CN110540972B

CN110540972B - Human legionella pneumophila surface protein monoclonal antibody and antigen capture ELISA kit

Info

Publication number: CN110540972B
Application number: CN201811596382.3A
Authority: CN
Inventors: 胡征
Original assignee: Hubei Yunlu Bioengineering Co ltd
Current assignee: Hubei Yunlu Bioengineering Co ltd
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-04-13
Anticipated expiration: 2038-12-20
Also published as: CN110540972A

Abstract

The invention discloses a human legionella pneumophila surface protein monoclonal antibody and an antigen capture ELISA kit. The human legionella pneumophila surface protein monoclonal antibody is prepared from a monoclonal antibody with a preservation number of CCTCCNO: c2017216. The human legionella pneumophila surface protein monoclonal antibody can be used for detecting human legionella pneumophila. The invention also discloses a human legionella pneumophila surface protein capture ELISA kit based on the monoclonal antibody.

Description

Human legionella pneumophila surface protein monoclonal antibody and antigen capture ELISA kit

Technical Field

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

Background

Legionella is a gram-negative pathogenic bacterium, often pollutes water environment, causes acute respiratory tract infection of people, and can be used as a biological warfare agent. Legionella is aerobic gram negative bacillus, has no bacteria membrane, produces no acid, produces no gas, has one to several terminal flagella or side flagella, and is movable. Currently, legionella has found 49 serotypes, more than 70 serotypes, of which at least 20 are associated with human disease. Common are legionella pneumophila (l.pneumophila, LP), legionella maurensis, legionella boehmeri, legionella phenanthrea, legionella dolichi, legionella longbeach, etc., wherein legionella pneumophila is an important pathogenic bacterium causing acquired pneumonia. The bacteria can be widely existed in natural water source, and can also be existed in cold and hot water pipeline system. The aerosol generated by soil, dust, river water, ditches, cooling water, air-conditioning condensate water, water for hospitals, ventilators and the like can be detected, and can be transmitted in an aerosol mode.

The outbreak range of the legionella pneumophila disease is wide, and the legionella pneumophila disease poses serious threats to human health, so that people are concerned more and more. Although legionella pneumophila is spread worldwide, there are very few standardized commercial reagents available for laboratory diagnostics. The existing method for detecting the pathogenic microorganisms mainly adopts the traditional method, namely a separation identification method, the method needs long time, generally 2-3 days, the accuracy is 25-75%, 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 colloidal gold detection methods and the like having proprietary intellectual property rights.

The selection of the antigenic component is critical to the preparation of antibodies for specific detection. PAL protein is one of the important outer membrane proteins of Legionella pneumophila. Engleberge et al found that serum from a legionella infected patient can specifically recognize legionella PAL and clone pneumophilia militaryRelative molecular mass of clitocybe is 19 multiplied by 10³The outer membrane lipoprotein gene of (1), wherein the gene encoding PAL is 528bp and comprises 176 amino acids, and a characteristic 22 amino acids are present in the sequence, which is identical to the sequence recognized by peptidase II, indicating that the antigen is a lipoprotein. However, the role of the outer membrane lipoproteins in the pathogenesis of legionnaires' disease is not clear enough. Sequence analysis shows that the PAL gene has high conservation in various legionella pneumophila, so the PAL protein can be used as a specific diagnostic antigen of legionella pneumophila. The PAL gene sequence is analyzed by bioinformatics and immunoinformatics software to remove the gene sequence with high similarity with other bacteria, so that the cross property of the PAL gene sequence with other bacteria antigens is reduced, the specificity of PAL protein is further ensured, and thus, the PAL gene sequence can be used for preparing high-specificity monoclonal antibodies. The research selects surface protein PAL with interspecies specificity as antigen, prepares monoclonal antibody with good specificity, and applies the monoclonal antibody to the preparation of human legionella pneumophila ELISA detection kit.

