Preparation method of recombinant protein of porcine intracellular lawsonia LsaA and monoclonal antibody thereof

Abstract

The application belongs to the technical field of veterinary biological products, and particularly relates to a preparation method of a Lawsonia intracellularis LsaA recombinant protein and a monoclonal antibody thereof. The application provides an optimized LsaA protein prokaryotic high-efficiency expression system, a high-specificity monoclonal antibody preparation method and application thereof, and meets the needs of rapid and accurate diagnosis and prevention and treatment of swine ileitis.

Description

Technical Field

[0001] This invention belongs to the field of veterinary biological product technology, specifically relating to a method for preparing a recombinant protein of Lawsonia intracellularis LsaA and its monoclonal antibody. Background Technology

[0002] Lawsonia intracellularis ( Lawsonia intracellularis Lawsonia intracellularis (LsaA) is an obligate intracellular parasitic bacterium that primarily resides within the epithelial cells of the intestinal mucosa, causing proliferative enteritis in pigs. Clinically, it leads to decreased appetite, diarrhea, and stunted growth in affected pigs, resulting in significant economic losses for the pig farming industry. Since its initial report in 1931, porcine proliferative enteritis has become prevalent in major pig-producing countries worldwide. However, due to the relatively low mortality rate caused by this disease, there is limited attention paid to Lawsonia intracellularis infection in my country, and a comprehensive and clear understanding of its prevalence in Chinese pig populations remains lacking. The main reason for this situation, besides insufficient attention, is the lack of adequate detection methods. LsaA is a surface antigen of Lawsonia intracellularis, synthesized during bacterial infection and involved in bacterial adhesion and invasion. Studies have demonstrated that oral inoculation of pigs and mice with Lawsonia intracellularis can detect antibodies against the LsaA protein in their plasma. Utilizing this protein as a detection antigen would provide a direct indication for the detection of Lawsonia intracellularis infection and is an ideal target for developing diagnostic reagents and subunit vaccines.

[0003] Currently, the diagnosis of Lawsonia intracellularis mainly relies on methods such as PCR and immunofluorescence, but these methods suffer from problems such as complex operation, high cost, or insufficient sensitivity. Preparing recombinant LsaA protein using a prokaryotic expression system and then using it to develop a monoclonal antibody for highly specific diagnosis is of great significance for the precise prevention and control of this disease. Summary of the Invention

[0004] To address the above problems, this invention provides a method for preparing recombinant Lawsonia intracellularis LsaA protein, comprising the following steps: (1) Synthesize the LsaA gene and amplify and recover the LsaA gene using primer pairs; (2) The gene obtained in step (1) is integrated into the pCold III plasmid vector by homologous recombination to obtain a recombinant plasmid; as a preferred embodiment, the plasmid vector is pCold III, which carries a His tag and a cold shock inducible promoter, and uses low temperature induction to reduce the protein from folding too quickly to form inclusion bodies, thereby increasing the soluble expression of the protein. (3) Transform the recombinant plasmid constructed in step (2) into the host bacterium BL21 to obtain bacterial culture; (4) Screening for recombinant bacteria carrying the LsaA gene and expressing LsaA protein in large quantities under IPTG induction; (5) Western blot detection of LsaA protein expression; (6) The purified LsaA protein was obtained by purifying it using His-tagged nickel affinity chromatography.

[0005] Furthermore, the base sequences of the primer pairs described in step (1) are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0006] This invention also provides a method for preparing an LsaA monoclonal antibody, comprising the following steps: (1) Animal immunization: The purified LsaA protein was emulsified with an equal volume of Freund's adjuvant and injected into 8-week-old female BALB / c mice at multiple sites. The mice were immunized once every two weeks for a total of 3 times. (2) Cell fusion: Spleen cells from mice that have been boosted with immunization for 3 days are mixed with SP2 / 0 cells in the logarithmic growth phase at a ratio of 5:1. Cell fusion is carried out under the action of PEG1500. The fused cell mixture is added to the feeder cells prepared one day in advance and cultured in HAT medium. (3) Subcloning screening: Positive wells with strong antibody secretion ability were screened by indirect ELISA method, and subcloning was performed by limiting dilution method. After three subclonings, a cell line that stably secretes antibodies was obtained. (4) Large-scale preparation of monoclonal antibodies: LsaA protein mAb was prepared in large quantities using the in vivo ascites induction method.

