Functional domain and determination method for the specific binding of Mycoplasma penetrant P35 lipoprotein to receptor protein on SV-HUC-1 cell membrane

Abstract

This invention belongs to the fields of pathogenic biology and medical technology, specifically relating to the functional domain and method for determining the specific binding of Mycoplasma penetratingis P35 lipoprotein to receptor proteins on the SV-HUC-1 cell membrane. This invention elucidates the mechanism by which Mpe adheres to and invades host cells via P35, identifies the ACTG1 protein as the main receptor for Mpe-P35 protein adhesion to SV-HUC-1 cells, and further identifies the actin binding site of Mpe-P35, specifically amino acids 35-42 and 179-186 of the Mycoplasma penetratingis P35 lipoprotein, providing the specific peptide sequence. This provides a theoretical basis for further research on the treatment or prevention of Mycoplasma penetratingis.

Description

Technical Field

[0001] This invention belongs to the fields of pathogenic biology and medical technology, and specifically relates to the functional domain and method for determining the specific binding of Mycoplasma penetrant P35 lipoprotein to receptor protein on SV-HUC-1 cell membrane. Background Technology

[0002] Mycoplasma penetratingense (Mpe) is a mycoplasma species first isolated from the urine of a human immunodeficiency virus (HIV) infected individual. This mycoplasma possesses a unique elongated flask-like morphology and pointed structure, and both in vitro and in vivo experiments have confirmed its ability to invade cells. The connection between Mpe and host target cells is via lipid-associated membrane proteins (LAMPs), particularly the P35 lipoprotein exposed on the cell surface. P35 lipoprotein is an immunodominant antigen and a major candidate antigen for the serological diagnosis of Mpe infection. Therefore, P35 lipoprotein plays a crucial role in Mpe infection and host adhesion. However, research on the receptor protein and binding domain of P35 lipoprotein is currently limited. This study aimed to screen for the receptor protein of Mpe-P35 on the membrane of human urethral epithelial cells (SV-HUC-1 cells) and identify its interaction with the receptor protein and its binding domain. Summary of the Invention

[0003] This invention expresses and purifies recombinant protein P35 (rP35) containing all amino acids of P35, and screens for receptor proteins that interact with rP35 from membrane proteins of human urethral epithelial cells (SV-HUC-1) using a modified virus-on-plasma binding assay (VOPBA). High-performance liquid chromatography-mass spectrometry (HPLC-MS) of the screened proteins revealed that γ-actin (ACTG1) and keratin 8 (KRT8) are likely the main receptor proteins. Subsequently, four independent experimental techniques confirmed that rP35 specifically binds to the ACTG1 protein on the host cell membrane without interacting with KRT8. Adhesion-adhesion inhibition assays further demonstrated that ACTG1 is the main receptor for Mpe-P35 protein adhesion to SV-HUC-1 cells. To further understand the main binding regions with host cells, the actin binding site of Mpe-P35 was identified using a truncated Mpe-P35 molecule. The results confirmed two possible actin binding sites located between amino acids 35-42 and 179-186 of the Mpe-P35 molecule. This study elucidates the mechanism by which Mpe adheres to and invades host cells via P35, laying an experimental foundation for the effective prevention and treatment of Mpe infection.

[0004] The specific technical solution of the present invention is: the use of amino acids 35-42 of the penetrant mycoplasma p35 lipoprotein as a functional domain that specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane, wherein the peptide sequence of amino acids 35-42 is SENNGNGN.

[0005] The use of amino acids 179-186 of the penetrant mycoplasma p35 lipoprotein as a functional domain that specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane, wherein the peptide sequence of amino acids 179-186 is PNLKLNNG.

[0006] The beneficial effects of this technical solution are as follows: it elucidates the mechanism by which Mycoplasma penetrating our bodies adhere to and invade host cells via P35, identifies the ACTG1 protein as the main receptor for Mpe-P35 protein adhesion to SV-HUC-1 cells, and further identifies the actin binding site of Mpe-P35, specifically amino acids 35-42 and 179-186 of the Mycoplasma penetrating our bodies P35 lipoprotein, and provides the specific peptide sequence. This provides a theoretical basis for further research on the treatment or prevention of Mycoplasma penetrating our bodies. Attached Figure Description

[0007] Figure 1 The results of the expression and purification of recombinant P35 protein;

[0008] Figure 2 The experimental results are for the preparation and purification of rabbit polyclonal antibodies against recombinant P35 protein;

[0009] Figure 3 The results of adhesion and adhesion inhibition experiments of Mpe and recombinant P35 proteins;

[0010] Figure 4 Experimental results for the identification of P35-specific binding proteins using improved VOPBA and HPLC-MS;

[0011] Figure 5 The results of experiments for the identification of the target protein and its distribution in cells;

[0012] Figure 6 The results are from an indirect ELISA experiment;

[0013] Figure 7 The results are from an immunofluorescence co-localization experiment.

[0014] Figure 8 Experimental results showing the effects of ACTG1 and its antibody on the adhesion of rP35 and Mpe;

[0015] Figure 9The experimental results show the effects of ACTG1 interference on the adhesion of Mpe and rP35 to SV-HUC-1 cells.

[0016] Figure 10 Analysis of the secondary structure and hydrophilicity / hydrophobicity of Mpe-P35;

[0017] Figure 11 To analyze the binding of the truncated Mpe-P35 peptide to ACTG1;

[0018] Figure 12 To indirectly detect the adhesion of rP35 to SV-HUC-1 cells treated with synthetic peptides using immunofluorescence. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. However, the scope of the present invention is not limited to the following embodiments.

[0020] The present invention aims to identify the specific receptor protein on the SV-HUC-1 cell membrane that interacts with Mycoplasma penetratingis P35 lipoprotein, and further determine the functional domain in which Mycoplasma penetratingis P35 lipoprotein specifically binds to the receptor protein. The overall approach is as follows:

[0021] Step 1. Expression and purification of Mycoplasma penetrant P35 lipoprotein: Induced expression and purification of recombinant protein P35 containing all amino acids of P35, the obtained recombinant protein P35 was concentrated and its concentration was identified;

[0022] Step 2. Preparation of recombinant protein P35 antibody: Recombinant protein P35 was mixed with Freund's adjuvant and used to immunize rabbits. Rabbit serum was collected after immunization. The immunoglobulin fraction was crudely purified by continuous ammonium sulfate precipitation. Then, the antibody specific to recombinant protein P35 was purified by coupling and elution with cyanogen bromide activated agarose 4B.

[0023] Step 3. Determine whether P35 is an adhesion-related protein of Mycoplasma penetrating to SV-HUC-1 cells: Culture SV-HUC-1 cells and Mycoplasma penetrating to cells separately; detect the adhesion of recombinant protein P35 and Mycoplasma penetrating to SV-HUC-1 cells by indirect immunofluorescence assay, and detect whether the recombinant protein P35 antibody prepared in step 2 can inhibit the adhesion of Mycoplasma penetrating to SV-HUC-1 cells;

[0024] Step 4. Analyze the receptor proteins in the SV-HUC-1 cell membrane that may specifically bind to recombinant protein P35: SDS-PAGE analysis was performed on SV-HUC-1 cell membrane proteins, and then HPLC-MS analysis was performed on the strips near 40kDa to 55kDa corresponding to the obvious bands in the modified VOPBA experiment. The receptor proteins in the SV-HUC-1 cell membrane that may specifically bind to recombinant protein P35 were identified as ACTG1 and KRT8.

[0025] Step 5. Identify the presence of receptor proteins ACTG1 and KRT8 on the SV-HUC-1 cell membrane and determine their distribution: Western blotting was used to identify the receptor proteins, and their distribution in the SV-HUC-1 cell membrane was observed by indirect immunofluorescence to determine the localization of receptor proteins ACTG1 and KRT8 on SV-HUC-1 cells.

[0026] Step 6. Verify whether the two receptor proteins ACTG1 and KRT8 can interact with recombinant protein P35: Four independent experimental methods, namely Far-western blotting, indirect ELISA, immunoprecipitation and immunofluorescence co-localization, were used to determine that recombinant protein P35 can specifically bind to ACTG1 but does not interact with KRT8.

