CN111793130B

CN111793130B - Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody

Info

Publication number: CN111793130B
Application number: CN201910214363.8A
Authority: CN
Inventors: 王湘如; 陈启超
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2019-03-20
Filing date: 2019-03-20
Publication date: 2021-09-14
Anticipated expiration: 2039-03-20
Also published as: CN111793130A

Abstract

The invention discloses a haemophilus parasuis CdtB hybridoma cell and application of a monoclonal antibody, belonging to the field of detection, diagnosis and treatment of animal infectious diseases. The preservation number of the haemophilus parasuis CdtB hybridoma cell is CCTCC NO: c201910, the subclass of the generated monoclonal antibody is IgG1, the light chain type is kappa, and the amino acid sequence of the recognized epitope is highly conserved in Haemophilus parasuis. The monoclonal antibody has high titer, can identify all serotype haemophilus parasuis, also has neutralization activity aiming at CdtB, can be widely applied to etiological diagnosis, serological diagnosis, immunological detection, disease prevention and treatment, wild virus infection and vaccine distinguishing and the like of the haemophilus parasuis, and is used for preparing products related to the applications.

Description

Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody

Technical Field

The invention relates to the field of detection, diagnosis and treatment of animal infectious diseases, in particular to a haemophilus parasuis CdtB hybridoma cell and application of a monoclonal antibody.

Background

Haemophilus Parasuis (HPS) is a gram-negative bacterium belonging to the genus Haemophilus of the family Pasteurellaceae, a causative agent of respiratory diseases in pigs, and it parasitizes in the upper respiratory tract of healthy pigs, a common respiratory symbiotic bacterium in pigs. In the case of disease, it can lead to the disease of pig Glasser's, characterized by multiple serositis, meningitis, arthritis in pigs, which occurs mainly in piglets of 5-8 weeks of age. Currently, haemophilus parasuis disease in pigs has caused serious economic losses on a global scale.

The traditional diagnosis method of haemophilus parasuis mainly comprises the following steps: according to autopsy of dead pigs and separation and identification of bacteria, biochemical identification, serological identification, detection identification based on PCR and the like. However, since the growth of HPS is highly NAD-dependent, its in vitro culture is difficult, the separation rate from clinical disease is low, and other bacterial growth is likely to occur; and the phenotype of HPS is susceptible to mutation, resulting in typing errors, which limits the clinical application of traditional diagnostic methods. In addition, serological diagnosis results of existing haemophilus parasuis are inconsistent and inaccurate. Complement fixation assay (CF), Indirect Hemagglutination Assay (IHA), latex agglutination assay (LAT), and enzyme-linked immunosorbent assay (ELISA) have been used to detect antibodies to Haemophilus parasuis. In complement fixation assays, complement-binding antibodies were detected in acutely (within one week) infected pigs, but cross-reactivity between the various serotypes of HPS was observed (Nielsen R. pathway and immunity students of Haemophilus parasuis serovars [ J ]. Acta Vet Scand,1993,34, 193-198.). In the indirect hemagglutination test, specific antibodies were detected based on the supernatant after ultrasonication of HPS cells or boiling-lysed cells as coating antigens, which were adsorbed on sheep erythrocytes. However, the IHA method still detects negative results when detecting antibodies in vaccine-protected pigs, and is considered to be incapable of being used as a method for detecting antibodies in vaccine-protected pigs (Miniats OP, Smart NL, Ewert E.Vaccination of cognitive primary specific vaccines-free pigs against diseases in Haemophilus parauis [ J ] Can J vector Res,1991a,55, 33-36.). Chua Xuanwang et al used mixed

HPS type

4, 5 as antigen to sensitize erythrocytes and establish IHA diagnostic methods, but this method had cross-reactivity to porcine infectious pleuropneumoniae, streptococcus, infectious atrophic rhinitis (Chua Xuanwang Haemophilus parasuis isolation and diagnostic methods and inactivated vaccine research [ D ] university of agriculture in Huazhong, 2006.).

To date, 15 serotypes of HPS have been identified separately with large differences in virulence. In addition to the identified serotypes, a high proportion of HPS remains unidentified. The commercial haemophilus parasuis vaccine in the domestic market is mainly an inactivated vaccine and achieves certain effect. However, because of the numerous serotypes of haemophilus parasuis, inactivated vaccines prepared using one or more of the serotypes have poor protection against different serotypes of the strain, even have very limited cross-protection against isolates of different regions of the same serotype, and no reliable method for distinguishing vaccine immunity from wild virus infection exists, and the lack of detection methods causes difficulties in the prevention and diagnostic treatment of haemophilus parasuis.