[0007] The present invention has the following beneficial effects: This invention provides an optimized prokaryotic high-efficiency expression system for LsaA protein, a method for preparing highly specific monoclonal antibodies, and their applications, meeting the needs for rapid and accurate diagnosis and prevention of porcine ileitis. Attached Figure Description

[0008] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0009] Figure 1 SDS-PAGE electrophoresis detection of LsaA protein induced expression.

[0010] Figure 2 SDS-PAGE electrophoresis was performed on purified LsaA protein.

[0011] Figure 3Chart showing the results of antiserum titer assay in mice immunized with LsaA protein.

[0012] Figure 4 Chart for screening LsaA monoclonal antibody cell lines.

[0013] Figure 5 Western blot identification of LsaA monoclonal antibody.

[0014] Figure 6 . Schematic diagram of the variable region sequence of the IgG heavy chain in LsaA mice.

[0015] Figure 7 . Schematic diagram of the variable region sequence of the IgG light chain in LsaA mice. Detailed Implementation

[0016] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Example 1: Construction of prokaryotic expression plasmid for LsaA protein Based on the LsaA genome sequence published in GenBank, Qingke Biotechnology Co., Ltd. was commissioned to synthesize the LsaA target gene after codon optimization according to the Escherichia coli host. Specific primers were designed, and the designed upstream and downstream primers each carried approximately 15-20 bp homologous arms of the pCold III vector. The LsaA gene was cloned into the prokaryotic expression vector pCold III using the 2×BasicAssembly Mix homologous recombinase of Total Gold. The cloned gene was transformed into competent Escherichia coli DH5α cells. After plasmid sequencing verification, the recombinant expression strain carrying the LsaA gene was obtained by transforming the prokaryotic expression strain BL21.

[0022] The primer pairs for amplifying the LsaA gene are as follows: SEQIDNO.1:AATCACAAAGTGCATatggctcacatagtaatggttgatg SEQIDNO.2:ATTCGGATCCCTCGAGCtcagtggtggtggtggtggtgctcgagcgggatatattcgg SEQ ID NO.3: ATGGCTCACATAGTAATGGTTGATGCATATAAGCCGACGAAAGGTGGCGGCGGCAGCAAGAAAAGTATCAAAGAGTACCTGATCGAGAAAAACTACAGCAATGCTGAGATCCTCATTCGTTCCGGTCAGGTTAAAATTAACAATGAGGTTGTTTTTACCCCGAGCTTCCTGATCAAGGACAGCGATAAAATCTTGATTGAGCAAAAAAAGGAGTTCGTCAGCCGTGGTGCGTATAAATTGCTGGCGGCGATAGAAGCCTTTAAACTTGATTTTGAAAACAAAGTCATTCTGGATATTGGTGCGAGCACTGGTGGCTTCAGCCAAGTTAGCCTGAATTATGGTGCGAAATTAGTTTACGCACTGGACGTTGGTAATAACCAGCTGGACTATAAACTGCGTCAAAACAGCAAGATTAAGTCCTTGGAACAAACCAATCTGAAGTCCATTTCCAAGCAGATGTTTGAAGTGGAAATTGACAAGGTCGTGTGCGATGTGTCCTTCATCAGCTTGAAGGAGGTGTACAAGGTGGTAGAGCATATTCTTAAGACCAACGATGATTGGATCGTTCTGCTGAAACCACAGTTCGAGGCATCGAGCAAGTACGTGGAGAAAGGCGGCTTCGTGCTGGAACAGTTCCACCCGTTTCTCATCGACCGCAGCATCTCTCAGGCTTCTGAACACAACTTCAAGTTTATCGACAAGATCACCTCTCCGATTAAAGGTCAAAAATCAAAGAACGCCGAATATATCCCGctcgagcaccaccaccaccaccactga Example 2 Expression and purification of LsaA protein (1) The recombinant expression bacteria constructed in Example 1 were activated and transferred to LB liquid medium containing ampicillin and cultured overnight in a shaker (37 ℃, 220 rpm). The bacterial culture was then transferred to new LB medium containing ampicillin at a volume ratio of 1:100 and cultured in a shaker until the OD600 value was ≈0.8. After cooling at room temperature for 30 min, IPTG was added to a final concentration of 0.5 mM and expression was induced at 16 ℃ and 220 rpm for 24 h. The bacterial cells were collected by centrifugation at 4 ℃ and 8000 rpm for 10 min. The bacterial cells were washed twice with PBS and resuspended with 1 / 10 volume of lysis buffer. The bacterial culture was sonicated at 200 W for 3 s and rest for 6 s until the bacterial culture was clear. The supernatant and precipitate were separated by centrifugation at 4 ℃ and 12000 rpm for 20 min. 100 μL of the supernatant was taken for SDS-PAGE electrophoresis to verify the protein expression. The results showed that the recombinant LsaA protein (approximately 33 kDa) could be expressed in E. coli BL21, as shown in the following figures. Figure 1 As shown.

