CN107253987B

CN107253987B - Receptor protein interacting with mycoplasma genitalium MgPa, and separation method and application thereof

Info

Publication number: CN107253987B
Application number: CN201710432872.9A
Authority: CN
Inventors: 游晓星; 邓湘赢; 戴佩
Original assignee: Nanhua University
Current assignee: Nanhua University
Priority date: 2017-06-09
Filing date: 2017-06-09
Publication date: 2020-11-24
Anticipated expiration: 2037-06-09
Also published as: CN107253987A

Abstract

The invention obtains the receptor protein histone H2B on the surface of the human urothelium cell membrane by screening. The protein is separated from cell membrane protein of human urothelial cells, the nucleotide sequence of the protein is shown in SEQ ID NO.1, and the protein consists of 126 amino acids. The invention screens the receptor of MgPa on the epithelial cell membrane of human urethra by using an improved VOPBA method, further clarifies possible Mg adhesion and pathogenic mechanisms, further designs a receptor mimic molecule and an antagonist molecule to prevent Mg from adhering or invading host cells, and provides an early experimental basis for Mg infection prevention and treatment. Therefore, the receptor protein histone H2B can be used for preparing a targeted medicament for treating or preventing infectious diseases of mycoplasma genitalium.

Description

Receptor protein interacting with mycoplasma genitalium MgPa, and separation method and application thereof

Technical Field

The invention relates to a receptor protein interacting with Mycoplasma genitalium MgPa, namely Histone H2B (Histone H2B), and also provides a separation method and application of the protein, belonging to the technical field of biological engineering and disease diagnosis and control.

Background

Mycoplasma genitalium (Mg) is a urogenital infectious Mycoplasma first isolated from the urinary secretions of a patient with nongonococcal urethritis. Clinical research shows that female infection with Mg can cause cervicitis, pelvic inflammation, infertility and the like; while male infection can cause acute and chronic nongonococcal urethritis and infertility; in addition, Mg infection is also closely related to opportunistic infections of Human Immunodeficiency Virus (HIV).

Binding of the pathogen to the corresponding receptor on the host cell membrane is the first step in the infection of the host by the pathogen and is critical to its ability to infect the host cell. Studies have shown that Mg must adhere to urogenital or respiratory mucosal epithelial cells first to infect the host and colonize the site of infection or invade the cell.

Mycoplasma has no cell wall, but there are more membrane proteins on the membrane of Mg. Mg infects the disease through membrane proteins on the cell membrane mediating its colonization to the host cell surface. Mycoplasma genitalium adhesion protein (MgPa) is a membrane protein with the highest Mg content, is the most important adhesion protein, and plays a crucial role in mediating Mg adhesion and even invading host epithelial cells. Spontaneous cell adhesion-free Mg mutants lack MgPa. Iverson-Cabral SL et al also showed that during the persistent infection with Mg, the body produced antibodies directed mainly against the conserved C-terminal variable region of MgPa, suggesting that the C-terminal of MgPa is closely related to its immunogenicity. Dehon PM and other researches show that an antibody aiming at the C terminal of MgPa can partially inhibit the adhesion of Mg to Hep-2 cells, and the result shows that the MgPa is an important adhesion protein and is mainly adhered to host epithelial cells through the C terminal of the MgPa. Since MgPa is involved in adhesion to Mg, pathogenesis of infection, receptor proteins that interact with MgPa may be present on the host cell. However, to date, there has been no report on what receptor molecules on the host cell membrane interact with Mg via MgPa, resulting in its adhesion or invasion to the host epithelial cells.

Receptors on the surface of host cells are key factors in determining whether mycoplasma genitalium is susceptible to host cells, and are one of the major factors in determining the route of invasion, the mode of transmission, and the pathological characteristics of the host. Therefore, the research on the mycoplasma genitalium receptor is helpful to elucidate the pathogenic mechanism of the mycoplasma genitalium and the interaction relationship between the mycoplasma genitalium and host cells from the molecular level, and the artificial expression of the receptor protein is utilized to block the combination of the mycoplasma genitalium and the receptor thereof, so that the research is an important way for preventing and treating the infection of the mycoplasma genitalium.