The cell lethal swollening toxin (CDT) is a secreted toxin protein produced by gram-negative bacteria, is the only AB2 type toxin with DNase activity discovered so far, wherein CdtB is the main functional subunit of CDT, and can cause DNA fragmentation reaction of eukaryotic cells, resulting in irreversible cell cycle arrest and apoptosis. The toxin is found in Escherichia coli supernatant by Johnson and Lior for the first time in 1987, and according to literature reports, CDT is sensitive to various epithelial cells, endothelial cells, fibroblasts and lymphocytes and exerts different toxic effects on different cells. CDT can cause significant swelling of epithelial and fibroblasts, causing apoptosis of B-, T-, and dendritic cells. After the CDT is transferred to the nucleus, it causes DNA damage and thus a series of cellular responses. To protect against the enormous damage that DNA damage inflicts on a genome, cells activate a complex set of mechanisms to repair or minimize damage to the genome, including causing cell cycling arrest to facilitate DNA repair and, if damage is too severe to repair, promoting apoptosis. CDT has been shown to be an important virulence factor for a variety of bacteria, playing an important role in the pathogenic process of pathogenic bacteria: CDT in Haemophilus parasuis can cause PIEC cell swelling death, cause slight cellulose exudation of liver and spleen of guinea pig, cause massive effusion of thoracic cavity, abdominal cavity and pericardium of dead pig, and massive cellulose exudation of lung and liver; CDT in campylobacter jejuni can cause persistent infection of the mouse digestive tract, which can exacerbate the cecal mucosal inflammatory response; CDT in dysentery bacilli can cause watery diarrhea or severe intestinal disease in mice; CDT in Haemophilus ducreyi can cause rabbit skin ulceration; CDT in Actinobacillus actinomycetemcomitans can destroy physiological balance of human periodontal tissue to cause periodontitis; helicobacter pylori CDT can accelerate hepatocyte proliferation by activating a proinflammatory reaction pathway NF-kB, so that infectious hepatitis is converted to cancer.

Disclosure of Invention

The invention aims to solve the problems in the prior art, provides a monoclonal antibody for resisting a haemophilus parasuis CdtB protein, a hybridoma secreting the monoclonal antibody and application of the monoclonal antibody.

The purpose of the invention is realized by the following technical scheme:

a monoclonal antibody 4F10 for resisting the CdtB protein of Haemophilus parasuis is prepared from hybridoma cell strain 4F 10. The monoclonal antibody subclass is IgG1, the light chain type is kappa, the recognition epitope is located in GVGFPIDEYVWNLGTRSRPN region of CdtB protein, and the amino acid sequence of the region is highly conserved in Haemophilus parasuis.

The hybridoma cell strain 4F10 is deposited in China center for type culture Collection (address: China, Wuhan university) in 2018, 12 months and 26 days, is classified and named as hybridoma cell strain 4F10, and the preservation number is CCTCC NO: and C201910.

The monoclonal antibody 4F10 has neutralizing activity against CdtB. The haemophilus parasuis cell lethal swelling toxin (CDT) is one of the main virulence factors, and the immunogenicity is strong. CdtB is a CDT active subunit, and participates in various cell injuries in the process of Haemophilus parasuis infection and causes animal morbidity, so that a CdtB neutralizing antibody has an important role in disease treatment and pathogenesis research; each serotype HPS strain of haemophilus parasuis has CdtB, so that the CdtB can be used as a specific diagnosis target of HPS; in addition, as the CdtB is a secreted protein but can be secreted by live bacteria instead of the inherent structural outer membrane protein, and commercial vaccines used in the market are inactivated vaccines, the detection of the CdtB can be used as a method for distinguishing wild virus infection from vaccine immunity. Based on this, monoclonal antibody 4F10 with CdtB neutralizing activity and polypeptide fragment GVGFPIDEYVWNLGTRSRPN of CdtB protein can be used to prepare the following products: the kit comprises a product for pathogenic diagnosis of the haemophilus parasuis, a product for serological diagnosis of the haemophilus parasuis, a product for immunological detection of the haemophilus parasuis, a product for preventing and treating haemophilus parasuis disease, and a product for distinguishing wild virus infection of the haemophilus parasuis and vaccine immunity.

The monoclonal antibody of the invention has the following advantages and beneficial effects:

(1) the conservation is high, the universality is good, and all serotype haemophilus parasuis including non-typed strains can be identified.

(2) The specificity is good, and the cross reaction with the following pathogenic bacteria which seriously affect the live pig breeding diseases does not exist: escherichia coli of pig, Streptococcus suis, Klebsiella pneumoniae of pig, Pasteurella suis, Bordetella suis, Staphylococcus aureus of pig, Actinobacillus pleuropneumoniae of pig, and Salmonella suis.

(3) Has the neutralization activity of the active functional subunit CdtB of the expansion lethal toxin of the haemophilus parasuis cells

(4) Easy mass production; the titer is high, and the antibody titer can reach 1/1024000 after purification.

(5) Can be widely applied to the pathogenic diagnosis, serological diagnosis, immunological detection, disease prevention and treatment, wild virus infection and vaccine distinguishing, pathogenic mechanism research and the like of the haemophilus parasuis.

Drawings

FIG. 1 is a SDS-PAGE analysis and WB identification of His-CdtB protein.

FIG. 2 is a SDS-PAGE analysis and WB identification of GST-CdtB protein.

FIG. 3 is a graph showing the results of indirect ELISA assay of serum titers of immunized mice, in which M1, M2, M3 and M4 are numbers of four immunized mice, respectively, and Con is a non-immunized negative mouse.