Disclosure of Invention

In order to solve the technical problems in the background art, the invention provides a human legionella pneumophila 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 a surface protein of human legionella pneumophila is characterized in that: the hybridoma cell strain for producing the human legionella pneumophila surface protein monoclonal antibody is preserved by China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: c2017216 hybridoma cell strain Lp-2 #.

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

1) the recombinant human legionella pneumophila surface protein PAL is taken as an antigen, 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 adopting an indirect ELISA method by taking human legionella pneumophila thalli as an envelope antigen; determination of OD Using an enzyme-Linked Detector₄₅₀Positive if the P/N value is more than 2.1; 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 include mycoplasma pneumoniae of human, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, haemophilus influenzae, streptococcus pyogenes, staphylococcus aureus, streptococcus pneumoniae of human, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida.

Preferably, the preparation process of the recombinant human legionella pneumophila surface protein PAL in the step 1) is as follows:

1.1) cloning expression of the PAL Gene of human Legionella pneumophila

Performing bioinformatics analysis on the surface protein PAL gene of the human legionella pneumophila, optimizing the DNA coding sequence of the surface protein PAL gene of the human legionella pneumophila by combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, introducing an enzyme cutting site NdeI into 5 ' of the surface protein PAL gene of the human legionella pneumophila, introducing a termination signal TAA and an enzyme cutting site EcoRI into 3 ' end of the surface protein PAL gene of the human legionella pneumophila, chemically synthesizing into a whole gene sequence, and connecting to a vector pUC57, and marking as PAL '; 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 PAL 'gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional method, transforming Escherichia coli TOP10, and constructing a pET-PAL' expression vector; enzyme digestion and sequence determination prove that the construction of the expression vector is correct; the vector expresses recombinant PAL-His fusion protein;

1.2) purification of human Legionella pneumophila PAL 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 human legionella pneumophila surface protein is characterized in that: the monoclonal antibody of the surface protein of the human legionella pneumophila is a monoclonal antibody secreted by the hybridoma cell strain Lp-2# in claim 1.

A preparation method of a human legionella pneumophila 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 human legionella pneumophila surface Protein monoclonal antibody Lp-2 #.

The application of the human legionella pneumophila surface protein monoclonal antibody in the detection of human legionella pneumophila, in particular to the application in the preparation of a reagent for detecting human legionella pneumophila, and most preferably to the application in the preparation of an ELISA kit for detecting the surface protein capture of human legionella pneumophila.

An ELISA kit for detecting surface protein capture of human legionella pneumophila is characterized in that: the ELISA kit for detecting the surface protein capture of the human legionella pneumophila comprises an ELISA plate and a human legionella pneumophila surface protein monoclonal antibody Lp-2 #; the ELISA plate is a solid phase carrier coated with a polyclonal antibody of the surface protein of the human legionella pneumophila.

The preparation method of the human legionella pneumophila surface protein polyclonal antibody comprises the following steps:

a) immunizing a new zealand pure rabbit by adopting a recombinant human legionella pneumophila surface protein PAL, and detecting the serum antibody level by an indirect ELISA method;

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

The invention has the following advantages and effects:

firstly, the invention successfully obtains the soluble recombinant PAL 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 bacterial PAL protein and has no cross reaction with other common respiratory pathogens.

Finally, a specificity experiment is carried out by using 7 strains of the human legionella pneumophila and 17 strains of the non-human legionella pneumophila standard strain (containing most common respiratory pathogens), and the result shows that the kit has good specificity and stability, can detect all tested human legionella pneumophila strains, and has no cross reaction with all non-human legionella pneumophila standard strains. Secondly, the sensitivity test result shows that the detection sensitivity of the kit is 1 multiplied by 10³CFU/hole is obviously higher than the traditional microorganism detection method, and 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 human Legionella pneumophila surface protein PAL

