CN113444175B - Recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof - Google Patents

Abstract

The invention discloses a recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof, wherein a heavy chain variable region of the monoclonal antibody comprises HCDR1, HCDR2 and HCDR3, and amino acid sequences of the monoclonal antibody are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the light chain variable region of the monoclonal antibody comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the light chain variable region are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6. The recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody prepared by the invention has high purity, good specificity and high sensitivity, has no cross reactivity with other intestinal pathogenic bacteria, can be used for ELISA and IFA detection, and has important significance for clinical LI diagnosis and research and development of laboratory LI diagnosis technology.

Description

Recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof.

Background

Lawsonia Intracellularis (LI) is an important intestinal pathogenic bacterium, and mainly causes clinical symptoms such as acute hemorrhagic diarrhea, intermittent diarrhea, appetite decrease and slow growth and development of 6-20-week-old growing and fattening pigs, the loss caused by ileitis is reported to reach 1 hundred million dollars per year, subclinical ileitis causes economic loss of 4 dollars for each pig, and the economic loss is brought to the pig industry. Has become an important intestinal tract disease in the modern pig industry. It is ubiquitous in pig farms all over the world, is more serious in developing countries, and finds that the positive rate of the disease accounts for 98% by investigating 54 fattening pigs in the pig farms in China (Wang et al, 2006). About 70% of pigs in a pig farm are positive only in the late weaning period, which indicates that Chinese pigs are in an environment infected by the disease only in the early weaning period. In China, the African swine fever is suffered in recent years, the live pig breeding industry is stricken greatly, and the prevention and the treatment of the disease of LI which jeopardizes the breeding efficiency are particularly important in the rejuvenation and reproduction stage.

LI was first discovered in 1931 that the bacterium could not grow on conventional cell-free media, even though the bacterium could grow on a particular cell line, the infected cell monolayer generally showed no cytopathic effect (CPE) and was not readily observable, and thus it was difficult to diagnose by conventional bacteriological methods. In view of the severe condition of PPE harm caused by LI and the mixed infection of the PPE harm and other intestinal diseases, it is very important to establish a high-sensitivity, high-specificity, fast and simple diagnostic method.

Currently, the diagnostic methods generally used internationally are mainly divided into two major categories, specific diagnosis and non-specific diagnosis. The nonspecific diagnosis method has high requirements on the histopathology knowledge and the section staining technique of detection personnel, the staining background and the subjective judgment of the detection personnel influence the result, and the accurate diagnosis of recessive infection and mild symptom cases is difficult. Specific diagnosis such as ELISA, IFA and IHC is the most common method for differential diagnosis at present, and compared with polyclonal antibodies, the monoclonal antibodies have higher specificity and sensitivity and are particularly suitable for detecting samples with complex microbial environments such as intestinal tracts, excrement and the like; the monoclonal antibody can be expressed in large quantity by amplifying hybridoma cells without obtaining the antibody through immunizing animals for many times, and compared with the polyclonal antibody, the monoclonal antibody has stable and uniform quality and is more suitable for commercial large-scale production; at present, the research on LI is less, the types of commercial detection kits are less, the price is high, and the preparation of the LI monoclonal antibody has important significance on the laboratory research, clinical diagnosis and detection kit research and development of LI.

There are few reports on LI studies, only a few proteins such as LsaA protein, LatA protein and LhlyA protein are reported, and many protein functions have not been studied. The LI Hsp60 protein is a molecular chaperone protein of Lawsonia intracellularis, which is rarely studied in LI, and no report related to the preparation of a Lawsonia intracellularis Hsp60 protein monoclonal antibody exists at home and abroad so far.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the technical problems, the invention provides a recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof, and the prepared anti-LI monoclonal antibody lays a foundation for establishment of a lawsonia intracellularis detection method and development of epitope vaccine.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a recombinant monoclonal antibody of Lawsonia intracellularis Hsp60 protein, wherein the heavy chain variable region of the monoclonal antibody comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences of the monoclonal antibody are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the light chain variable region of the monoclonal antibody comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the light chain variable region are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8. The heavy chain variable region and the light chain variable region of the monoclonal antibody both have the structures of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4.

A nucleic acid molecule for coding the recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody, wherein the nucleotide sequence of the nucleic acid molecule for coding the heavy chain variable region of the recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody is shown as SEQ ID NO. 9; the nucleotide sequence of the variable region of the light chain of the monoclonal antibody of the nucleic acid molecule coding recombinant Lawsonia intracellularis Hsp60 protein is shown in SEQ ID NO. 10.

A vector comprising the nucleic acid molecule described above.

A host cell comprising the nucleic acid molecule or vector described above.

A kit, which comprises the recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody.

The preparation method of the recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody adopts a hybridoma technology for preparation.

