseptically taken, the spleen cells are separated. 1*10 8 spleen cells are mixed with 2.5*10 7 SP2/0 myeloma cells at a ratio of 4:1 for cell fusion with 1 ml of PEG1000, and dropping ends within 1 minute. 1 ml of a 1640 culture medium preheated to 37° C. is then added dropwise into a cell suspension within 1 minute while stirring. 1 ml of the 1640 culture medium preheated to 37° C. is added dropwide into the cell suspension within 3 minutes while stirring. Finally, 10 ml of the 1640 culture medium at 37° C. is slowly added into the cell suspension, and the whole process is operated in a 37° C. water bath. The cell suspension is centrifuged at 1000 r/min for 10 minutes, the supernatant is discarded, and the cells are resuspended with an HAT culture medium, spread evenly on a 96-well cell culture plate pre-spread with feeder cells, and placed in a 5% CO 2 incubator for culture at 37° C.

3. Screening and Cloning of Positive Hybridoma Cell Strains

The Ts-ZH68 indirect ELISA method is used to screen positive hybridoma cell strains, positive hybridoma cells are subjected to expanded culture, and the positive hybridoma cells are subcloned for the first time by using a limiting dilution method. With reference to the ES indirect ELISA method described in OIE Terrestrial Manual 2017 Chapter 2.1.20-Trichinellosis to screen the positive hybridoma cell strains again, the positive hybridoma cells are subjected to expanded culture, the positive hybridoma cells are the subcloned by using a limiting dilution method at least 3 times, and subcloned positive hybridoma cells are frozen and stored in time. A hybridoma cell strain, ZH68-2A4, for stable secretion of an anti-Ts-ZH68 protein monoclonal antibody is obtained. This cell strain is deposited in the China General Microbiological Culture Collection Center on Aug. 15, 2019, the address is No. 3, No. 1 courtyard, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the strain accession number of deposit is CGMCC No. 18317, and the monoclonal antibody secreted therefrom is named ZH68-2A4-Ab, hereinafter referred to as 2A4.

Each 12-week-old healthy BALB/c mouse is injected with 0.5 ml of paraffin oil intraperitoneally and 1*10 6 hybridoma cells prepared in Example 2 intraperitoneally one week later, and after 7-10 days, when the abdominal cavity of each mouse is extremely dilated, ascites is extracted once every 2 days and then collected. The ascites is purified by using a protein G affinity chromatography medium. Before purification, the ascites is centrifuged at 10,000 rpm/min for 10 minutes to remove red blood cells and fat, and the supernatant is collected for purification. First, a chromatography column is connected with a syringe by an adapter, 5-10 ml of a binding buffer is injected into the chromatography column at a flow rate of 1 mL/min, the prepared ascites is injected into the chromatography column, the column is washed with 10-15 mL of a washing buffer to elute contaminant protein, and finally the column is eluted with 5 mL of an elution buffer, the solution is collected in a centrifuge tube, and SDS-PAGE analysis ( FIG. 2 ) confirms that the monoclonal antibody 2A4 is successfully purified and stored at −20° C. for use.

Identification of a Monoclonal Antibody:

1. Subclass Identification of a Monoclonal Antibody

The monoclonal antibody obtained in Example 2 is subjected to subclass identification according to the operating instructions of an antibody subclass identification kit. The results show that the heavy chain of the monoclonal antibody 2A4 of the disclosure is IgG2a, and the light chain is kappa chain.

2. A Ts-ZH68 Competition ELISA Test

The coating and blocking method is the same as the Ts-ZH68 indirect ELISA method described in Example 2. From 1:1, the negative serum and positive serum of pigs infected with T. spiralis are subjected to twice doubling dilution, 50 μl of the diluted serum to be tested is taken separately and mixed with 50 μl of the anti-ZH68 monoclonal antibody supernatant in equal volume, 100 μl of a mixed solution is added into a microwell plate for reaction at 37° C. for 1 hour, and the remaining steps are the same as those of indirect ELISA to analyze whether the monoclonal antibody supernatant can compete with the positive serum of pigs infected with T. spiralis for binding to the Ts-ZH68 antigen.