1) Cloning expression of PAL gene of human legionella pneumophila

Bioinformatics analysis is carried out on the surface protein PAL (the access number in the NCBI protein database is YP _096059) gene of the legionella pneumophila, the DNA coding sequence is optimized by combining the consideration of GC content, codon preference, secondary structure of mRNA, RNA unstable motif, mRNA free energy stability and the like, meanwhile, the 5 ' end of the PAL gene is introduced with enzyme cutting site NdeI, the 3 ' end of the PAL gene is introduced with termination signal TAA and enzyme cutting site EcoRI, and then the complete gene sequence is chemically synthesized (the complete sequence synthesis is completed by the Kinsley Biotechnology Limited company, and the artificially synthesized gene fragment is connected to the pUC57 when being delivered), which is marked as PAL '. 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. Meanwhile, NdeI and EcoRI are adopted to carry out double enzyme digestion on the vector pET-28a (+), and PAL 'gene obtained after double enzyme digestion is connected into the pET-28a (+) vector according to a conventional method, and escherichia coli TOP10 is transformed to construct pET-PAL' expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant PAL-His fusion protein.

2) Purification of human legionella pneumophila PAL 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. Individual colonies transformed with pET-PAL and having the ability to express foreign proteins were 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 were inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, and cultured at 37 ℃ to OD₆₀₀When the concentration is 0.6, 1mol/L IPTG is added to a final concentration of 0.6mmol/L, and the mixture is 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 resulting mixture was diluted with 50mL Buffer A (50mM Na)₃PO₄0.5M NaCl; pH7.4) was washed 3 times and 50mL of loading buffer (50mM Na)₃PO₄0.5M NaCl; 5mM imidazole, pH7.4) followed by resuspension, sonication, operating 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 PAL protein was found to be present in the bacterial cells in a solubilized form. Thin-layer scanning shows that the recombinant protein accounts for more than 30 percent of the total protein of the bacteria, which indicates that higher protein is obtained after gene optimizationThe expression level. 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 obtaining the recombinant human legionella pneumophila surface protein PAL.

EXAMPLE 2 preparation of polyclonal antibody against surface protein of human Legionella pneumophila

1) Immunization of New Zealand pure rabbits

The recombinant PAL 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, the emulsion formed by the recombinant PAL protein and Freund's incomplete adjuvant is used for immunization once every two weeks for 5 times, 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 PAL protein is used as coating antigen, the coating concentration is 10 mug/ml, each hole is coated with 100 mug, and the level of serum antibody is detected by 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 human legionella pneumophila is prepared.

EXAMPLE 3 preparation of monoclonal antibody Lp-2# of human Legionella pneumophila surface protein

1) Immunization of animals

5 BALB/c mice of 8 weeks old were immunized with the recombinant human Legionella pneumophila surface protein PAL prepared in example 1 as 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: using PBS (8.5g/L NaCl, 1.4g/L Na)₂HPO₄，0.2g/L NaH₂PO₄pH7.4) was diluted to 3. mu.g/mL, added to a 96-well plate at 100. mu.L/well, and incubated at 37 ℃ for 2 hours or overnight at 4 ℃. Using PBST (8.5g/L NaCl, 1.4g/L Na)₂HPO₄，0.2g/L NaH₂PO₄0.5% (v/v) Tween-20, pH7.4) the plate was photographed after 3 washes. 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 ℃. Adding 100 μ L of TMB chromogenic substrate solution prepared freshly into each well, reacting at 37 deg.C for 10min, adding 100 μ L of 1M hydrochloric acid into each well to terminate reaction, and determining OD with enzyme-linked detector₄₅₀nm values, reading and observing the results. 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, rapidly taking out liquid nitrogen frozen myeloma cells, placing in 37 deg.C water bath, slightly shaking to melt rapidly, paying attention to the fact that frozen tube orifice can not touch water to avoid pollution, transferring cells into 24-well plate containing 2ml RPMI-1640 complete medium (RPMI-1640 medium containing 20% fetal calf serum purchased from Shanxi Runsheng Dai Industrial biological materials Co., Ltd.), placing in 37 deg.C and 5% CO₂Culturing in incubator for half an hour, timely changing liquid when cells are all adherent to growth, subculturing once every 3 days, and regulating cellsThe cells are made to be at the optimum growth density, and when the cells reach certain activity, the cells are counted and prepared for fusion. Cells were passaged 1: 4 2 days before cell fusion and the cell concentration per flask was adjusted to 1X 10 with fresh medium⁵/ml。