Preferably, the preparation method of the recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody comprises the following steps:

step 1: constructing a recombinant expression vector pet-28a-Hsp60/BL 21; (ii) a

Step 2: induced expression, purification and immunoreactivity analysis of the recombinant Hsp60 protein;

and step 3: immunization of Balb/c mice;

and 4, step 4: screening, identifying and subcloning positive hybridoma cells;

and 5: preparing ascites and identifying subtypes;

step 6: purifying ascites;

and 7: and (5) analyzing the characteristics of the monoclonal antibody.

The invention finally provides application of the recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody in lawsonia intracellularis detection.

Preferably, the application comprises the following steps:

the Hsp60 protein monoclonal antibody is used as a primary antibody, a FITC-labeled goat anti-mouse antibody is used as a secondary antibody, and the Lawsonia intracellularis is detected by adopting an IFA detection method.

Preferably, the application comprises the following steps:

culturing IPEC-J2 cells, adding bacterial liquid containing Lawsonia intracellularis LJS19052, and setting up negative control; adding the Hsp60 protein monoclonal antibody, and incubating; adding a goat anti-mouse antibody labeled with FITC, and incubating; adding DAPA staining solution, reacting at room temperature in a dark place, washing, taking a picture under a fluorescence inverted microscope, and observing the result; and (4) IFA result judgment: specific green fluorescence exists in the cytoplasm of the IPEC-J2 cell, namely the cell is positive, and non-specific green fluorescence exists in the cytoplasm, namely the cell is negative.

The invention extracts DNA from ileitis positive pathological material, obtains recombinant Hsp60 protein through PCR, plasmid construction and prokaryotic expression, prepares the monoclonal antibody aiming at Hsp60 through hybridoma technology, and lays a foundation for further research and diagnosis method establishment of Lawsonia intracellularis.

Has the advantages that: compared with the prior art, the invention has the following advantages:

1. according to the invention, by optimizing the induced expression conditions of the Hsp60 protein, the recombinant Hsp60 protein is finally expressed in Escherichia coli in an upper clear and soluble expression form, the correct two-tertiary structure of the Hsp60 protein is reserved, and the protein has natural biological activity;

2. in the method, the His tag carried by the recombinant Hsp60 protein only consists of 6 histidines, has small molecular weight, does not change the biological structure of the protein basically, is not easy to cause immune reaction in a mouse body, and simultaneously, three monoclonal antibodies prepared by Western-Blot verification do not react with the His tag;

3. the LI Hsp60 monoclonal antibody prepared by the invention has high specificity, does not have cross reaction with common intestinal pathogenic bacteria of pigs, and is suitable for detecting samples with complex microbial environments such as intestinal tracts, excrement and the like;

4. the titer of the LI Hsp60 monoclonal antibody prepared by the invention reaches 2^18～19The kit can be established by a plurality of detection methods such as ELISA, IFA, IHC and the like;

5. the invention can express a large amount of monoclonal antibodies by amplifying the frozen hybridoma cells without obtaining the antibodies by immunizing animals for many times, has stable and uniform quality compared with the polyclonal antibodies, and is more suitable for commercial large-scale production.

Drawings

FIG. 1 shows PCR amplification of LIHsp60 gene; m: DL2000 Marker; 1: an Hsp60 gene;

FIG. 2 is a double-restriction enzyme identification chart of recombinant vector pet-28a-Hsp 60; m: DL2000 Marker; 1: recombinant plasmid is obtained after enzyme digestion;

FIG. 3 is a SDS-PAGE analysis of recombinant Hsp60 protein after induced expression; m: protein molecular weight standard; 1: IPTG inducing pet-28a no-load; 2: not inducing pet-28a-Hsp 60; 3: IPTG induction of pet-28a-Hsp 60;

FIG. 4 is an SDS-PAGE analysis of recombinant Hsp60 protein after disruption and purification; m: protein molecular weight standard; 1: crushing the whole strain; 2: crushing and then clearing the supernatant; 3: crushing the inclusion body; 4: purified Hsp 60;

FIG. 5 shows the Western-Blot results of His tag of recombinant Hsp60 protein; m: protein molecular weight standard; 1: recombinant Hsp60 protein;

FIG. 6 shows the OD detection of 3 monoclonal antibodies using the mouse monoclonal antibody subtype identification kit_450nmReading results;

FIG. 7 is a SDS-PAGE analysis of mouse ascites after purification; m: protein molecular weight standard; 1: 3E monoclonal antibody before purification; 2: purified 3E antibody; 3: is 4E monoclonal antibody before purification; 4: purified 4E antibody; 5: 9G antibody before purification; 6: purified 9G antibody;

FIG. 8 shows the result of Western-Blot for monoclonal antibody reactivity after purification; m: protein molecular weight standard; 1: other His-tag proteins; 2: purified monoclonal antibodies 3E, 4E, 9G, respectively;

FIG. 9 shows the result of ELISA detection of monoclonal antibody titer after purification;

FIG. 10 shows the result of the specific ELISA assay of the purified monoclonal antibody;

FIG. 11 shows the results of IFA detection of cells infecting Lawsonia intracellularis IPEC-J2 using purified monoclonal antibodies 3E, 4E, 9G; a: 9G; b: 3E; c: 4E; d: and (5) negative control.

