CN110540969A - monoclonal antibody of surface protein of mycoplasma hyopneumoniae and antigen capture ELISA kit - Google Patents

Abstract

The invention discloses a human mycoplasma pneumoniae surface protein monoclonal antibody and an antigen capture ELISA kit. The monoclonal antibody of the surface protein of the mycoplasma pneumoniae is prepared by the following components in percentage by weight, wherein the preservation number is CCTCC NO: c2017217. The human mycoplasma pneumoniae surface protein monoclonal antibody can be used for detecting mycoplasma pneumoniae. The invention also discloses a human mycoplasma pneumoniae surface protein capture ELISA kit based on the monoclonal antibody.

Description

Monoclonal antibody of surface protein of mycoplasma hyopneumoniae and antigen capture ELISA kit

Technical Field

The invention belongs to the field of immunology, and relates to a human mycoplasma pneumoniae surface protein monoclonal antibody, a hybridoma cell strain and an antigen capture ELISA kit.

Background

Mycoplasma pneumoniae (Mp) is the causative agent of mycoplasma pneumoniae in humans. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchopneumonia, and are called primary atypical pneumonia. Mainly through droplet infection, the incubation period is 2-3 weeks, and the incidence rate is the highest for teenagers. It can occur all the year round, but mostly in autumn and winter. The clinical manifestations of mycoplasmal pneumonia and chest X-ray examination are not characteristic and no diagnosis can be made on the basis of the clinical manifestations and chest X-ray examination alone. Detection of pathogens is required for a definitive diagnosis.

The existing method for detecting mycoplasma pneumoniae in respiratory tract mainly adopts the traditional method, namely a separation identification method, the method needs long time, generally 2-3 days, and the requirement of quick identification is difficult to meet; the PCR technology developed in recent years is a quick, sensitive and specific technology, but at present, the technology still depends on the previous enrichment step of the traditional method, and PCR inhibitors are often contained in the enrichment liquid, so that the amplification effect is influenced. Meanwhile, the technology also needs professional detection equipment, and is not suitable for bedside detection. Antibody-based immunological detection has become an indispensable important technical means for the detection of human pathogenic microorganisms. Various specific immunoassay techniques, such as Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Chemiluminescence Immunoassay (CIA), immunoprecipitation, immunoagglutination, ELISA detection kit, immune colloidal gold test strip, immune latex detection reagent, and the like, have been developed. Among them, ELISA detection kits, immune colloidal gold test strips and other antibody-based immunological detection techniques have become an indispensable important means for detecting pathogenic microorganisms due to their characteristics of simplicity, rapidness, sensitivity, accuracy and practicality. Therefore, research and development of antibodies against pathogenic microorganisms having proprietary intellectual property rights are the basis for development of ELISA detection methods, colloidal gold detection methods, and the like having proprietary intellectual property rights.

The choice of antigenic component is critical to the specificity of the assay. Research shows that the adhesion of a special cell adsorber with a tip-like structure to human cells is the first step of Mp infection, and the special cell adsorber is composed of a plurality of adhesion proteins including P1, P30, P116, HMVI-3 and the like, wherein P1 and P30 with the highest sequence conservation are main adhesion factors. Layh-Schmitt et al found that the Mycoplasma pneumoniae lacking the P1 or P30 protein loses the ability to adhere to epithelial cells and becomes an avirulent strain, and therefore, it is a hot spot to select the P1 or P30 protein as a novel antigen component with high specificity to study. The Pl adhesion protein is a good immunogen and can induce an organism to generate immune response, and the P1 gene is discovered to contain more stop codons UGA through sequence analysis research, so that the expression of the full length P1 of the mycoplasma pneumoniae is difficult to realize, and the fragment expression is the key for solving the problem. In conclusion, P1 is a good target for antigen detection due to its good conservation and surface accessibility.

in the research, a surface protein P1 with interspecies specificity is selected as an antigen, a monoclonal antibody with good specificity is prepared, and the monoclonal antibody is applied to the preparation of the Mycoplasma pneumoniae ELISA detection kit.

Disclosure of Invention

in order to solve the technical problems in the background art, the invention provides a human mycoplasma pneumoniae surface protein monoclonal antibody and an antigen capture ELISA kit which have good specificity and do not have cross reaction with other common respiratory pathogens.

In order to achieve the purpose, the invention adopts the following technical scheme:

A hybridoma cell strain for producing a monoclonal antibody of surface protein of mycoplasma pneumoniae is characterized in that: the hybridoma cell strain for producing the mycoplasma hyopneumoniae surface protein monoclonal antibody is preserved by China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: c2017217 hybridoma cell strain Mp-7 #.

A preparation method of hybridoma cell strain for generating monoclonal antibody of surface protein of mycoplasma pneumoniae is characterized by comprising the following steps: the preparation method comprises the following steps:

1) The recombinant mycoplasma pneumoniae surface protein P1 is used as an antigen, and after the BALB/c mouse of 8 weeks old is immunized, the antiserum titer is detected by adopting an indirect ELISA method;

2) Recovering and culturing SP2/0 myeloma cells;

3) preparing feeder cells;

4) Preparing immune spleen cell suspension;

5) preparing a SP2/0 myeloma cell suspension;

6) Fusing cells;

7) Screening and cloning culture of positive clones, and detecting by using mycoplasma pneumoniae thalli as a coating antigen by adopting an indirect ELISA method; determining OD450 with enzyme-linked detector to satisfy P/N value > 2.1 as positive; selecting continuous positive cloning holes to perform subcloning for 2-3 times, and screening monoclonal hybridoma cells; carrying out amplification culture on the monoclonal hybridoma cells to obtain cell culture solution supernatant containing a monoclonal antibody; respectively coating an enzyme label plate with respiratory tract pathogenic bacteria, carrying out ELISA detection on the specificity of the screened antibody, eliminating monoclonal antibodies having positive reactions with the pathogens, and screening out qualified cell strains; the respiratory pathogenic bacteria comprise legionella pneumophila, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, haemophilus influenzae, streptococcus pyogenes, staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida.