FIG. 4 is a graph showing the results of titer determination of the purified monoclonal antibody 4F 10.

FIG. 5 is a graph showing the WB results of the reaction of monoclonal antibodies 2E3, B16, 5A10, 7G10 with a serotype strain of Haemophilus parasuis 15.

FIG. 6 is a WB result of the monoclonal antibody 4F10 reacting with various strains, in which Hps1-15 is Haemophilus parasuis strains type 1 to 15 in order from left to right.

FIG. 7 is an immunofluorescence chart of the monoclonal antibody 4F10 for detecting HPS infected animal tissues.

FIG. 8 is a graph showing the result of detecting the CdtB protein DNA cleavage activity of monoclonal antibody 4F10 by agarose gel electrophoresis.

FIG. 9 is a graph showing the results of cytomorphological staining to identify that the monoclonal antibody 4F10 inhibits the CdtB protein to cause cell swelling, wherein Con is an untreated blank under the same conditions.

FIG. 10 is a graph showing the results of flow analysis of the cell cycle arrest caused by CdtB protein inhibition by monoclonal antibody 4F10, wherein Control is an untreated Control under the same conditions.

FIG. 11 is a schematic diagram of the CDtB protein divided into three regions, A1, A2 and A3.

FIG. 12 is a graph showing the results of WB identifying the position of the epitope recognized by monoclonal antibody 4F10 in the three regions A1, A2 and A3.

FIG. 13 is a schematic diagram of the full sequence of the proteins A1-A2 divided into three regions B1, B2 and B3.

FIG. 14 is a graph showing the results of WB identifying the positions of the epitope recognized by monoclonal antibody 4F10 in the three regions B1, B2 and B3.

FIG. 15 is a schematic diagram of a fragment B2 divided into five regions S1-S5.

FIG. 16 is a graph showing the results of indirect ELISA determination of the region where the epitope recognized by monoclonal antibody 4F10 was located between S1 and S5.

Detailed Description

The following examples are intended to further illustrate the invention but should not be construed as limiting it. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

TABLE 1 primer sequences

EXAMPLE 1 screening and application of monoclonal antibody 4F10

Cloning of Haemophilus parasuis CdtB gene

According to a CdtB gene sequence of a Haemophilus parasuis SH0165 strain published by GenBank, two pairs of PCR amplification primers CdtB30a-F, CdtB30a-R and CdtB6p1-F, CdtB6p1-R are designed by using Primer 5 software, EcoRI enzyme cutting sites and XhoI enzyme cutting sites are respectively introduced into an upstream Primer and a downstream Primer, and the primers are synthesized by Nanjing Kingsler Biotech limited.

The extracted genome DNA of the H.parasuis SH0165 strain is used as a template to carry out PCR amplification on a target gene, and the reaction system of the PCR is as follows: prime Star 25. mu.L, 1. mu.L each of the upstream and downstream primers, 1. mu.L of template, ddH₂And O is supplemented to 50 mu L. The PCR reaction program is: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 15s, and 33 cycles; finally, extension is carried out for 10min at 72 ℃. The PCR product was checked for yield and specificity by electrophoresis on a 1% agarose gel and purified using a DNA purification kit.

Second, construction of recombinant expression strains

The PCR product and plasmids pET-30a and pGEX-6p-1 are subjected to double digestion by EcoRI and XhoI, and the digestion product is recovered by a kit after agarose gel electrophoresis.

And respectively connecting the recovered PCR enzyme digestion products with enzyme digestion plasmids pET-30a and pGEX-6p-1 (a product corresponding to a primer CdtB30a-F, CdtB30a-R is connected with pET-30a, a product corresponding to a primer CdtB6p1-F, CdtB6p1-R is connected with pGEX-6 p-1), converting the connection product into large intestine DH5 alpha, further screening to obtain recombinant expression plasmids pET-30a-CdtB and pGEX-6p-1-CdtB, and sequencing the recombinant expression plasmids pET-30a-CdtB and pGEX-6 p-1-CdtB.

The recombinant expression plasmid and the no-load plasmid (as a control) with correct sequencing are transformed into escherichia coli BL21(DE3) and then coated on an LB plate containing kanamycin and ampicillin to obtain recombinant expression strains pET-30a-CdtB, pGEX-6p-1-CdtB and corresponding no-load control strains.

Expression of recombinant proteins

The recombinant expression strain and the no-load control strain are streaked on an LB plate, after the strain is cultured at the constant temperature of 37 ℃ overnight, a single colony is picked by a sterilized gun head, inoculated in 4mL of LB liquid culture medium containing corresponding antibiotics, and shake cultured at the constant temperature of 37 ℃ overnight (10-12 h). The next day, inoculating the bacterial liquid into LB liquid culture medium containing corresponding antibiotics according to a ratio of 1:100, placing at 37 ℃ for shake culture for 1-2h, and culturing to OD₆₀₀The value is between 0.5 and 0.6, IPTG is added to make the final concentration 0.8mM/L for protein induction expression. Wherein, the recombinant expression strain pET-30a-CdtB and the corresponding no-load contrast strain are placed at the constant temperature of 37 ℃ for shake culture for 5 hours, and the recombinant expression strain pGEXPlacing the-6 p-1-CdtB and the corresponding no-load control strain at the constant temperature of 30 ℃ for shake culture for 4 h.

