CN113999305A

CN113999305A - Preparation and application of monoclonal antibody of streptococcus equi zooepidemicus SzM protein

Info

Publication number: CN113999305A
Application number: CN202111297080.8A
Authority: CN
Inventors: 马喆; 张钰
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-11-03
Filing date: 2021-11-03
Publication date: 2022-02-01

Abstract

The invention discloses a 3-strain streptococcus equi zooepidemicus SzM protein monoclonal antibody which is prepared and used for evaluating immunoreactivity of the monoclonal antibody under different application scenes. BALB/c mice were immunized with expression purified SzM protein, and a panel of candidate hybridoma cell lines was obtained based on hybridoma cell technology. 3 hybridoma cells producing the monoclonal antibody against the SzM protein are obtained by screening through ELISA, Western Blot and other methods. The Western Blot result shows that the monoclonal antibodies secreted by the 3 hybridoma cell strains can react with wild SzM protein which is artificially expressed or crude extracted from bacteria, and the antibodies are all suitable for immunoassay methods such as Dot Blot, IFA and the like. The 3 monoclonal antibodies are all antibodies of IgG1 subtype, kappa light chain, and the binding region is located in the constant region of SzM protein. The purified antibody has good opsonophagocytic activity through in vitro opsonophagocytic experiments.

Description

Preparation and application of monoclonal antibody of streptococcus equi zooepidemicus SzM protein

The specification is as follows:

the technical field is as follows:

the invention relates to preparation and application effect evaluation of 3 anti-streptococcus equi subsp zooepidemicus SzM protein monoclonal antibodies. The antibody is secreted and produced by 3 hybridoma cells which produce the anti-SzM protein monoclonal antibody respectively, can be used for immunological detection methods such as ELISA, Western-Blot, Dot-Blot, IFA and the like, can identify SzM protein constant regions, and has good specificity and wide applicability.

Background art:

the Streptococcus equi is divided into group C Streptococcus according to Lancefield grouping, mainly comprising subspecies equi, subspecies zooepidemicus and subspecies equi, wherein Streptococcus equi Subspecies Epidemicus (SEZ) ATCC35246 strain is one of the main pathogens causing the Streptococcus suis disease, large-area epidemics occur in Sichuan, Guangdong provinces and other provinces in China, and in 2019 to 2020, highly homologous strains (with homology of 99.7%) of SEZATCC35246 cause the Streptococcus suis disease outbreak in North America. The SEZ can cause the clinical symptoms of septicemia, arthritis, meningitis and the like of pigs, can cause acute death of hosts when being serious, and seriously threatens the healthy development of the pig industry.

The prevention and treatment of the streptococcus suis disease caused by SEZ in the market are mainly based on vaccines and antibiotics. In recent years, the breeding industry in China begins to limit the use of antibiotics; in addition, it takes a certain time for the animal body to generate antibodies to resist the invasion of pathogens after vaccination, and the animals can also generate SEZ infection before the completion of the immunization procedure or under the condition of complete non-immunization, so that other strategies for preventing and treating the SEZ infection are urgently needed to be developed.

Besides artificial active immunization methods such as vaccines, the animal body can be protected by artificial passive immunization. The artificial passive immunity has the characteristics of quick response, strong targeting property and the like. In passive immunization, the monoclonal antibody has the characteristics of higher titer, better specificity, less anaphylactic reaction and the like, and has better practical value for preventing and treating the antibacterial infectious diseases. In recent years, monoclonal antibodies have been only in the exploratory stage against bacterial infections, and the potential of monoclonal antibodies for antibacterial use is enormous, since they usually target bacterial virulence proteins rather than proteins required for survival, and hardly cause the bacteria to acquire resistance; and the monoclonal antibody can accurately target a certain pathogen, so that the disorder of the normal flora in the body is not easy to cause.