Preferably, the recombinant mycoplasma hyopneumoniae surface protein P1 in step 1) is prepared by the following steps:

1.1) cloning and expression of Mycoplasma pneumoniae P1 Gene

Performing bioinformatics analysis on the surface protein P1 gene of the mycoplasma pneumoniae, optimizing the DNA coding sequence of the surface protein P1 gene of the mycoplasma pneumoniae by combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, introducing a restriction enzyme cutting site NdeI into 5 ' of the surface protein P1 gene of the mycoplasma pneumoniae, introducing a termination signal TAA and an enzyme cutting site EcoRI into 3 ' end of the surface protein P1 gene of the mycoplasma pneumoniae, chemically synthesizing a whole gene sequence, and connecting the whole gene sequence to a vector pUC57, wherein the whole gene sequence is marked as P1 '; carrying out double enzyme digestion on a vector pUC57 containing the artificially synthesized DNA fragment by NdeI and EcoRI, and then recovering a target fragment for later use according to a conventional method; carrying out double enzyme digestion on a vector pET-28a (+) by NdeI and EcoRI, connecting a P1 'gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional method, transforming Escherichia coli TOP10, and constructing a pET-P1' expression vector; enzyme digestion and sequence determination prove that the construction of the expression vector is correct; the vector expresses recombinant P1-His fusion protein;

1.2) purification of the human Mycoplasma pneumoniae P1 protein

Identifying the correct positive clone bacteria, culturing, extracting plasmid, transferring into competent E.coli BL21(DE3) plysS according to the conventional technology, coating the bacterial liquid on LB plate containing 50 ug/mL kanamycin after the conversion is finished, screening expression strains according to the conventional method, culturing, and purifying.

A monoclonal antibody of surface protein of mycoplasma pneumoniae of human, which is characterized in that: the monoclonal antibody of the surface protein of mycoplasma pneumoniae is the monoclonal antibody secreted by the hybridoma cell strain Mp-7# in claim 1.

A preparation method of a human mycoplasma pneumoniae surface protein monoclonal antibody is characterized by comprising the following steps: the method comprises the following steps:

Carrying out expanded culture on the screened positive monoclonal hybridoma cells; preparing monoclonal antibody ascites by conventional ascites in vivo induction method, collecting monoclonal antibody ascites and purifying the ascites by Protein A affinity chromatography to obtain mycoplasma pneumoniae surface Protein monoclonal antibody Mp-7 #.

The application of the mycoplasma hyopneumoniae surface protein monoclonal antibody in detecting mycoplasma hyopneumoniae, especially in preparing reagent for detecting mycoplasma hyopneumoniae, and is especially the application in preparing surface protein capture ELISA kit for detecting mycoplasma hyopneumoniae.

a surface protein capture ELISA kit for detecting mycoplasma hyopneumoniae is characterized in that: the surface protein capture ELISA kit for detecting the mycoplasma pneumoniae comprises an ELISA plate and a mycoplasma pneumoniae surface protein monoclonal antibody Mp-7 #; the ELISA plate is a solid phase carrier coated with a polyclonal antibody of the surface protein of the mycoplasma pneumoniae.

The preparation method of the polyclonal antibody against the surface protein of the mycoplasma pneumoniae comprises the following steps:

a) Immunizing a new-Zealand pure rabbit by adopting a recombinant mycoplasma pneumoniae surface protein P1, and detecting the level of a serum antibody by an indirect ELISA method;

b) purifying and extracting the polyclonal antibody by using a GE-HiTrap Protein A HP pre-packed column to prepare the human mycoplasma pneumoniae surface Protein polyclonal antibody.

The invention has the following advantages and effects:

Firstly, the invention successfully obtains the soluble recombinant P1 protein by adopting the modes of gene optimization and the like, the protein structure is natural, and the protein is a good material for preparing the antibody as an immune source.

Secondly, the monoclonal antibody obtained by the invention can specifically recognize the extracellular region of the thallus P1 protein, and has no cross reaction with other common respiratory tract pathogens.

Finally, the results of specificity experiments carried out by using 7 strains of mycoplasma hyopneumoniae and 17 strains of mycoplasma nonhuman pneumoniae standard strains (containing most common pathogens in respiratory tract) show that the kit has good specificity and stability, can detect all tested mycoplasma hyopneumoniae strains, and has no cross reaction with all the mycoplasma hyopneumoniae standard strains. Secondly, the sensitivity test result shows that the detection sensitivity of the kit is 1 multiplied by 103 CFU/hole, which is obviously higher than the traditional detection method of microorganism, and the kit has the advantages of rapidness, high efficiency and the like.

Detailed Description

The methods used in the following examples are conventional methods unless otherwise specified.

Example 1 preparation of recombinant Mycoplasma pneumoniae surface protein P1

1) Cloning and expression of Mycoplasma pneumoniae P1 gene

the bioinformatics analysis of the surface protein P1 of Mycoplasma pneumoniae (the access number in NCBI protein database is AAK92040.1) gene was performed, the DNA coding sequence was optimized in consideration of GC content, codon preference, secondary mRNA structure, RNA instability motif, mRNA free energy stability and the like, and the whole gene sequence was chemically synthesized after introducing a cleavage site NdeI at the 5 'end, a termination signal TAA at the 3' end and an cleavage site EcoRI at the 3 'end (the whole sequence synthesis was completed by King Murray Biotech Co., Ltd., and the artificially synthesized gene fragment was linked to the vector pUC57 at delivery), which was designated as P1'. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. The vector pUC57 containing the artificially synthesized DNA fragment was digested with NdeI and EcoRI, and the target fragment was recovered by a conventional method and used. And carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and EcoRI, connecting the P1 'gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional method, transforming Escherichia coli TOP10, and constructing a pET-P1' expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant P1-His fusion protein.

2) purification of Mycoplasma hyopneumoniae P1 protein

Identifying the correct positive clone bacteria, culturing, extracting plasmid, transferring into competent E.coli BL21(DE3) plysS according to the conventional technology, coating the bacterial liquid on LB plate containing 50 ug/mL kanamycin after the conversion is finished, and screening expression strains according to the conventional method. A single colony transformed with pET-P1 and having the ability to express a foreign protein was picked and inoculated into 100mL of LB medium and cultured overnight at 37 ℃. After taking out the bacterial liquid, the bacterial liquid is prepared according to the following steps of 1: 100 was inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, and when the cells were cultured at 37 ℃ until OD600 became 0.6, 1mol/L of IPTG was added to a final concentration of 0.6mmol/L, and the cells were cultured with shaking at 37 ℃ to induce expression of the fusion protein. After induction for 4h, the thalli are collected by centrifugation for 10min at 8000 r/min. The cells were washed 3 times with 50mL Buffer A (50mM Na3PO4, 0.5M NaCl; pH7.4) and resuspended in 50mL loading Buffer (50mM Na3PO4, 0.5M NaCl; 5mM imidazole, pH7.4) and sonicated under the following conditions: the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30 min. After the ultrasonic treatment is finished, the mixture is centrifuged at 12000g for 15min, and then the precipitate and the supernatant are respectively collected for electrophoresis detection. The recombinant P1 protein was found to be present in the bacterial cells in dissolved form. Thin-layer scanning shows that the recombinant protein accounts for more than 30% of the total protein of the bacteria, which indicates that higher expression level is obtained after gene optimization. The sonicated supernatant obtained above was filtered through a 0.45 μm filter and purified by His Trap affinity columns (GE healthcare Co.) according to the method described in the specification. The specific method comprises the following steps:

Connecting a chromatography system, wherein the system comprises a sample inlet tube, a peristaltic pump, a chromatography column and an ultraviolet detector (Shanghai Huxi analytical instrument factory, model HD1), the column volume is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable; and (5) correcting T%: adjusting a brightness knob to display 100%; rotate the sensitivity to the appropriate position, typically 0.2A; equilibrating the chromatography system with the loading buffer until the reading stabilizes and then rotating "zero" to show "000"; applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid; washing away unbound protein with a loading buffer, recording the reading during the process until the reading does not change any more, and collecting the eluate; eluting with Buffer A +10mM imidazole, and collecting an elution peak; eluting with Buffer A +20mM imidazole, and collecting an elution peak; eluting with Buffer A +40mM imidazole, and collecting an elution peak; eluting with Buffer A +100mM imidazole, and collecting an elution peak; eluting with Buffer A +150mM imidazole, and collecting an elution peak; taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis; as a result, it was found that the target protein was eluted at 100mM imidazole and had a purity of 90% or more, and the concentration was adjusted to 0.2mg/mL for use after the protein concentration was measured with a bradford kit. Thus, the recombinant Mycoplasma pneumoniae surface protein P1 is prepared.

EXAMPLE 2 preparation of polyclonal antibody against surface protein of Mycoplasma pneumoniae

1) immunization of New Zealand pure rabbits

The recombinant P1 protein prepared in example 1 was mixed with Freund's complete adjuvant, emulsified and used as immunogen to immunize 2 male New Zealand rabbits, each rabbit was injected subcutaneously in a total amount of 2ml, and the total amount of antigen was 2 mg/rabbit. And then immunizing once every two weeks by using an emulsion formed by the recombinant P1 protein and Freund's incomplete adjuvant, wherein the immunization is performed for 5 times in total, and the dosage of the antigen is the same as that of the primary immunization. Large amount of blood is taken 3-5 days after five-immunization, placed at 37 ℃ for 1 hour, then placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.

2) Determination of polyclonal antibody titer

the recombinant P1 protein is used as a coating antigen, the coating concentration is 10 mu g/ml, each well is coated with 100 mu l, and the serum antibody level is detected by an indirect ELISA method. The serum dilution times of the experimental groups are 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1: 102400, 1: 204800; the ELISA plate is coated with bovine serum albumin as a negative control, and an enzyme-linked detector is used for measuring OD450, so that the positive result is obtained when the P/N value is more than 2.1. The results showed that the serum antibody titers of 2 rabbits all reached 1: 102400, it is indicated that the immune effect is better.

3) Extraction of polyclonal antibodies

the antibodies were purified using a GE-HiTrap Protein A HP pre-packed column as described, in the following manner:

1.5 mL of antiserum was added with 0.5mL of 1M Tris (pH8.0) to adjust to pH8.0, and the mixture was centrifuged at 20000g for 20min to remove the precipitate.

2. after loading, the column was washed with 10 column volumes of buffer A (100mM Tris-Cl, pH8.0) and then with 10 column volumes of buffer B (10mM Tris-Cl, pH 8.0).

3. IgG was eluted with approximately three column volumes of IgG elution buffer (100mM glycine, pH 3.0). (0.1 mL IgG-neutralizing buffer (1M Tris-Cl, pH8.0) was preloaded into the collection tube, 0.9mL of eluent was added to each tube)

4. Dialysis was performed with 50 volumes of Tris (10mM Tris-Cl, pH 8.0).

5. after ultrafiltration and concentration, the concentration was adjusted to 1mg/ml and the mixture was stored at-70 ℃ for further use. Thus, the polyclonal antibody of the surface protein of the mycoplasma pneumoniae is prepared.

EXAMPLE 3 preparation of Mycoplasma pneumoniae surface protein monoclonal antibody Mp-7#

1) Immunization of animals

5 BALB/c mice 8 weeks old were immunized with the recombinant Mycoplasma pneumoniae surface protein P1 prepared in example 1 as an antigen, and 2 mice not immunized were used as negative controls. After fully emulsifying the primary immune antigen with equivalent Freund's complete adjuvant, the mice were immunized by subcutaneous multiple injections at the back, 100. mu.g/mouse. Then, the antigen with the same dose is fully emulsified with Freund's incomplete adjuvant at an interval of three weeks and then injected into the abdominal cavity for the second immunization, and the antigen with the same dose is fully emulsified with Freund's incomplete adjuvant at an interval of 2 weeks and then injected into the abdominal cavity for the third immunization. Blood is collected from tail vein 15 days after the third immunization, and the antiserum titer is detected.

2) Antiserum potency detection

The antiserum titer adopts an indirect ELISA method: the antigen concentration was diluted to 3. mu.g/mL with PBS (8.5g/L NaCl, 1.4g/L Na2HPO4, 0.2g/L NaH2PO4, pH7.4), added to a 96-well plate, 100. mu.L/well, and incubated at 37 ℃ for 2h or at 4 ℃ overnight. The plate was washed 3 times with PBST (8.5g/L NaCl, 1.4g/L Na2HPO4, 0.2g/L NaH2PO4, 0.5% (v/v) Tween-20, pH7.4) before plate-picking. 1% bovine serum albumin was dissolved in PBS solution at 250. mu.L/well and blocked at 37 ℃ for 1h or overnight at 4 ℃. The plates were photographed after 3 washes with PBST. 4 immune mouse serum was diluted with PBS gradient and added to corresponding wells at 100. mu.L/well, blank control was PBS solution, and negative control was plate washing after the mouse serum was coated for 1h at 37 ℃ before immunization. HRP-labeled goat anti-mouse IgG was expressed as 1: plates were washed after 3000 fold dilution addition, 100 μ L/well, 1h coating at 37 ℃. 100 mu L of a freshly prepared TMB chromogenic substrate solution is added into each well, 100 mu L of 1M hydrochloric acid is added into each well after the reaction is carried out for 10min at 37 ℃, the reaction is stopped, an OD450nm value is measured by an enzyme-linked immunosorbent assay, and the result is read and observed. The titer was highest (titer 51200), boosting was performed after one month interval for the third immunization, and 5 days later splenocytes from mice were taken for cell fusion.