Fourthly, purification of recombinant protein and WB identification

1) pET-30a-CdtB inclusion body protein His-CdtB purification: after the induction, the bacterial solution was centrifuged at 8000rpm for 5min at 4 ℃ for collection, resuspended by PBS centrifugation, washed 3 times, resuspended by Buffer A (Tris-base 50mM, EDTA 0.5mM, NaCl 50mM, glycerol 5%, DTT 0.05mM), and crushed under pressure for 40 min. And centrifuging the crushed bacterial liquid at 4 ℃ and 7500rpm for 20min, discarding the supernatant, centrifugally resuspending the bacterial liquid by PBS, washing the bacterial liquid for 3 times, and centrifugally collecting the precipitate. The protein pellet was resuspended in Buffer A and DTT solution was added to a final concentration of 0.5 mM. After mixing, SKL (20g/mL) stock solution was added, and the mixture was dissolved by vigorous shaking and allowed to stand at 4 ℃ overnight. Centrifuging at 7500rpm for 40min at 4 deg.C in the next day, collecting supernatant, discarding precipitate, adding PEG400 to final concentration of 0.2%, adding oxidized glutathione to final concentration of 1mM, adding reduced glutathione to final concentration of 2mM, mixing, and standing at 4 deg.C for 3 hr. The liquid after standing was filled into a prepared dialysis bag, and 1L was made by volume using 1 XTE Buffer (100mM Tris-HCl, 10mM EDTA), dialyzed at 4 ℃ for 3 days, the dialysate was changed at intervals, and the purity was analyzed by SDS-PAGE electrophoresis.

2) pGEX-6p-1-CdtB soluble protein GST-CdtB purification: after induction, the cells were collected using a precooled 1 XPBS Buffer (137mM NaCl, 2.7mM KCl, 10mM Na)₂HPO₄，2mM KH₂PO₄，ddH₂O constant volume to 1L) for three times, performing pressure crushing, centrifuging, collecting the crushed supernatant, and adding the crushed supernatant into a GST adsorption column (Glutathione Sepharose 4B) which is cleaned and balanced for adsorption. Washed with PBS and then with GST Elution buffer (15mL of 1M Tris-HCl, 3.5g NaCl, 0.9219g reduced glutathione, ddH)₂O to 300mL, ph8.0), and 1mL was collected in one tube and analyzed for purity by SDS-PAGE electrophoresis.

3) The purified proteins His-CdtB, GST-CdtB were subjected to SDS-PAGE followed by Western-Blotting assay. After the electrophoresis is finished, the gel is taken out to be subjected to membrane transfer to transfer the protein to a PVDF membrane, and after the membrane transfer is finished, the PVDF membrane is taken out and subjected to membrane transfer in TBST (8.8 g of NaCl, 1.21g of Tris-base, 1.05 percent of Tween-200.05 percent of ddH)₂O is added to 1L with constant volume, the pH value is 8.0), then the solution is added with a confining liquid (TBST containing 5% BSA), the solution is sealed overnight at 4 ℃, a PVDF membrane is taken out of the confining liquid, the solution is rinsed with TBST and then is put into primary antibody (His-tag, GST-tag mouse monoclonal antibody) diluted by TBST, and the solution is placed on a shaking table to act for 1 hour at room temperature. The PVDF membrane is taken out and rinsed by TBST, then is put into a secondary antibody (goat anti-mouse HRP-IgG enzyme labeled secondary antibody) diluted by TBST, and is horizontally placed on a shaking table to act for 1h at room temperature. The PVDF membrane was subjected to chromogenic exposure by adding ECL after rinsing with TBST.

The results (FIGS. 1 and 2) show that His-CdtB and GST-CdtB are correctly expressed and have high purity.

Fifth, mouse immunization and potency determination

Diluting the recombinant protein His-CdtB to 500 mu g/mL by PBS buffer, adding equivalent volume of Freund complete adjuvant for full emulsification, injecting 50 mu g of protein into the neck and back of each 6-week-old female BALB/c mouse at multiple points for primary immunization, then respectively carrying out secondary immunization and tertiary immunization by using the same amount of protein emulsified by the Freund incomplete adjuvant, wherein each immunization is separated by 14 days, tail vein blood collection is carried out 14 days after the tertiary immunization, serum is separated, and the titer is determined by indirect ELISA. Mice with the highest assay titer (over 1/100000) were boosted once immediately with 100 μ g of unadjuvanted His-CdtB protein by intraperitoneal injection, and fused 3 days later.