Disclosure of Invention

The invention utilizes an ultrasonic disruption method to extract membrane protein of human urothelial cells (SV-HUC-1 cells), and applies an improved virus overlay protein blotting technology (VOPBA), namely, a receptor protein-histone H2B positioned on the surface of the human urothelial cell membrane is obtained by utilizing prokaryotic expression and purified recombinant protein rMgPa to directly incubate with a cell membrane protein component transferred to a PVDF membrane and screening. The protein is separated from cell membrane protein of human urothelial cells, the nucleotide sequence of the protein is shown in SEQ ID NO.1, and the protein consists of 126 amino acids.

The invention also provides a separation and preparation method of the receptor protein histone H2B, which comprises the following steps:

(1) expressing and purifying a membrane protein MgPa of the mycoplasma genitalium to prepare a recombinant protein rMgPa;

(2) extracting membrane protein of human urothelial cells by an ultrasonic disruption method;

(3) screening the membrane protein specifically combined with the recombinant protein rMgPa in the membrane protein prepared in the step (2) by adopting an improved virus spreading western blot technology to obtain a protein with 14 KDa;

(4) and (3) carrying out mass spectrometry identification on the 14KDa protein obtained in the step (3) to obtain the receptor protein histone H2B.

In the step (2), taking human urothelial cells with good culture form, digesting with trypsin for 5min, adding a culture medium to terminate digestion, centrifuging for 6min, and collecting cell precipitates; then adding PBS to slightly blow and beat cell sediment, and washing for three times; adding cell lysate into the mixture, performing ice bath for 20min, adding a protease inhibitor PMSF, performing ultrasonic disruption for 10W, 5 s/time, performing intermittent 15s, totaling 30 times, then centrifuging for 10min at 2500rpm/min, taking supernatant, centrifuging for 30min, and resuspending the precipitate with PBS (phosphate buffer solution), and storing at-80 ℃ to obtain the membrane protein extract of the human urethral epithelial cells.

The specific steps of the step (3) are as follows:

a、SDS-PAGE

and (3) adding the human urothelial cell membrane protein extracted in the step (2) into a loading buffer solution (5 x) after the concentration is determined, uniformly mixing, boiling for 5min, centrifuging for 1min at 1000g, taking 10 mu L of loading sample, and performing SDS-PAGE separation, wherein the separation gel is 12%, the concentration gel is 5%, and the electrophoresis conditions are as follows: concentrating gel at 80V, and separating gel at 120V; simultaneously setting a comparison group;

b. transfer film and seal

(i) Treatment of PVDF membrane and equilibration of gel: cutting a PVDF membrane slightly larger than the gel fragment, soaking the PVDF membrane in methanol for 10-15s, rinsing the PVDF membrane with deionized water for 10s, balancing the PVDF membrane with a membrane-transferring buffer solution for 10min, and after electrophoresis is finished, placing the target fragment in the separation gel in the membrane-transferring buffer solution for balancing for 10 min;

(ii) in a semi-dry membrane converter, the balanced filter paper, PVDF membrane, gel and filter paper are sequentially superposed from bottom to top, air bubbles are removed by a glass rod, and membrane conversion is carried out under the condition of constant pressure: the condition is 15V, 30 min;

(iii) immersing the PVDF membrane in a sealing solution, and rinsing with TBST for 5 times (5 min each time) after overnight at 4 ℃;

c. immunoblotting

(i) Incubation of rMgPa protein: adding rMgPa protein, incubating at 4 deg.C overnight, and rinsing with TBST for 5 times (5 min each time);

(ii) incubating the primary antibody: adding rabbit anti-rMgPa polyclonal antibody diluted at a ratio of 1:1000, incubating at 37 ℃ for 2h, and rinsing with TBST for 5 times, each time for 5 min;

(iii) incubation of secondary antibody: adding HRP-labeled goat anti-rabbit IgG diluted to 1:5000, incubating at 37 ℃ for 1h, and rinsing with TBST for 5 times, each time for 5 min;

(iv) and (6) developing.

