CN110540963A

CN110540963A - Human streptococcus pneumoniae surface protein monoclonal antibody and application

Info

Publication number: CN110540963A
Application number: CN201811563393.1A
Authority: CN
Inventors: 胡征
Original assignee: Hubei Numeihua Antibody Drug Technology Co ltd
Current assignee: Hubei Numeihua Antibody Drug Technology Co ltd
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2019-12-06

Abstract

The invention provides a human streptococcus pneumoniae surface protein monoclonal antibody and a human streptococcus pneumoniae immunochromatographic assay test strip. Immunizing a Balb/c mouse by using the recombinant streptococcus pneumoniae PspA protein to obtain a hybridoma cell strain Sp-18 secreting the human streptococcus pneumoniae surface protein monoclonal antibody with the preservation number of CCTCC NO: c2017209; the monoclonal antibody secreted by the cell strain is used for preparing the colloidal gold immunochromatographic test strip, the monoclonal antibody secreted by the hybridoma cell strain Sp-18# is used as a labeled monoclonal antibody, and the anti-PspA protein polyclonal antibody is used as a coating antibody, so that the obtained test strip has the characteristics of high detection speed, high specificity, high sensitivity, strong stability, simplicity and convenience in manufacturing and the like. The result is clear and easy to judge, and the clinical diagnosis of the streptococcus pneumoniae infection can be efficiently assisted.

Description

human streptococcus pneumoniae surface protein monoclonal antibody and application

Technical Field

The invention belongs to the field of immunology, and relates to a human streptococcus pneumoniae surface protein monoclonal antibody, a hybridoma cell strain and application.

background

streptococcus pneumoniae was first isolated from patient sputum in 1881 by Pasteur (Louis Pasteur) and g.m. sternberg, respectively, in france and in the united states. It is gram staining positive bacteria, the thallus is similar to spearhead shape, is diplococcus arranged in duplex or short chain shape, the toxic strain has a capsule with polysaccharide as the chemical component outside the thallus. 5-10% of normal people carry the bacterium in the upper respiratory tract. Virulent strains are important pathogenic bacteria causing human diseases. Of the pyogenes, streptococcus pneumoniae is second only to staphylococcus aureus in virulence. Streptococcus pneumoniae mainly causes lobar pneumonia, tracheitis, otitis media, meningitis, pleuritis, endocarditis, septicemia and other diseases. An acute lung infection caused by streptococcus pneumoniae is streptococcus pneumoniae pneumonia. Bronchopneumonia is caused if the lungs of infants under 3 years old are infected. The pneumonia of children is the most serious problem of respiratory infectious diseases, the number of deaths of the respiratory infectious diseases is greater than the sum of AIDS, measles, malaria and other diseases, and the death rate of the children is very high. There is data showing that over 1 million and 5 million children under 5 years of age suffer pneumonia in developing countries worldwide, with over 150 million children dying due to pneumonia. The pneumonia death rate of China, which is a large-population country, is very high, more than 40 percent of pneumonia deaths occur in Asia globally, and the pneumonia death rate is second to India in China. In addition, data statistics show that the incidence rate and the death rate of sequelae caused by the streptococcus pneumoniae disease are obviously higher than other common pathogenic bacteria of children. In severe pneumonia bacteria 50% belong to streptococcus pneumoniae, and the lethal cases are more than 50% according to statistics. In conclusion, the rapid diagnosis of the streptococcus pneumoniae infection can realize the early detection and treatment, and has very important clinical significance.

The existing method for detecting the pathogenic microorganisms in the respiratory tract mainly adopts the traditional method, namely a separation identification method, the method needs long time, generally takes 2 to 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 colloidal gold detection methods and the like having proprietary intellectual property rights.

The research selects a surface protein PspA with interspecific specificity as an antigen, prepares a monoclonal antibody with good specificity, and applies the monoclonal antibody to the preparation of the test paper strip for immunochromatography detection of the human streptococcus pneumoniae.

Disclosure of Invention

In order to solve the technical problems in the background art, the invention provides the human streptococcus pneumoniae surface protein monoclonal antibody which has strong specificity, high sensitivity and clear and easily-judged result and application thereof.

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

a hybridoma cell strain for producing a human streptococcus pneumoniae surface protein monoclonal antibody is characterized in that: the hybridoma cell strain for producing the streptococcus pneumoniae surface protein monoclonal antibody is a hybridoma cell strain which is preserved by China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: c2017209 hybridoma cell strain Sp-18 #.