[0027] Step 7. Verify whether ACTG1 affects the adhesion of Mycoplasma penetratingus and recombinant protein P35 to SV-HUC-1 cells: Adhesion-adhesion inhibition assay and indirect immunofluorescence were used to observe the effects of ACTG1 and its antibody on the adhesion of Mycoplasma penetratingus and recombinant protein P35 to SV-HUC-1 cells. Finally, it was determined that ACTG1 protein is the main receptor for Mpe-P35 protein adhesion to SV-HUC-1 cells.

[0028] Step 8. Identify the binding domain of recombinant protein P35 to ACTG1: Synthesize all truncated P35 peptides that may be associated with binding. Indirect ELISA and Far-western blotting are used to detect the binding of ACTG1 to the synthesized peptides. Indirect immunofluorescence is used to detect the effect of the synthesized peptides on the adhesion of rP35 to SV-HUC-1 cells. Finally, the functional domain that specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane of Mycoplasma penetrant P35 lipoprotein is identified.

[0029] The specific implementation method is as follows: passage and culture of 1SV-HUC-1 cells

[0030] SV-HUV-1 cells (human immortalized urethral epithelial cells) were adherent cells and cultured in F-12K (Gibco, 21127022) medium.

[0031] 1.1 Cell resuscitation

[0032] Remove the SV-HUV-1 cells frozen at -80℃ and gently agitate them in a 37℃ water bath for about 1 minute. Once the liquid in the cryovial has thawed to the size of rice grains, transfer the liquid to a 15mL sterile centrifuge tube containing 2mL of F-12K complete medium under aseptic conditions. Gently mix. Centrifuge at 1200rpm for about 7 minutes, discard the supernatant, add 3mL of F-12K complete medium to resuspend the cells, and then transfer them to a 25cm² cell culture flask. Incubate at 37℃ in a 5% CO2 incubator.

[0033] 1.2 Cell passage

[0034] After culturing SV-HUV-1 cells at 37℃ in a 5% CO2 incubator for 12 hours, cell adhesion was observed. Fresh F-12K complete culture medium was then added. When cell adhesion reached 80%–90%, the cells needed to be digested and passaged. The specific steps were as follows: Discard the old culture medium in the cell culture flask, wash the cells twice with 2 mL of PBS, add 800 μL of trypsin digestion solution, and incubate the cells at 37℃ for 5 minutes. Observe under a microscope when the cells become rounded and a few cells are floating. Add 2 mL of complete culture medium to stop the digestion. Gently blow away the cells from the flask wall, and after the suspension is mixed, aliquot the cells into cell culture flasks at a 1:1 ratio. Add 2 mL of complete culture medium to each flask and continue culturing in the incubator.

[0035] 1.3 Cell cryopreservation

[0036] Cells in good condition and in the logarithmic growth phase were cryopreserved. The digestion procedure was the same as in 3.2.1. After digestion was terminated with complete culture medium, the cells were resuspended and transferred to a sterile centrifuge tube. The tube was centrifuged at 1200 rpm for 7 min, the supernatant was discarded, 1 mL of serum-free cell cryopreservation solution was added and mixed well. The cells were then transferred to a sterile cryopreservation tube, labeled, and quickly stored at -80°C.

[0037] 2. Passage and culture of Mpe

[0038] The Mpe GTU-54-6Al standard strain was cultured using SP-4 medium. Mpe samples were removed from a -80℃ freezer and allowed to thaw at room temperature. Then, in a clean bench, they were subcultured at a medium-to-bacterial-solution ratio of 10:1, i.e., 5 mL of SP-4 medium and 500 μL of Mpe bacterial solution were added to a 10 mL sterile test tube. The tubes were then incubated at 37℃. After approximately 6–8 days of growth in the incubator, the SP-4 medium changed from red to orange-yellow, indicating that the Mpe had entered the logarithmic growth phase. At this point, the Mpe samples were collected for further experiments.

[0039] 3. Expression and purification of recombinant p35 protein

[0040] 3.1 Induced expression of recombinant P35 protein

[0041] 1) RosettaDE3 bacteria containing pET-30a / P35 prokaryotic plasmid were inoculated onto solid LB plates containing kanamycin and incubated overnight at 37°C.

[0042] (2) Use an inoculation loop to scrape a single colony into 6 mL of LB liquid medium (containing kanamycin) and incubate at 37°C and 220 rpm for at least 12 h with constant temperature shaking.

[0043] (3) Transfer the cultured bacterial solution to 20 mL of LB liquid medium (containing kanamycin) and allow it to undergo OD200 reaction. 600 ≈0.2, incubate at 37℃ with shaking at 220 rpm until OD 600 ≈0.6;

[0044] (4) IPTG added to OD 600 In a bacterial culture with a concentration of approximately 0.6, IPTG was added to a final concentration of 0.5 mmol / L, and expression was induced for 4 h at 37°C and 180 rpm.

[0045] (5) Collect the bacterial solution at 4℃ and 10000rpm, wash with PBS 3 times, resuspend the precipitate in 300μL of PBS containing 0.1mmol / L PMSF, and sonicate at low temperature (40% power, vibrate for 8s, pause for 8s) until the bacterial solution becomes clear.

[0046] (6) Centrifuge the bacterial culture at 12000 rpm for 15 min at 4℃ and collect the supernatant and precipitate. Resuspend the precipitate in 100 μL PBS.

[0047] (7) Take 20 μL of the precipitate and the supernatant respectively, mix them with 5×SDS loading buffer, boil for 10 min, and take 10 μL for SDS-PAGE analysis.

[0048] 3.2 Protein purification

[0049] (1) Induce expression in 600 mL of bacterial culture according to the method in 3.3.1, and collect the supernatant of the lysate;

[0050] (2) Fill the chromatography column with 2 mL of nickel column, wash with 10 mL of deionized water and then wash with pre-equilibration solution. Add 5 mL of target protein supernatant to the chromatography column and mix thoroughly with the nickel column. Incubate on ice with shaking for 6 h.

[0051] (3) Collect the eluent and elute the target protein twice with 20 mmol / L imidazole elution buffer. Then elute once with gradients of 60, 80, 90, 100 and 150 mmol / L imidazole elution buffers respectively. Collect the eluent.

[0052] (4) Take 20 μL of the elution solution after each concentration and mix it with 5×SDS loading buffer. Boil for 10 min and perform SDS-PAGE electrophoresis to analyze the optimal elution concentration.

[0053] 3.3 Protein Concentration and Concentration Identification

[0054] (1) Rinse the 10kD ultrafiltration tube with 15mL of sterile pre-cooled PBS, add 15mL of protein sample, and centrifuge at 4℃ and 4000rpm for 20min.

[0055] (2) Add 8 mL of sterile PBS to wash the protein, centrifuge at 4000 rpm for 20 min at low temperature, remove the filtrate, repeat this step 2-3 times, and extend the ultrafiltration time appropriately for the last time to concentrate the protein to 1 mL;

[0056] (3) Collect the liquid in the ultrafiltration tube into a 1.5 mL EP tube, add 1 μL of polymyxin B with a concentration of 100 mg / mL, mix well, and place on ice and shake for 2 hours to remove endotoxins.

[0057] (4) After detecting the protein concentration using the BCA kit, the protein was sterilized, filtered, aliquoted, and frozen to -80°C for storage.

[0058] Experimental results of expression and purification of recombinant p35 protein:

[0059] The pET-30a(+) vector containing the complete Mpe P35 gene was transformed into RosettaDE3 bacteria. The bacterial culture was induced with 0.5 mmol / L IPTG for 4 h. SDS-PAGE analysis was performed on the precipitate and supernatant after ultrasonic lysis. Results are as follows: Figure 1 As shown in Figure -A, compared with the blank control group, the bacterial culture with added IPTG showed a clear band around 35 kDa in both the supernatant and precipitate. The recombinant P35 protein was induced to express in large quantities, purified using a nickel column, eluted with imidazole, and the eluent was analyzed by SDS-PAGE. The results are shown in Figure -A. Figure 1As shown in -B, the protein purity was high when the concentration of eluting imidazole reached 60 mmol / L. The rP35 eluent was ultrafiltered, concentrated, and subjected to SDS-PAGE; the results are as follows. Figure 1 As shown in Figure -C, a clear protein band of high purity is visible at 35 kDa. Subsequently, the ultrafiltered sample was transferred to a membrane via electrophoresis and Western blotting was performed using 6X-His antibody as the primary antibody. The results are shown in Figure -C. Figure 1 As shown in Figure D, compared with the empty E. coli suspension (D-1) and the group without IPTG induction (D-3), the purified rP35 group (D-2) showed a clear band at a molecular weight of 35 kDa, indicating that the recombinant P35 protein with the His tag was successfully expressed and purified. BCA assay showed that the concentration of rP35 protein after ultrafiltration reached 1.8 mg / mL.