Detailed Description

In order that the invention may be more readily understood, the invention will now be further described with reference to specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention, and that specific experimental procedures not mentioned in the following examples are generally conducted according to conventional experimental procedures.

Sources of materials for use in the present invention

Salmonella pullorum, Salmonella typhimurium, Escherichia coli and Lawsonia intracellularis are stored in the laboratory; wherein, the lawsonia intracellularis strain LJS19051 is preserved in China center for type culture Collection, the preservation place is Wuhan university in Wuhan, the preservation date is 2020, 7 and 21, and the preservation number is CCTCC NO: v202046, classified and named Lawsonia intracellularis strain LJS 19051.

His-tagged protein purification column, available from GE Healthcare;

PreScission Protease was purchased from Biyuntian Biotech;

freund's Complete Adjuvant (FCA) and Freund's Incomplete Adjuvant (FIA) are products of Sigma company;

female BALB/c (6-8 weeks old) mice were purchased from the comparative medicine center of Yangzhou university;

DMEM high-glucose medium and fetal bovine serum were purchased from GIBCO company;

SP2/0 cells were maintained in the laboratory;

HAT, HT Media Supplement (50X) from Sigma;

HRP-labeled goat anti-mouse antibody purchased from warrior, tebuch bioengineering, ltd;

TMB color developing solution purchased from Beyotime corporation.

A high-sensitivity ECL chemiluminescence detection kit (ready-to-use type) is purchased from Vazyme company;

the HiTrap Protein G HP purification column is a product of GE company;

the mouse monoclonal antibody subtype identification kit is a product of proteintech company;

FITC-labeled goat anti-mouse antibody was purchased from Sigma;

other reagents not listed are commercially available from regular sources;

example 1: expression and purification of lawsonia intracellularis Hsp60 protein

1) PCR amplification of the Hsp60 Gene

Analyzing by DNASAR biological software according to the sequence of Lawsonia intracellularis Hsp60 gene in GenBank, selecting a region with good antigenicity, entrusting Shanghai Bioengineering Co., Ltd to synthesize an Hsp60 gene sequence with EcoR I and Sal I enzyme cutting sites, wherein the expected target fragment length is 1647bp, and the sequence of an Hsp60 gene specific primer is F15 'CG-GAATTC-ATGGCTTCTAAAGAAATC 3'; r15 'GC-GTCGAC-CTAGTACATACC GTCCAT 3' (underlined indicates EcoR I and Sal I cleavage sites, respectively). 25 μ L PCR reactionThe system is as follows: ileitis positive pig manure DNA2 μ L, Mix 12.5 μ L, 10 μmol/L upstream and downstream primers each 1 μ L, ddH₂O8.5. mu.L. The PCR amplification conditions were: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 ℃ for 10 min. The PCR products were detected by 1% agarose gel electrophoresis (see FIG. 1).

2) Ligation and transformation of PCR products with pet-28a

And recovering and purifying the PCR product of the Hsp60 gene by using a gel recovery kit, performing double enzyme digestion by using EcoR I and Sal I, and connecting the product with pet-28a subjected to the same enzyme digestion. The connecting system is as follows: hsp60/(EcoRI, Sal I)4.0 μ L, pet-28a/(BamH I, Sal I)1.0 μ L, 10 XT₄ DNA ligase Buffer 1.0μL、T₄ DNA ligase 1.0μL、ddH₂O3.0. mu.L, each component was gently blown and mixed, and then the mixture was put in a metal bath at 16 ℃ overnight.

Taking out 1 tube of E.coliDH5a competent cells from a refrigerator at the temperature of-80 ℃, quickly putting the E.coliDH5a competent cells on ice, adding all the ligation products after the E.coliDH5a competent cells are melted, gently mixing the cells uniformly, placing the cells in an ice-water bath for 30min, carrying out heat shock at the temperature of 42 ℃ for 60s, then carrying out 3min in the ice-water bath, subsequently adding the cells into 1000 mu L of LB liquid culture medium, activating the cells at the temperature of 37 ℃ and 180rpm for 1h, coating the transformation solution on an LB solid plate containing 100 mu g/mL of ampicillin, carrying out culture at the temperature of 37 ℃ overnight, picking out a single colony the next day, extracting recombinant plasmids, and carrying out double enzyme digestion and sequencing identification (see figure 2). The correct pet-28a-Hsp 6-recombinant plasmid was subsequently transformed into e.coli BL21(DE3) competent cells as described above, while the empty plasmid pet-28a was transformed as a negative control.