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

1) Preparation of recombinant human streptococcus pneumoniae PspA protein:

Performing bioinformatics analysis on a human streptococcus pneumoniae surface protein PspA gene, wherein the accession number in an NCBI protein database of the human streptococcus pneumoniae surface protein PspA is AAF27703.1, combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, optimizing a DNA coding sequence of the human streptococcus pneumoniae surface protein PspA, introducing an enzyme cutting site NdeI into the 5 ' end of the DNA coding sequence of the human streptococcus pneumoniae surface protein PspA, introducing a termination signal TAA and an enzyme cutting site XhoI into the 3 ' end of the DNA coding sequence of the human streptococcus pneumoniae surface protein PspA, and then chemically synthesizing a complete gene sequence, wherein the chemically synthesized complete gene sequence is connected to a vector pUC57 and is marked as PspA '; the complete gene sequence and the coded amino acid sequence are shown in a sequence table; cloning PspA' into a prokaryotic expression vector pET-28a (+) according to a conventional method, inducing recombinant escherichia coli expression by IPTG in a low-temperature expression mode, and purifying soluble recombinant human streptococcus pneumoniae PspA protein by Ni2+ affinity chromatography;

2) Screening anti-human streptococcus pneumoniae surface protein positive hybridoma cell strain:

a) Preparing immune spleen cells: immunizing 5 BALB/c mice of 8 weeks old by taking the recombinant human streptococcus pneumoniae PspA protein prepared in the step 1) as an antigen, and setting 2 mice which are not immunized as negative controls; fully emulsifying the primary immune antigen with equivalent Freund's complete adjuvant, and injecting immune mice 100 μ g/mouse subcutaneously at multiple points on the back; then fully emulsifying the antigen and Freund's incomplete adjuvant with the same dose of antigen at an interval of three weeks, and then carrying out intraperitoneal injection for the second immunization, and fully emulsifying the antigen and Freund's incomplete adjuvant with the same dose of antigen at an interval of 2 weeks, and then carrying out intraperitoneal injection for the third immunization; collecting blood from tail vein after the third immunization for 15 days, and detecting the titer of antiserum; taking a mouse spleen, washing the mouse spleen with RPMI-1640 washing liquor, puncturing the spleen carefully by using a syringe needle which is sucked with 5ml of RPMI-1640 culture medium to wash out splenocytes, then sieving the splenocytes to ensure that the splenocytes are extruded into solution through meshes as much as possible, transferring the splenocyte suspension into a centrifuge tube, centrifuging the centrifuge tube at 1100rpm for 5min, removing supernatant, and centrifuging and washing the centrifuge tube twice; gently resuspending the splenocytes with RPMI-1640 culture medium, and counting for later use;

b) cell fusion: mixing the suspension containing 1 × 108 spleen cells and the suspension containing 1 × 107 myeloma cells, supplementing a culture medium washing solution to 40ml, and fully and uniformly mixing; centrifuging at 1200rpm for 5 minutes, and discarding the supernatant; the cell blocks are loosened and uniform to be pasty; taking out the prepared 50% PEG (MW1450) and RPMI-1640 washing solution, placing in a water bath at 37 ℃, and pre-warming for later use; 0.8ml of 50 percent PEG is absorbed, slowly added into a centrifugal tube while stirring, and the time is controlled within 60 seconds plus or minus 5 seconds; then, 40ml of preheated RPMI-1640 washing liquid is gradually added for 60 seconds to dilute the PEG and lose the function of promoting melting; centrifuging the fused cells at room temperature, centrifuging at 1000rpm for 5min, and removing the supernatant; adding HAT culture solution, gently sucking, and resuspending the precipitated cells; adding the fused cell suspension into a 96-well culture plate containing feeder cells, culturing at 50 μ l/well in an incubator at 37 deg.C and 5% CO 2;

c) Screening and cloning culture of positive clones: observing the growth condition of cells in each hole every day from the 3 rd day after fusion, and immediately treating the cells with sodium azide if the cells are polluted; replacing the HT culture medium at 7d after fusion; the next day after the liquid change, the supernatant of the wells in which the clones appeared was aspirated for specific detection; and (3) subcloning and cloning the detected holes with strong positive by using a limiting dilution method, and after cloning operation is performed for 3 times, determining that the positive rate of all the cloned cell holes is 100 percent, thus determining the hybridoma cell strain secreting the monoclonal antibody.

A monoclonal antibody of surface protein of human streptococcus pneumoniae, which is characterized in that: the human streptococcus pneumoniae surface protein monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell strain Sp-18 #.

A method for preparing a human streptococcus pneumoniae surface protein monoclonal antibody is characterized by comprising the following steps: the method comprises the following steps:

1) Carrying out expanded culture on hybridoma cell strains capable of producing the human streptococcus pneumoniae surface protein monoclonal antibody to obtain cell culture solution supernatant containing the monoclonal antibody;

2) 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 mycoplasma pneumoniae, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, legionella pneumophila, streptococcus pyogenes, staphylococcus aureus, haemophilus influenzae, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida; preparing monoclonal antibody ascites from the screened hybridoma cells according to a conventional ascites in vivo induction method, and purifying the antibody by Protein A Sepharose affinity chromatography to obtain the human streptococcus pneumoniae surface Protein monoclonal antibody.

the application of the human streptococcus pneumoniae surface protein monoclonal antibody in detecting human streptococcus pneumoniae is preferably the application in preparing a reagent for detecting the human streptococcus pneumoniae, and more preferably the application in preparing an immunochromatography detection test strip for detecting the human streptococcus pneumoniae; preferably, the monoclonal antibody is an labeled monoclonal antibody of the surface protein of the human streptococcus pneumoniae, which is obtained by secreting the hybridoma cell strain Sp-18 #.