[0060] 4. Preparation and purification of recombinant rabbit polyclonal antibody against p35 protein

[0061] 4.1 Immunized Animals

[0062] Three 8-week-old female New Zealand rabbits weighing 2.5 kg were purchased and immunized after 4 days of rearing. 250 μL of rP35 protein (2 mg / mL) was mixed with 250 μL of Freund's complete adjuvant. A control group was also established, where equal volumes of PBS and Freund's complete adjuvant were mixed and vortexed for 20 minutes, then allowed to stand for 1 minute; no stratification indicated complete emulsification. Blood was collected from the ear arteries of the New Zealand rabbits to collect pre-immunization serum. Two rabbits were then immunized with the rP35 protein-adjuvant mixture (experimental group), and the other two were immunized with the PBS-adjuvant mixture. The rabbits were immunized subcutaneously and intramuscularly at multiple sites (100 μL of the mixture was injected into each thigh muscle, and 50 μL was injected subcutaneously into the back of the neck, achieving a total protein concentration of 200–250 μg / rabbit). Two weeks later, the experimental group was immunized with a mixture of Freund's incomplete adjuvant and rP35, while the control group was immunized with a mixture of Freund's incomplete adjuvant and PBS. The immunization method was the same as before, and immunization was continued for 3 times. Blood from the ear artery was collected before each immunization. Two weeks after the fourth immunization, the New Zealand rabbits underwent carotid artery cannulation, and serum was collected.

[0063] 4.2 Detection of rabbit polyclonal antibody titer for rP35 protein

[0064] (1) Dilute rP35 to 10 μg / mL with 1× coating buffer, take 150 μL / well to coat a 96-well plate, seal it and incubate overnight at 4°C in a humidified chamber.

[0065] (2) Discard the coating solution in the plate, pat dry thoroughly on absorbent paper, add 200 μL / well PBST and wash 4 times, 3 min each time;

[0066] (3) After tapping the plate, add 150 μL / well of 5% skim milk (1 g skim milk + 20 mL PBST) and block at 37°C for 2 h;

[0067] (4) Wash three times with 200 μL / well PBST, 3 min each time;

[0068] (5) Dilute the collected rabbit serum with blocking buffer at a ratio of 1:2500, and then perform serial dilutions until 1:1280000. Add the serum to each well of the ELISA plate in sequence, with three replicates per group, and incubate at 37°C for 2 hours.

[0069] (6) Wash 6 times with 200 μL / well PBST, 4 min each time;

[0070] (7) Add HRP-conjugated goat anti-rabbit secondary antibody diluted 1:5000 and incubate at 37°C for 1 h;

[0071] (8) Wash 6 times with 200 μL / well PBST, 4 min each time;

[0072] (9) Add 100 μL of TMB chromogenic solution to each well, incubate at 37°C in the dark for 15 min, then stop the reaction by adding 50 μL of stop solution per well. Detect the A in each well using a microplate reader. 450 value.

[0073] 4.3 Purification of rabbit-derived polyclonal antibody against rP35 protein

[0074] 4.3.1 Preliminary purification of antiserum (rP35 polyclonal antibody)

[0075] (1) Centrifuge the rabbit serum in the experimental group at 4℃ and 12000rpm for 30min. Take 20mL of supernatant and mix it with an equal amount of sterile PBS and put it into a beaker. Then add 40mL of saturated ammonium sulfate solution to the beaker to prepare a 50% ammonium sulfate solution. Stir thoroughly during the addition process. After stirring evenly, dispense into 50mL centrifuge tubes and place at 4℃ to precipitate overnight.

[0076] (2) After precipitation, centrifuge at 3000 rpm for 20 min at low temperature; discard the supernatant, add 24 mL of sterile PBS to dissolve all the precipitate, and after the precipitate is completely dissolved, add 12 mL of saturated ammonium sulfate solution to prepare a 33% ammonium sulfate solution. Precipitate at 4℃ for more than 3 hours.

[0077] (3) Repeat step (2) 2 to 3 times;

[0078] (4) After precipitation, centrifuge at 3000 rpm for 20 min at low temperature; discard the supernatant and add 12 mL of sterile PBS to dissolve the precipitate. At this time, the preliminarily purified rP35 polyclonal antibody was obtained.

[0079] 4.3.2 Purification of antiserum (rP35 polyclonal antibody)

[0080] (1) Weigh 1g of agarose 4B powder activated by cyanogen bromide and put it into the chromatography column. Add 2mL of HCl (pH=2) solution to the column and allow the agarose to swell at 4℃ for 30min. Then wash thoroughly with HCl solution 3 times.

[0081] (2) After washing, add 3 mL of 0.1 mol / L NaHCO3 solution (containing 0.5 mol / L NaCl) at pH 8.3 mixed with rP35 protein, mix well and couple overnight at 4℃;

[0082] (3) After the reaction is complete, wash off the excess ligand with 5 times the volume of sterile PBS;

[0083] (4) Add 3 mL of Tris-HCl buffer (pH 8.0) to the washed gel and block it overnight at 4°C to block residual active groups;

[0084] (5) Wash with blocking buffer containing 0.5 mol / L NaCl and 0.1 mol / L acetate buffer (pH 4.0) alternately, then wash twice with sterile PBS. After washing, add the preliminarily purified antibody and incubate at 4°C overnight.

[0085] (6) Wash the chromatography column 4 times with sterile PBS, add 4 mL of 0.1 mol / L Gly-HCl buffer (pH 2.4) to the column for desorption for 5 min, and immediately neutralize with 1 mol / L NaHCO3;

[0086] (7) Take 20 μL of desorbed protein and mix it with 5 μL of protein loading buffer (5×). Boil for 10 min and then perform SDS-PAGE and Western blotting analysis.

[0087] Experimental results on the preparation and purification of recombinant p35 protein rabbit polyclonal antibody:

[0088] To prepare rP35 antibodies, New Zealand rabbits were immunized every two weeks with a mixture of rP35 and adjuvant. Rabbit serum was collected via the marginal ear artery, and antibody titers were detected using indirect ELISA. Results are as follows: Figure 2 As shown in Figure A, after four immunizations of New Zealand rabbits, the specific antibody titer in the serum reached 1:2,560,000. Rabbit serum was collected after four immunizations via carotid artery cannulation. The immunoglobulin fraction was crudely purified by continuous ammonium sulfate precipitation, followed by cyanogen bromide-activated agarose gel coupling and elution. The eluted antibodies were detected by SDS-PAGE, and the results are shown below. Figure 2As shown in Figure -B, after reduction with 2-mercaptoethanol, the rP35 polyclonal rabbit antibody showed heavy and light chains at molecular weights of approximately 50 kDa and 25 kDa, indicating good antibody purification. Subsequently, the recombinant protein rP35 was transferred to a membrane via electrophoresis, and the specificity of the antibody was verified by Western blotting using the purified antibody as the primary antibody. The results are as follows... Figure 2 As shown in Figure C-C, compared with the empty E. coli suspension group (C-1), the rP35 group (C-2) after induction expression and purification showed a clear band at a molecular weight of 35 kDa, indicating that the rabbit-derived rP35 polyclonal antibody was successfully prepared and purified.

[0089] Adhesion and adhesion inhibition experiments of 5Mpe and recombinant p35 proteins

[0090] (1) Plating: Take a bottle of SV-HUC-1 cells in good growth condition (cell mass of 1x10⁻¹) 7 After digestion, add 4 mL of complete culture medium and mix well by pipetting; add 200 μL of cell suspension to the wells of a 24-well plate with sterile climbing smears, then add 800 μL of LF12-K complete culture medium, mix well and incubate at 37°C in a 5% CO2 incubator for more than 8 hours.