3) Inducible expression of recombinant Hsp60 protein

Single colonies of pet-28a-Hsp60/BL21 were picked and inoculated in LB liquid medium containing ampicillin and chloramphenicol, and shake-cultured overnight at 37 ℃. The next day, the cells were transferred to 3mL of LB medium (containing ampicillin and chloramphenicol) at a ratio of 1:100, and shake-cultured at 37 ℃ and 180rpm/min to OD_{600 nm}The value is 0.5, 1mmol/L IPTG is added to the mixture, and the mixture is induced and expressed for 18h at 16 ℃ and 90 rpm/min. Collecting the expression product, centrifuging at 12000rpm/min at 4 deg.C for 5min, collecting thallus precipitate, washing with 0.01mol/L PBS (pH 7.4) for 3 times, and re-suspending the thallus precipitate with cell lysis buffer solutionRepeatedly freezing and thawing the thallus for 3 times at the temperature of minus 80 ℃, carrying out ultrasonic crushing and cracking (200W work for 4s and 6s at intervals) until the thallus is not viscous any more, centrifuging for 20min at the temperature of 4 ℃ and 12000rpm/min, and obtaining supernatant which is a soluble part and a precipitate which is an inclusion body part. The supernatant fraction and the inclusion body fraction were subjected to SDS-PAGE to detect the expression of recombinant Hsp60 protein. Meanwhile, the synchronous induction of pet-28a empty vector plasmid transformation E.coli BL21 was set as a blank control (see FIGS. 3 and 4).

4) Purification of recombinant Hsp60 protein

And (3) purifying the Hsp60 protein in the supernatant after the disruption by using a Ni-Charged Resin affinity chromatography column, wherein the specific operation steps are carried out according to the instruction. After the purified protein is concentrated and desalted for 3 times by an ultrafiltration concentration tube, the purity and concentration of the protein are detected by SDS-PAGE electrophoresis and a trace nucleic acid protein determinator (see figure 4).

5) Western-Blot detection of recombinant Hsp60 protein His tag

Hsp60 protein was separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride membrane (PVDF), blocked with 5% skimmed milk powder at 4 ℃ overnight, washed with 0.05% PBST for 3 times, diluted 1:1000 times with mouse anti-His tag primary antibody, incubated at 37 ℃ for 2h, washed with 0.05% PBST for 6 times, 10min each, added with 1:6000 HRP-labeled goat anti-mouse IgG, incubated at room temperature for 1h, washed with 0.05% PBST for 6 times, 10min each, and developed with ECL chemiluminescence solution to detect the expression of recombinant 60 protein (see FIG. 5).

Example 2: preparation of monoclonal antibody of anti-lawsonia intracellularis Hsp60 protein

1) Immunogen preparation and animal immunization procedure

And mixing the purified Hsp60 protein and Freund's complete adjuvant in a ratio of 1:1, and emulsifying to obtain the immunogen. The immunogen is used for immunizing a 7-week-old Bab/C mouse in a back subcutaneous multipoint injection mode, the immunization dose is 50 mu g/mouse, the immunization is boosted for 1 time respectively on 14 th and 21 th days after primary immunization, and the immunization dose and the immunization route are the same as that of primary immunization. 10 days after the three-immunization, the orbital venous plexus of the mouse is subjected to blood collection, serum is separated, and the antibody level in the serum is determined by an indirect ELISA method. The mice with the highest antibody level are selected to be subjected to impact immunization 3 days before fusion, namely, no adjuvant is added into immunogen, and purified Hsp60 protein is directly injected into the abdominal cavity at 50 mu g/mouse.

2) Cell fusion

Preparation of feeder cells

(1) Taking Bab/C mice 8 weeks old to sacrifice cervical vertebra, soaking in 75% alcohol for 3-5 min;

(2) in a biosafety cabinet, the skin is shaved with sterilized ophthalmic scissors, exposing the peritoneum. The syringe is used for sucking 10mL of DMEM culture solution and injecting the DMEM culture solution into the abdominal cavity, so that the intestinal canal is prevented from being punctured, the peritoneum is repeatedly massaged, and the culture solution is sucked out.

(3) The cells were put into a 10mL centrifuge tube, centrifuged at 1000rpm/min for 10min, and the supernatant was discarded to perform cell counting.

(4) Suspending with DMEM culture solution containing 15% fetal calf serum, adjusting cell number to 1 × 10⁵Per mL, add 96 well cell plates, 2 drops per well, place at 37 deg.C, 5% CO₂Culturing in an incubator.