An immunochromatography test strip for detecting human streptococcus pneumoniae, which is characterized in that: the immunochromatographic detection test strip for detecting the human streptococcus pneumoniae comprises a nitrocellulose membrane detection layer and a binding pad; the binding pad is sprayed with a colloidal gold marker of a human streptococcus pneumoniae surface protein monoclonal antibody secreted by hybridoma cell strain Sp-18 #; and the nitrocellulose membrane is also sprayed with a detection line for the anti-PspA protein polyclonal antibody.

A preparation method of an immunochromatographic detection test strip for detecting human streptococcus pneumoniae is characterized by comprising the following steps: the method comprises the following steps:

1) Preparation of a nitrocellulose membrane detection layer:

1.1) preparation of polyclonal anti-PspA protein antibody:

a) Immunizing a new-Zealand pure rabbit by adopting a recombinant human streptococcus pneumoniae PspA protein, and detecting the serum antibody level 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 an anti-PspA Protein polyclonal antibody;

1.2) coating:

Diluting the anti-PspA protein polyclonal antibody to 2.0mg/ml, diluting the goat anti-mouse IgG antibody to 0.3mg/ml, filling the diluted anti-PspA protein polyclonal antibody into a sprayer of a film scratching instrument, and spraying the diluted anti-PspA protein polyclonal antibody on a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line; loading the diluted goat anti-mouse IgG into a sprayer of a membrane scribing instrument, and spraying the diluted goat anti-mouse IgG onto a nitrocellulose membrane in an amount of 1.0 mu l/cm to serve as a quality control line, wherein the distance between the quality control line and a detection line is 0.5 cm; drying the sprayed nitrocellulose membrane at 37 ℃ in vacuum for more than 12h, sealing and drying at 4 ℃ for storage; storing for later use;

2) Preparation of the bonding pad:

2.1) preparation of colloidal gold solution: the colloidal gold used by the detection test strip is prepared by a trisodium citrate reduction method;

2.2) preparing, purifying and concentrating the human streptococcus pneumoniae surface protein monoclonal antibody secreted by the hybridoma cell strain Sp-18# by colloidal gold;

2.3) marking the load of the monoclonal antibody of the surface protein of the human streptococcus pneumoniae secreted by the hybridoma cell strain Sp-18# by colloidal gold;

3) Preparation of sample pad: preparing a sample pad treatment solution; taking one glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for at least 2h, drying in a vacuum drying oven at 37 ℃, cutting to obtain a sample pad, sealing at room temperature, drying and storing;

4) Preparing the absorbent pad: cutting the absorbent paper to obtain an absorbent pad;

5) preparing a bottom plate: cutting the PVC plate to obtain a bottom plate;

6) the nitrocellulose membrane detection layer, conjugate pad, sample pad, absorbent pad and base plate were assembled according to the prior art.

The invention has the following advantages and effects:

Firstly, the invention successfully obtains the soluble recombinant PspA protein by adopting the modes of gene optimization, low-temperature induced expression and the like, has natural protein structure, and is a good material for preparing the antibody by taking the protein as an immune source. Secondly, the monoclonal antibody obtained by the invention can specifically recognize the extracellular region of the thallus PspA protein, and has no cross reaction with other common respiratory tract pathogens. Finally, specific experiments carried out by using the streptococcus pneumoniae bacterial strain and 17 non-human streptococcus pneumoniae standard bacterial strains (comprising most common respiratory pathogens) show that the kit has good specificity and stability, can detect the tested streptococcus pneumoniae bacterial strain, and has no cross reaction with all the non-human streptococcus pneumoniae standard bacterial strains. Secondly, sensitivity experiment results show that the detection sensitivity of the kit is 1 multiplied by 104CFU/mL, which is obviously higher than that of the traditional microorganism detection method, and the kit has the advantages of rapidness, high efficiency and the like.

Drawings

FIG. 1 is a schematic structural diagram of a test strip for immunochromatography detection of human Streptococcus pneumoniae;

Wherein:

1-sample pad; 2-a conjugate pad; 3-detection layer; 4-detection line; 5-quality control line; 6-absorbent pad; 7-rigid polyvinyl chloride back bottom plate.

Detailed Description

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

example 1 preparation of recombinant human Streptococcus pneumoniae surface protein PspA and polyclonal antibody thereto

(1) Cloning and expression of the PspA Gene of human Streptococcus pneumoniae

Bioinformatics analysis is carried out on the surface protein PspA (the access number of the surface protein PspA in the NCBI protein database is AAF27703.1) gene of human streptococcus pneumoniae, 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, and a complete gene sequence is chemically synthesized after enzyme cutting site NdeI is introduced into the 5 ' end and termination signal TAA and enzyme cutting site XhoI are introduced into the 3 ' end (complete sequence synthesis is finished by Kinsley Biotechnology Limited company, and artificially synthesized gene segments are connected to a vector pUC57 when delivered), which is marked as PspA '. 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 XhoI, and the desired fragment was recovered by a conventional method. And simultaneously carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and XhoI, connecting the PspA 'gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional method, transforming Escherichia coli TOP10, and constructing the pET-PspA' expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses a recombinant PspA-His fusion protein.