The receptor is the first molecule encountered when the mycoplasma genitalium infects host cells, and the type and the structure of the receptor are revealed, which is helpful for further clarifying the pathogenic mechanism of the mycoplasma genitalium. The invention screens the receptor of MgPa on the epithelial cell membrane of human urethra by using an improved VOPBA method, further clarifies possible Mg adhesion and pathogenic mechanisms, further designs a receptor mimic molecule and an antagonist molecule to prevent Mg from adhering or invading host cells, and provides an early experimental basis for Mg infection prevention and treatment. Therefore, the receptor protein histone H2B can be used for preparing a targeted medicament for treating or preventing infectious diseases of mycoplasma genitalium.

Drawings

FIG. 1 is an SDS-PAGE analysis of human urothelial cell membrane proteins;

FIG. 2 is a diagram of rMgPa binding protein screened by the improved VOPBA technique;

FIG. 3 is a diagram of indirect ELISA for identifying specific binding of rMgPa to H2B;

FIG. 4 is a diagram for identifying the binding of rMgPa to H2B in cell membrane protein by Western blotting;

FIG. 5 is a map of the localization of H2B protein on human urothelial cells;

FIG. 6 is a graph of an indirect immunofluorescence adhesion inhibition assay of rMgPa treated with H2B;

FIG. 7 is a graph of an indirect immunofluorescence adhesion inhibition assay after Mg treatment with H2B.

Detailed Description

At present, mass spectrometry techniques have played an important role in protein and proteomics research. It can use the difference of protein molecule charge-to-mass ratio (M/Z) to separate different proteins, so as to analyze and identify unknown protein. The invention carries out mass spectrum analysis on the 14kDa protein, and the result shows that the 14kDa receptor protein has stronger correlation with histone H2B.

The method for obtaining the receptor protein and the receptor protein antibody is beneficial to researching the characteristics and functions of the receptor protein, and the interaction between the rMgPa and the receptor protein is researched by ELISA and Far-western blotting methods by further utilizing the H2B protein and the H2B antibody. The result shows that the rMgPa can interact with H2B to be specifically bound; a specific protein in the target band of about 14kDa also binds rMgPa, indicating that the protein of about 14kDa contains histone H2B. The invention utilizes indirect immunofluorescence test to detect the location of histone H2B in human urothelial cell. The results show that a part of the fluorescence signal is on the membrane and a part of the fluorescence signal is in the cytoplasm, which indicates that the histone H2B is distributed on the cell membrane of the human urothelium, and the receptor of the pathogen is mainly concentrated on the surface of the cell membrane of the host, so the results of the immunofluorescence assay reflect that the histone H2B protein is probably the receptor capable of interacting with the rMgPa from the side.

The invention further verifies that the histone H2B is a receptor of rMgPa, namely the histone H2B can mediate rMgPa and Mg to be adhered to human urothelial cells, and an adhesion inhibition test proves that the histone H2B can inhibit the adhesion of the rMgPa and the Mg to the human urothelial cells, which indicates that the histone H2B can be used for treating infectious diseases caused by the Mg.

The specific embodiments described above are as follows:

1. preparation of recombinant protein rMgPa

According to the conditions previously discovered by the inventor, the recombinant protein rMgPa is expressed, purified, concentrated by ultrafiltration, identified and subjected to endotoxin removal. The protein concentration was approximately 2000. mu.g/mL as determined by the BCA kit.