[0091] (2) Divide the cells into groups for experiments:

[0092] ①rP35 adhesion group: After replacing the culture medium with fresh one, add 50 μg / mL of rP35 to the well and incubate overnight at 37°C;

[0093] ②Mpe adhesion group: After replacing with fresh culture medium, add 1×10 to the wells 7 Mpe at CCU / mL, incubated overnight at 37°C;

[0094] ③ Antibody adhesion inhibition group: Before incubating with SV-HUC-1 cells, Mpe and rP35 specific rabbit anti or pre-immunization serum were pre-incubated at 37°C for 2 hours, and then incubated with cells after changing to fresh culture medium.

[0095] ④ Blank control group: Only fresh culture medium was used, and no other treatment was performed;

[0096] (3) Fixation: Wash cells with PBS 3 times for 2 min each time. After washing, add about 150 μL of 4% paraformaldehyde to each well and fix cells at 4℃ for 30 min.

[0097] (4) Blocking: Wash 4 times with PBS for 3 min each time. After washing, add about 150 μL of F-12K complete medium to each well and block at 37℃ for 1 h.

[0098] (5) Primary antibody: Wash 3 times with PBS for 3 minutes each time, and add different primary antibodies according to cell grouping;

[0099] ①rP35 adhesion group: Add 150μL of rP35 rabbit antibody (1:50) and incubate at 37℃ for 2h;

[0100] ②Mpe adhesion group: Add 150 μL of Mpe rabbit anti-antibody (1:10000) and incubate at 37℃ for 2 h;

[0101] ③ Antibody adhesion inhibition group: Add 150 μL of Mpe rabbit antibody (1:10000) and incubate at 37℃ for 2 h;

[0102] ④ Blank control group: Add 150 μL of rP35 rabbit antibody (1:50) or Mpe rabbit antibody (1:10000) and incubate at 37℃ for 2 h;

[0103] (5) Secondary antibody: Wash 5 times with PBS for 4 min each time. After washing, add 150 μL of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) and stain at 37°C for 1 h in the dark.

[0104] (6) Nuclear staining: Wash 5 times with PBS for 4 min each time. After washing, add DAPI (150 μL / well) and incubate at 37°C for 10 min in the dark.

[0105] (7) Slide preparation: Wash with PBS 5 times, 4 min each time. Drop 1 μL of anti-fluorescence quencher onto the slide, take out the slide, place the cell side on the anti-fluorescence quencher, and seal the slide with nail polish. Observe with an inverted fluorescence microscope.

[0106] Results of adhesion and adhesion inhibition experiments of Mpe and recombinant P35 proteins:

[0107] To verify whether p35 acts as an adhesion-associated protein mediating the adhesion of Mpe to SV-HUC-1 cells, indirect immunofluorescence was used to observe the adhesion of rP35 and Mpe to cells. The results showed that: ① In the rP35 adhesion experiment to SV-HUC-1 cells, obvious red fluorescence was visible on the surface of SV-HUC-1 cells incubated with rP35 (…). Figure 3 -B), while no obvious red fluorescence was observed on the cell surface of the blank control group that was not incubated with rP35 ( Figure 3 -A) indicates that rP35 can adhere to the surface of SV-HUC-1 cells and that the antibody does not undergo non-specific binding. ② In the adhesion assay of SV-HUC-1 cells to Mpe, obvious red fluorescence was visible on the surface of SV-HUC-1 cells incubated with Mpe ( Figure 3 -D), no obvious red fluorescence was observed on the cell surface of the blank control group that was not incubated with Mpe ( Figure 3-C), indicating that Mpe can adhere to the surface of SV-HUC-1 cells and that the antibody does not undergo non-specific binding. ③ Adhesion inhibition assay to detect whether rP35 rabbit antibody can prevent the binding of Mpe to SV-HUC-1 cells. Compared with the Mpe adhesion group incubated with Mpe ( Figure 3 Compared to pre-immunized serum and Mpe-pretreated SV-HUC-1 cell membranes, no significant change was observed in the red fluorescence of the cell membrane. Figure 3 -E), while the red fluorescence on the surface of SV-HUC-1 cell membranes pretreated with rP35 rabbit anti-Mpe was significantly reduced ( Figure 3 -F). The mean integrated optical density of images from the Mpe adhesion group and the antibody adhesion inhibition group was obtained using ImageJ, and then the staining intensity of rP35 or Mpe was quantitatively analyzed. The results are as follows: Figure 3 As shown in Figure G, there was no statistically significant difference between pre-immune serum and the Mpe pretreatment group (F) and the Mpe adhesion group (D) (P > 0.05), while there was a statistically significant difference between rP35 rabbit antibody and the Mpe pretreatment group (E) and the Mpe adhesion group (D) (P < 0.0001), indicating that rP35 antibody can inhibit Mpe adhesion to SV-HUC-1 cells. These experimental results demonstrate that P35 is a protein related to Mpe adhesion to SV-HUC-1 cells.

[0108] 6. Improved VOPBA and HPLC-MS for the identification of P35-specific binding proteins

[0109] 6.1 Extraction of SV-HUV-1 cell membrane proteins

[0110] (1) Take 12 bottles of cells in good growth condition, digest them with trypsin, collect the cell suspension into centrifuge tubes, and centrifuge at 1200 rpm for 8 min.

[0111] (2) Wash the cells twice with PBS, then add 1 mL of a mixture of cell lysis buffer and PMSF at a ratio of 100:1, and lyse the cells by horizontal shaking on ice for 20 min.

[0112] (3) Ultrasonic fragmentation on ice for 8 minutes, power at 30%, over 5 seconds, stop for 15 seconds;

[0113] (4) Centrifuge at 4℃ and 2000 rpm for 10 min, and collect the supernatant;

[0114] (5) Centrifuge the collected supernatant (16000g) for 30 min, carefully remove the supernatant, resuspend the precipitate with 200μLPBS, determine the protein concentration of the resuspended liquid and perform SDS-PAGE analysis.

[0115] 6.2 Improved VOPBA screening for cell membrane proteins that specifically bind to rP35

[0116] (1) After determining the concentration, add the protein to 5× protein loading buffer, mix well, boil for 10 min, take 10 μL for SDS-PAGE, and set up a control group at the same time.

[0117] (2) Cut two PVDF membranes of 6.5×2cm in size, soak them in methanol for 20 seconds, and then place them in transfer buffer for 10 minutes to equilibrate.

[0118] (3) After electrophoresis, cut the target band gel and place it in the transfer buffer. In the semi-dry transfer instrument, place the transfer filter paper, PVDF membrane, gel, and transfer filter paper from bottom to top for transfer. Avoid generating air bubbles during the process. Transfer conditions: 15V, 30min.

[0119] (4) After the transfer, rinse the PVDF membrane twice with TBST and place it in 5% skim milk prepared by TBST. Shake horizontally at room temperature for 2 hours.

[0120] (5) Wash twice with TBST, 4 min each time; incubate overnight at 4°C with 1 mg / mL rP35;

[0121] (6) Wash 6 times with TBST, 4 min each time; incubate with purified rP35 rabbit-derived antibody (1:50) at 37℃ for 2 h;

[0122] (7) Wash 6 times with TBST, 4 min each time; incubate with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) at 37℃ for 1 h;

[0123] (8) Wash with TBST 6 times, 4 min each time; after slightly blotting off the moisture with filter paper, add ultrasensitive ECL developing solution and take pictures in a chemiluminescence imaging system.

[0124] 6.3 HPLC-MS identification of target bands

[0125] Take 20 μL of boiled SV-HUV-1 cell membrane protein sample for SDS-PAGE. Take out all the gel and put it into Coomassie Brilliant Blue staining solution for 2 h. After staining, put it into destaining solution for 3 h. Cut out the target protein corresponding to the obvious band in the modified VOPBA method and put it into a 1.5 mL Eppendorf tube. Add a small amount of deionized water and seal it. Send it to Guangzhou Huijun Biotechnology Co., Ltd. for identification.