Indirect ELISA method: with coating solution (1.59g NaCO)₃，2.93g NaHCO₃Adding appropriate amount of ddH₂O dissolved and then made to volume of 1L) His-CdtB was diluted to a concentration of 1 μ g/mL, and ELISA reaction plates were coated in 100 μ L per well and refrigerated overnight at 4 ℃. Discarding antigen solution, PBST (2.9g Na)₂HPO₄·12H₂O，8.0g NaCl，0.2g KH₂PO₄0.2g KCl, 0.5mL Tween-20, adding appropriate amount of ddH₂Dissolving O, metering to 1L), washing for 3 times, adding 200 μ L per well, and gently shaking at room temperature for 5 min. Liquid in the holes is thrown away as far as possible. Blocking with 5% skim milk in PBS at 200. mu.L/well overnight at 4 ℃. The blocking solution was discarded, PBST was washed 3 times, and the immunized and blank mouse sera were diluted with PBS in a gradient, added to the ELISA reaction plate at 100. mu.L/well, and allowed to react at 37 ℃ for 1 h. Serum was discarded and PBST washedAdding 1/5000 diluted goat anti-mouse HRP-IgG enzyme labeled secondary antibody at 100 μ L/hole, and keeping constant temperature at 37 deg.C for 30 min. Enzyme-labeled secondary antibody is discarded, PBST is washed for 3 times, and finally liquid in the holes is sequentially dried as much as possible. Adding TMB developing solution, developing for 10min at room temperature in dark place, terminating the reaction with stop solution, and measuring OD value at 630nm wavelength. The assay (figure 3) showed that the immunized mice with M1 titers of up to more than 1/102400 were selected for fusion.

Sixthly, cell fusion

Taking immunized BALB/c mice eyeball blood, taking cervical dislocation and killing, the collected serum can be used as positive control serum, soaking the mice in 75% alcohol for 5min, then taking the mice on an ultra-clean workbench, fixing the mice, carefully cutting the abdominal skin, carrying out blunt separation to completely expose the peritoneum, cutting the left peritoneum, taking out the spleen, placing the spleen in a homogenizer containing 10mL of 1640 basic medium, carrying out light extrusion to enable spleen cells to enter the 1640 basic medium, standing for 2min, sucking the upper layer cell suspension by a suction pipe, adding the upper layer cell suspension into a sterilized centrifuge tube, simultaneously adding 5mL of the basic medium 1640 into the homogenizer, standing for 2min, sucking the upper layer cell suspension by the suction pipe, and adding the upper layer cell suspension into the sterilized centrifuge tube. Centrifugation is carried out at 1000rpm for 10min, centrifugal washing is carried out for 1-2 times by using 1640 basic medium, and then cells are resuspended in 10mL of 1640 basic medium for use.

Will be 1 × 10⁸Immune spleen cell and 5X 10⁷SP2/0 myeloma cells were mixed in a 50mL centrifuge tube, supplemented with 1640 basal medium to 30mL, and mixed well. Centrifuging at 1000rpm for 10min, discarding the supernatant, and sucking the liquid at the bottom of the centrifuge tube as dry as possible. Lightly flicking the bottom of the centrifugal tube with fingers to loosen and homogenize the cells settled at the bottom of the centrifugal tube, and preheating in a 40 ℃ water bath. 1mL of 50% PEG1450, preheated to 40 deg.C, was added to the centrifuge tube over 1min using a 1mL pipette with gentle stirring. 30mL of 1640 basal medium, pre-warmed to 37 ℃, was added to the centrifuge tube over 90 seconds using a10 mL pipette, again with gentle stirring. After the addition, the mixture is kept stand at room temperature for 10 min. Centrifuge at 1000rpm for 5min and discard the supernatant. Adding 5mL HAT culture medium into the centrifugal tube, gently blowing up the precipitated cells to make them suspended and uniform, and supplementingHAT medium for feeder cells to 100 mL. Subpackaging 96-well cell culture plates with 150 mu L of each well; subpackaging with 24-well plate (1.5 mL per well), placing the culture plate in 5% CO₂And culturing at 37 ℃ in an incubator. After 5 days, 1/2 well medium was changed out with HAT medium. HAT medium was changed to HT medium after 7 days, and ordinary 1640 complete medium was used after 14 days.

Seventhly, screening and subcloning hybridoma cells

The GST-CdtB coating concentration is determined to be 6.25 mug/mL by square matrix titration, and 100 mug of cell supernatant is taken for specific antibody detection about 10 days after cell fusion. Specifically, the diluted GST-CdtB protein is added into a 96-hole enzyme label plate, 100 mu L of each hole is coated overnight at 4 ℃. On the following day, PBST was washed 3 times, blocking solution was added, the mixture was incubated at 37 ℃ for 1 hour and washed three times, culture supernatant of hybridoma was added, incubated at 37 ℃ for 1 hour, washed three times with washing solution, goat anti-mouse IgG-HRP was added, the mixture was reacted at 37 ℃ for 1 hour, washed three times, TMB substrate solution was added for color development for 10min, and after the reaction was stopped with stop buffer, OD was measured at a wavelength of 630 nm. Meanwhile, positive serum of an immunized mouse is taken as a positive control, culture supernatant of SP2/0 cells is taken as a negative control, and OD is taken₆₃₀ Samples 2 times greater than the negative control were positive.