Preparation of splenocytes

(1) According to the indirect ELISA detection result, selecting a mouse with the highest antiserum titer, picking eyeballs and bleeding to prepare positive serum for later use; killing the mouse by cervical dislocation, and soaking in 75% alcohol for 3-5 min;

(2) fixing the mouse on a dissection plate in a biological safety cabinet, cutting the right side position of the abdomen of the mouse by a sterilized ophthalmic scissors, exposing the spleen, taking out the mouse, placing the mouse into a plate with 1640 culture medium, and stripping the capsule;

(3) placing a 100-mesh cell screen on a 50mL disposable centrifuge tube, cutting the spleen into small pieces, taking out and placing the small pieces on the centrifuge tube, sucking about 10mL of culture medium, edging the edge of an inner core (sterilization) of a 1mL glass syringe, dripping the culture medium, centrifuging at 1500rpm, discarding supernatant, resuspending with a small amount of DMEM basic culture medium, and staining with 0.4% trypan blue; note that: the day before fusion SP2/0 cells were guaranteed to grow logarithmically.

Cell fusion

(1) The above counted spleen cells and myeloma cells were counted according to 5: 1, adding 10mL of DMEM culture solution, uniformly mixing by blowing, centrifuging at 1000rpm for 8min, and completely sucking the supernatant;

(2) grinding the cells into paste on the air holes at the edge of an operation table beside a safety cabinet, putting the centrifuge tube into warm water at 37 ℃, shaking the centrifuge tube, dripping PEG1450 preheated at 37 ℃, and adding 1mL in 1 min. Continuously placing the mixture in a 37 ℃ warm bath for 90 s;

(3) adding 25mL of DMEM medium within 5min to rapidly dilute PEG and lose effect, adding 1mL within the first 1min, adding 4mL within the middle 2min, and finally adding 20mL after 2min, and standing for 5-10 min;

(4) centrifuging at 1500rpm for 5min, discarding supernatant, resuspending HAT medium to 45mL, adding to 96-well plate containing feeder cells, 2 drops per well, standing at 37 deg.C and 5% CO₂Culturing in an incubator;

(5) after 5 days, 15% serum HAT medium was used for medium replacement, the medium was aspirated after about 8 days, and the medium was replaced with 15% serum HT DMEM.

3) Screening and identification of hybridoma cells

Observing whether each hole has a string-shaped cell mass or not at about 10d of cell fusion, recording the hole with cells, sucking cell supernatant of each hole when the cells grow to 1/2-1/3 of the whole hole bottom, and determining a positive cell hole by indirect competition ELISA, wherein if the hole without protein and the hole with protein are OD₄₅₀Very different wells are positive, and a larger difference indicates a better sensitivity of the well to the protein. If the two values are similar, the result is negative.

The indirect ELASA method is as follows:

(1) coating antigen: 50ug/mL protein, 100 uL/well coated ELISA plate, 4 ℃ refrigerator overnight;

(2) PBST washing for 3 times, sealing with 5% skimmed milk powder at 37 deg.C for 2h, PBST washing for 3 times, and patting to dry;

(3) adding cell supernatant: diluting cell supernatant by 100 times with PBS, dividing into two equal parts, adding one part directly into the well, mixing the other part with 100ng/mL protein, adding into the well, setting positive, negative and blank controls, and acting in 37 deg.C incubator for 1 h;

(4) washing is carried out as above;

(5) adding an enzyme-labeled secondary antibody: PBST (basic-positive potential contrast) dilutes the enzyme-labeled secondary antibody, and reacts for 1h at the temperature of 37 ℃ in a 100 uL/hole manner;

(6) washing is carried out as above;

(7) color development: adding 100uL of TMB color developing solution into each well, reacting for 15min at 37 ℃ in the dark, and adding 50uL of 2mol/L H into each well₂SO₄Terminating the reaction;

(8) OD measurement by enzyme-linked immunosorbent assay (ELISA)₄₅₀The value is obtained.

Positive cell subcloning

And selecting the wells sensitive to the protein and having better titer, and subcloning according to a limiting dilution method until the wells of the cells cloned at the last time are all positive.

The limiting dilution method operates as follows:

(1) feeder cell plating was prepared the day before cloning;

(2) counting the cloned cells, and diluting the cells to about 2000 cells/mL by using HT culture solution;

(3) taking 0.1mL of cell suspension, and adding 4mL of HT medium;

(4) two rows of A, B of 2.4mL were added to a 96-well cell culture plate containing feeder cells at 100 uL/well of approximately 5 cells per well at a cell concentration of 50 cells/mL;

(5) adding 2.4mL of HT medium into the rest 1.6mL, adding 2.4mL of HT medium into C, D two rows, and adding 2 cells in each hole;

(6) the remaining 1.6mL of HT medium was added to 3.4mL, and the remaining four rows were added, 0.5 cells per well, 37 deg.C, 5% CO₂Culturing in an incubator;

(7) and 7d, screening the subcloned cells, carrying out expanded culture on the positive monoclone, carrying out subcloning continuously according to the method, and freezing and storing the positive target cell for later use during each subcloning.