[0126] Results of improved VOPBA and HPLC-MS identification of P35-specific binding proteins:

[0127] To screen for rP35 receptor proteins on the SV-HUC-1 cell membrane, SDS-PAGE analysis and a modified VOPBA assay were performed on SV-HUC-1 cell membrane proteins. SDS-PAGE analysis showed that the molecular weights of SV-HUC-1 cell membrane proteins were mainly distributed in the range of 10–100 kDa. Figure 4-1 The modified VOPBA experiment showed that the PVDF film incubated with rP35 exhibited two distinct bands in the molecular weight range of 40 kDa to 55 kDa. Figure 4-2 The control group showed no obvious bands. Figure 4-3 The results suggest that SV-HUC-1 cell membrane proteins in the 55kDa–40kDa range may be target proteins that specifically bind to rP35.

[0128] To identify the main protein components in the SV-HUC-1 cell membrane that specifically bind to rP35, membrane proteins were analyzed by SDS-PAGE. Then, strips corresponding to the prominent bands in the modified VOPBA experiment, ranging from 40kDa to 55kDa, were analyzed by HPLC-MS. Protein matching and comparison searches were performed using the NCBI database. The results showed that the strip near 50kDa, KRT8 (Keratin, type II cytoskeletal 8), had the highest score (Table 1-1), and the strip near 40kDa, ACTG1 (ActinGamma 1), had the highest score (Table 1-2). Therefore, it is inferred that KRT8 and ACTG1 are likely the proteins in the SV-HUC-1 cell membrane that specifically bind to rP35.

[0129] Table 1. Protein components in the 55kDa–40kDa range analyzed by LC-MS

[0130]

[0131] 7. Identification of the target protein and its distribution in cells

[0132] 7.1 Western blotting to confirm the target protein

[0133] (1) Gel running: Take 20 μL of SV-HUC-1 cell membrane protein suspension and mix it with 5 μL of protein loading buffer (5×), boil for 10 min, and load 10 μL of the sample into each well for SDS-PAGE.

[0134] (2) Transfer: Place the transfer filter paper, PVDF membrane, gel, and transfer filter paper in the order of bottom to top on the rapid transfer apparatus to transfer the membrane, avoiding the generation of air bubbles during the process; transfer the membrane under constant pressure conditions: 1.3V, 5min;

[0135] (3) Sealing: After rinsing the PVDF membrane twice with TBST, place it in 5% skim milk (1g skim milk powder + 20mL TBST) and seal at room temperature for 2 hours;

[0136] (4) Primary antibody: Wash PVDF membrane twice with TBST for 4 min each time; incubate with 1:2000 diluted ACTG1 and KRT8 rabbit antibodies at 4℃ overnight, and set up a control group at the same time;

[0137] (5) Secondary antibody: Wash PVDF membrane 6 times with TBST, 4 min each time; incubate with HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) at 37℃ for 1 h;

[0138] (6) Development: Wash the PVDF membrane 6 times with TBST for 4 minutes each time. After slightly blotting off the moisture with filter paper, add ultrasensitive ECL developing solution and take pictures in the chemiluminescence imaging system.

[0139] 7.2 Localization of ACTG1 and KRT8 proteins in SV-HUC-1 cells

[0140] (1) The specific method is the same as in 3.5. After culturing the cells in plates for 12 hours, fix and block them.

[0141] (2) Primary antibody: Wash 3 times with PBS for 3 min each time. After washing, add 150 μL of ACTG1 antibody and KRT8 antibody (1:200) diluted with complete culture medium respectively, and incubate at 37℃ for 2 h.

[0142] (3) Secondary antibody: Wash 5 times with PBS for 4 min each time. After washing, add 150 μL of Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:200) and incubate at 37°C for 1 h in the dark.

[0143] (4) Nuclear staining: Wash 5 times with PBS for 4 min each time. After washing, add DAPI (150 μL / well) and incubate at 37°C for 10 min in the dark.

[0144] (5) Slide preparation and observation: Wash with PBS 5 times, 4 min each time, add anti-fluorescence quenching agent and seal with nail polish, and observe with an inverted fluorescence microscope.

[0145] Experimental results on the identification of the target protein and its distribution in cells:

[0146] To verify the presence of ACTG1 and KRT8 proteins on the SV-HUC-1 cell membrane, cell membrane proteins were extracted and subjected to SDS-PAGE. Then, strips near 42 kDa and 53 kDa were analyzed by Western blotting. The results are as follows: Figure 5As shown in Figure A, after incubation with ACTG1 and KRT8 antibodies, distinct bands appeared near 42 kDa (A-2) and 53 kDa (A-4), while no bands appeared in the control groups (A-1 and A-3), indicating the presence of ACTG1 and KRT8 proteins in the SV-HUC-1 cell membrane. Indirect immunofluorescence was used to detect the localization of ACTG1 and KRT8 proteins in SV-HUC-1 cells. The results showed that the green fluorescence representing ACTG1 (…)… Figure 5 -B) and the green fluorescence representing KRT8 ( Figure 5 -C) is present both on the cell membrane and in the cytoplasm, indicating that ACTG1 and KRT8 proteins may exist on the cell membrane and in the cytoplasm of SV-HUC-1 cells.

[0147] 8. Detection of the interaction between the target protein and recombinant P35 protein

[0148] 8.1 Far-Western blotting

[0149] 8.1.1 Identification of direct binding of rP35 to ACTG1 and KRT8

[0150] (1) After boiling the rP35 protein sample, perform gel running, transfer, and blocking according to the method in 3.7.1;

[0151] (2) Protein incubation: Wash twice with TBST for 4 min each time, and incubate the PVDF membrane with ACTG1 protein (100 μg / mL) and KRT8 protein (100 μg / mL) at 4°C overnight;

[0152] (3) Primary antibody: Wash 6 times with TBST for 4 min each time, and incubate with ACTG1 rabbit antibody (1:2000) and KRT8 rabbit antibody (1:2000) at 37℃ for 2 h respectively;

[0153] (4) Secondary antibody: Wash 6 times with TBST for 4 min each time, and incubate with HRP-conjugated goat anti-rabbit IgG diluted 1:5000 at 37℃ for 1 h;

[0154] (5) Wash with TBST 6 times, 4 minutes each time. After slightly blotting off the moisture with filter paper, add ultrasensitive ECL developing solution and take pictures in a chemiluminescence imaging system.

[0155] 8.1.2 Identification of the binding of rP35 to ACTG1 and KRT8 proteins on the SV-HUC-1 cell membrane

[0156] (1) After boiling the rP35 protein sample, perform gel running, transfer, and blocking according to the method in 3.7.1;

[0157] (2) Wash twice with TBST for 4 min each time, and incubate PVDF membrane with SV-HUC-1 cell membrane protein suspension at 4°C overnight;

[0158] (3) Wash TBST 6 times, 4 min each time, and incubate with ACTG1 rabbit antibody (1:2000) and KRT8 rabbit antibody (1:2000) at 4℃ for more than 12 h;

[0159] (4) Wash TBST 6 times, 4 min each time, and incubate with HRP-conjugated goat anti-rabbit IgG diluted 1:5000 at 37℃ for 1 h;

[0160] (5) Wash with TBST 6 times, 4 min each time; after slightly blotting off the moisture with filter paper, add ultrasensitive ECL developing solution and take pictures in the chemiluminescence imaging system.

[0161] Experimental results of Far-Western blotting:

[0162] First, recombinant protein P35 was isolated using SDS-PAGE. P35 was then transferred to a PVDF membrane via electrophoresis. The PVDF membrane was incubated with ACTG1, KRT8, and BSA proteins, respectively, with the BSA incubation group serving as a negative control. Far-western blotting was then performed using ACTG1 and KRT8 specific antibodies. Figure 6 As shown in Figure A, the PVDF membrane incubated with ACTG1 protein and then with ACTG1 antibody showed a distinct band at 35 kDa, while the control group showed no band. This suggests that ACTG1 may directly bind to rP35. However, the PVDF membrane incubated with KRT8 protein and then with KRT8 antibody did not show a distinct band, consistent with the control group. This suggests that KRT8 protein may not directly bind to rP35.