The SEZ surface has an important virulence factor SzM protein, the N end and the C end of the protein respectively have a signal peptide and a specific sortase recognition sequence (LPXTG), and the secondary structure is predicted to be an alpha-helical structure; the protein can bind fibrinogen in animal blood, resist phagocytosis of phagocytes, and stimulate strong protective antibody response in animal body. SzM is an important virulence factor in SEZ, and previous experiments also prove that immunity SzM stimulates the production of specific antibodies by the body to help the body resist SEZ infection at a certain level. SzM is also an effective diagnostic target for SEZ infection, and gene detection by PCR or antibody detection by serology can help to diagnose SEZ infection.

Based on the above considerations, the present inventors prepared monoclonal antibodies C7, D4, B11 against SEZ surface antigen SzM protein. So as to provide an effective monoclonal antibody for the development of an artificial passive immunotherapy method and provide necessary biological materials for the development of a serological diagnosis technology of SEZ infection.

The invention content is as follows:

the 3 anti-streptococcus equi subsp zooepidemicus SzM protein monoclonal antibodies prepared by the invention can be used for evaluating immunoreactivity effects in different application scenes, such as ELISA, Western-Blot, Dot-Blot, IFA and the like.

The 3 monoclonal antibodies prepared by the invention are all IgG1 subtype kappa light chain antibodies, and the binding region is located in a SzM protein constant region. The purified antibody is proved to have good opsonophagocytic activity through an in vitro opsonophagocytic experiment.

The hybridoma cell strain secreting the monoclonal antibody is kept by a cell preservation room of animal medical college of Nanjing agriculture university, and the preservation numbers are NJAUC No.2020010, NJAUC No.2020011 and NJAUC No.2020012 respectively.

The application of any antibody in the development of serological detection technology belongs to the protection scope of the invention.

The application of any antibody in the development of streptococcus equi subsp zooepidemicus or other pathogenic infection treatment technologies belongs to the protection scope of the invention.

The development and application of any antibody as a commercial reagent in the field of biological research belong to the protection scope of the invention.

The invention has the advantages and beneficial effects that:

the 3 anti-streptococcus equi subsp zooepidemicus SzM protein monoclonal antibodies are generally applicable to serological technical applications such as ELISA, Western-Blot, Dot-Blot, IFA and the like. The 3 monoclonal antibodies are IgG1 subtype antibodies, kappa light chain antibodies, and are the most common effective antibody types participating in humoral immunity; the antigen site binding region is positioned in the SzM protein constant region, and has good binding effect on different strain SzM proteins, so that the application range of the antigen site binding region is wider. 3 the monoclonal antibodies show better in-vitro opsonophagocytosis activity and have the potential of being developed as artificial passive immune medicaments.

Drawings

FIG. 1 is a schematic diagram showing the expression and purification of recombinant protein SzM according to example 1 of the present invention;

FIG. 2 shows the antibody titer in the serum of the immunized mouse according to example 2 of the present invention;

FIG. 3 shows the identification of the reactogenicity of the monoclonal antibody of example 2 of the present invention with recombinant SzM protein and lysates of Streptococcus equi subsp.

FIG. 4 is IFA identification of monoclonal antibodies of example 2 of the invention

FIG. 5 shows the results of identifying the class, subclass and type of monoclonal antibody in example 3 of the present invention

FIG. 6 shows the purification results of the monoclonal antibody of example 4 of the present invention

FIG. 7 shows the opsonophagocytic activity assay of the mAbs of example 5 of the invention

Detailed Description

Unless otherwise specified, the raw materials and chemical reagents used in the examples are all conventional commercial products, and the technical means used are conventional means known to those skilled in the art.

The materials, reagents, apparatus and methods used in the following examples, which are not specifically illustrated, are all conventional in the art and are commercially available.