(2) purification of recombinant human Streptococcus pneumoniae PspA 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-PspA' having the ability to express a foreign protein 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 was inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, cultured at 30 ℃ until OD600 became 0.6, added with 1mol/L of IPTG to a final concentration of 0.6mmol/L, and cultured with shaking at 18 ℃ to induce expression of the fusion protein. After 12h of induction, 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 PspA protein was found to be present in the bacterial cells in solubilized 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 0.5mg/mL for use after the protein concentration was measured with a bradford kit. Thus obtaining the recombinant human streptococcus pneumoniae surface protein PspA.

(3) Preparation of polyclonal antibody against PspA protein

1) Immunization of New Zealand pure rabbits

The recombinant PspA 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 PspA protein and Freund's incomplete adjuvant, wherein the immunization is carried out 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 PspA 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:

1.5mL 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, an anti-PspA protein polyclonal antibody was prepared.

EXAMPLE 2 preparation of monoclonal antibody Sp-18# of human Streptococcus pneumoniae surface protein

1) Immunization of animals

5 BALB/c mice of 8 weeks old were immunized with the recombinant human Streptococcus pneumoniae surface protein PspA 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 tapping. 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. Human streptococcus pneumoniae (ATCC49619, 1 x 108 bacteria/hole) is used as a coating antigen and is detected by 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 d; screening positive single colony wells by ELISA, and subcloning again; after 2-3 times of continuous subcloning, making the subcloned cell colony holes all 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. Mycoplasma hominus (ATCC15531, 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), Legionella pneumophila (ATCC33152, 1 × 108 cells/well), Streptococcus pyogenes (ATCC19615, 1 × 108 cells/well), Staphylococcus aureus (ATCC25923, 1 × 108 cells/well), Haemophilus influenzae (ATCC49247, 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 albicans (ATCC1 × 108 cells/well), 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: specificity at 2000 dilution, the results showed that all 8 antibodies bound only to the PspA protein among the human streptococcus pneumoniae whole cell proteins.

(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 added to 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 a coating antibody and a gold-labeled antibody of the colloidal gold test strip to carry out pairing experiments and screen out the optimal combination. Finally, through a plurality of experiments, the monoclonal antibody secreted by the hybridoma cell strain named Sp-18# is determined to be preferably a coating antibody and a gold-labeled antibody. This monoclonal antibody was named Sp-18 #.

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 Sp-18#, preservation number CCTCC NO: c2017209; address: wuhan university in Wuhan, China.

Example 3 immunochromatography test strip for detecting human streptococcus pneumoniae

the human streptococcus pneumoniae immunochromatographic assay test strip (shown in figure 1) consists of a sample pad 1, a combination pad 2, a detection layer 3, a detection line 4, a quality control line 5, a water absorption pad 6 and a hard polyvinyl chloride back bottom plate 7;

The nitrocellulose membrane detection layer 3 is pasted on the rigid polyvinyl chloride back bottom plate 7, the combination pad 2 and the sample pad 1 are sequentially pasted on the nitrocellulose membrane detection layer 3 from bottom to top by the left side, and the water absorption pad 6 is arranged on the nitrocellulose membrane detection layer 3 by the right side. The combination pad 2 is made of a glass cellulose membrane, and the monoclonal antibody Sp-18# marked by colloidal gold (hybridoma cell strain Sp-18#, preservation number CCTCC NO: C2017209 secretion) is sprayed on the combination pad 2. The detection line 4 is arranged on the upper side of the nitrocellulose membrane detection layer 3 close to the left side, the quality control line 5 is arranged on the right side, the interval distance between the two lines is 5mm, and the detection line 4 and the quality control line 5 are respectively sprayed with anti-PspA protein polyclonal antibody and goat anti-mouse antibody.

when the detected sample contains the human streptococcus pneumoniae, the human streptococcus pneumoniae can be respectively combined with the gold-labeled anti-human streptococcus pneumoniae surface protein monoclonal antibody, then combined with the antibody on the corresponding detection line under the chromatography action to form a macroscopic color band after aggregation, and then judgment is carried out according to the color development result.

example 4 preparation method of test strip for immunochromatography detection of human Streptococcus pneumoniae

The preparation method of the test strip for detecting the streptococcus pneumoniae by immunochromatography comprises the following steps:

Preparation of nitrocellulose membrane detection layer

the nitrocellulose membrane (material Sidoilies CN140) was cut into a size of 30 cm. times.2 cm. Diluting an anti-PspA protein polyclonal antibody to a final concentration of 2.0mg/ml, diluting a goat anti-mouse IgG (purchased from Beijing Saichi biological technology Co., Ltd.) to 0.3mg/ml by using PBS buffer (8.5g/L NaCl, 1.4g/L Na2HPO4, 0.2g/L NaH2PO4, pH7.4), filling the diluted anti-PspA protein polyclonal antibody into a membrane scribing instrument (Shanghai gold marking biological technology Co., Ltd., XYZ three-dimensional membrane scribing gold instrument, model HM3030) spray head 3, setting the volume of 1.0 mu L/cm, and spraying the diluted anti-PspA protein polyclonal antibody onto a nitrocellulose membrane to form a detection line; and (3) filling the diluted goat anti-mouse IgG into a sprayer 1 of a membrane scribing instrument, setting the quantity of 1.0 mu l/cm, spraying the diluted goat anti-mouse IgG on a nitrocellulose membrane to be used as a quality control line, and setting the distance between the diluted goat anti-mouse IgG and the detection line to be 0.5 cm. And (4) drying the sprayed nitrocellulose membrane at 37 ℃ in vacuum for more than 12h, and sealing, drying and storing at 4 ℃. Storing for later use.

Preparation of (II) bonding pad

1) Preparation of colloidal gold solution

The colloidal gold used by the product is prepared by a trisodium citrate reduction method, and the specific method is as follows:

a) 99ml of ultrapure water was poured into a 250ml round-bottom flask, and 1ml of 1% chloroauric acid (final concentration: 0.01%) was added thereto. The round bottom flask was placed in an oil bath set at 100 ℃ and heated to boiling at 100 ℃.

b) Rapidly adding 1.5ml 1% trisodium citrate, heating for 10min, observing the color of the solution changing from yellow to black to purple, and finally changing to stable wine red, stopping heating, stirring in water bath, and cooling.

c) And (3) carrying out 400-600nm ultraviolet scanning on the cooled colloidal gold solution by using an ultraviolet spectrophotometer, determining the wavelength of the maximum absorption peak, and observing the peak type and the peak width of the maximum absorption peak. The colloidal gold has a single absorption peak at 520-530 nm.

2) Preparation, purification and concentration of colloidal gold-labeled Sp-18#

a) 10ml of colloidal gold solution is put into a 50ml round bottom flask with a stirrer, 20ul of potassium carbonate solution with the concentration of 0.2M is added, the flask is placed in a magnetic stirrer to start stirring for 5 minutes (the rotating speed is based on no generation of bubbles), and after the antibody Sp-18# to be marked is slowly dripped according to the amount of 10ug/ml of final concentration (the dripping speed is based on 5 minutes of 1mg of protein), the flask is stirred for 45 minutes at a low speed.

b) Adding PEG (10% PEG20000 prepared by pure water) 200ul at constant speed, stirring for 15min, and storing at 4 deg.C.

c) and (3) subpackaging the marked colloidal gold liquid into 2ml centrifuge tubes, centrifuging for 5 minutes at 2500g, and removing coagulation. The supernatant was centrifuged at 15000g for 30min and discarded. The pellet was resuspended in an amount of 3/40 parts of the original volume of the gold solution and stored at 4 ℃ until use. (washing solution formulation: 0.02M Tris, 5% sucrose, 0.3% polyvinylpyrrolidone, pH8.7)

3) Load of gold labeled Sp-18#

a) The conjugate pad (obtained from Shanghai gold-labeled Biotech Co., Ltd., type: glass fiber film CB08) was immersed in the treatment solution for 2 hours, taken out, and vacuum-dried at 37 ℃ for 12 hours. Stored in a clean, dustless and dry place for later use. (conjugate pad treatment formulation: 20mM Tris, 0.3% casein, 1% (v/v) Tween 20, 5% sucrose, 0.3% polyvinylpyrrolidone, pH8.0)

b) Spraying gold: and (3) spraying gold-labeled antibody on the treated bonding pad by using a film-scratching gold-spraying instrument (XYZ three-dimensional film-scratching gold-spraying instrument HM3030 of Shanghai gold-labeled company), wherein the gold-spraying amount is 8ul/cm, vacuum drying is carried out at 37 ℃ for 12 hours, and the bonding pad is dried, cleaned and placed for later use.

(III) preparation of sample pad

Sample pad treatment fluid (20mM Tris, 0.3% casein, 1% tween 20, 5% sucrose, 0.3% polyvinylpyrrolidone, pH8.0) was prepared. A piece of glass fiber membrane (type: glass fiber membrane CB08, available from Shanghai gold Biotech Co., Ltd.) was cut into a size of 4cm × 1.5 cm. Soaking the sample in the sample pad treatment solution for at least 2h, drying in a vacuum drying oven at 37 deg.C to obtain sample pad, sealing at room temperature, drying, and storing.

(IV) preparation of absorbent pad

Absorbent paper (available from Shanghai gold-labeled Biotechnology Co., Ltd., model CH 37) was cut into 4cm × 1.5cm pieces to obtain absorbent pads.