3) Resuscitation and culture of SP2/0 myeloma cells

Thawing frozen myeloma cells (SP2/0) in advance, quickly taking out the myeloma cells frozen by liquid nitrogen, placing the myeloma cells in a water bath at 37 ℃ to slightly shake to quickly melt the myeloma cells, paying attention to the fact that the mouth of the frozen tube cannot touch water so as to avoid pollution, transferring the cells into a 24-hole plate containing 2ml of RPMI-1640 complete culture medium (RPMI-1640 culture medium containing 20% fetal calf serum purchased from Shanxi Runsheng Dai biomaterial Co., Ltd.), placing the 24-hole plate into a 37 ℃ C., 5% CO2 culture box to culture for half an hour, timely replacing the cells when the cells are all adherent to the skin, carrying out passage once every 3 days later, adjusting the cells to be in the optimal growth density, and counting the cells to prepare for fusion when the cells reach certain activity. Cells were passaged 1 to 4 days before cell fusion and the cell concentration per flask was adjusted to 1X 105/ml with fresh medium.

4) Preparation of feeder cells

4.1) the BALB/c mouse eyeball is bled, then the neck is pulled to be killed, the ball is completely soaked in 75% alcohol for 5min, and the ball is moved into a plate of an ultra-clean workbench, so that the abdomen of the ball faces upwards.

4.2) the skin of the thoracoabdominal region of the mouse was lifted up with forceps, a small opening was cut with scissors, the skin was torn open with two forceps to a larger opening, the peritoneum of the mouse was lifted up, the spleen of the mouse was found, the spleen was carefully taken out with the forceps and the small scissors, placed in a disposable dish, the fat, connective tissue and the like attached to the spleen were carefully peeled off, 5ml of RPMI-1640 medium (purchased from Hyclone, cat # SH30809.01) was added, the spleen was punctured with the needle of a syringe with 5ml of RPMI-1640 medium, splenocytes were carefully washed out, followed by sieving, the spleen cell suspension was added to a 10ml centrifuge tube, centrifuged at 1100rpm for 5min, the supernatant was discarded, and centrifuged and washed twice with the RPMI-1640 medium.

4.3) cells were gently resuspended and mixed well with 5ml HAT medium, counted and supplemented with HAT medium to a cell concentration of 1X 105/ml.

4.4) dropping the cell suspension into a 96-well cell culture plate, and culturing in a 5% CO2 incubator at 37 ℃ at a concentration of 130. mu.l/well.

5) Preparation of immune spleen cell suspension

5.1) after 5 days of booster immunization, BALB/c mice with highest serum titer were selected, the eyeballs were removed, blood was bled, and serum was collected and isolated as a positive control for antibody detection.

5.2) the mouse is soaked in 75% alcohol for 5min after being killed by breaking the neck, and the mouse is taken out and placed in a dish of a sterile ultra-clean workbench, so that the abdomen of the mouse faces upwards.

5.3) the skin of the chest and abdomen of the mouse is lifted by forceps, a small opening is cut by scissors, then the skin is torn open by two forceps to a larger opening, then the peritoneum of the mouse is lifted by a new forceps, the spleen of the mouse is found by cutting, the spleen is carefully taken out and put in a disposable plate, and the fat and connective tissues are carefully removed.

5.4) washing with RPMI-1640 washing solution, adding new RPMI-1640 washing solution, puncturing spleen with a needle of a syringe sucked with 5ml of RPMI-1640 culture medium to wash out splenocytes carefully, sieving to press the splenocytes into the solution through meshes as much as possible, transferring the splenocyte suspension into a centrifuge tube, centrifuging at 1100rpm for 5min, discarding supernatant, and centrifuging and washing twice.

5.5) gently resuspend the splenocytes in RPMI-1640 medium and count for use.

6) Preparation of SP2/0 myeloma cell suspension

6.1) 2 bottles of myeloma cells cultured in T75 flasks (liquid changed the day before fusion, cells should be in logarithmic growth phase at the time of fusion) were collected in 50ml centrifuge tubes.

6.2) centrifugation at 1000rpm for 5 minutes, and discarding the supernatant.

6.3) adding 30ml of RPMI-1640 washing liquor into the precipitate, gently suspending, uniformly mixing, and centrifugally washing once again by the same method.

6.4) the splenocytes were gently resuspended in 10ml of RPMI-1640 medium and mixed well and counted for future use.

7) Cell fusion

7.1) the suspension containing 1X 108 spleen cells and the suspension containing 1X 107 SP2/0 myeloma cells were mixed in a 50ml centrifuge tube, supplemented with medium to 40ml and mixed well.

7.2) centrifugation at 1200rpm for 5 minutes, the supernatant was discarded and the supernatant was removed as much as possible.

7.3) lightly flicking the bottom of the centrifugal tube by hand to ensure that cell masses are loosened, uniform and pasty.

7.4) the prepared 50% PEG (MW1450), RPMI-1640 lotion was taken out of the 4 ℃ freezer and placed in a 37 ℃ water bath, pre-warmed for use.

7.5) 0.8ml of 50% PEG (MW1450) was pipetted with a 1ml pipette and slowly added to the centrifuge tube with stirring for a controlled period of 60 seconds.

7.6) then 40ml of pre-warmed RPMI-1640 wash was added gradually over a period of 60 seconds to dilute the PEG and lose its fusogenic effect.

7.7) centrifuge at 1000rpm for 5min and discard the supernatant.

7.8) add 400mL HAT medium (purchased from Sigma, cat # H0262), gently aspirate, and resuspend the pelleted cells.

7.9) the fused cell suspension was added to a 96-well plate containing feeder cells at a concentration of 50. mu.l/well, and 20 plates were spread in total, and then the plate was incubated in an incubator at 37 ℃ with 5% CO 2.