The specific implementation case is as follows:

example 1: preparation of recombinant SzM protein

1.1 construction of expression vectors for the 1.1SzM antigen

Based on the sequence of SzM gene of Streptococcus equi subsp zooepidemicus ATCC35246 strain (GenBank: CP002904), a pair of primer sequences aiming at SzM is designed, and the primer sequences are as follows:

SzMF：5’-GTGCGGCCGCAAGCTTTGACCAGCTTTAGCAGTTGTAGTGCCT-3’

SzMR：3’-CGCGGATCCGAATTAGGAGCGGCTGTAAAGGC-5’

szM gene is amplified by taking streptococcus equi subsp zooepidemicus ATCC35246 genome as a template, the target gene is connected to a vector pET-28a by a homologous recombination method, and then the recombinant vector is transferred into a competent cell BL 21. The recombinant bacterium pET-28a-szM/BL21 is constructed and verified to be correct by sequencing. After the sequenced strains are recovered, selecting a monoclonal antibody, inoculating the monoclonal antibody into an LB liquid culture medium containing kanamycin, collecting thalli, and extracting plasmids by using a plasmid extraction kit.

1.2 expression and purification of 1.2SzM antigen

Culturing streptococcus equi subsp zooepidemicus ATCC35246SzM recombinant expression bacterium pET-28a-szM/BL21 to logarithmic phase; adding IPTG (Isopropyl Thiogalactoside) inducer with the final concentration of 1mmol/L, and continuing shake culture at 37 ℃ for 4 h; after the induced thallus is resuspended by PBS, the thallus is subjected to ultrasonic disruption; and after 1h, centrifuging the crushed bacterial liquid for 10min at 8000rpm and 4 ℃, respectively taking supernatant and precipitate to perform SDS-PAGE, and determining that the recombinant protein is expressed in a large amount after the bacterial crushed lysate supernatant. Inducing BL21 Escherichia coli expression strain, treating by centrifuging, washing, resuspending, and crushing, centrifuging the crushed bacterial lysate, filtering the supernatant with 0.45ul filter, filtering the supernatant, and purifying the protein

The pure protein rapid protein purification system is matched with a His trap Ni + protein purification prepacked column for affinity chromatography purification. After the protein with the his label is adsorbed on a nickel column in an affinity manner, eluting by using an Elution Buffer containing high-concentration imidazole, adopting a linear Elution method when eluting to obtain a purer band, collecting the liquid at the highest position of an outflow peak, thus obtaining the antigen, and carrying out SDS-PAGE identification. (see FIG. 1, FIG. 1A. SDS-PAGE analysis of recombinant protein SzM protein; FIG. 1B. SDS-PAGE analysis of elution and sampling during purification of SzM protein; M. protein standard; 1.pET-28a-szM/BL21 was not induced; 2.pET-28a-szM/BL21 induction; 3.pET-28a-szM/BL21 induction sonication supernatant; 4.pET-28a-szM/BL21 induction sonication precipitation; 5-11 recombinant protein SzM was sampled after elution during purification).

Example 2: and (3) preparing an anti-SzM protein monoclonal antibody.

2.1 animal immunization

SPF grade 6-8 week old female BALB/c mice were selected and the immunization program is shown in Table 1. During priming, adding the purified protein into an adjuvant ISA206 according to the volume ratio of 1: 1 for emulsification, and carrying out back subcutaneous multipoint injection on the emulsified SzM protein, wherein the injection dose is 200ug/0.2 mL/protein; the time of the secondary immunization and the time of the tertiary immunization are 14d and 21d after the primary immunization respectively, and the emulsified SzM protein is also used for carrying out subcutaneous multi-point injection on the back, so that in order to avoid immunological tolerance, the dose of the secondary immunization and the tertiary immunization is reduced to some extent and is 100ug/0.2 mL/mouse; after the secondary and tertiary immunization, blood is collected and serum is separated 5d, the titer of a mouse is measured by an indirect ELISA method, and the result is shown in figure 2 (figure 2: the titer of the antibody of the serum of the mouse after the immunization, and 1-4 are the serial numbers of the mouse), the mouse with the highest titer is selected for impact immunization, namely SzM protein which is emulsified without adjuvant is injected into the abdominal cavity at a dose of 80ug/0.1 mL/mouse; splenocytes from this mouse were taken for fusion within 96 hours after the challenge immunization.