In the cell wells with positive specific antibody detection, cells are selected according to factors such as the growth state and the size of cell colonies. The resuspended cells were gently blown up with a pipette tip, counted and diluted with HT medium containing 20% serum to a dilution of 10 cells per mL. The diluted cell suspension was added to a 96-well cell culture plate containing feeder cells at 100. mu.L/well. When the cell colony grows to the cell well 1/3 on days 7-9 after cloning, antibody detection is performed again to eliminate the negative cell strain. Continuously cloning for 3-4 times, selecting single cell colony wells continuously secreting antibody in good state each time, cloning until the cloned cells are 100% positive to obtain cell strains capable of stably secreting antibody against single epitope, and finally selecting five hybridoma cells with high titer and good state, wherein the hybridoma cells are named as 2E3, 4F10, B16, 5A10 and 7G10 respectively.

Preparation and purification of monoclonal antibody in large scale

Selecting female BALB/c mice of 8 weeks old, injecting Freund's incomplete adjuvant into abdominal cavity 0.5 mL/mouse, collecting hybridoma cells with vigorous growth and good morphology 7 days later, and adjusting to 2 × 10 with RPMI-1640 base solution⁶Each mouse is injected with 0.5mL of abdominal cavity, and ascites can be collected after the abdomen of the mouse expands about 7-10 days after the cells are inoculated. Immediately after collecting ascites, the mixture was centrifuged at 1500 rpm for 10min at 4 ℃ and the supernatant was collected. Standing at 4 deg.C overnight, centrifuging at 12000r/min at 4 deg.C for 5min, removing upper fat layer, centrifuging the ascites supernatant to be purified at 12000r/min for 5min, adding the supernatant into 50mL beaker, adding acetic acid buffer solution (0.06mol/L, pH 4.5) with four times volume, and stirring on magnetic stirrer. And (3) fully and uniformly mixing the ascites and the acetic acid buffer solution, calculating the total volume according to the proportion of adding 33 mu L of caprylic acid into each mL, adding an appropriate amount of caprylic acid into the mixture, and slowly stirring the mixture on a magnetic stirrer while adding the caprylic acid. After the addition, the mixture was stirred for 30min and then centrifuged at 12000r/min at 4 ℃ for 30 min. The centrifuged supernatant was filtered through neutral filter paper, and the pH of the filtrate was adjusted to 7.0 to 7.4. After the pH value is adjusted, precipitating the antibody by using Saturated Ammonium Sulfate (SAS) with the total volume being less than or equal to 45%, placing the beaker on a magnetic stirrer, slowly dropwise adding the saturated ammonium sulfate and slowly stirring, continuously stirring for 30min after the addition, and standing for 4-5h at 4 ℃. After standing, the suspension is centrifuged for 30min at 12000r/min at 4 ℃, the supernatant is aspirated and the same volume of Tris-base solution with the pH value of 9.010 mM as the ascites is taken for re-suspension precipitation. After the pellet was resuspended, the pellet was placed in a dialysis bag and dialyzed against a pH 7.220mM sodium phosphate solution for 24 hours. After dialysis, centrifugation is carried out for 30min at 12000r/min at 4 ℃, insoluble precipitates are removed, and the supernatant containing the monoclonal antibody is separately packaged at-80 ℃ for later use.

Ninth, monoclonal antibody potency assay

The measurement was carried out by indirect ELISA. And (3) taking the purified monoclonal antibody for 2-fold dilution, taking the highest dilution multiple when the P/N is more than or equal to 2.1 as a titer judgment end point, and taking SP2/0 cell culture supernatant as a negative control. The assay results (fig. 4) show that the 4F10 monoclonal antibody titer after purification can be as high as 1/1024000, while the titers of the remaining four antibodies are determined to be lower than that of the 4F10 antibody, which is 2E 3: 1/256000, B16: 1/256000, 5A 10: 1/64000, 7G 10: 1/256000.

Ten, identification of conservation and specificity of monoclonal antibody

A Haemophilus parasuis type 1-15 standard strain (strain numbers 1: HS145, type 2: SW140, type 3: SW114, type 4: SW124, type 5: Nagasaki, type 6: 131, type 7: 174, type 8: C5, type 9: D74, type 10: H367, type 11: H465, type 12: H425, type 13: IA-84-17975, type 14: IA-84-22113, type 15: SD-84-15995), two undifferentiated Haemophilus parasuis Hps-N-1942, Hps-N-1805111 (clinical isolate), Staphylococcus aureus, Salmonella suis, Klebsiella pneumoniae, Pasteurella suis, Streptococcus suis pleuropneumoniae, Escherichia coli, Bordetella bronchiseptica PAGE, and whole bacterial protein were subjected to SDS-electrophoresis, and then transferred to a PVDF membrane. The primary antibody is a purified monoclonal antibody, and the secondary antibody is goat anti-mouse IgG marked by HRP (horse radish peroxidase), and Western blot verification is carried out. The results demonstrated (fig. 5, 6) that none of the four antibodies 2E3, B16, 5a10, 7G10 identified all 15 standard serotype HPS strains, and only antibody 4F10 reacted with all 15 standard serotype HPS strains, with the unshaped HPS strain and without cross-reactivity with other control bacteria. The monoclonal antibody 4F10 can be used as a Western blot antibody for specifically diagnosing haemophilus parasuis.