Expanded culture and cryopreservation of hybridoma cells

(1) Selecting a well with better sensitivity to the immune protein, sucking cell supernatant, slightly blowing the cells with a fresh HT culture medium and adding the cells into a 24-well plate for culture when the cells grow to the bottom of the well;

(2) then, continuously carrying out amplification culture in a 6-well plate according to the method, and finally transferring to a cell bottle for culture;

(3) when the density of the cells in the bottle reaches more than 80% and the cells are in logarithmic growth, pouring out the culture solution, and blowing down the cells attached to the wall of the bottle by using fresh culture solution;

(4) centrifuging at 1500rpm for 10min, discarding the supernatant, resuspending the precipitate with 2mL of culture solution containing 20% DMSO, and packaging 1 mL/tube in a freezing tube;

(5) 30min at 4 ℃, 2h at-20 ℃ and overnight at-80 ℃, and storing in liquid nitrogen for a medium-long time.

4) Monoclonal antibody subtype identification

And collecting the supernatant of the monoclonal cell strain, identifying by using an antibody typing kit, and according to the detection result, obtaining three monoclonal antibody subtypes which are IgG (shown in figure 6).

Preparation of ascites

(1) Before inoculating the mouse for 10-14 days, fixing the mouse by hand, inclining the head downwards to retreat the intestinal canal, positioning and injecting 0.5ml of liquid paraffin on the right side of the midline of the lower abdomen, slowly withdrawing the needle, and performing pre-stimulation;

(2) culturing hybridoma cells in an enlarged manner before 1-2 days, blowing off, counting cells, centrifuging at 800rpm for 5min, collecting precipitate, adding PBS to make the number of cells 1 × 10⁶About one/ml is suitable;

(3) injecting hybridoma cell suspension 0.5ml into abdominal cavity of each mouse with sterile needle, pre-stimulating, observing health status and ascites generation every other day, collecting ascites three times after 7-10 days, and removing neck to kill mouse;

monoclonal antibody purification

(1) Centrifuging the mouse ascites obtained before at 3500rpm for 15min to ensure complete precipitation of impurities, and sucking 800. mu.L of supernatant into a new 1.5mL EP tube;

(2) boiling the hose and plug matched with the antibody purification column with deionized water for 5min, and cooling to room temperature;

(3)10mL Binding Buffer equilibrated column at about 18 rpm;

(4) the column was washed using 5mL of Elution Buffer at approximately 18 rpm;

(5) again using Binding Buffer to balance the column, the rotation speed is about 18 rpm;

(6) passing the supernatant collected previously through a column (one time for 0.8mL) at about 8rpm, and after passing through the column, horizontally placing the purification column in a refrigerator at 4 ℃ for at least 1h to sufficiently bind to the antibody;

(7) then passing through 20mL Binding Buffer for eluting the hybrid protein, wherein the rotating speed is about 18 rpm;

(8) eluting the antibody with Elution Buffer, inoculating 7 tubes, and inoculating 1mL tubes (before inoculation, 100 μ L of neutralizing Buffer is added into each tube to neutralize the acidic environment of the eluent and protect the purified antibody), wherein the rotation speed is about 8 rpm;

(9) then passing through a 10mL Binding Buffer balance column;

(10) the purification effect was identified by using 20% ethanol to block the column and performing SDS-Page protein electrophoresis on the purified antibody (see FIG. 7).

Reactivity of monoclonal antibody

Hsp60 protein was separated by 10% SDS-PAGE, transferred to polyvinylidene fluoride membrane (PVDF), blocked with 5% skimmed milk powder at 37 ℃ for 1h, washed with 0.05% PBST for 3 times, diluted 1:200 times with purified monoclonal antibody, incubated overnight at 4 ℃, washed with 0.05% PBST for 4 times, 10min each, added with 1:6000 HRP-labeled goat anti-mouse IgG, incubated at room temperature for 1h, washed with 0.05% PBST for 4 times, 10min each, developed with ECL chemiluminescence solution and analyzed for reactivity of the purified monoclonal antibody (see FIG. 8).