[0163] In addition to direct binding verification using purified protein, the binding of rP35 to ACTG1 and KRT8 proteins on the SV-HUC-1 cell membrane was also verified. SDS-PAGE was performed on rP35 protein, and PVDF membranes were incubated with SV-HUC-1 cell membrane proteins and BSA, with the BSA incubation group serving as a negative control. Subsequently, Far-western blotting was performed using ACTG1 and KRT8 antibodies, respectively. Figure 6 As shown in Figure B, a distinct band appeared at 35 kDa on the PVDF membrane of the ACTG1 antibody group, while no band was observed in the KRT8 antibody group and the control group. This is consistent with the results of the direct binding verification experiment, indicating that rP35 can interact with the ACTG1 protein on the SV-HUC-1 cell membrane without interacting with KRT8.

[0164] 8.2 Indirect ELISA

[0165] (1) Following the same steps as in 3.4.2, coat rP35 onto an ELISA plate, wash and block;

[0166] (2) Wash the plate twice with PBST for 4 min each time, and incubate with 150 μL of ACTG1 protein (0.2 mg / mL) or KRT8 protein (0.2 mg / mL) at 37°C for 2 h. At the same time, a control group was set up.

[0167] (3) Wash the plate with PBST 4 times, 4 min each time. The experimental groups were incubated with ACTG1 rabbit anti (1:2000 dilution) or KRT8 rabbit anti (1:2000 dilution) at 37℃ for 2 h.

[0168] (4) Wash the plate 4 times with PBST, 4 min each time, and incubate with HRP-conjugated goat anti-rabbit IgG diluted 1:5000 at 37℃ for 1 h;

[0169] (5) Wash the plate 6 times with PBST, 4 min each time. After color development, use a microplate reader to detect the A in each well. 450 value.

[0170] Results of indirect ELISA experiments:

[0171] Indirect ELISA was used to detect the binding of ACTG1 and KRT8 proteins to immobilized rP35 in a 96-well plate. Results are as follows: Figure 6 As shown in Figure C, the absorbance of the rP35 incubation group with ACTG1 was greater than 2.0, the absorbance of the positive control rP35 antibody incubation group was also greater than 2.0, and the absorbance of the blank control group was less than 0.5, which was statistically significant (P<0.0001); while... Figure 6 D shows the incubation group A of rP35 and KRT8. 450 The absorbance values ​​of both the rP35 and the blank control group were less than 0.5, which was not statistically significant. The ELISA results were consistent with those of the Far-western blotting experiment. These results indicate that rP35 specifically binds to ACTG1 without interacting with KRT8.

[0172] 8.3 Immunoprecipitation

[0173] (1) Mix 200 μL of SV-HUC-1 cell membrane protein suspension with 100 μL of rP35 protein (10 μg / mL) and place on ice. Shake on a horizontal shaker overnight. After overnight, add 40 μL of 50% Protein A / G agarose bead working solution and continue shaking on ice for 2 hours to remove non-specific proteins.

[0174] (2) Centrifuge at 4℃ and 14000rpm for 15min and collect the supernatant;

[0175] (3) Divide the supernatant into 5 EP tubes. Add 5X protein loading buffer to one tube, boil for 10 min and store at -20℃. Add rP35 rabbit antibody (1:25), ACTG1 mouse antibody (1:50), mouse IgG (1:50) and rabbit IgG (1:50) to the other four tubes respectively, and shake on ice overnight.

[0176] (4) After overnight incubation, add 10 μL of 50% Protein A / G agarose bead working solution to each tube to capture antigen-antibody conjugates, and shake on ice overnight.

[0177] (5) Centrifuge at 14000rpm for 15 seconds at 4℃, wash 4 times with PBS, discard the supernatant and resuspend the precipitate with 60μL of PBS. After resuspending, add 12.5μL of protein electrophoresis loading buffer (5×), boil for 10min, and perform Western blotting analysis.

[0178] Results of the immunoprecipitation experiment:

[0179] SV-HUC-1 cell lysates were mixed with rP35 protein and then immunoprecipitated with specific antibodies against ACTG1 or rP35. The precipitate was subsequently subjected to SDS-PAGE electrophoresis, and the formation of the ACTG1-rP35 complex was identified by Western blotting using specific antibodies. Results are as follows: Figure 7 As shown in Figure A, the anti-rP35 antibody precipitated sample showed a clear band at 42 kDa (A3), and the anti-ACTG1 antibody precipitated sample also showed a clear band at 35 kDa (A6), while the control IgG precipitated group showed no clear bands (A1 and A4). This indicates that the complex formed by rP35 and ACTG1 can be precipitated by either rP35 or ACTG1 antibody, and that rP35 interacts with the ACTG1 protein.

[0180] 8.4 Immunofluorescence co-localization

[0181] (1) The specific method is the same as in 3.5. After culturing the cells in plates for 12 hours, fix and block them.

[0182] (2) After washing three times with PBS, add 150 μL of rP35 (50 μg / mL) or Mpe (1×10⁻⁶) to the well. 7 CCU / mL), incubate at 37℃ for 2 hours;

[0183] (3) After washing with PBS 4 times, add 100 μL of ACTG1 mouse antibody (1:100) and 100 μL of rP35 rabbit antibody (1:1) or 100 μL of ACTG1 mouse antibody and 100 μL of LMpe rabbit antiserum (1:10000) to the wells and incubate overnight at 4°C.

[0184] (4) After washing with PBS 4 times, add 150 μL of a mixture of Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody and Cy3-labeled goat anti-mouse secondary antibody, and stain at 37°C in the dark for 1 h.

[0185] (5) After washing 5 times with PBS, stain the nuclei with DAPI for 10 min, wash the slides, and observe them under an inverted fluorescence microscope. Results of immunofluorescence co-localization experiments:

[0186] SV-HUC-1 cells were pre-incubated with rP35, then incubated with mouse anti-ACTG1 antibody and rabbit anti-rP35 antibody. Indirect immunofluorescence assays were performed using combined staining with goat anti-mouse secondary antibody (red) and goat anti-rabbit secondary antibody (green). The results are as follows: Figure 7 The green fluorescence representing r-P35 and Mpe shown in Figures B and 7C largely overlaps with the red fluorescence representing ACTG1, indicating that rP35 and Mpe may interact with the ACTG1 protein on SV-HUC-1 cells. In summary, these results demonstrate that Mpe-P35 can specifically bind to the ACTG1 protein on the SV-HUC-1 cell membrane.

[0187] 9. Effects of ACTG1 on rP35 and Mpe adhesion to SV-HUC-1 cells

[0188] 9.1 Effects of ACTG1 and its antibody on the adhesion of rP35 and Mpe

[0189] (1) Refer to steps 3.5 for fixing and sealing;

[0190] (2) Divide the cells into groups for experiments:

[0191] ① Positive control group: Cells were washed and then directly reacted with 150 μL of rP35 (50 μg / mL) or Mpe (1×10⁻⁶). 7 Incubate with CCU / mL at 37℃ for 2 h; after washing, incubate with 150 μL of rP35 rabbit antibody (1:50) or Mpe rabbit antibody (1:100000) at 37℃ for 2 h; after washing, stain with 150 μL of Cy3-labeled goat anti-rabbit secondary antibody (1:150) at 37℃ for 1 h, and observe the nuclear staining slides under an inverted fluorescence microscope;

[0192] ②ACTG1 protein pretreatment group: rP35 and Mpe were first incubated with ACTG1 protein (0.25 mg / mL) at 37℃ for 2 h, and then incubated with SV-HUC-1 cells in well plates at 37℃ for 2 h; subsequently, they were incubated with rP35 rabbit anti or Mpe rabbit anti at 37℃ for 2 h; staining was performed with Cy3-labeled goat anti-rabbit secondary antibody at 37℃ for 1 h, and nuclear staining was performed and observed under an inverted fluorescence microscope;

[0193] ③ACTG1 antibody pre-incubation group: SV-HUC-1 cells in the well plate were first incubated with 150 μL of ACTG1 antibody (1:200) at 37℃ for 2 h, washed, and then incubated with rP35 or Mpe at 37℃ for 2 h; then incubated with rP35 rabbit antibody or Mpe rabbit antibody at 37℃ for 2 h; stained with Cy3-labeled goat anti-rabbit secondary antibody at 37℃ for 1 h, and observed under an inverted fluorescence microscope after nuclear staining and slide preparation.