[0023] (2) Purification of the supernatant obtained in (1) using a His-tagged nickel affinity chromatography column. The protein supernatant was added to a His-tagged nickel affinity chromatography column and incubated overnight at 4 °C; impurities were eluted with 20 mM imidazole washing buffer, and the target protein was eluted with 200 mM; high-purity LsaA protein was obtained by ultrafiltration and centrifugation to remove imidazole. The protein purification was detected by SDS-PAGE, and the results showed that recombinant LsaA protein could be effectively purified. The results are as follows. Figure 2 As shown.

[0024] Example 3: Detection of animal immunity and serum titer (1) Animal immunization The immunization dose was 50 µg antigen per mouse. The first administration was a plantar injection of the mouse after thoroughly mixing Freund's complete adjuvant with an equal volume of antigen. Subsequently, two booster injections were given after thoroughly mixing Freund's incomplete adjuvant with an equal volume of antigen. The injection sites were multiple injections on the back of the mouse for immunization, once every two weeks. To ensure the effectiveness of immunization, the mice could be given an intraperitoneal bolus immunization of LsaA protein (50 µg / mouse) three days before fusion.

[0025] (2) Antiserum titer detection After immunization, blood was collected from the tail vein, and the serum titers of the two mice were detected by indirect ELISA.

[0026] Dilute LsaA protein to 0.5 µg / mL using coating buffer, and add 100 µL / well to each well of a 96-well microplate. Coat overnight at 4°C, wash three times with PBST, and blot dry. Add 200 µL / well of freshly prepared 5% skim milk powder, block at 37°C for 2 h. Add 100 μL / well of serum at a 1:1000 initial serial dilution (1000, 2000, 4000, 8000, 16000, 32000, 64000), incubate at 37°C for 1 h, and wash three times with PBST. Add 100 μL of diluted goat anti-mouse HRP secondary antibody to each well, incubate at 37°C for 1 h, wash three times with PBST, develop with TMB, and read the OD. 450 Value. Based on the ELISA results, OD... 450 The dilution ratio corresponding to a value >0.2 is the titer of the mouse. A higher dilution ratio indicates a better titer. Figure 3 .