2. Ultrasonic crushing method for extracting human urothelial cell membrane protein

(1) Taking human urothelial cell with good culture form about 10⁷Performing trypsinization for 5min, adding a culture medium to stop digestion, performing centrifugation for 6min at 1000g, and collecting cell precipitates;

(2) adding PBS to gently blow and beat cell sediments, and washing for three times;

(3) adding cell lysate, performing ice bath for 20min, adding protease inhibitor PMSF, performing ultrasonication (10W, 5 s/time, 15s intermittent, 30 times in total), centrifuging at 2500rpm/min for 10min, collecting supernatant 16000 g, centrifuging for 30min, and resuspending the precipitate with 300 μ L PBS at-80 deg.C;

SDS-PAGE analysis is carried out after human urothelial cell membrane protein is extracted by using an ultrasonication method, the result is shown in figure 1, obvious strips appear in the range of 12-55 KDa, the concentration of the resuspended membrane protein after ultracentrifugation is 600 mug/mL through a BCA method, and the successful extraction of the membrane protein of human urothelial cells is shown.

3. Improved VOPBA method for screening cell membrane protein interacting with rMgPa

3.1 SDS-PAGE

And (3) measuring the concentration of the extracted human urothelial cell membrane protein, adding 20 mu L of the extracted human urothelial cell membrane protein into 5 mu L of loading buffer solution (5X), uniformly mixing, boiling for 5min, centrifuging for 1min at 1000g, and taking 10 mu L of the loading solution for SDS-PAGE separation. The separation gel is 12 percent, the concentration gel is 5 percent, and the electrophoresis conditions are as follows: concentrating gel at 80V, and separating gel at 120V; a control group was also set.

3.2 transfer of film and sealing

(1) Treatment of PVDF membrane and equilibration of gel: cutting a PVDF membrane slightly larger than the gel segment, soaking in methanol for 10-15s, rinsing with deionized water, and balancing the PVDF membrane with membrane transfer buffer solution for 10 min. After electrophoresis, the target fragment was placed in a membrane transfer buffer and equilibrated for 10 min.

(2) In a semi-dry membrane converter, the balanced filter paper, PVDF membrane, gel and filter paper are sequentially superposed from bottom to top, and the membrane is converted under the condition of constant pressure after air bubbles are removed by a glass rod: the conditions were 15V, 30 min.

(3) PVDF membrane was immersed in blocking solution, rinsed 5 times with TBST after overnight at 4 ℃ for 5min each time.

3.3 immunoblotting

(1) Incubation of rMgPa protein: adding rMgPa protein, incubating at 4 deg.C overnight, and rinsing with TBST for 5 times (5 min each time);

(2) incubating the primary antibody: adding polyclonal antibody of rMgPa diluted at a ratio of 1:1000, incubating at 37 deg.C for 2h, and rinsing with TBST for 5 times, each time for 5 min;

(3) incubation of secondary antibody: adding HRP-labeled goat anti-rabbit IgG diluted to 1:5000, incubating at 37 ℃ for 1h, and rinsing with TBST for 5 times, each time for 5 min;

(4) and (6) developing.

The results are as follows:

after the extracted membrane protein is analyzed by SDS-PAGE and is converted into a membrane, the membrane protein is incubated with rMgPa, a rabbit anti-rMgPa antibody is a primary antibody, and a goat anti-rabbit IgG labeled by HRP is a secondary antibody. The results indicated that there was a clear band at a molecular weight of about 14kDa in the lane incubated with rMgPa, while no band was evident in the lane not incubated with rMgPa, as shown in FIG. 2, indicating that a protein of about 14kDa was likely the target protein for interaction with rMgPa.

4. Identification of target bands by mass spectrometry

(1) Adding 2.5 μ L (5 Xprotein electrophoresis loading buffer) into 10 μ L of human urothelial cell membrane protein after concentration determination, mixing, boiling for 5min, centrifuging for 1min at 1000g, and performing SDS-PAGE separation on 10 μ L of loading. The separation gel is 12 percent, the concentration gel is 5 percent, and the electrophoresis conditions are as follows: 80V of concentrated gel and 120V of separation gel.

(2) Removing glue with a mask and a film glove;

(3) dyeing with Coomassie brilliant blue staining solution for 3h, and decolorizing with decolorizing solution for 2h until the target band is clearly visible;

(4) cleaning a blade for cutting the rubber, and washing the cut strip with deionized water;

(5) putting the target strip into an inlet EP pipe, sealing the EP pipe by using a sealing film, and transporting at normal temperature;

(6) LC-MS identification was performed by Jun Biotech.