Potency of monoclonal antibody

(1) Coating antigen: recombinant Hsp60 protein, 50ug/mL, 100 uL/well coated ELISA plate, refrigerator at 4 deg.C overnight;

(2) PBST washing for 3 times (5 min each time), sealing with 5% skimmed milk powder at 37 deg.C for 2h, PBST washing for 4 times (5 min each time), and patting to dry;

(3) diluting the purified monoclonal antibody by PBST 2 times to 100 mu L/hole, respectively setting positive, negative and blank controls, and acting in a constant temperature incubator at 37 ℃ for 1 h;

(4) washing is carried out as above;

(5) adding an enzyme-labeled secondary antibody: PBST enzyme-labeled secondary antibody was subjected to 1:6000 dilution, 100 uL/hole, reacting for 1h at 37 ℃;

(6) washing is carried out as above;

(7) color development: adding 100uL of TMB color development liquid into each well, and carrying out light-shielding reaction at 37 DEG C50uL of 2mol/L H should be added into each hole for 15min₂SO₄Terminating the reaction;

(8) OD measurement by enzyme-linked immunosorbent assay (ELISA)₄₅₀A value;

(9) as a result: when the purified monoclonal antibody is diluted to 2¹³When the detection result is positive, the ELISA detection result is still positive; when diluted to 2¹⁴And the ELISA result is negative, and the result shows that the titer of the purified monoclonal antibody reaches 2¹³(see FIG. 9).

Specificity of monoclonal antibody

(1) Coating antigen: recombinant Hsp60 protein, porcine Escherichia coli, porcine salmonella, PEDV and TEGV pathogenic bacteria total protein, 50 mu g/mL, 100 uL/hole coating ELISA plate, and refrigerating overnight at 4 ℃; (ii) a

(2) PBST washing for 3 times (5 min each time), sealing with 5% skimmed milk powder at 37 deg.C for 2h, PBST washing for 4 times (5 min each time), and patting to dry;

(3) PBS 1: diluting 200 parts of purified monoclonal antibody, adding into the hole, setting positive, negative and blank controls at 100 mu L/hole respectively, and acting in a constant temperature incubator at 37 ℃ for 1 h;

(4) washing is carried out as above;

(5) adding an enzyme-labeled secondary antibody: PBST enzyme-labeled secondary antibody was subjected to 1:6000 dilution, 100 uL/hole, reacting for 1h at 37 ℃;

(6) washing is carried out as above;

(7) color development: adding 100uL of TMB color developing solution into each well, reacting for 15min at 37 ℃ in the dark, and adding 50uL of 2mol/L H into each well₂SO₄Terminating the reaction;

(8) OD measurement by enzyme-linked immunosorbent assay (ELISA)₄₅₀A value;

(9) as a result: except that the wells coated with Hsp60 protein were positive, the wells inoculated with E.coli, Salmonella, PEDV, and TGEV were all negative, indicating that the purified monoclonal antibody had good specificity (see FIG. 10).

Sequencing of monoclonal antibodies

The monoclonal antibody 9G prepared above was sequenced (consignment: Jinzhi Biotech, Suzhou) as follows: the heavy chain variable region and the light chain variable region both have the structures of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3; the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences are respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Application of lawsonia intracellularis Hsp60 protein monoclonal antibody

IPEC-J2 cells were digested and centrifuged to prepare a gel containing 1.0X 10⁵Cell suspension of 500. mu.L/well was seeded into 24-well cell culture plates containing cell slide, placed at 37 ℃ in 5% CO₂Culturing in an incubator; the next day, after the cells are completely attached to the wall, the culture solution is discarded, 500 mu L of Lawsonia intracellularis LJS19051 bacterial solution is added into each hole, and a negative control with only DMEM culture solution is set; place the cell plate at 37 ℃ and 8% O₂，8.8％CO₂And 83.2% N₂Culturing in a three-gas culture box, and continuously culturing after replacing with fresh culture solution after 3 hours; taking out the 96-well plate after 24 hours; washing with PBS for 3 times, adding 500 μ L-20 deg.C pre-cooled anhydrous methanol into each well, and fixing at room temperature for 15 min; removing the anhydrous methanol, and washing with PBST for 3 times (5 min/time); sealing each hole with 5% skimmed milk powder at 500 μ L/hole at 37 deg.C for 2 hr; PBST washing 5 times, 5 min/time, patting dry; add 1: incubating 3E, 4E and 9G monoclonal antibodies diluted by 200 times at 37 ℃ for 1 h; PBST washing 5 times, 5 min/time, patting dry; 500 μ L of FITC-labeled goat anti-mouse antibody diluted at 1:500 was added to each well and incubated at 37 ℃ for 1 h; PBST washing 5 times, 5 min/time, patting dry; add 500. mu.L DAPA staining solution into each well, react for 13min in the dark at room temperature, wash 5 times with PBST, 5 min/time, photograph under a fluorescence inverted microscope and observe the result. And (4) IFA result judgment: specific green fluorescence exists in the cytoplasm of the IPEC-J2 cell, namely the cell is positive, and non-specific green fluorescence exists in the cytoplasm, namely the cell is negative. IFA results are shown in FIG. 11, and when compared with the negative control group, the primary antibodies were 3E, 4E and 9G monoclonal antibodies, which showed specific green fluorescence in IPEC-J2 cell cytoplasm and were consistent with the presence of Lawsonia intracellularis in the cell, indicating that the 3 monoclonal antibodies against Lawsonia intracellularis Hsp60 protein prepared in this study could be combined with the monoclonal antibodies on pig intestineSpecific binding occurs to the whole lawsonia intracellularis in the skin cells.