8) Screening and cloning culture of Positive clones

Starting on day 3 after fusion, the cells in each well were observed daily for growth and were immediately treated with sodium azide if contamination was present. Total replacement with HT medium at 7d after fusion (HT, 50 ×, purchased from Sigma, cat # H0137). The next day after the change of fluid, the supernatant from the wells in which the clones appeared was aspirated for specific detection. The detection is carried out by taking mycoplasma pneumoniae thalli (ATCC15531, 1 × 108 thalli/hole) as a coating antigen and adopting an indirect ELISA method. The OD450 is measured by an enzyme-linked detector, and the positive result is that the P/N value is more than 2.1. And (3) replacing the positive holes with HT culture medium, performing ELISA detection on the positive holes again the next day, selecting continuous positive cloning holes, performing subcloning for 2-3 times, and screening monoclonal hybridoma cells. Subcloning the specific steps: firstly, blowing and uniformly mixing the hybridoma cells with the positive fusion holes, and measuring the cell concentration; preparing feeder cells in advance, suspending the feeder cells in an HT culture medium at 130 ul/hole, spreading the feeder cells in a 96-hole plate, and placing the culture plate in an incubator at 37 ℃ and 5% CO2 for later use; taking the hybridoma cells with the positive fusion wells, and evenly dispersing the hybridoma cells into the 96-well plate obtained in the step II; fourthly, placing the culture plate in an incubator with 37 ℃ and 5% CO2 for culture for 7-8 days; screening positive single colony wells by ELISA, and subcloning again; after continuous subcloning for 2-3 times, making the subcloned cell colony holes positive and similar numerical values to obtain monoclonal hybridoma cells; and carrying out amplification culture on the monoclonal hybridoma cells to obtain a cell culture solution supernatant containing the monoclonal antibody. Legionella pneumophila (ATCC33152, 1 × 108 cells/well), Pseudomonas aeruginosa (ATCC27853, 1 × 108 cells/well), Moraxella catarrhalis (ATCC25240, 1 × 108 cells/well), Acinetobacter baumannii (ATCC19606, 1 × 108 cells/well), Haemophilus parainfluenzae (ATCC7901, 1 × 108 cells/well), Haemophilus influenzae (ATCC49247, 1 × 108 cells/well), Streptococcus pyogenes (ATCC19615, 1 × 108 cells/well), Staphylococcus aureus (ATCC25923, 1 × 108 cells/well), Streptococcus pneumoniae (ATCC49619, 1 × 108 cells/well), Klebsiella pneumoniae (ATCC 700603, 1 × 108 cells/well), Enterobacter cloacae (ATCC13047, 1 × 108 cells/well), Escherichia coli (ATCC25922, 1 × 108 cells/well), Candida (ATCC1, 1 × 108 cells/well) and other respiratory tract pathogenic bacteria are respectively coated on an enzyme label plate, the specificity of the screened antibody is detected by ELISA, and monoclonal antibodies which have positive reactions with the pathogens are eliminated. In this step, 8 qualified cell lines were selected.

9) Ascites preparation and cell preservation

And performing expanded culture on the finally screened 8 strains of positive monoclonal hybridoma cells. Preparing the monoclonal antibody ascites by a conventional ascites in vivo induction method. (1) Western blot detection of mAb: and (3) determining the ascites by adopting a conventional Western blot experiment method in the ratio of 1: the results of the specificity at 2000 dilution showed that all 8 antibodies bound only P1 protein among the whole cell proteins of Mycoplasma pneumoniae. (2) Antibody purification and potency determination: the ascites fluid was purified by Protein a affinity chromatography, the procedure was as follows:

The antibodies were purified using a GE-HiTrap Protein A HP pre-packed column as described, in the following manner:

a. 5mL of hybridoma supernatant was added with 0.5mL of 1M Tris (pH8.0) to adjust to pH8.0, and the mixture was centrifuged at 20000g for 20min to remove the precipitate.

b. After loading, the column was washed with 10 column volumes of buffer A (100mM Tris-Cl, pH8.0) and then with 10 column volumes of buffer B (10mM Tris-Cl, pH 8.0).

c. IgG was eluted with approximately three column volumes of IgG elution buffer (100mM glycine, pH 3.0). (0.1 mL IgG-neutralizing buffer (1M Tris-Cl, pH8.0) was preloaded into the collection tube, 0.9mL of eluent was loaded into each tube);

d. Dialysis was performed with 50 volumes of PBS (8.5g/L NaCl, 1.4g/L Na2HPO4, 0.2g/L NaH2PO4, pH 7.4).

e. After concentration by ultrafiltration, the concentration was adjusted to 1mg/ml with PBS and stored at-70 ℃ for further use.

The 8 purified antibodies were diluted by a multiple ratio and then the titer was measured by indirect ELISA. The purity of the antibody is more than 95% through SDS-PAGE analysis, and the ELISA titer is 1: 1000000 or more. The purified antibody was adjusted to a concentration of 1mg/ml and stored at-70 ℃ until use.

The 8 antibodies are respectively used as labeled antibodies for ELISA detection, and monoclonal antibody-polyclonal antibody pairing experiments are carried out to screen out the optimal combination. Finally, through a plurality of experiments, the monoclonal antibody secreted by the hybridoma cell strain named as Mp-7# is preferably a labeled antibody. This antibody was designated monoclonal antibody Mp-7 #.

The hybridoma cell strain secreting the antibody is sent to China center for type culture Collection for preservation in 2018, 10 months and 15 days, and is classified and named as follows: hybridoma cell strain Mp-7#, preservation number CCTCC NO: c2017217; address: wuhan university in Wuhan, China.

Example 4 Mycoplasma pneumoniae surface protein Capture ELISA kit

1) mycoplasma hyopneumoniae surface protein capture ELISA kit composition

the solid phase carrier coated with the polyclonal antibody of the surface protein of the mycoplasma pneumoniae is an enzyme label plate, the monoclonal antibody Mp-7# of the surface protein of the mycoplasma pneumoniae, the goat anti-mouse IgG marked by horseradish peroxidase, substrate reaction liquid, positive and negative controls of enzyme, and a washing liquid and a reaction stopping liquid jointly form the capture ELISA kit for the surface protein of the mycoplasma pneumoniae of adults.

Enzyme label plate coating multi-antibody:

The polyclonal antibody against the surface protein of Mycoplasma pneumoniae (prepared as described in example 2) was diluted to a concentration of 10. mu.g/mL with PBS buffer, and coated with a 96-well EIA high-efficiency binding microplate at 50. mu.L/well for 2 hours at 37 ℃. Taking out, washing the plate with washing liquid for three times, and spin-drying. Using a washing solution containing 1% BSA as a blocking solution, 250. mu.L/well of the blocking solution was applied to an ELISA plate, and the plate was blocked at 37 ℃ for 1 hour. Taking out, washing the plate with washing solution for 3 times, one minute each time, spin-drying, sealing, and storing.

wherein, the PBS buffer solution: 1.4g of disodium hydrogen phosphate, 0.2g of sodium dihydrogen phosphate, 8.5g of sodium chloride and 1000mL of deionized water with the pH value of 7.4; wash solution (PBST): PBS aqueous solution containing 0.01% Tween-20, pH 7.4; sealing liquid: aqueous washing containing 1% BSA, pH 7.4.