Eleven, monoclonal antibody immunofluorescence

The tissue section infected with haemophilus parasuis is prepared, the slide is wiped dry by absorbent paper, an immunohistochemical painting brush is used for drawing circles around the tissue, 5% diluted normal goat serum is dripped, the tissue section is sealed for 30min at room temperature to reduce nonspecific staining, excess liquid is thrown off and not washed, and then diluted monoclonal antibody 4F10 with the concentration of 1/250 is dripped to be incubated overnight in a 4 ℃ wet box. Washing the section with PBST for 3 times, each time for 3min, wiping the section with absorbent paper, dripping diluted Fluorescent (FITC) labeled goat anti-mouse IgG, incubating at 37 ℃ in a wet box for 1h, washing the section with PBST for 4 times, each time for 3min, dripping DAPI, incubating in dark for 5min, staining the specimen, washing off excessive DAPI 5min × 4 times with absorbent paper, wiping the liquid on the section with absorbent paper, sealing the section with sealing liquid containing an anti-fluorescence quencher, and observing and acquiring images under a fluorescence microscope. The results of the sectioning (fig. 7) show that antibody 4F10 can recognize native proteins and specifically recognize and diagnose haemophilus parasuis infection as an immunofluorescent or immunohistochemical antibody.

Twelve, monoclonal antibody neutralization activity identification

1) Neutralizing DNA cleavage activity: buffer H (HEPES 25mM, MgCl) in 20. mu.L system₂ 10mM，CaCl₂5mM) is added with 4 mu g of GST-CdtB protein and 2 mu g of pET-28a plasmid, the mixture is incubated at 37 ℃ for 12 hours, and then the protein DNA cutting activity is detected by agarose gel electrophoresis; GST-CdtB protein and monoclonal antibody 4F10 were pre-incubated at 37 ℃ for 1H at a ratio of 1:1, and then 4. mu.g of pre-incubated GST-CdtB protein and 2. mu.g of pET-28a plasmid were incubated at 37 ℃ for 12H in 20. mu.L of buffer H, and then the neutralizing effect of the antibody on protein DNA cleavage was detected by agarose gel electrophoresis.

The results (FIG. 8) show that the CdtB protein is able to cleave plasmid DNA and antibody 4F10 is able to inhibit the DNA cleavage activity of CdtB. The antibody 4F10 has neutralizing activity against CdtB protein, and can protect DNA damage caused by the Hps CdtB protein.

2) Neutralizing cell morphological changes: laying newborn pig trachea epithelial cells (NPTr) in a 6-well plate, changing to starving in a serum-free culture medium for 12h when the cells grow to 80% -90%, respectively adding GST-CdtB protein and GST-CdtB protein which is pre-incubated with monoclonal antibody 4F10 for 1h (the concentration is 1:1) at 37 ℃, wherein the final concentration of the protein is 500ng/mL, acting for 24h, then rinsing the cells for 3 times by PBS, fixing the cells for 20min by PBS containing 50% methanol, changing to 100% methanol solution for fixing for 20min, discarding the methanol solution, staining for 2-3min by Giemsa staining solution, washing off the staining solution, and observing under a mirror.

The result is shown in fig. 9, the observation of cell morphology shows that the cell morphology is obviously expanded after the CdtB protein is added, the cell expansion effect caused by the CdtB protein can be obviously blocked after the antibody 4F10 is incubated with the CdtB protein, the antibody 4F10 has the neutralization activity aiming at the expansion death of the CdtB protein, and the damage of the organism caused by the haemophilus parasuis can be protected to a certain extent.

3) Neutralization of cell cycle arrest: laying newborn pig trachea epithelial cells (NPTr) in a 3.5cm plate, changing to starvation in a serum-free culture medium for 12h when the cells grow to 80% -90%, respectively adding GST-CdtB protein and GST-CdtB protein which is pre-incubated with monoclonal antibody 4F10 for 1h (the concentration is 1:1) at 37 ℃, wherein the final concentration of the protein is 500ng/mL, acting for 24h, then rinsing the cells for 3 times by PBS, adding pancreatin (without EDTA) in the 3.5cm plate, placing for 5-10min at 37 ℃, then blowing down adherent cells by PBS, replacing the blown adherent cells in respective 15mL centrifuge tubes, and horizontally centrifuging for 5min at 3000 rpm. After the supernatant was aspirated, 1mL of precooled PBS was added for resuspension, and the mixture was horizontally centrifuged at 3000rpm for 5 min. After the supernatant was aspirated, it was resuspended in a 15mL centrifuge tube and 1mL pre-cooled 70% ethanol, fixed at 4 ℃ for 12h, and centrifuged horizontally at 3000rpm for 5 min. After the supernatant was aspirated, 1mL of precooled PBS was added for resuspension, and the mixture was horizontally centrifuged at 3000rpm for 5 min. After the supernatant was aspirated, 500. mu.L of propidium iodide staining solution (content of each sample: 500. mu.L of staining buffer, 25. mu.L of propidium iodide staining solution, 10. mu.L of RNase A) was added to a 15mL centrifuge tube and used as it was, and the cell pellet was slowly and sufficiently resuspended, and incubated at 37 ℃ for 30min in the dark. The cells can then be stored at 4 ℃ in the dark. Add 200. mu.L PBS to resuspend the cells in a centrifuge tube, flow detect and analyze.