(V) preparation of the sole plate

PVC board (from Shanghai gold-labeled Biotechnology Co., Ltd., model number SM31-40) was cut into 4cm × 6cm size to obtain a base plate.

(VI) Assembly

(1) Sequentially sticking the combination pad, the sample pad, the detection layer and other auxiliary materials according to the structure shown in figure 1, assembling the cut test strip into a complete PVC card during assembly, and then pressing the card by using a card pressing machine, wherein the test strip is required to be inspected to determine whether the scratch and the stain exist or not and whether the width meets the requirement or not during strip installation; the PVC card has clean and smooth appearance, no deformation and good bottom surface matching property, and the tightness can be adjusted by pressing the card to avoid crushing or deforming the card.

Note: the length of the test strip is 60mm, and the width is 4 mm. 15mm for sample pad, 6mm for combined pad, 20mm for nitrocellulose membrane and 25mm for absorbent paper. The sample pad is combined with the combination pad for 2mm, the combination pad is combined with the NC membrane for 2mm, and the absorbent pad is combined with the NC membrane for 2 mm. The detection line is 24mm away from the left edge of the bottom plate, and the quality control line is 29mm away from the left edge of the bottom plate.

(2) Cutting: and cutting the assembled test paper into test paper strips with the width of 4mm in an automatic shearing machine to obtain finished products.

Example 5 method of Using the test strip for immunochromatography detection of Streptococcus pneumoniae

Pretreatment of a sample to be detected: the pharyngeal swab of the person to be detected is obtained by the conventional method, and is inserted into a soft plastic tube filled with 500 mu L of physiological saline, and the tube wall of the plastic tube is squeezed to fully dissolve the 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.

And (3) detection: about 100 mu L of the crushing liquid is dropped on the sample pad of the test paper of the invention, and the result is observed after 15 min.

And (4) judging a result: when the red color appears on the quality control line and the detection line, the sample is positive for the human streptococcus pneumoniae, namely the sample contains the human streptococcus pneumoniae, and the detection line does not appear the red color and is negative for the human streptococcus pneumoniae, namely the sample does not contain the human streptococcus pneumoniae. If the quality control line does not have a red line, the test paper is invalid.

Example 6 specificity and sensitivity assay for the test strip for immunochromatography of human Streptococcus pneumoniae

1) Specific assay

In order to verify the specificity of the test strip for immunochromatography detection of human streptococcus pneumoniae of the present invention, human streptococcus pneumoniae strains and 17 non-human streptococcus pneumoniae standard strains were tested according to the composition and use method of the test strip described in examples 3 and 5, and are shown in table 1. The result shows that the detection result of the kit of the invention on the human streptococcus pneumoniae bacterial strain is positive, and the detection results on other 17 common pathogenic microorganisms of respiratory tract are negative. The kit showed good specificity.

TABLE 1

Strain name	Results of the detection	Strain name	Results of the detection
				Streptococcus pneumoniae ATCC49619	Positive for	legionella pneumophila ATCC33152	Negative of
mycoplasma pneumoniae ATCC15531	Negative of	Streptococcus pyogenes ATCC19615	negative of
				Pseudomonas aeruginosa ATCC27853	Negative of	Staphylococcus aureus ATCC25923	negative of
Moraxella catarrhalis ATCC25240	Negative of	human Haemophilus influenzae ATCC49247	Negative of
				Acinetobacter baumannii ATCC19606	negative of	Klebsiella pneumoniae ATCC700603	Negative of
Haemophilus parainfluenza ATCC790	Negative of	Enterobacter cloacae ATCC13047	Negative of
				Adenovirus ATCCVR-3	negative of	Escherichia coli ATCC25922	Negative of
influenza A virus ATCCVR-1743	Negative of	candida ATCC10231	Negative of
				influenza B virus ATCCVR-790	Negative of	Respiratory syncytial virus ATCCVR26	Negative of

2) Sensitivity assay

The Streptococcus pneumoniae ATCC49619 strain is inoculated into a sheep blood chocolate culture medium, after 48 hours of culture at 37 ℃, the gradient dilution is carried out by 10 times by normal saline, meanwhile, the plate counting is carried out, the thallus solution with the thallus concentration of 108-103CFU/mL is obtained, 100 mu L of the thallus solution is taken, and the detection is carried out according to the composition and the method of the test paper strip described in the embodiment 3 and the embodiment 5. The result shows that the detection sensitivity of the test strip is 104 CFU/mL.