2) Monoclonal antibody Mp-7# of surface protein of mycoplasma hyopneumoniae

The monoclonal antibody Mp-7# of the surface protein of Mycoplasma pneumoniae human as described in example 3 at a concentration of 1mg/mL was dispensed in an amount of 20. mu.L per vial, and was used by diluting 1000-fold with PBS buffer.

3) Horse radish peroxidase labeled goat anti-mouse IgG

horse radish peroxidase-labeled goat anti-mouse IgG is a common commercial product, which is purchased from Beijing seichi Biotechnology Co., Ltd in this example under the product number 030006-G, and is diluted 3000 times with PBS buffer when used.

4) Substrate reaction solution for enzyme

The preparation process of the substrate reaction solution of the enzyme is as follows:

Solution A: configured quantity 1L)

1. 3.14g of citric acid (containing 1 molecule of crystal water and having a molecular weight of 210.14g) and 11.56g of sodium acetate (containing 3 molecules of crystal water and having a molecular weight of 136.0) were weighed and dissolved in 970mL of double distilled water to prepare an aqueous solution of sodium acetate having a pH of 5.0.

2. Weighing 0.08g of phenacetin, adding 30mL of double distilled water, heating to 100 ℃, and adding the mixture into the solution in the first step after completely dissolving.

3. then 0.5g of carbamide peroxide is added and mixed evenly.

And B, liquid B: configured quantity 1L)

adding 500mL of methanol into a 2L beaker, adding 1.27g of 3, 3, 5, 5-tetramethylbenzidine TMB (SIGMA), heating at 60 ℃ to dissolve, and adding 500mL of glycerol.

A, B liquid 1: 1, mixing the two solutions to prepare a substrate reaction solution of the enzyme.

5) Positive and negative controls

Positive control: recombinant Mycoplasma pneumoniae surface protein P1, extracted as described in example 1, at 10. mu.g/mL.

Negative control: serum from normal mice without the recombinant mycoplasma hyopneumoniae surface protein P1 was diluted 200-fold with PBS buffer.

6) The washing solution, PBST solution, was prepared as described in (1).

7) The reaction termination solution was 1M HCl solution prepared with double distilled water.

Example 5 methods of Using the Mycoplasma pneumoniae surface protein Capture ELISA kits

1) Treatment of samples to be examined

A pharyngeal swab of a subject is obtained by a conventional method, and the pharyngeal swab is inserted into a soft plastic tube containing 500. mu.L of a washing solution (PBST), and the tube wall of the plastic tube is pressed to sufficiently dissolve a sample on the swab. The sample is subjected to ultrasonic disruption (Ningbo Xinzhi JY96-IIN type ultrasonic cell disrupter, 30% power, 15min) to prepare a disruption solution.

2) Adding control and sample to be tested

Taking 100 mu L of a to-be-detected crushing liquid sample, adding the sample into corresponding enzyme labeled holes, adding 1 hole of a positive control (100 mu L/hole) and 4 holes of a negative control (100 mu L/hole), incubating for 1 hour at 37 ℃, washing the plate for 3 times by using 250 mu L of PBST washing liquid, and drying by spin.

3) adding monoclonal antibody Mp-7#

add 50. mu.L/well of the M.pneumoniae surface protein monoclonal antibody Mp-7# working solution (blocking solution 1: 1000 dilution) described in example 4, incubate for 1 hour at 37 ℃, wash the plate 3 times with 250. mu.L PBST wash solution, and spin-dry.

4) Adding enzyme-labeled antibody

Horseradish peroxidase-labeled goat anti-mouse IgG (described in example 4) working solution (blocking solution 1: 3000 diluted) was added at 50. mu.L/well, incubated at 37 ℃ for 1 hour, and then washed with 250. mu.L of PBST washing solution 3 times, and spun-dried.

5) Adding substrate to develop color

50. mu.L/well of the freshly prepared substrate reaction solution for the enzyme described in example 4 was added, and after sufficient color development (15 minutes).

6) Adding a reaction stopping solution

The reaction was terminated by adding 50. mu.L of a reaction terminator to each well.

7) Measuring OD450nm value

The microplate was placed in a microplate reader to determine the OD450nm value.

8) Determination of results

Respectively reading the OD450nm values of the 4-hole negative quality control sample and the 1-hole positive quality control sample; the sum of the average value of the OD450nm readings of the 4-well negative control samples and the 3-fold standard deviation is the CUT-OFF value; if the detection OD450nm value of the human pharynx swab sample is larger than the CUT-OFF value, the antigen of the mycoplasma hyopneumoniae in the clinical pharynx swab is judged to be positive, otherwise, the antigen of the mycoplasma hyopneumoniae in the human pharynx swab sample is judged to be negative; if the OD450nm value of the positive quality control sample is less than the CUT-OFF value, the kit is invalid.

Example 6 determination of specificity and sensitivity of Mycoplasma pneumoniae surface protein Capture ELISA kits

1) Specific assay

In order to verify the specificity of the mycoplasma hyopneumoniae surface protein capture ELISA kit of the present invention, 7 mycoplasma hyopneumoniae strains and 17 mycoplasma hyopneumoniae standard strains were tested according to the kit composition and method described in example 4 and example 5, and are shown in Table 1. The result shows that the detection results of the kit are positive for all 7 strains of the mycoplasma pneumoniae of the human, and the detection results of the kit are negative for other 17 strains of the common pathogenic microorganisms of the respiratory tract. The kit showed good specificity.