2.2 establishment of Indirect ELISA screening method

By optimizing the reaction conditions, the indirect ELISA antibody detection method is established as follows: diluting the purified SzM protein to 8ug/mL with carbonate buffer solution (pH 9.6), adding 100ul per well into 96-well enzyme label plate, and acting at 4 deg.C overnight to coat antigen; washing with PBST 3 times the next day; adding 5% skimmed milk prepared from PBST (Poly-p-phenylene benzobisoxazole), 200 ul/hole, and sealing at 37 deg.C for 2 h; PBST wash 3 times; adding 50-100ul of hybridoma cell culture supernatant, setting positive serum of an immunized SzM mouse as a positive control hole, and incubating for 1h at 37 ℃; PBST wash 5 times; adding HRP-goat anti-mouse IgG secondary antibody diluted by 1: 100000, incubating for 1h at 37 ℃ in 100 ul/hole; PBST wash 5 times; adding TMB color development solution, 100 ul/hole, and developing at 37 deg.C in dark for 15 min; 2M sulfuric acid, 50 ul/well, was added, the color development was stopped, and OD450nm values were read on a microplate reader.

2.3 preparation of monoclonal antibodies

And fusing the mouse spleen cells with high OD450nm value detected by ELISA with myeloma cells SP2/0 to form hybridoma cells. And carrying out continuous 3-4 times of subcloning on the positive hybridoma cell hole by adopting a limiting dilution method, and finally obtaining the cell strain stably secreting the antibody for cryopreservation. Finally, 3 monoclonal antibody cell strains resisting SzM protein, namely C7, D4 and B11, are obtained. The accession numbers are NJAUC No.2020010, NJAUC No.2020011 and NJAUC No.2020012, respectively.

Example 3 serological identification of monoclonal antibodies C7, D4, B11

3.1 Western Blot method for identifying reactogenicity of monoclonal antibody and Streptococcus equi subsp zooepidemicus SzM antigen

And transferring the purified SzM recombinant protein and the SEZ ATCC35246 bacterial lysate to a PVDF membrane after SDS-PAGE, and carrying out Western blot identification by using the prepared ascites as a primary antibody and using HRP-goat anti-mouse IgG as a secondary antibody.

The results are shown in FIG. 3, where all 3 monoclonal antibodies reacted with SzM protein and SEZ ATCC35246 bacterial lysate. (see FIG. 3, FIG. 3A. C7; FIG. 3B. D4; FIG. 3℃ B11; M. protein molecular weight standards; SEZ ATCC35246 bacterial lysate; 2. recombinant SzM protein).

3.2 Dot Blot method for identifying reactogenicity of monoclonal antibody and Streptococcus equi subsp zooepidemicus SzM antigen

Determining whether the antibody binds to SEZ ATCC35246 in ascites by Dot Blot; SEZ 35246 Δ SzM as a control strain; the method is as follows.

1) And (3) bacterial culture: SEZ ATCC35246 and delta SzM were inoculated into THY liquid medium, cultured to logarithmic phase, centrifuged to discard the supernatant, washed 1-2 times, resuspended in PBS, and OD600 adjusted to about 1.5 as stock solution. The stock solution was diluted twice more than twice with PBS for four concentration gradients.

2) Preparing an NC membrane: after the membrane was cut, it was activated in methanol for one minute. After activation, the cells were equilibrated in PBS. The membrane was then placed on filter paper and allowed to air dry.

3) And (3) taking 10 mu L of each of two bacterial liquids with 5 dilutions on an NC membrane, and placing the NC membrane in a super clean bench for natural air drying. The bubble was then fixed in 70% ethanol for 5 minutes.