Mass spectrometric identification results

Through LC-MS identification, the NCBI database is searched, the measured protein is subjected to secondary mass spectrometry, and through protein comparison matching analysis, the histone H2B protein has high score and is probably a receptor protein capable of interacting with the rMgPa.

5. Indirect ELISA for identifying the binding condition of rMgPa and H2B

(1) Diluting rMgPa to 100. mu.g/mL with TBS, coating ELISA plate with 150. mu.L, and wet-packing overnight at 4 ℃; simultaneously setting a comparison group;

(2) throwing off the coating solution, washing for 3 times by using TBST, filling the blocking solution, and blocking for 2 hours at 4 ℃;

(3) washing the plate for 3-5 times, diluting H2B protein (1:1000) with blocking solution at 100 μ L/hole, 37 deg.C for 2H;

(4) washing the plate for 3-5 times, diluting the H2B protein antibody (1:1000) with blocking solution at 100 μ L/hole, 37 deg.C for 2H;

(5) washing the plate for 6 times, adding HRP-labeled goat anti-rabbit IgG (1:5000), 100 μ L/well, 37 deg.C, 1 h;

(6) washing for 6-8 times by TBST after 1h at 37 ℃;

(7) adding A and B color developing agents, and incubating for 1h at 37 ℃ in a dark place;

(8) adding stop solution, and measuring light absorption value A at 450nm by using an enzyme-labeling instrument.

The results are as follows:

the specific binding of the rMgPa and the histone H2B is detected by indirect ELISA, and the result shows that: compared with the blank control group, the absorbance value of the incubation group of the rMgPa and the histone H2B is more than 1.0, as shown in figure 3, the absorbance value of the incubation group of the positive control rabbit anti-rMgPa antibody is also more than 1.0, and the absorbance value of the blank control group is less than 0.5, which has statistical significance (P <0.05), and the results show that the rMgPa can interact with the H2B to generate specific binding.

6. Western blotting identification of specific binding of rMgPa and histone H2B

6.1 identification of direct binding of rMgPa to Histone H2B

(1)SDS-PAGE

Adding 20 μ L of rMgPa into 5 μ L (5 Xprotein electrophoresis loading buffer), mixing, boiling for 5min, centrifuging for 1min at 1000g, adding 10 μ L of loading sample per well, and performing SDS-PAGE separation.

(2) Transfer film and seal

Performing film transfer under a constant current condition: the conditions are 0.03A and 30min, and the rest operation steps are shown in 3.2.

(3) Immunoblotting

Incubation of histone H2B protein: H2B protein (1:1000) was added and incubated at 4 ℃ for 6H or overnight, and rinsed 5 times for 5min each in TBST.

Incubation of primary antibody: histone H2B antibody was added at a dilution of 1:1000, incubated at 37 ℃ for 2H, and rinsed 5 times in TBST for 5min each.

③ incubating secondary antibody: diluted HRP-labeled goat anti-rabbit IgG (1:5000) was added, incubated at 37 ℃ for 1h, and rinsed 5 times in TBST for 5min each.

Development

6.2 identification of binding of rMgPa to cell Membrane proteins

(1)SDS-PAGE

(2) Transfer film and seal

(3) Immunoblotting

Human urothelial cell membrane protein incubation: membrane proteins were added, incubated at 4 ℃ for 4h or overnight, and rinsed 5 times in TBST for 5min each.

And fourthly, developing.

6.3 binding of the approximately 14kDa target protein to the Histone H2B antibody

(1)SDS-PAGE

Adding 20 μ L cell membrane protein into 5 μ L (5 × protein electrophoresis loading buffer), mixing, boiling for 5min, centrifuging for 1min at 1000g, and adding 10 μ L loading per well for SDS-PAGE separation.