[0027] Example 4: Screening of LsaA monoclonal antibody cell lines (1) Hybridoma cell preparation One week prior to fusion, SP2 / 0 cells were resuscitated and cultured normally to the logarithmic growth phase. Mouse number 1 was selected for fusion. One day before fusion, a healthy, unimmunized Kunming mouse was euthanized by cervical insemination, and feeder cells were added to a 96-well plate. On the day of fusion, mouse number 1 was euthanized by enucleation and blood loss, and serum was collected as a positive control. The spleen was harvested, and spleen cells were collected and counted according to standard procedures.

[0028] Myeloma cells and spleen cells were mixed at a ratio of 1:5, and cell fusion was performed using standard procedures. The cells were then cultured in HATDMEM complete medium. Hybridoma cells were visible 4 days after fusion, and selection and detection began approximately 10 days post-fusion. Cell fusion results: After fusion, the cells were cultured in HAT selective medium, and multiple growing hybridoma cells were observed under a microscope, confirming the success of the fusion operation.

[0029] (2) Antibody titer detection method Prepare ELISA plates coated with antigen (0.5 µg / mL). Aspirate 100 µL of cell supernatant per well for indirect ELISA detection, including positive and negative controls. Incubate at 37 °C for 1 h. Wash the plate three times with PBST and blot dry. Dilute goat anti-mouse IgG-HRP 1:5000 with PBS and add 100 µL per well to a 96-well microplate. Incubate at 37 °C for 1 h. Wash five times with PBST and blot dry. Add 100 μL of TMB reaction solution per well and incubate at room temperature for 10 min. Add 50 μL of 2 M H₂SO₄ stop solution per well. Immediately after adding the stop solution, quickly measure the OD of each well using a microplate reader. 450 Value, record the data. If the OD of the hole to be measured... 450A P / N value greater than or equal to 2.1 times that of the negative control (i.e., P / N value > 2.1) is considered a positive result. Figure 4 A, the red-marked holes are positive.

[0030] (3) Subcloning screening For the positive wells of monoclonal cell clusters selected by indirect ELISA, subcloning was performed using the limiting dilution method. After three subcloning processes, two stable antibody-secreting cell lines were obtained, namely mAb-2E2 and mAb-3D4. Figure 4 BC, select the red markers for expanded culture to proceed to the next experiment.

[0031] (4) Large-scale preparation of monoclonal antibodies Large-scale preparation of LsaA protein mAb was performed using an in vivo ascites induction method. BALB / c mice were intraperitoneally injected with 0.5 mL of ascites adjuvant two weeks prior to preparation. Cells in logarithmic growth phase were resuspended and counted, and each female mouse was intraperitoneally injected with 1 x 102 cells. 6 Each mouse was tested for ascites. Approximately 5 mL of ascites fluid was collected after 5-7 days. The ascites fluid was purified using a Protein G affinity chromatography column to obtain high-purity monoclonal antibodies.

[0032] Example 5: Identification of Monoclonal Antibodies The recombinant LsaA protein was subjected to SDS-PAGE electrophoresis and transferred to a membrane. High-titer antibodies against mAb-2E2 and mAb-3D4 strains were selected as primary antibodies and incubated overnight at 4 °C. Western blot analysis showed that the mAbs specifically recognized the recombinant LsaA protein, such as... Figure 5 A, 5B.

[0033] Example 6: Light and heavy chain sequencing of the prepared monoclonal antibody. After the final subclone-positive mAb-2E2 hybridoma cell line was expanded and cultured until the cell number reached approximately 1 x 10⁷, it was centrifuged at 1000 rpm for 10 min, the cells were collected, and sent to a sequencing company for sequencing. The LsaA mAb-2E2 heavy and light chain sequences are shown below. The sequencing results are as follows. Figure 6-7 .