The test results confirm that the monoclonal antibody 2A4 prepared by the disclosure can compete with the positive serum of pigs infected with T. spiralis for binding to the Ts-ZH68 protein, as shown in FIG. 3 .

3. Western Blot Identification

T. spiralis muscle larvae are washed 3 times with ddH 2 O, a small amount of ddH 2 O is added, the larvae are ground into fragments with a tissue grinder in an ice bath, and then the larva fragments are crushed with an ultrasonic cell crusher in an ice bath. The larva fragments are subjected to ultrasonic treatment at a voltage of 300 V for 5 seconds at an interval of 9 seconds 5 times in 3 cycles, when it is observed under a light microscope that the larvae are crushed into small fragments, the larvae are subjected to alternate freezing and thawing at 4° C. and −20° C. 5 times, the larvae after freezing and thawing are centrifuged at 1600 g for 30 minutes at 4° C., and the supernatant is collected to obtain a soluble antigen. After treatment, SDS-PAGE is carried out, then the protein is transferred to a nitrocellulose membrane by electrotransfering, the membrane is blocked with 5% skim milk at 4° C. overnight, the monoclonal antibody is added for incubation at room temperature for 1 hour, and the membrane is washed with PBST 3 times, then incubated with a 3000-fold diluted HRP-labeled goat anti-mouse IgG antibody at room temperature for 1 hour, washed with PBST 3 times and then developed with an ECL luminescent substrate. The test results confirm that the monoclonal antibody 2A4 prepared by the disclosure can specifically react with a soluble antigen of T. spiralis muscle larvae, as shown in FIG. 4 .

1. Segmented Expression of Ts-ZH68 Protein

The gene sequence of the recombinant plasmid pET28a-ZH68 is used as a template, 3 pairs of primers are designed, ZH68-1-up and ZH68-1-down are used for amplification of Ts-ZH68-1, ZH68-2-up and ZH68-2-down are used for amplification of Ts-ZH68-2, and ZH68-3-up and ZH68-3-down are used for amplification of Ts-ZH68-3. After PCR amplification, the primers are connected to pET28a to construct recombinant plasmid and named pET28a-ZH68-1, pET28a-ZH68-2 and pET28a-ZH68-3 respectively. The recombinant bacterial containing pET28a-ZH68-1, pET28a-ZH68-2 and pET28a-ZH68-3 are subjected to induced expression separately according to the method in Example 1, and subjected to SDS-PAGE electrophoresis analysis to confirm the successful expression of short peptides ( FIG. 5 ). SDS-PAGE is carried out after treatment of three segments of proteins, and the three segments of proteins are separately subjected to a western blot test with the monoclonal antibody according to the method in Example 3. SDS-PAGE analysis shows that all three proteins are successfully expressed. It is identified by western blot that the monoclonal antibody can react specifically with Ts-ZH68-2 and has high reactivity. The test results show that the B cell epitope targeted by the monoclonal antibody is located on the pET28a-ZH68-2 segmented protein ( FIG. 6 ).

2. Identification of Monoclonal Antibody Epitope

The biological software DNAstar is used to predict the hydrophilicity and antigenicity of ZH68, the epitope prediction programs ABpred and Bepipred are used for epitope prediction, the following short peptides (Table 3) are synthesized by using a PepScan technology, and then an indirect ELISA test is carried out with the coating quantity of 0.25 μg/well. The results show that 2A4 reacts specifically with the Ts-ZH68-W5 peptide, it is inferred that the Ts-ZH68 protein B cell epitope recognized by 2A4 is initially located at 222 GVDRSATCQGDSGGP 236 in

combination with the western blot results, and a (W5 in Table 4) nucleic acid sequence:

In summary, a monoclonal antibody against Ts-ZH68 protein is prepared and identified in the disclosure. The monoclonal antibody of the disclosure and the Ts-ZH68 protein B cell epitope polypeptide recognized by the monoclonal antibody can be used to prepare a reagent or a vaccine for diagnosing or preventing infection of T. spiralis , laying the foundation for establishment of a serological diagnosis method of T. spiralis.