Sequence listing

<110> lake Bei Nuomeua antibody pharmaceutical technology Co., Ltd

<120> human streptococcus pneumoniae surface protein monoclonal antibody and application

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1083

<212> DNA

<213> PspA' Gene Sequence (Artificial Sequence)

<400> 1

catatgaaca aaaaaaaaat gattttaaca tctttagcat ctgtagcaat tttaggtgca 60

ggtttagtaa catctcaacc aacagtagta cgtgcagaag aatctccagt agcatctcaa 120

tctaaagcag aaaaagatta tgatgcagca gtaaaaaacg caacagcagc aaaaaaagca 180

gcagaagatg cacaacgtgc attagatgaa gcaaaagcag cacaaaaaaa atatgatgaa 240

gatcaaaaaa aaacagaaga aaaagcaaaa gaagtaaaaa aagcatctga agaagaacaa 300

gcagcaaact taaaatatca acaagaatta gtaaaatata ttaaatatac acgtgaaaac 360

aactctacaa aacgtacaga agcagaaaaa gtaatgacag cagcaaaaaa agaacatgaa 420

aaaaaacaaa cagaattagc aaaagtatta gcaaaagtaa ttccatctgc agaagaatta 480

gaaaacacac gtcaaaaagc agaaaaagca aaagaaaaag aaccagaatt aacaaaaaaa 540

ttagaagaag caaaagcaaa atctgaagaa gcagaaaaaa aagcaacaga agcaaaacaa 600

aaagtagatg cagaacatgc agaagaagta gtaccacaag caaaaattgc agaattagaa 660

aacgaagtac aaaaattaga aaaagattta aaagaaattg atgaatctga ttctgaagat 720

tatgtaaaag aaggtttacg tgcaccatta caatctgaat tagatgcaaa acaagcaaaa 780

ttatctaaat tagaagaatt atctgataaa attgatgaat tagatgcaga aattgcaaaa 840

ttagaaaaaa acgtagaaga tttcaaaaac tctaacggtg aacaagcaga acaatatcgt 900

gcagcagcag gtgaagattt agcagcaaaa caagcagaat tagaaaaaac agaagcagat 960

ttaaaaaaag cagtaaacga accagaaaaa ccagcaccag caccagaaac accagcacca 1020

gaagcaccag cagaacaacc aaaaccagca ccagcaccac aaccagcacc agcataactc 1080

gag 1083

<210> 2

<211> 357

<212> PRT

<213> PspA' protein Sequence (Artificial Sequence)

<400> 2

Met Asn Lys Lys Lys Met Ile Leu Thr Ser Leu Ala Ser Val Ala Ile

1 5 10 15

Leu Gly Ala Gly Leu Val Thr Ser Gln Pro Thr Val Val Arg Ala Glu

20 25 30

Glu Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp Ala

35 40 45

Ala Val Lys Asn Ala Thr Ala Ala Lys Lys Ala Ala Glu Asp Ala Gln

50 55 60

Arg Ala Leu Asp Glu Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu Asp

65 70 75 80

Gln Lys Lys Thr Glu Glu Lys Ala Lys Glu Val Lys Lys Ala Ser Glu

85 90 95

Glu Glu Gln Ala Ala Asn Leu Lys Tyr Gln Gln Glu Leu Val Lys Tyr

100 105 110

Ile Lys Tyr Thr Arg Glu Asn Asn Ser Thr Lys Arg Thr Glu Ala Glu

115 120 125

Lys Val Met Thr Ala Ala Lys Lys Glu His Glu Lys Lys Gln Thr Glu

130 135 140

Leu Ala Lys Val Leu Ala Lys Val Ile Pro Ser Ala Glu Glu Leu Glu

145 150 155 160

Asn Thr Arg Gln Lys Ala Glu Lys Ala Lys Glu Lys Glu Pro Glu Leu

165 170 175

Thr Lys Lys Leu Glu Glu Ala Lys Ala Lys Ser Glu Glu Ala Glu Lys

180 185 190

Lys Ala Thr Glu Ala Lys Gln Lys Val Asp Ala Glu His Ala Glu Glu

195 200 205

Val Val Pro Gln Ala Lys Ile Ala Glu Leu Glu Asn Glu Val Gln Lys

210 215 220

Leu Glu Lys Asp Leu Lys Glu Ile Asp Glu Ser Asp Ser Glu Asp Tyr

225 230 235 240

Val Lys Glu Gly Leu Arg Ala Pro Leu Gln Ser Glu Leu Asp Ala Lys

245 250 255

Gln Ala Lys Leu Ser Lys Leu Glu Glu Leu Ser Asp Lys Ile Asp Glu

260 265 270

Leu Asp Ala Glu Ile Ala Lys Leu Glu Lys Asn Val Glu Asp Phe Lys

275 280 285

Asn Ser Asn Gly Glu Gln Ala Glu Gln Tyr Arg Ala Ala Ala Gly Glu

290 295 300

Asp Leu Ala Ala Lys Gln Ala Glu Leu Glu Lys Thr Glu Ala Asp Leu

305 310 315 320

Lys Lys Ala Val Asn Glu Pro Glu Lys Pro Ala Pro Ala Pro Glu Thr

325 330 335

Pro Ala Pro Glu Ala Pro Ala Glu Gln Pro Lys Pro Ala Pro Ala Pro

340 345 350

Gln Pro Ala Pro Ala

355

Claims

1. A hybridoma cell strain for producing a human streptococcus pneumoniae surface protein monoclonal antibody is characterized in that: the hybridoma cell strain for producing the streptococcus pneumoniae surface protein monoclonal antibody has a preservation number of CCTCC NO: c2017209 hybridoma cell strain Sp-18 #.