The PBS buffer in the above examples was obtained by the following steps:

taking 3.63g of disodium hydrogen phosphate dodecahydrate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride and 8g of sodium chloride; mixing the above components, dissolving in 1000mL ultrapure water, adjusting pH to 7.4-7.6 with 0.1M HCl, filtering with 0.22 μ M filter membrane, and storing at 4 deg.C.

The cell lysis buffer in the above examples was obtained by the following steps:

5mL of 1mol/L Tris-HCl with pH 8.0, 2.5mL of 0.5mol/L EDTA and 10mg of lysozyme are uniformly mixed, ultrapure water is added to 100mL, and the mixture is stored at 4 ℃.

The PBST solution in the above example was obtained by the following steps:

taking 3.63g of disodium hydrogen phosphate dodecahydrate, 0.24g of potassium dihydrogen phosphate, 0.2g of potassium chloride and 8g of sodium chloride; mixing the above components, dissolving in 1000mL of ultrapure water, adjusting pH to 7.4-7.6 with 0.1M HCl, adding 500 μ L Tween-20, mixing, and storing at room temperature.

The confining liquid in the above examples was obtained by the following steps:

5g of skim milk is dissolved in 100ml of LPBS solution and is mixed evenly for standby.

The SP2/0 cell complete culture solution in the above example was obtained by the following steps:

HDMEM culture solution 90V/V%

Fetal bovine serum 10V/V%

HAT selection medium in the above examples was obtained by the following steps:

HDMEM culture solution 88V/V%

Fetal bovine serum 10V/V%

HAT 2V/V％

The HT selection medium of the above examples was obtained by the following steps:

HDMEM culture solution 88V/V%

Fetal bovine serum 10V/V%

HT 2V/V％

In conclusion, the lawsonia intracellularis Hsp60 monoclonal antibody prepared by the invention has high purity, good specificity and high sensitivity, has no cross reactivity with other intestinal pathogens, can be used for ELISA and IFA detection, and has important significance for clinical LI diagnosis and research and development of laboratory LI diagnosis technology.

The above embodiments are merely illustrative of the present invention and are not to be construed as limiting the invention. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that various combinations, modifications or equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and the technical solution of the present invention is covered by the claims of the present invention.

Sequence listing

<110> Nanjing university of agriculture

<120> recombinant swine lawsonia intracellularis Hsp60 protein monoclonal antibody and application thereof

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tttccaggaa acaaactgga gtggatgggc tacataacct acactggtga cactagctac 180

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Claims (9)

1. A recombinant pig lawsonia intracellularis Hsp60 protein monoclonal antibody is characterized in that a heavy chain variable region of the monoclonal antibody comprises HCDR1, HCDR2 and HCDR3, and amino acid sequences of the monoclonal antibody are respectively shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3; the light chain variable region of the monoclonal antibody comprises LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the light chain variable region are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

2. The recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody of claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID No.7, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID No. 8.

3. A nucleic acid molecule encoding the recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody of claim 1, wherein the nucleic acid molecule encodes the heavy chain variable region of the recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody as shown in SEQ ID No. 9; the nucleotide sequence of the variable region of the light chain of the monoclonal antibody of the nucleic acid molecule coding recombinant Lawsonia intracellularis Hsp60 protein is shown in SEQ ID NO. 10.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A kit comprising the recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody of claim 1.

7. The use of the recombinant lawsonia intracellularis Hsp60 protein monoclonal antibody of claim 1 in the preparation of a reagent for detecting lawsonia intracellularis.

8. Use according to claim 7, characterized in that it comprises the following steps:

the Hsp60 protein monoclonal antibody is used as a primary antibody, a FITC-labeled goat anti-mouse antibody is used as a secondary antibody, and the Lawsonia intracellularis is detected by adopting an IFA detection method.

9. Use according to claim 7, characterized in that it comprises the following steps:

culturing IPEC-J2 cells, adding bacterial liquid containing Lawsonia intracellularis LJS19052, and setting up negative control; adding the Hsp60 protein monoclonal antibody, and incubating; adding a goat anti-mouse antibody labeled with FITC, and incubating; adding DAPA staining solution, reacting at room temperature in a dark place, washing, taking a picture under a fluorescence inverted microscope, and observing the result; and (4) IFA result judgment: specific green fluorescence exists in the cytoplasm of the IPEC-J2 cell, namely the cell is positive, and non-specific green fluorescence exists in the cytoplasm, namely the cell is negative.

Images