TABLE 1

Strain name	Results of the detection	Strain name	Results of the detection
				Mycoplasma pneumoniae ATCC15531	Positive for	Human Haemophilus influenzae ATCC49247	negative of
Mycoplasma pneumoniae ATCC39505	Positive for	Streptococcus pyogenes ATCC19615	Negative of
				Mycoplasma pneumoniae ATCC29342	Positive for	Staphylococcus aureus ATCC25923	Negative of
Mycoplasma pneumoniae ATCC15293	Positive for	Streptococcus pneumoniae ATCC49619	Negative of
				Mycoplasma pneumoniae ATCC15492	Positive for	klebsiella pneumoniae ATCC700603	Negative of
Mycoplasma pneumoniae ATCC15377	Positive for	Enterobacter cloacae ATCC13047	Negative of
				Mycoplasma pneumoniae ATCC29343	Positive for	Escherichia coli ATCC25922	negative of
Legionella pneumophila ATCC33152	Negative of	Candida ATCC10231	Negative of
				Pseudomonas aeruginosa ATCC27853	Negative of	Influenza A virus ATCCVR-1743	Negative of
Moraxella catarrhalis ATCC25240	Negative of	Influenza B virus ATCCVR-790	Negative of
				Acinetobacter baumannii ATCC19606	Negative of	Respiratory syncytial virus ATCCVR26	negative of
Haemophilus parainfluenza ATCC7901	Negative of	Adenovirus ATCCVR-3	Negative of

2) Sensitivity assay

The mycoplasma pneumoniae ATCC15531 strain is inoculated in an OXOID mycoplasma culture medium, after 15 days of culture at 37 ℃, 10-fold gradient dilution is carried out by normal saline, meanwhile, plates are counted, a thallus solution with the thallus concentration of 108-. The result shows that the detection sensitivity of the kit is 103 CFU/mL.

Sequence listing

<110> Hubei Yunluo bioengineering Co Ltd

<120> human mycoplasma pneumoniae surface protein monoclonal antibody and antigen capture ELISA kit

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1224

<212> DNA

<213> P1 Gene Sequence (Artificial Sequence)

<400> 1

catatggcgt acggtgaagt aaacggtttc ttcaaccctg cttttgtgga aacttatttc 60

ggcaacacta gcgctactgg taccggcacc aacactacca ccaccccggg catcggcttc 120

aaaattccgg aacagaccgg taccaacacc accactaaag ctgttttcat caccccgggc 180

tttgcgtgga ctccgcagga tgttggcaac ttcgtagtta caggcaccac tttcaccttc 240

cagttcggtg gttggtttgt aaccttcact gactttatca agccgtctgc tggctacttc 300

ggtttccaat ttaccggctt tgatgctact gacgcaacac agagcgcgtt catctgggct 360

ccatccccgt gggctgcatt ttctggtact tgggttaact ccttcggctc tgttgaaaca 420

gtttgggatt tcaaaggtgt atgggcggac caggctcaga ccgataccca gggcacgacc 480

accactgcga cctctgacgc gtttccagaa cacccgaacg ctttcgcgtt ccaggttact 540

gttgttgaag caaccgctta caagccgaac accaccaccg gtcagactca gaccaccaac 600

actaccccgt actttcattt tgttaaaccg aaaaaagtta ttcagaccga caaattcgac 660

gatgacttca aaaacttctt cgacccgaac caggtttcta ccaaattcag ccagaccttc 720

ggcactgacc acaccaccca gccgcagccg cagactttca agaccactac cccaatcttt 780

ggtactacca ccggtaactt cactaccgtg ttcactggcg gcggcgctgg tggcggtact 840

accggcaccg gtcagactgg cgttgacttc accccggttg aaaaagtaac cggttggttc 900

gtgggtcagt tcccaactac caccgacggt aacaccacca ctaccaacaa cttcgctccg 960

aacactaaca ccggcaacga tgttgtgggt gttggctctt tcaccgaaac caacgcagct 1020

aaaatgaacg acgacgtaga cggtatcgta agcaccccgt ttgccgagtt ctttgatggt 1080

gaaggccaga ctgcagacac cggtccgcag accgttaaat tcaaaactcc agaccagatc 1140

gactttaact ccttcttcac ccacccagta accgactttt tcgaccctgt tactatgttc 1200

gtatacgacc agtacatcct cgag 1224

<210> 2

<211> 405

<212> PRT

<213> P1 protein Sequence (Artificial Sequence)

<400> 2

Met Ala Tyr Gly Glu Val Asn Gly Phe Phe Asn Pro Ala Phe Val Glu

1 5 10 15

Thr Tyr Phe Gly Asn Thr Ser Ala Thr Gly Thr Gly Thr Asn Thr Thr

20 25 30

Thr Thr Pro Gly Ile Gly Phe Lys Ile Pro Glu Gln Thr Gly Thr Asn

35 40 45

Thr Thr Thr Lys Ala Val Phe Ile Thr Pro Gly Phe Ala Trp Thr Pro

50 55 60

Gln Asp Val Gly Asn Phe Val Val Thr Gly Thr Thr Phe Thr Phe Gln

65 70 75 80

Phe Gly Gly Trp Phe Val Thr Phe Thr Asp Phe Ile Lys Pro Ser Ala

85 90 95

Gly Tyr Phe Gly Phe Gln Phe Thr Gly Phe Asp Ala Thr Asp Ala Thr

100 105 110

Gln Ser Ala Phe Ile Trp Ala Pro Ser Pro Trp Ala Ala Phe Ser Gly

115 120 125

Thr Trp Val Asn Ser Phe Gly Ser Val Glu Thr Val Trp Asp Phe Lys

130 135 140

Gly Val Trp Ala Asp Gln Ala Gln Thr Asp Thr Gln Gly Thr Thr Thr

145 150 155 160

Thr Ala Thr Ser Asp Ala Phe Pro Glu His Pro Asn Ala Phe Ala Phe

165 170 175

Gln Val Thr Val Val Glu Ala Thr Ala Tyr Lys Pro Asn Thr Thr Thr

180 185 190

Gly Gln Thr Gln Thr Thr Asn Thr Thr Pro Tyr Phe His Phe Val Lys

195 200 205

Pro Lys Lys Val Ile Gln Thr Asp Lys Phe Asp Asp Asp Phe Lys Asn

210 215 220

Phe Phe Asp Pro Asn Gln Val Ser Thr Lys Phe Ser Gln Thr Phe Gly

225 230 235 240

Thr Asp His Thr Thr Gln Pro Gln Pro Gln Thr Phe Lys Thr Thr Thr

245 250 255

Pro Ile Phe Gly Thr Thr Thr Gly Asn Phe Thr Thr Val Phe Thr Gly

260 265 270

Gly Gly Ala Gly Gly Gly Thr Thr Gly Thr Gly Gln Thr Gly Val Asp

275 280 285

Phe Thr Pro Val Glu Lys Val Thr Gly Trp Phe Val Gly Gln Phe Pro

290 295 300

Thr Thr Thr Asp Gly Asn Thr Thr Thr Thr Asn Asn Phe Ala Pro Asn

305 310 315 320

Thr Asn Thr Gly Asn Asp Val Val Gly Val Gly Ser Phe Thr Glu Thr

325 330 335

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

340 345 350

Phe Ala Glu Phe Phe Asp Gly Glu Gly Gln Thr Ala Asp Thr Gly Pro

355 360 365

Gln Thr Val Lys Phe Lys Thr Pro Asp Gln Ile Asp Phe Asn Ser Phe

370 375 380

Phe Thr His Pro Val Thr Asp Phe Phe Asp Pro Val Thr Met Phe Val

385 390 395 400

Tyr Asp Gln Tyr Ile

405

Claims (10)

1. A hybridoma cell strain for producing a monoclonal antibody of surface protein of mycoplasma pneumoniae is characterized in that: the hybridoma cell strain for producing the mycoplasma hyopneumoniae surface protein monoclonal antibody has a preservation number of CCTCC NO: c2017217 hybridoma cell strain Mp-7 #.