[0194] Experimental results on the effects of ACTG1 and its antibody on the adhesion of rP35 and Mpe:

[0195] To verify whether ACTG1 protein affects the adhesion of rP35 and Mpe to SV-HUC-1 cells, an adhesion-adhesion inhibition assay was used to observe the adhesion of rP35 and Mpe to cells. Indirect immunofluorescence results showed that after SV-HUC-1 cells were incubated with rP35 and Mpe, respectively, a greater amount of rP35 (…) was absorbed. Figure 8 A) and Mpe( Figure 8 D) Both can adhere to the cell membrane surface. However, after SV-HUC-1 cells were incubated with rP35 and Mpe treated with ACTG1, rP35 ( Figure 8 B) and Mpe( Figure 8 E) reduced cell adhesion. Similarly, rP35 on the cell surface pretreated with ACTG1 antibody reduced cell adhesion. Figure 8 C) and Mpe( Figure 8 F) Adhesion was also reduced. The average integrated optical density of all groups was obtained using ImageJ, and the staining intensity of rP35 or Mpe was then quantitatively analyzed. The results showed that, compared with the positive control group, the changes in average fluorescence intensity in the ACTG1 protein pretreatment group and the ACTG1 antibody pre-incubation group were statistically significant (P<0.001). These results confirm that after ACTG1 protein pretreatment, the adhesion of rP35 and Mpe to SV-HUC-1 cells decreased, indicating that ACTG1 protein may be closely related to the adhesion of rP35 and Mpe to cells.

[0196] 9.2 Effects of ACTG1 interference on Mpe and rP35 adhesion to SV-HUC-1 cells

[0197] 9.2.1 ACTG1-siRNA transfection

[0198] (1) After digestion, SV-HUC-1 cells were seeded into 6-well plates and cultured for 1 day until the cell number reached 10. 5 ~10 6 Replace with fresh serum-free F-12K medium at intervals and incubate overnight.

[0199] (2) The ACTG1-siRNA was dissolved to a concentration of 20 μmol / L in a biosafety cabinet. The siRNA sequence is as follows:

[0200]

[0201] (3) Take 5 μL of dissolved siRNA and 120 μL of Opti-MEM. TM Mix the culture medium thoroughly and let it stand for 5 minutes;

[0202] (4) Take 10 μL of Lipo3000 and 490 μL of Opti-MEM. TM Mix the culture medium thoroughly and let it stand for 5 minutes;

[0203] (5) Take 125 μL of Lipo3000 and mix it with each tube of diluted siRNA, and let it stand for 20 min;

[0204] (6) Discard the liquid in the original 6-well plate and add 750 μL of Opti-MEM. TM The culture medium was then added, followed by the Lipo3000 and siRNA mixture. After gently shaking to mix, the mixture was placed in a 37°C cell culture incubator.

[0205] (7) After culturing for 8 hours, the medium was replaced with fresh F12K medium and cultured for another 48 hours. The total protein in the cells was then extracted and the expression level of ACTG1 protein was detected by Western blotting.

[0206] 9.2.2 Observation of changes in adhesion amount using indirect immunofluorescence

[0207] (1) The cells were seeded into 24-well plates and transfected according to the results of Western blotting.

[0208] (2) Fix and seal the cells according to the steps in 3.6.2;

[0209] (3) Add 150 μL of rP35 (50 μg / mL) or Mpe (1×10⁻⁶) to the well. 7 CCU / mL), incubate at 37℃ for 2 hours;

[0210] (4) After washing 4 times with PBS, incubate overnight at 4°C with 150 μL of rP35 rabbit antibody (1:1) or Mpe rabbit antibody (1:10000);

[0211] (5) After washing 4 times with PBS, stain with 150 μL of Cy3-labeled goat anti-rabbit fluorescent secondary antibody (1:150) at 37℃ for 1 h;

[0212] (6) After washing with PBS 5 times, stain the nuclei with DAPI for 10 min, and observe under an inverted fluorescence microscope after washing.

[0213] Experimental results on the effects of ACTG1 interference on Mpe and rP35 adhesion to SV-HUC-1 cells:

[0214] To more directly observe whether decreased ACTG1 protein expression affects the adhesion of Mpe and rP35 to SV-HUC-1 cells, siRNA was used to reduce ACTG1 protein expression in SV-HUC-1 cells, and adhesion experiments of Mpe and rP35 to cells were performed. The results are as follows: Figure 9 As shown, the expression level of ACTG1 protein in SV-HUC-1 cells transfected with siRNA was significantly decreased. Furthermore, adhesion assays indicated that the adhesion of Mpe and rP35 to the interfered SV-HUC-1 cells was significantly reduced compared to the untreated group. The average integrated optical density of all groups was obtained using ImageJ, and then the staining intensity of rP35 or Mpe was quantitatively analyzed. The results showed that the changes in the average fluorescence intensity of rP35 or Mpe adhering to SV-HUC-1 cells with decreased ACTG1 protein expression levels were statistically significant compared to the untreated group. This indicates that the decrease in ACTG1 protein expression can partially inhibit the adhesion of Mpe and rP35 to cells, and that ACTG1 protein is the main adhesion receptor for Mpe-P35 adhesion to SV-HUC-1 cells.

[0215] Analysis of the binding sites of 10 Mpe-P35 and ACTG1

[0216] Secondary structure and hydrophilicity / hydrophobicity analyses were performed on the complete amino acid sequence of Mpe-P35. Based on the antigenic epitopes of each synthetic Mpe-P35 peptide and their recognition rate with human serum, five peptides potentially associated with binding were screened. Truncation-length Mpe-P35 peptides were synthesized by Nanjing GenScript Co., Ltd., and each peptide was tagged with a Flag tag (amino acid sequence: DYKDDDDK). The peptides and Flag tags were linked using a flexible Linker (amino acid sequence: GGGGSGGGGS). A negative control peptide (amino acid sequence: GGGGSGGGGSDYKDDDDK) was also synthesized.

[0217] Specifically, this refers to the amino acid sequence analysis of the MpeP35 protein, the research of Neyrolles et al., and the secondary structure of Mpe-P35. Figure 10 -A) and its hydrophobicity analysis ( Figure 10 -B), synthesized 5 truncated P35 peptides and negative control peptides that may be associated with P35 and ACTG1 binding (Table 2).

[0218] Table 2. Peptides used in this study

[0219]

[0220]

[0221] 10.1 Synthesis, purification and identification of truncated rP35 peptides

[0222] The synthesis, purification, and identification of the truncated Mpe-P35 peptide were completed by Nanjing GenScript Co., Ltd., and mass spectrometry identification was performed.

[0223] 10.2 Indirect ELISA to identify the interaction between truncated rP35 and ACTG1

[0224] (1) Same as step 3.4.2, coat the microplate with 10 μg / mL ACTG1, wash and block;

[0225] (2) After washing the plate, incubate with 150 μL of each truncated rP35 peptide (0.25 mg / mL) at 37 °C for 2 h. Use full-length rP35 as a positive control and control peptide as a negative control.

[0226] (3) After washing the plate, incubate it with 150 μL of rabbit anti-rP35 antibody (1:200) or rabbit anti-Flag antibody (1:2000) at 37°C for 2 h.

[0227] (4) After washing the plate, 150 μL of goat anti-rabbit IgG (1:5000) conjugated with HRP was incubated at 37°C for 1 h.

[0228] (5) After washing and developing the color, use an ELISA reader to detect the A in each well. 450 value.

[0229] Experimental results of indirect ELISA identification of the interaction between truncated rP35 and ACTG1:

[0230] To identify the binding region between Mpe-P35 and ACTG1, an indirect ELISA assay was used to detect the binding of truncated rMpe-P35 molecules to ACTG1 immobilized in a 96-well plate. The results are as follows: Figure 11 As shown in -A, the absorbance of the rP35-2, rP35-3, and rP35-5 polypeptide groups was almost the same as that of the negative control polypeptide group, while the absorbance of the rP35-1 and rP35-4 polypeptide groups was significantly higher than that of the negative control group, indicating that the truncated polypeptides rP35-1 and rP35-4 in Mpe-P35 may bind to the ACTG1 protein in vitro.