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) gently resuspending the cells with 5ml HAT medium and mixing well, counting, and supplementing HAT medium to a cell concentration of 1X 10⁵/ml。

4.4) dropping the cell suspension into a 96-well cell culture plate at a concentration of 130. mu.l/well, placing at 37 ℃ and 5% CO₂Culturing in an incubator.

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) will contain 1X 10⁸Spleen cell suspension and 1X 10-containing suspension⁷A suspension of SP2/0 myeloma cells was 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) adding the fused cell suspension to a 96-well plate containing feeder cells at a concentration of 50. mu.l/well, spreading 20 plates in total, and then placing the plates at 37 ℃ under 5% CO₂Cultured in an incubator.

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. Human Legionella pneumophila (ATCC33152, 1X 10)⁸Individual thallus/hole) as coating antigen, and adopting indirect ELISA method to detect. Determination of OD Using an enzyme-Linked Detector₄₅₀Positive results showed P/N values > 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; ② preparing feeder cells in advance, suspending in HT medium, 130 ul/well, spreading in 96-well plate, then placing the plate at 37 deg.C, 5% CO₂The incubator is reserved; 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 at 37 ℃ and 5 percent CO₂Culturing for 7-8 days in the incubator; 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. Prepared from human mycoplasma pneumoniae (ATCC15531, 1 × 10)⁸Individual thallus/well), pseudomonas aeruginosa (ATCC27853, 1 × 10)⁸Individual cell/well), Moraxella catarrhalis (ATCC25240, 1X 10)⁸Individual thallus/pore), abaloneAcinetobacter mansonii (ATCC19606, 1X 10)⁸Individual cell/well), haemophilus parainfluenza (ATCC7901, 1X 10)⁸Individual cell/well), Haemophilus influenzae (ATCC49247, 1X 10)⁸Individual cell/well), Streptococcus pyogenes (ATCC19615, 1X 10)⁸Individual cell/well), Staphylococcus aureus (ATCC25923, 1X 10)⁸Individual thallus/well), human streptococcus pneumoniae (ATCC49619, 1X 10)⁸Individual thallus/well), Klebsiella pneumoniae (ATCC 700603, 1X 10)⁸Individual cell/well), Enterobacter cloacae (ATCC13047, 1X 10)⁸Individual cell/well), Escherichia coli (ATCC25922, 1X 10)⁸Individual bacteria/well), Candida (ATCC10231, 1X 10)⁸Individual bacteria/pores) and other respiratory tract pathogenic bacteria are respectively coated on an enzyme label plate, the ELISA detection is carried out on the specificity of the screened antibody, 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 to PAL protein from the whole-cell protein of human Legionella pneumophila. (2) Antibody purification and potency determination: the ascites fluid was purified by Protein a affinity chromatography, the procedure was as follows:

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. 50 volumes of PBS (8.5g/L NaCl, 1.4g/L Na) were used₂HPO₄，0.2g/L NaH₂PO₄pH7.4) was subjected to dialysis.

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 Lp-2# is determined to be preferably a labeled antibody. This antibody was designated as monoclonal antibody Lp-2 #.

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 Lp-2#, preservation number CCTCC NO: c2017216; address: wuhan university in Wuhan, China.

Example 4 surface protein Capture ELISA kit for human Legionella pneumophila

1) Human legionella pneumophila surface protein capture ELISA kit composition

The solid phase carrier coated with the polyclonal antibody of the surface protein of the human legionella pneumophila is an enzyme label plate, the monoclonal antibody Lp-2# of the surface protein of the human legionella pneumophila, the goat anti-mouse IgG marked by the horseradish peroxidase, a substrate reaction solution, a positive control and a negative control of the enzyme, and a washing solution and a reaction stopping solution jointly form the ELISA kit for capturing the surface protein of the human legionella pneumophila.