(2) Transfer film and seal

Film transfer was performed under constant pressure conditions: the conditions were 15V, 30min, and the rest of the operating steps were 3.2.

(4) Immunoblotting

Incubating a primary antibody: histone H2B antibody was added at a dilution of 1:1000, incubated at 37 ℃ for 2H, and rinsed 5 times in TBST for 5min each.

Incubation of secondary antibody: diluted HRP-labeled goat anti-rabbit IgG (1:5000) was added, incubated at 37 ℃ for 1h, and rinsed 5 times in TBST for 5min each.

And developing.

Identification of analysis results

The rMgPa can interact with histone H2B in the cell membrane protein to generate specific binding

After SDS-PAGE and membrane conversion is carried out on the rMgPa, Western blotting is used for detecting whether the rMgPa can be combined with H2B in the cell membrane protein, and the result is shown in figure 4, when the rMgPa and the membrane protein are incubated, a histone H2B antibody is added, a secondary antibody is added for color development, and a specific band appears at a position of about 37 kDa; no band appeared in the control group, indicating that rMgPa can be specifically combined with histone H2B in the cell membrane protein.

The indirect immunofluorescence test is used for detecting the positioning of the H2B protein in the human urothelial cells, and the result is shown in figure 5, and as can be seen from figure 5, the cell membrane, cytoplasm and nucleus areas of the human urothelial cells have red fluorescence, and the cell nucleus is blue; indicating the presence of the H2B protein both within and on the cell membrane.

7. Inhibition of rMgPa and Mg adhesion to human urothelial cells by histone H2B

(1) Taking a bottle of human urothelial cells which are in a good growth state and are paved on 80-90% of the bottom of a cell culture bottle, cleaning 3 times by using a cell cleaning solution, digesting by using trypsin, then adding 5mL of a culture medium, and uniformly blowing and beating;

(2) add 500. mu.L of culture medium to wells (with cell slide) of 24-well plate, then aspirate 50. mu.L of suspended human urothelial cells into each well, place at 37 ℃ with 5% CO₂Culturing in an incubator;

(3) rMgPa (30. mu.g) was pre-incubated with histone H2B alone at 4 ℃ for 2H;

(4)100 μ L of Mg suspension (1X 10)⁷CCU/mL) was pre-incubated with H2B alone at 37 ℃ for 30 min;

(5) the cells were washed 3 times with PBS (pH7.4), then fixed with 4% paraformaldehyde at 4 ℃ for 30 min;

(6) discarding 4% paraformaldehyde, washing with PBS for 3 times, adding F-12K culture medium, sealing, and keeping at 37 deg.C for 1 hr;

(7) removing the blocking solution, washing with PBS for 3 times, adding histone H2B antibody into the hole of a 24-hole plate, placing at 37 ℃, and incubating for 2H;

(8) adding the rMgPa protein pre-incubation mixture and 100 μ L Mg suspension pre-incubation mixture into the wells of a 24-well plate, respectively, and placing at 37 ℃ for 2 h;

(9) the procedure of adding primary and secondary antibodies and observation of the samples were the same as for the adhesion test of rMgPa and Mg.

The results are as follows:

in order to further verify that the histone H2B is a receptor protein capable of interacting with rMgPa, the rMgPa and the histone H2B are preincubated, and then the adhesion condition of the rMgPa to human urothelial cells is detected. The results are shown in FIG. 6: compared with a control group, red fluorescence on the surface of the cell membrane of the human urothelium is obviously reduced, which indicates that specific binding occurs between part of rMgPa and histone H2B during pre-incubation, and the adhesion of the rMgPa to the human urothelium is reduced. These results indicate that histone H2B is capable of partially inhibiting the adhesion of rMgPa to human urothelial cells.

Then, after Mg and histone H2B are pre-incubated, whether the histone H2B can inhibit the adhesion of Mg to human urothelial cells is detected by indirect immunofluorescence, and the following observation results are observed under a fluorescence microscope: the red fluorescence at the cell membrane surface and in the cytoplasm of human urothelium in the test group was significantly reduced compared to the control group, indicating a reduction in Mg adhesion and invasion to human urothelium cells, as shown in FIG. 7. These results indicate that the histone H2B protein can partially inhibit the adhesion and invasion of Mycoplasma genitalium to human urothelial cells.