4) And (3) sealing: and soaking the fixed membrane in 5% skim milk, and sealing for 1 h.

5) Primary antibody incubation: after blocking, the membranes were placed in ascites fluid diluted with 5% skim milk and F598 and incubated for 2h at room temperature.

6) Washing: wash 3 times with PBST for 10 minutes each.

7) And (3) secondary antibody incubation: the washed membrane was incubated with Goat Anti-Mouse IgG H & L (HRP) and Goat Anti-Human IgG H & L (HRP) secondary antibody diluted in 5% skim milk for 1H at room temperature.

8) Washing: the same as 6.

9) Exposure: finally, the ECL luminous liquid is covered on the NC film and exposed by an exposure instrument.

The results are shown in FIG. 4, where all of these 4 monoclonal antibodies reacted with SzM protein and SEZ ATCC35246 bacterial lysate. (see FIG. 4, 1.SEZ ATCC 35246; 2.SEZ ATCC 35246. DELTA. SzM; A. ascites diluted 500-fold).

3.3 IFA identification of monoclonal antibodies

To determine whether the binding site of the monoclonal antibody to SEZ ATCC35246 was on the outer membrane of the bacterium, an indirect immunofluorescence experiment was performed, setting ATCC35246 Δ SzM as a control group. Colonies ATCC35246 and Δ SzM were scraped from the THY plate and spread evenly onto the slides. 0.1% TritonX-100 was used for permeation for 3 minutes at room temperature to prepare 1% BSA as blocking solution, and blocking was carried out for 45 minutes at room temperature. After diluting the monoclonal antibody 100-fold with PBS, it was incubated at room temperature for 1.5h and washed 3 times with PBS, each for 5 minutes. The second antibody, Goat Anti-Mouse IgG H&L(

488) Diluted with 1% BSA, incubated for 45 min at room temperature, and washed 3 times with PBS, 5min each. The slides were stained with a mounting medium containing DAPI and coverslipped and observed under a fluorescent microscope.

As a result, as shown in FIG. 5, it was observed from a fluorescence microscope that all of the three monoclonal antibodies reacted well with the wild strain and did not react with the deletion strain. FIG. 3A, B, C is an enlarged detail of the area where the white frame is circled with positive fluorescence, and shows that the monoclonal antibody binds to the membrane surface of the bacteria to form a hollow circular fluorescence ring, consistent with the property that SzM protein is a surface protein of the bacterial membrane. From the above, it was found that these three monoclonal antibodies were all monoclonal antibodies against membrane surface protein SzM of SEZ ATCC 35246.

Example 4 Western Blot identification of the antigenic regions bound by monoclonal antibodies directed against SzM protein in ascites

The conserved and variable region truncation mutant proteins cSzM and vSzM of SzM were successfully expressed. Ascites prepared from three hybridoma cells is diluted by skim milk by 500 times and respectively reacted with antigen SzM and truncated protein of derivative mutant thereof. Subsequently, the binding condition of the three monoclonal antibodies and the four proteins is identified by a Dot Blot experiment, the four proteins are respectively incubated by the antibodies, and no-load pET-28a-BL21 bacterial liquid is used as a control. The results are shown in FIG. 6, where the three monoclonal antibodies hardly bound to pET-28a-BL21, indicating that the antibodies are not directed against other proteins on the carrier, but bind weakly to vSzM, indicating that the binding regions of the three monoclonal antibodies are likely to be dominated by the constant regions of the antigen. (FIG. 6A shows the result of Western Blot identification, FIG. 6B shows the result of Dot Blot identification, A (Western Blot) M.protein, 1.rSzM, 2.cSzM, 3.vSzM, a.C7, b.D4, c.B11. B (Dot Blot) 1.rSzM, 2.tSzM, 3.vSzM, 4.cSzM, 5.pET-28a-BL21, d.C7, e.D4, f.B11. phi.100 ng/uL, 10 ng/uL, and 1 ng/uL

Example 5: opsonophagocytic Activity characterization of monoclonal antibodies

5.1 identification of monoclonal antibody class and subclass.