Enzyme label plate coating polyclonal antibody

The polyclonal antibody (prepared as in example 2) to the surface protein of human Legionella pneumophila was diluted to a concentration of 10. mu.g/mL with PBS buffer, coated with 96-well EIA high-efficiency binding ELISA plates at 50. mu.L/well, and incubated at 37 ℃ for 2 hours. 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) Human legionella pneumophila surface protein monoclonal antibody Lp-2#

The human Legionella pneumophila surface protein monoclonal antibody Lp-2# as described in example 3 was dispensed at a concentration of 1mg/mL in an amount of 20. mu.L per serving, and was diluted 1000-fold with PBS buffer at the time of use.

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: the recombinant human legionella pneumophila surface protein PAL, extracted as described in example 1, was 10. mu.g/mL.

Negative control: serum from normal mice not immunized with recombinant human legionella pneumophila surface protein PAL 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 method of Using the human Legionella pneumophila surface protein Capture ELISA kit

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 Lp-2#

Adding 50 mu L/hole of the human Legionella pneumophila surface protein monoclonal antibody Lp-2# working solution (sealing solution 1: 1000 dilution) described in example 4, incubating at 37 ℃ for 1 hour, washing the plate with 250 mu L of PBST washing solution for 3 times, and spin-drying.

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 OD₄₅₀Value of nm

Placing the ELISA plate in an ELISA reader for determining OD₄₅₀And (5) nm value.

8) Determination of results

Respectively reading OD of 4-hole negative quality control sample and 1-hole positive quality control sample₄₅₀The value of nm; OD of 4-well negative quality control sample₄₅₀The sum of the average value of the nm readings and the 3-fold standard deviation is the CUT-OFF value; if the detection OD of the human throat swab sample is₄₅₀If the nm value is larger than the CUT-OFF value, the antigen of the human legionella pneumophila in the clinical throat swab is judged to be positive, otherwise, the antigen of the human legionella pneumophila in the human throat swab sample is judged to be negative; OD of positive quality control sample₄₅₀And when the nm value is less than the CUT-OFF value, the kit is invalid.

Example 6 determination of specificity and sensitivity of the human Legionella pneumophila surface protein Capture ELISA kit

1) Specific assay

In order to verify the specificity of the ELISA kit for capturing the surface protein of the human legionella pneumophila, 7 human legionella pneumophila strains and 17 non-human legionella pneumophila 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 disclosed by the invention are positive for all 7 human legionella pneumophila strains, and the detection results of the kit are negative for other 17 common respiratory pathogenic microorganisms. The kit showed good specificity.

TABLE 1

2) Sensitivity assay

Inoculating Legionella pneumophila ATCC33152 strain to activated charcoal yeastCulturing in mother extract agar culture medium at 37 deg.C for 72 hr, diluting with 10 times of normal saline, and counting to obtain thallus concentration of 10⁸-10³CFU/mL of the cell solution, 100. mu.L of the cell solution was dropped on the microplate, and detection was performed according to the kit composition and method described in examples 4 and 5. The result shows that the detection sensitivity of the kit is 10³CFU/mL。

Sequence listing

<110> Hubei Yunluo bioengineering Co Ltd

<120> human legionella pneumophila surface protein monoclonal antibody and antigen capture ELISA kit

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 384

<212> DNA

<213> PAL' Gene Sequence (Artificial Sequence)

<400> 1

catatgcagg agccaggtga atcctatacc acccaggctc cgcataacca actgtacctg 60

ttcgcatacg atgattctac tctggcgagt aaatacctgc cgagcgtaaa cgcacaggca 120

gaatacctga aaacccaccc gggtgctcgt gttatgatcg caggccatac tgatgagcgt 180

ggttctcgcg aatataacgt tgctctgggt gagcgtcgtg cagacacggt tgccgagatc 240

ctgcgtatgg caggcgtttc tcgtcagcaa atccgtgttg ttagctatgg taaagagcgt 300

ccggcaaact atggccacga cgaagcgtcc catgctcaga accgtcgtgt cgaattcatc 360

tacgaagcga cccgttaaga attc 384

<210> 2

<211> 124

<212> PRT

<213> PAL' protein Sequence (Artificial Sequence)