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

1) Preparation of recombinant human streptococcus pneumoniae PspA protein:

Performing bioinformatics analysis on a human streptococcus pneumoniae surface protein PspA gene, wherein the accession number in an NCBI protein database of the human streptococcus pneumoniae surface protein PspA is AAF27703.1, combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, optimizing a DNA coding sequence of the human streptococcus pneumoniae surface protein PspA, introducing an enzyme cutting site NdeI into the 5 ' end of the DNA coding sequence of the human streptococcus pneumoniae surface protein PspA, introducing a termination signal TAA and an enzyme cutting site XhoI into the 3 ' end of the DNA coding sequence of the human streptococcus pneumoniae surface protein PspA, and then chemically synthesizing a complete gene sequence, wherein the chemically synthesized complete gene sequence is connected to a vector pUC57 and is marked as PspA '; cloning PspA' into a prokaryotic expression vector pET-28a (+) according to a conventional method, inducing recombinant escherichia coli expression by IPTG in a low-temperature expression mode, and purifying soluble recombinant human streptococcus pneumoniae PspA protein by Ni2+ affinity chromatography;

b) cell fusion: mixing the suspension containing 1 × 108 spleen cells and the suspension containing 1 × 107 myeloma cells, supplementing a culture medium washing solution to 40ml, and fully and uniformly mixing; centrifuging at 1200rpm for 5 minutes, and discarding the supernatant; the cell blocks are loosened and uniform to be pasty; taking out the prepared 50% PEG and RPMI-1640 lotion, placing in a water bath at 37 ℃, and pre-heating for later use; the PEG is MW 1450; 0.8ml of 50 percent PEG is absorbed, slowly added into a centrifugal tube while stirring, and the time is controlled within 60 seconds plus or minus 5 seconds; then, 40ml of preheated RPMI-1640 washing liquid is gradually added for 60 seconds to dilute the PEG and lose the function of promoting melting; centrifuging the fused cells at room temperature, centrifuging at 1000rpm for 5min, and removing the supernatant; adding HAT culture solution, gently sucking, and resuspending the precipitated cells; adding the fused cell suspension into a 96-well culture plate containing feeder cells, culturing at 50 μ l/well in an incubator at 37 deg.C and 5% CO 2;

3. A monoclonal antibody of surface protein of human streptococcus pneumoniae, which is characterized in that: the human streptococcus pneumoniae surface protein monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell strain Sp-18# in claim 1.

4. A method of producing a human streptococcus pneumoniae surface protein monoclonal antibody according to claim 3, comprising: the method comprises the following steps:

2) 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 mycoplasma pneumoniae of human, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, legionella pneumophila, streptococcus pyogenes, staphylococcus aureus, haemophilus influenzae, klebsiella pneumoniae, enterobacter cloacae, escherichia coli and candida; preparing monoclonal antibody ascites from the screened hybridoma cells according to a conventional ascites in vivo induction method, and purifying the antibody by Protein A Sepharose affinity chromatography to obtain the human streptococcus pneumoniae surface Protein monoclonal antibody.

5. The use of the human streptococcus pneumoniae surface protein monoclonal antibody according to claim 3 in detecting human streptococcus pneumoniae.

6. the use of the human streptococcus pneumoniae surface protein monoclonal antibody according to claim 3 in the preparation of a reagent for detecting human streptococcus pneumoniae.

7. The use of the human streptococcus pneumoniae surface protein monoclonal antibody according to claim 3 in the preparation of an immunochromatographic assay test strip for detecting human streptococcus pneumoniae.

8. The application of the human streptococcus pneumoniae surface protein monoclonal antibody in preparing an immunochromatographic assay test strip for detecting human streptococcus pneumoniae according to claim 3, wherein the human streptococcus pneumoniae surface protein monoclonal antibody obtained by secretion of hybridoma cell strain Sp-18# is a labeled monoclonal antibody.

9. An immunochromatography test strip for detecting human streptococcus pneumoniae, which is characterized in that: the immunochromatographic detection test strip for detecting the human streptococcus pneumoniae comprises a nitrocellulose membrane detection layer and a binding pad; the binding pad is sprayed with a colloidal gold marker of a human streptococcus pneumoniae surface protein monoclonal antibody secreted by hybridoma cell strain Sp-18 #; and a detection line for anti-PspA protein polyclonal antibody is sprayed on the nitrocellulose membrane.

10. A preparation method of an immunochromatographic detection test strip for detecting human streptococcus pneumoniae is characterized by comprising the following steps: the method comprises the following steps:

1) Preparation of a nitrocellulose membrane detection layer:

1.1) preparation of polyclonal anti-PspA protein antibody:

1.2) coating:

2) Preparation of the bonding pad:

5) Preparing a bottom plate: cutting the PVC plate to obtain a bottom plate;