The result is shown in figure 10, and the flow analysis result shows that the cell cycle of NPTr is obviously blocked after being treated by CtdB protein, cells at the G2 stage are obviously increased, and the antibody 4F10 can obviously inhibit the cell cycle block caused by CdtB protein and can protect the damage of the organism caused by haemophilus parasuis to a certain extent.

The hybridoma cell strain 4F10 capable of secreting monoclonal antibody 4F10 has been deposited in China center for type culture Collection (address: China, Wuhan university) in 12 months and 26 days in 2018, is classified and named as hybridoma cell strain 4F10, and the preservation number is CCTCC NO: C201910.

EXAMPLE 2 identification of monoclonal antibody 4F10 subclass and epitope recognized by same

Identification of monoclonal antibody 4F10 subclass

The subtype rapid identification kit (Roche) of the monoclonal antibody is used for identification, and the operation process refers to the kit specification. After the monoclonal antibody 4F10 diluted by phosphate buffer solution is incubated with latex particles for 30min, a test strip is added to react with the latex particles for 5-10min, and the result shows that the subclass of the antibody is IgG1, and the light chain is kappa.

II, identification of epitope recognized by monoclonal antibody 4F10

1) According to software analysis (http:// tools. iedb. org/main/bcell /), the CdtB protein complete sequence (SEQ ID NO.1) is divided into three sections of A1(SEQ ID NO.2), A2(SEQ ID NO.3) and A3(SEQ ID NO.4) which are mutually overlapped for truncation expression (FIG. 11), and the monoclonal antibody 4F10 is used as an antibody for Western blotting identification to preliminarily determine the epitope recognized by the monoclonal antibody 4F10, and the identification result (FIG. 12) shows that the antibody 4F10 reacts with the two sections of A1 and A2, and the recognition sites of the antibody 4F10 are the cross regions of the two sections of A1 and A2.

2) According to the first identification result, the full sequences of the fragments A1-A2 proteins are divided into three segments of B1(SEQ ID NO.5), B2(SEQ ID NO.6) and B3(SEQ ID NO.7) which are overlapped with each other to be subjected to truncation expression (figure 13), and a monoclonal antibody 4F10 is used as an antibody to be subjected to Western blotting identification, so that the regions recognized by the monoclonal antibodies are further verified to be the regions of A1 and B2 which are two sections of crossing regions of A2 (figure 14).

The expression and purification of each of the above-mentioned truncated proteins were performed in the same manner as in example 1, the primers for amplifying each fragment are shown in Table 1, and the prokaryotic expression plasmid was pGEX-6 p-1.

3) The whole sequence of fragment B2 was separated into 5 fragments S1(SEQ ID NO.8), S2(SEQ ID NO.9), S3(SEQ ID NO.10), S4(SEQ ID NO.11) and S5(SEQ ID NO.12) (FIG. 15) by overlapping 10 amino acids every 20 amino acid fragments according to the second identification result, and peptide synthesis was performed by Nanjing Kinshire and ELISA identification was performed using monoclonal antibody 4F10 as an antibody, and as a result (FIG. 16), it was revealed that antibody 4F10 reacted only with fragment S2 and not with fragments S1 and S3. The monoclonal antibody 4F10 was finally determined to recognize the region as fragment S2, whose amino acid sequence was GVGFPIDEYVWNLGTRSRPN.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. A monoclonal antibody for resisting a Haemophilus parasuis CdtB protein is characterized in that: the preservation number is CCTCC NO: c201910.

2. A hybridoma cell producing the monoclonal antibody of claim 1, wherein: the preservation number is CCTCC NO: C201910.

3. use of the monoclonal antibody of claim 1 for the preparation of a product for the pathogenic diagnosis of haemophilus parasuis.

4. Use of the monoclonal antibody of claim 1 for the preparation of a serodiagnostic product for haemophilus parasuis.

5. The use of the monoclonal antibody of claim 1 in the preparation of an immunological detection product for haemophilus parasuis.

6. The use of the monoclonal antibody of claim 1 in the preparation of a product for the prevention and treatment of haemophilus parasuis disease.

7. Use of the monoclonal antibody of claim 1 for the preparation of a product for differentiating between wild virus infection of haemophilus parasuis and vaccine immunization.

The application of the polypeptide fragment of the CdtB protein in the preparation of a serological diagnosis product of haemophilus parasuis is characterized in that: the amino acid sequence of the polypeptide fragment of the CdtB protein is GVGFPIDEYVWNLGTRSRPN.

The application of the polypeptide fragment of the CdtB protein in the preparation of an immunological detection product of haemophilus parasuis is characterized in that: the amino acid sequence of the polypeptide fragment of the CdtB protein is GVGFPIDEYVWNLGTRSRPN.

The application of the polypeptide fragment of the CdtB protein in preparing a product for preventing and treating haemophilus parasuis disease is characterized in that: the amino acid sequence of the polypeptide fragment of the CdtB protein is GVGFPIDEYVWNLGTRSRPN.