<110 >: university of southern China

<120 >: receptor protein interacting with mycoplasma genitalium MgPa, and separation method and application thereof

<160>：1

<210>：1

<211>：126

<212>：Prt

<213 >: artificial sequences

<400>：1

Met Pro Glu Pro Ala Lys Ser Ala Pro Ala Pro Lys Lys Gly Ser Lys

1 5 10 15

Lys Ala Val Thr Lys Ala Gln Lys Lys Asp Gly Lys Lys Arg Lys Arg

20 25 30

Ser Arg Lys Glu Ser Tyr Ser Ile Tyr Val Tyr Lys Val Leu Lys Gln

35 40 45

Val His Pro Asp Thr Gly Ile Ser Ser Lys Ala Met Gly Ile Met Asn

50 55 60

Ser Phe Val Asn Asp Ile Phe Glu Arg Ile Ala Gly Glu Ala Ser Arg

65 70 75 80

Leu Ala His Tyr Asn Lys Arg Ser Thr Ile Thr Ser Arg Glu Ile Gln

85 90 95

Thr Ala Val Arg Leu Leu Leu Pro Gly Glu Leu Ala Lys His Ala Val

100 105 110

Ser Glu Gly Thr Lys Ala Val Thr Lys Tyr Thr Ser Ala Lys

115 120 125

Claims

1. A method for separating and preparing receptor protein interacting with Mycoplasma genitalium MgPa, wherein the protein is membrane protein separated from human urothelial cells, is histone H2B, has a nucleotide sequence shown in SEQ ID NO.1, and consists of 126 amino acids, and is characterized by comprising the following steps:

(1) expressing and purifying a membrane protein MgPa of the mycoplasma genitalium to obtain a recombinant protein rMgPa;

(3) screening the membrane protein specifically combined with the recombinant protein rMgPa in the membrane protein obtained in the step (2) by adopting an improved virus overlay western blot technology to obtain a 14KDa protein;

the method comprises the following specific steps:

a、SDS-PAGE

and (3) adding the human urothelial cell membrane protein extracted in the step (2) into a 5 x protein electrophoresis loading buffer solution after concentration determination, uniformly mixing, boiling for 5min, centrifuging for 1min at 1000g, taking 10 mu L of loading sample, and performing SDS-PAGE separation, wherein the separation gel is 12%, the concentration gel is 5%, and the electrophoresis conditions are as follows: concentrating gel at 80V, and separating gel at 120V; simultaneously setting a comparison group;

b. film transferring and sealing;

(iii) immersing the PVDF membrane in a sealing solution, standing overnight at 4 ℃, and rinsing 5 times for 5min by TBST;

c. immunoblotting;

(iv) developing;

(4) and (4) carrying out mass spectrometry identification on the 14KDa protein obtained in the step (3) to obtain the receptor protein histone H2B.

2. The method for separating and producing a receptor protein according to claim 1, wherein: in the step (2), taking human urothelial cells with good culture form, digesting with trypsin for 5min, adding a culture medium to terminate digestion, centrifuging for 6min, and collecting cell precipitates; then adding PBS to slightly blow and beat cell sediment, and washing for three times; adding cell lysate into the mixture, performing ice bath for 20min, adding a protease inhibitor PMSF, performing ultrasonic disruption for 10W, 5 s/time, performing intermittent 15s, totaling 30 times, then centrifuging for 10min at 2500rpm/min, taking supernatant, centrifuging for 30min, and resuspending the precipitate with PBS (phosphate buffer solution), and storing at-80 ℃ to obtain the membrane protein extract of the human urethral epithelial cells.

3. Use of the receptor protein according to claim 1, characterized in that: the application of the histone H2B in preparing a targeted medicine for treating or preventing mycoplasma genitalium infectious diseases.