And identifying three monoclonal antibodies by using an enzyme-labeled secondary antibody through identifying the class, the subclass and the type of the immunoglobulin. The monoclonal antibodies C7, D4 and B11 are all antibodies of IgG1 subclass and kappa type.

5.2 purification of monoclonal antibodies

Since all three antibodies were identified as subclasses IgG1, the antibodies were purified using Protein G affinity chromatography resin media which bind only the Fc fragment of IgG.

5.3 opsonophagocytic Activity identification of monoclonal antibodies

In order to determine whether the prepared monoclonal antibody has opsonizing activity, an opsonophagocytosis experiment was performed using RAW cells and SEZ bacterial fluid. RAW264.7 cells were plated on 2-well 12-well plates on average, and 7 treatment groups of antibodies were diluted with PBS and added to the wells at 37 ℃ with 5% CO₂The cells were incubated for 4h and the amount of antibody per group was 20 ug/well. Selecting SEZ ATCC35246 single colony in liquid medium, shake culturing at 37 deg.C to logarithmic phase, washing with PBS three times, and adjusting to 5×10⁸CFU/ml; after the antibiotic body induction is finished, 200ul of bacterial liquid is put into a cell hole and placed at 37 ℃ and 5% CO₂The cell culture box is acted for 2 hours. After the action is finished, penicillin (5ug/ml) and gentamicin (100ug/ml) are added into each well for 1h to kill non-phagocytized extracellular bacteria. After sterilization, the supernatant was discarded, washed three times with sterile PBS, 500ul of distilled water was added to lyse the cells, and 10ul of the cell lysate stock solution and 10-, 100-, and 1000-fold diluted solutions were dropped onto the THY plate, respectively, to count the cells.

Counting the number of phagocytosed bacteria in each 10ul of cell lysate, and as a result, as shown in fig. 7, C7 and B11 have obvious opsonization promoting effect in the three monoclonal antibodies, so that the amount of RAW phagocytosed bacteria is obviously different from that in the PBS group; in addition, the mixed phagocytosis promoting effect of the three monoclonal antibodies is more obvious, and the phagocytosis effect among all groups is optimal.

Claims

1.3 anti-streptococcus equi subsp zooepidemicus SzM protein monoclonal antibodies C7, D4 and B11 are characterized in that the monoclonal antibodies are secreted by artificially prepared murine hybridoma cell strains SzM _ C7, SzM _ D4 and SzM _ B11 respectively, and the accession numbers of the monoclonal antibodies are NJAUC No.2020010, NJAUC No.2020011 and NJAUC No.2020012 respectively.

2. The 3-strain streptococcus equi subsp zooepidemicus SzM Protein monoclonal antibodies C7, D4 and B11 of claim 1 are all purified by Protein G affinity chromatography resin.

3. The 3-strain anti-streptococcus equi subsp zooepidemicus SzM protein monoclonal antibody C7, D4 and B11 of claim 1 are all antibodies of an IgG1 subtype, kappa light chain, and the epitope binding region is located in a SzM protein constant region.

4. The monoclonal antibodies C7, D4 and B11 of 3 strains of Streptococcus equi subsp zooepidemicus SzM of claim 1 can be used for immunological detection methods such as ELISA, Western-Blot, Dot-Blot and IFA.

5. The 3-strain streptococcus equi subsp zooepidemicus SzM protein monoclonal antibodies C7, D4 and B11 of claim 1 show good opsonophagocytic activity against streptococcus equi subsp zooepidemicus in vitro.

6. Hybridoma cells of the monoclonal antibody of claim 1 are hybridoma cell lines SzM _ C7, SzM _ D4 and SzM _ B11 with accession numbers NJAUC No.2020010, NJAUC No.2020011 and NJAUC No. 2020012.