2. A preparation method of hybridoma cell strain for generating monoclonal antibody of surface protein of mycoplasma pneumoniae is characterized by comprising the following steps: the preparation method comprises the following steps:

1) The recombinant mycoplasma pneumoniae surface protein P1 is used as an antigen, and after the BALB/c mouse of 8 weeks old is immunized, the antiserum titer is detected by adopting an indirect ELISA method;

2) Recovering and culturing SP2/0 myeloma cells;

3) Preparing feeder cells;

4) Preparing immune spleen cell suspension;

5) Preparing a SP2/0 myeloma cell suspension;

6) Fusing cells;

7) Screening and cloning culture of positive clones, and detecting by using mycoplasma pneumoniae thalli as a coating antigen by adopting an indirect ELISA method; determining OD450 with enzyme-linked detector to satisfy P/N value > 2.1 as positive; selecting continuous positive cloning holes to perform subcloning for 2-3 times, and screening monoclonal hybridoma cells; carrying out amplification culture on the monoclonal hybridoma cells to obtain cell culture solution supernatant containing a monoclonal antibody; respectively coating an enzyme label plate with respiratory tract pathogenic bacteria, carrying out ELISA detection on the specificity of the screened antibody, eliminating monoclonal antibodies having positive reactions with the pathogens, and screening out qualified cell strains; the respiratory pathogenic bacteria comprise legionella pneumophila, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, haemophilus influenzae, streptococcus pyogenes, staphylococcus aureus, streptococcus pneumoniae, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida.

3. The method for preparing a hybridoma cell line producing a monoclonal antibody to a surface protein of mycoplasma pneumoniae according to claim 2, wherein the method comprises the following steps: the preparation process of the recombinant Mycoplasma pneumoniae surface protein P1 in the step 1) comprises the following steps:

1.1) cloning and expression of Mycoplasma pneumoniae P1 Gene

Performing bioinformatics analysis on the surface protein P1 gene of the mycoplasma pneumoniae, optimizing the DNA coding sequence of the surface protein P1 gene of the mycoplasma pneumoniae by combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, introducing a restriction enzyme cutting site NdeI into 5 ' of the surface protein P1 gene of the mycoplasma pneumoniae, introducing a termination signal TAA and an enzyme cutting site EcoRI into 3 ' end of the surface protein P1 gene of the mycoplasma pneumoniae, chemically synthesizing a whole gene sequence, and connecting the whole gene sequence to a vector pUC57, wherein the whole gene sequence is marked as P1 '; carrying out double enzyme digestion on a vector pUC57 containing the artificially synthesized DNA fragment by NdeI and EcoRI, and then recovering a target fragment for later use according to a conventional method; carrying out double enzyme digestion on a vector pET-28a (+) by NdeI and EcoRI, connecting a P1 'gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional method, transforming Escherichia coli TOP10, and constructing a pET-P1' expression vector; enzyme digestion and sequence determination prove that the construction of the expression vector is correct; the vector expresses recombinant P1-His fusion protein;

1.2) purification of the human Mycoplasma pneumoniae P1 protein

Identifying the correct positive clone bacteria, culturing, extracting plasmid, transferring into competent E.coli BL21(DE3) plysS according to the conventional technology, coating the bacterial liquid on LB plate containing 50 ug/mL kanamycin after the conversion is finished, screening expression strains according to the conventional method, culturing, and purifying.

4. A monoclonal antibody of surface protein of mycoplasma pneumoniae of human, which is characterized in that: the monoclonal antibody of the surface protein of mycoplasma pneumoniae is the monoclonal antibody secreted by the hybridoma cell strain Mp-7# in claim 1.

5. A preparation method of a human mycoplasma pneumoniae surface protein monoclonal antibody is characterized by comprising the following steps: the method comprises the following steps:

Carrying out expanded culture on the screened positive monoclonal hybridoma cells; preparing monoclonal antibody ascites by conventional ascites in vivo induction method, collecting monoclonal antibody ascites and purifying the ascites by Protein A affinity chromatography to obtain mycoplasma pneumoniae surface Protein monoclonal antibody Mp-7 #.

6. The use of the monoclonal antibody against the surface protein of Mycoplasma pneumoniae of claim 4 in the detection of Mycoplasma pneumoniae.

7. the use of the monoclonal antibody against the surface protein of Mycoplasma pneumoniae of claim 4 in the preparation of a reagent for detecting Mycoplasma pneumoniae.

8. The use of the monoclonal antibody against surface protein of Mycoplasma pneumoniae of claim 4 in the preparation of ELISA kit for detecting surface protein capture of Mycoplasma pneumoniae.

9. A surface protein capture ELISA kit for detecting mycoplasma hyopneumoniae is characterized in that: the surface protein capture ELISA kit for detecting the mycoplasma pneumoniae comprises an ELISA plate and a mycoplasma pneumoniae surface protein monoclonal antibody Mp-7 #; the ELISA plate is a solid phase carrier coated with a polyclonal antibody of the surface protein of the mycoplasma pneumoniae.

10. The capture ELISA kit for detecting Mycoplasma pneumoniae surface protein according to claim 9, wherein: the preparation method of the human mycoplasma pneumoniae surface protein polyclonal antibody comprises the following steps:

a) Immunizing a new-Zealand pure rabbit by adopting a recombinant mycoplasma pneumoniae surface protein P1, and detecting the level of a serum antibody by an indirect ELISA method;

b) Purifying and extracting the polyclonal antibody by using a GE-HiTrap Protein A HP pre-packed column to prepare the human mycoplasma pneumoniae surface Protein polyclonal antibody.