[0231] 10.3 Far-Western blotting to identify specific binding of truncated rP35 to ACTG1

[0232] (1) The steps are the same as in 3.8.1.1: ACTG1 protein is subjected to electrophoresis, transfer to a membrane, and blocking;

[0233] (2) After washing, the peptides were incubated overnight at 4°C with full-length rP35, each truncated rP35 peptide, and control peptide (100 μg / mL).

[0234] (3) After washing, incubate with rabbit-derived anti-rP35 antibody (1:200) or rabbit-derived anti-Flag antibody (1:2000) at 4°C for more than 14 hours;

[0235] (4) After washing, the goat anti-rabbit secondary antibody (1:5000) conjugated with HRP was incubated at 37℃ for 1 h;

[0236] (5) Develop after washing.

[0237] Far-Western blotting results for identifying the specific binding of truncated rP35 to ACTG1:

[0238] To further determine the binding region between Mpe-P35 and ACTG1, ACTG1 protein was subjected to SDS-PAGE. After being transferred to a PVDF membrane, ACTG1 was incubated with full-length recombinant P35 protein, various truncated P35 peptides, and a negative control peptide. Subsequently, it was incubated with anti-P35 rabbit anti- or anti-Flag antibodies for Far-western blotting. Results are as follows: Figure 11 As shown in Figure B, the PVDF membrane incubated with rP35 and rP35-4 peptides showed a distinct band at 42 kDa, while no distinct bands were observed in other peptides and the negative control peptide. This indicates that among the truncated peptides, rP35-4 peptide is most likely to bind to ACTG1 protein in vitro.

[0239] 10.4 Does truncated rP35 affect the adhesion of full-length rP35 and Mpe to SV-HUC-1 cells?

[0240] (1) Fix and seal the cells according to the steps in 3.6.2;

[0241] (2) After washing twice with PBS, 150 μL of each truncated rP35 peptide and control peptide (100 μg / mL) were added to the wells and incubated at 37°C for 2 h.

[0242] (3) After washing with PBS 4 times, add 150 μL of full-length rP35 (50 μg / mL) to the well and incubate at 37°C for 2 h;

[0243] (4) After washing with PBS 4 times, incubate with 150 μL of rP35 rabbit antibody at 37°C for 2 h;

[0244] (5) After washing with PBS 4 times, stain with Cy3-labeled goat anti-rabbit fluorescent secondary antibody (1:150) at 37℃ for 1 h; (6) After washing 6 times, stain the nucleus with DAPI for 10 min, and observe under a fluorescence microscope after washing.

[0245] Does truncated rP35 affect the adhesion of full-length rP35 and Mpe to SV-HUC-1 cells?

[0246] To verify whether pre-incubation of SV-HUC-1 cells with the truncated peptide in Mpe-P35 affected the adhesion of rP35 to cells, an indirect immunofluorescence assay was used to observe the adhesion of rP35 to treated SV-HUC-1 cells. Figure 12 As shown, a significant amount of rP35 adhered to the SV-HUC-1 cell membrane surface (12-A). Pre-incubation with rP35-1 peptide resulted in a relative decrease in rP35 adhesion (12-B), and pre-incubation with rP35-4 peptide significantly reduced rP35 adhesion (12-E). However, pre-incubation with rP35-2 (12-C), rP35-3 (12-D), rP35-5 (12-F), and the control peptide (12-G) did not significantly alter the amount of rP35 adhering to cells. Quantitative analysis of staining intensity also indicated that the change in the average fluorescence intensity of rP35 adhering to SV-HUC-1 cells after pre-incubation with rP35-1 and rP35-4 peptides was statistically significant compared to the control peptide pre-incubation group. This suggests that rP35-1 and rP35-4 may be important components of rP35 that adhere to SV-HUC-1 cells.

[0247] In summary, the truncated peptide rP35-4 can significantly interact with ACTG1, while peptide rP35-1 may exhibit a low-affinity interaction with ACTG1. In contrast, other truncated peptides used as control peptides did not bind to ACTG1. This suggests that amino acids 35-42 (represented by peptide rP35-1) and 179-186 (represented by peptide rP35-4) of the Mpe GTU-54-6A1 lipoprotein P35 may contain potential ACTG1 binding sites.

Claims

1. The use of amino acids 35-42 of the Mycoplasma penetrant P35 lipoprotein as a functional domain that specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane, wherein the peptide sequence of amino acids 35-42 is SENNGNGN.

2. The use of amino acids 179-186 of the Mycoplasma penetrant P35 lipoprotein as a functional domain that specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane, wherein the peptide sequence of amino acids 179-186 is PNLKLNNG.

3. A method for determining the functional domain by which the Mycoplasma penetratingis P35 lipoprotein of claim 1 or 2 specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane, characterized in that... Includes the following steps: Step 1. Expression and purification of Mycoplasma penetrant P35 lipoprotein: Induced expression and purification of recombinant protein P35 containing all amino acids of P35, the obtained recombinant protein P35 was concentrated and its concentration was identified; Step 2. Preparation of recombinant protein P35 antibody: Recombinant protein P35 was mixed with Freund's adjuvant and used to immunize rabbits. Rabbit serum was collected after immunization. The immunoglobulin fraction was crudely purified by continuous ammonium sulfate precipitation. Then, the antibody specific to recombinant protein P35 was purified by coupling and elution with cyanogen bromide activated agarose 4B. Step 3. Determine whether P35 is an adhesion-related protein of Mycoplasma penetrating to SV-HUC-1 cells: Culture SV-HUC-1 cells and Mycoplasma penetrating to cells separately; detect the adhesion of recombinant protein P35 and Mycoplasma penetrating to SV-HUC-1 cells by indirect immunofluorescence assay, and detect whether the recombinant protein P35 antibody prepared in step 2 can inhibit the adhesion of Mycoplasma penetrating to SV-HUC-1 cells; Step 4. Analyze the receptor proteins in the SV-HUC-1 cell membrane that may specifically bind to recombinant protein P35: SDS-PAGE analysis was performed on SV-HUC-1 cell membrane proteins, and then HPLC-MS analysis was performed on the strips near 40kDa to 55kDa corresponding to the obvious bands in the modified VOPBA experiment. The receptor proteins in the SV-HUC-1 cell membrane that may specifically bind to recombinant protein P35 were identified as ACTG1 and KRT8. Step 5. Identify the presence of receptor proteins ACTG1 and KRT8 on the SV-HUC-1 cell membrane and determine their distribution: Western blotting was used to identify the receptor proteins, and their distribution in the SV-HUC-1 cell membrane was observed by indirect immunofluorescence to determine the localization of receptor proteins ACTG1 and KRT8 on SV-HUC-1 cells. Step 6. Verify whether the two receptor proteins ACTG1 and KRT8 can interact with recombinant protein P35: Four independent experimental methods, namely Far-western blotting, indirect ELISA, immunoprecipitation and immunofluorescence co-localization, were used to determine that recombinant protein P35 can specifically bind to ACTG1 but does not interact with KRT8. Step 7. Verify whether ACTG1 affects the adhesion of Mycoplasma penetratingus and recombinant protein P35 to SV-HUC-1 cells: The adhesion-adhesion inhibition assay and indirect immunofluorescence were used to observe the effects of ACTG1 and its antibody on the adhesion of Mycoplasma penetratingus and recombinant protein P35 to SV-HUC-1 cells. Step 8. Confirm the binding functional domain site of recombinant protein P35 to ACTG1 protein: Synthesize all possible truncated P35 peptides related to binding, and use indirect ELISA and Far-western blotting to detect the binding of ACTG1 to the synthesized peptides. Use indirect immunofluorescence to detect the effect of the synthesized peptides on the adhesion of rP35 to SV-HUC-1 cells. Finally, identify the functional domain of Mycoplasma penetrant P35 lipoprotein that specifically binds to the receptor protein ACTG1 on the SV-HUC-1 cell membrane.

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