[0034] 1. Re-link information (1) The heavy chain variable region base sequence of the heavy chain sequencing: GAGGTTCAGCTGCAACAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTCTTTAACATTAAAGACGACTATATACACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAAAATGTTGATACTGA ATATGCCTCGAAGTTCCAGGGCAAGGCCACTATAACAGTAGACACATCCTCCAACACAGCCTACCTGCAATTAAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGACTACACCCAATGTCTGGTITGCTTTCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (2) The amino acid sequence of the heavy chain variable region of the heavy chain sequencing: EVOLQOSGAELVRPGASVKLSCTASGENIKDDYIHWVKORPEOGLEWIGWIDPENVDTEYASKFOGKATITVDTSSNTAYLOLSSLTSEDTAVYYCTTPNVWFAFWGOGTLVTVSA Compared with the mouse proteome, the above sequence is the variable region sequence of the mouse IgG heavy chain, and its specific structure is described in [reference needed]. Figure 6 .

[0035] exist Figure 6 middle: CDR1: GFNIKDDY CDR2: IDPENVDT CDR3: TTPNVWFAF 2 Light Chain Information (1) The light chain variable region base sequence result of the light chain sequencing is as follows: GATGTITTGCTGACCCAAATGCCACTCTTTCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCITGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCIGATCTACAAAGTT TCCAACCGATTTTCTGGGGTCCCAGACAGGGTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGATGGAGGCTGAGGATCTGGGAGTTTATTGTTTTCAAGGTTCACATGTACCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (2) The amino acid sequence of the light chain variable region of the light chain sequencing: DVLLTOMPLFLPVSLGDOASISCRSSOSIVHSNGNTYLEWYLOKPGOSPKLLIYKVSNRFSGVPDRVSGSGSGTDETLKISRVEAEDLGVYYCFOGSHVPYTFGGGTKLEIK Compared with the mouse proteome, the above sequence is the variable region sequence of the mouse IgG light chain, and its specific structure is described in [reference needed]. Figure 7 .

[0036] exist Figure 7 middle: CDR1: QSIVHSNGNTY CDR2: KVS CDR3: FQGSHVPYT

Claims

1. A method for preparing recombinant Lawsonia intracellularis LsaA protein, characterized in that, Includes the following steps: (1) Synthesize the LsaA gene and amplify and recover the LsaA gene using primer pairs; (2) The gene obtained in step (1) is integrated into the pCold III plasmid vector by homologous recombination to obtain a recombinant plasmid; as a preferred embodiment, the plasmid vector is pCold III, which carries a His tag and a cold shock inducible promoter, and uses low temperature induction to reduce the protein from folding too quickly to form inclusion bodies, thereby increasing the soluble expression of the protein. (3) Transform the recombinant plasmid constructed in step (2) into the host bacterium BL21 to obtain bacterial culture; (4) Screening for recombinant bacteria carrying the LsaA gene and expressing LsaA protein in large quantities under IPTG induction; (5) Western blot detection of LsaA protein expression; (6) The purified LsaA protein was obtained by purifying it using His-tagged nickel affinity chromatography.

2. The preparation method according to claim 1, characterized in that, The base sequences of the primer pair described in step (1) are shown in SEQ ID NO.1 and SEQ ID NO.

2.

3. A method for preparing an LsaA monoclonal antibody, characterized in that, Includes the following steps: (1) Animal immunization: The purified LsaA protein was emulsified with an equal volume of Freund's adjuvant and injected into 8-week-old female BALB / c mice at multiple sites. The mice were immunized once every two weeks for a total of 3 times. (2) Cell fusion: Spleen cells from mice that have been boosted with immunization for 3 days are mixed with SP2 / 0 cells in the logarithmic growth phase at a ratio of 5:

1. Cell fusion is carried out under the action of PEG1500. The fused cell mixture is added to the feeder cells prepared one day in advance and cultured in HAT medium. (3) Subcloning screening: Positive wells with strong antibody secretion ability were screened by indirect ELISA method, and subcloning was performed by limiting dilution method. After three subclonings, a cell line that stably secretes antibodies was obtained. (4) Large-scale preparation of monoclonal antibodies: LsaA protein mAb was prepared in large quantities using the in vivo ascites induction method.

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