<400> 2

Met Gln Glu Pro Gly Glu Ser Tyr Thr Thr Gln Ala Pro His Asn Gln

1 5 10 15

Leu Tyr Leu Phe Ala Tyr Asp Asp Ser Thr Leu Ala Ser Lys Tyr Leu

20 25 30

Pro Ser Val Asn Ala Gln Ala Glu Tyr Leu Lys Thr His Pro Gly Ala

35 40 45

Arg Val Met Ile Ala Gly His Thr Asp Glu Arg Gly Ser Arg Glu Tyr

50 55 60

Asn Val Ala Leu Gly Glu Arg Arg Ala Asp Thr Val Ala Glu Ile Leu

65 70 75 80

Arg Met Ala Gly Val Ser Arg Gln Gln Ile Arg Val Val Ser Tyr Gly

85 90 95

Lys Glu Arg Pro Ala Asn Tyr Gly His Asp Glu Ala Ser His Ala Gln

100 105 110

Asn Arg Arg Val Glu Phe Ile Tyr Glu Ala Thr Arg

115 120

Claims

1. A hybridoma cell strain for producing a monoclonal antibody of a surface protein of human legionella pneumophila is characterized in that: the hybridoma cell strain for producing the human legionella pneumophila surface protein monoclonal antibody has a preservation number of CCTCC NO: c2017216 hybridoma cell strain Lp-2 #.

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

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 adopting an indirect ELISA method by taking human legionella pneumophila thalli as an envelope antigen; determination of OD Using an enzyme-Linked Detector₄₅₀Positive if the P/N value is more than 2.1; 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 are mycoplasma pneumoniae, 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 human legionella pneumophila according to claim 2, wherein the method comprises the following steps: the preparation process of the recombinant human legionella pneumophila surface protein PAL in the step 1) is as follows:

1.1) cloning expression of the PAL Gene of human Legionella pneumophila

1.2) purification of human Legionella pneumophila PAL protein

Identifying the correct positive clone bacteria, extracting plasmid, and transferring into competence according to conventional techniqueE.coliBL21(DE3) plysS, after transformation, the bacterial suspension was spread on LB plate containing 50. mu.g/mL kanamycin, and the expression strain was selected by a conventional method, cultured and purified.

4. A monoclonal antibody of human legionella pneumophila surface protein is characterized in that: the monoclonal antibody of the surface protein of the human legionella pneumophila is a monoclonal antibody secreted by the hybridoma cell strain Lp-2# in claim 1.

5. A method for preparing the monoclonal antibody against the surface protein of human Legionella pneumophila according to claim 4, wherein: the method comprises the following steps:

6. The use of the monoclonal antibody against the surface protein of human legionella pneumophila according to claim 4 in the preparation of a reagent for detecting human legionella pneumophila.

7. The use of the monoclonal antibody against the surface protein of human legionella pneumophila according to claim 4 in the preparation of an ELISA kit for detecting the surface protein capture of human legionella pneumophila.

8. An ELISA kit for detecting surface protein capture of human legionella pneumophila is characterized in that: the ELISA kit for detecting the surface protein capture of the human legionella pneumophila comprises an ELISA plate and the human legionella pneumophila surface protein monoclonal antibody Lp-2# as claimed in claim 4; the ELISA plate is a solid phase carrier coated with a polyclonal antibody of the surface protein of the human legionella pneumophila.

9. The ELISA kit for detecting surface protein capture of human Legionella pneumophila according to claim 8, wherein: the preparation method of the human legionella pneumophila surface protein polyclonal antibody comprises the following steps: