CN110540965A - human haemophilus influenzae surface protein monoclonal antibody and application - Google Patents
human haemophilus influenzae surface protein monoclonal antibody and application Download PDFInfo
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- CN110540965A CN110540965A CN201811564870.6A CN201811564870A CN110540965A CN 110540965 A CN110540965 A CN 110540965A CN 201811564870 A CN201811564870 A CN 201811564870A CN 110540965 A CN110540965 A CN 110540965A
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- haemophilus influenzae
- monoclonal antibody
- human haemophilus
- surface protein
- protein
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Abstract
The invention provides a human haemophilus influenzae surface protein monoclonal antibody and a human haemophilus influenzae immunochromatography detection test strip. The recombinant haemophilus influenzae P6 protein is used for immunizing a Balb/c mouse to obtain a hybridoma cell strain Hi-17# secreting a human haemophilus influenzae surface protein monoclonal antibody with the preservation number of CCTCC NO: c2017214; 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 Hi-17# is used as a labeled monoclonal antibody, and the anti-P6 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 haemophilus influenzae infection can be efficiently assisted.
Description
Technical Field
the invention belongs to the field of immunology, and relates to a human haemophilus influenzae surface protein monoclonal antibody, a hybridoma cell strain and application.
Background
Haemophilus influenzae (Hi) is called Haemophilus influenzae for short, and belongs to the genus Haemophilus. The bacterium is named as Haemophilus because it must be added with fresh blood or blood components for growth. Haemophilus influenzae has generally six strains, which are called type a, type b (also called type B), type c, type d, type e and type f. Naturally occurring diseases caused by haemophilus influenzae occur only in humans. In infants and children, haemophilus influenzae b causes bacteremia and acute bacterial meningitis. Occasionally it causes cellulitis, osteomyelitis and joint infections. Since 1990, the prevalence of HiB disorders has decreased to 1.3 childhood infections per hundred thousand children in the United states after the use of HiB-conjugated vaccines. However, HiB remains the leading cause of lower respiratory tract disease in infants and children in developing countries. In addition, non-typeable haemophilus influenzae without pods can also cause ear infections (e.g. otitis media), eye infections (conjunctivitis) and sinusitis in children, and associated pneumonia, and this strain has no corresponding vaccine applications.
At present, the method for detecting pathogenic microorganisms in respiratory tract mainly adopts a traditional method, namely a separation identification method, the method needs long time, generally takes 2-3 days, and the requirement of quick identification is difficult to meet; the PCR technology developed in recent years is a quick, sensitive and specific technology, but at present, the technology still depends on the previous enrichment step of the traditional method, and PCR inhibitors are often contained in the enrichment liquid, so that the amplification effect is influenced. Meanwhile, the technology also needs professional detection equipment, and is not suitable for bedside detection. Antibody-based immunological detection has become an indispensable important technical means for the detection of human pathogenic microorganisms. Various specific immunoassay techniques, such as Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Chemiluminescence Immunoassay (CIA), immunoprecipitation, immunoagglutination, ELISA detection kit, immune colloidal gold test strip, immune latex detection reagent, and the like, have been developed. The immune colloidal gold test strip, the ELISA detection kit and other antibody-based immunological detection technologies have become indispensable important means for detecting pathogenic microorganisms due to the characteristics of simplicity, rapidness, sensitivity, accuracy and practicability. Therefore, research and development of antibodies against pathogenic microorganisms having proprietary intellectual property rights are the basis for development of colloidal gold detection methods, ELISA detection methods, and the like having proprietary intellectual property rights.
The choice of antigenic component is critical to the specificity of the assay. When 35 NTHi strains isolated from different specimens were subjected to outer membrane protein typing by Barenkamp et al in 1982 by SDS-PAGE, it was found that each strain had its own protein pattern but a band with a molecular weight of about 16kD was present in all the strains. Munson RJ discovered that 3 strains of Hib outer membrane protein show protein band spectrums with the same main bands after gel electrophoresis in 1983, and the 6 th main band with the minimum molecular weight of 16KD is named as P6.RFLP analysis results prove that the P6 gene and adjacent fragments thereof are highly conserved. The P6 protein has 100% homology of amino acid and 97% -99% homology of nucleotide. Its good conservation and surface accessibility make it a good target for antigen detection.
The research selects surface protein P6 with interspecies specificity as antigen to prepare monoclonal antibody with good specificity, and the monoclonal antibody is applied to preparing human haemophilus influenzae immunochromatography detection test paper strips.
Disclosure of Invention
In order to solve the technical problems in the background art, the invention provides the human haemophilus influenzae surface protein monoclonal antibody which has strong specificity, high sensitivity and clear result and is easy to judge, and the application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
A hybridoma cell strain for generating a human haemophilus influenzae surface protein monoclonal antibody is characterized in that: the hybridoma cell strain for generating the human haemophilus influenzae surface protein monoclonal antibody is preserved by China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: c2017214 hybridoma cell strain Hi-17 #.
a preparation method of hybridoma cell strain for generating monoclonal antibody of human haemophilus influenzae surface protein is characterized in that: the preparation method comprises the following steps:
1) Preparation of recombinant human haemophilus influenzae P6 protein:
bioinformatics analysis is carried out on a human haemophilus influenzae surface protein P6 gene, wherein the accession number in an NCBI protein database of the human haemophilus influenzae surface protein P6 is WP _005652235, the DNA coding sequence of the human haemophilus influenzae surface protein P6 is optimized by combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, meanwhile, a complete gene sequence is chemically synthesized after enzyme cutting site NdeI is introduced into 5 ' of the DNA coding sequence of the human haemophilus influenzae surface protein P6, and a termination signal TAA and enzyme cutting site XhoI are introduced into 3 ' end of the DNA coding sequence of the human haemophilus influenzae surface protein P6, and the chemically synthesized complete gene sequence is connected to a pUC57 and is marked as P6 '; the complete gene sequence and the coded amino acid sequence are shown in a sequence table; cloning P6' into 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 haemophilus influenzae P6 protein by Ni2+ affinity chromatography;
2) Screening anti-human haemophilus influenzae surface protein positive hybridoma cell strains:
a) Preparing immune spleen cells: immunizing 5 BALB/c mice with the age of 8 weeks by taking the recombinant human Haemophilus influenzae P6 protein prepared in the step 1) as an antigen, and setting 2 mice without being 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 human haemophilus influenzae surface protein monoclonal antibody is characterized in that: the human haemophilus influenzae surface protein monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell strain Hi-17 #.
A method for preparing a human haemophilus influenzae surface protein monoclonal antibody is characterized by comprising the following steps: the method comprises the following steps:
1) Carrying out expanded culture on a hybridoma cell strain capable of producing a human haemophilus influenzae surface protein monoclonal antibody to obtain a 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 of human, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, legionella pneumophila, streptococcus pyogenes, staphylococcus aureus, streptococcus pneumoniae of human, 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 haemophilus influenzae surface Protein monoclonal antibody.
The application of the human haemophilus influenzae surface protein monoclonal antibody in detecting human haemophilus influenzae is preferably the application in preparing a reagent for detecting human haemophilus influenzae, and more preferably the application in preparing an immunochromatography detection test strip for detecting human haemophilus influenzae; preferably, the monoclonal antibody is an labeled monoclonal antibody of the human haemophilus influenzae surface protein secreted by the hybridoma cell strain Hi-17 #.
An immunochromatography test strip for detecting human haemophilus influenzae is characterized in that: the immunochromatographic detection test strip for detecting the human haemophilus influenzae comprises a nitrocellulose membrane detection layer and a combination pad; the binding pad is sprayed with a colloidal gold marker of a human haemophilus influenzae surface protein monoclonal antibody secreted by a hybridoma cell strain Hi-17 #; and a detection line for the polyclonal antibody against the P6 protein is sprayed on the nitrocellulose membrane.
a preparation method of an immunochromatographic detection test strip for detecting human haemophilus influenzae 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-P6 protein antibody:
a) Immunizing a new Zealand pure rabbit by adopting recombinant human Haemophilus influenzae P6 protein, and detecting the level of a serum antibody by an indirect ELISA method;
b) Purifying and extracting the polyclonal antibody by using a GE-HiTrap Protein A HP pre-packed column to prepare an anti-P6 Protein polyclonal antibody;
1.2) coating:
Diluting the anti-P6 protein polyclonal antibody to 2.0mg/ml, diluting the goat anti-mouse IgG antibody to 0.3mg/ml, filling the diluted anti-P6 protein polyclonal antibody into a sprayer of a film scratching instrument, and spraying the diluted anti-P6 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 haemophilus influenzae surface protein monoclonal antibody secreted by the hybridoma cell strain Hi-17# by colloidal gold labeling;
2.3) marking the load of the monoclonal antibody of the surface protein of the human haemophilus influenzae secreted by the hybridoma cell strain Hi-17# with 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 P6 protein by adopting the modes of gene optimization, low-temperature induced expression and the like, the protein structure is natural, and the protein is a good material for preparing the antibody as an immune source. Secondly, the monoclonal antibody obtained by the invention can specifically recognize the extracellular region of the thallus P6 protein, and has no cross reaction with other common respiratory tract pathogens. Finally, the results of specificity experiments carried out by using 7 strains of human haemophilus influenzae and 17 strains of non-human haemophilus influenzae standard strains (containing most common respiratory pathogens) show that the kit disclosed by the invention has good specificity and stability, can detect all tested human haemophilus influenzae strains, and has no cross reaction with all non-human haemophilus influenzae standard 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 human Haemophilus influenzae immunochromatographic assay test strip;
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 Haemophilus influenzae surface protein P6 and polyclonal antibody thereto
(1) Cloning expression of human Haemophilus influenzae P6 gene
The human haemophilus influenzae surface protein P6 (the access number in NCBI protein database is WP _005652235) gene is subjected to bioinformatics analysis, the DNA coding sequence is optimized by combining the consideration of GC content, codon preference, mRNA secondary structure, RNA instability motif, mRNA free energy stability and the like, and the whole 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 (the whole sequence synthesis is completed by Kingsley Biotech Co., Ltd., and the artificially synthesized gene fragment is connected to a vector pUC57 during delivery), which is marked as P6 '. 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 carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and XhoI, connecting the P6 'gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional method, transforming Escherichia coli TOP10, and constructing a pET-P6' expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses recombinant P6-His fusion protein.
(2) purification of recombinant human Haemophilus influenzae P6 protein
Identifying the correct positive clone bacteria, culturing, extracting plasmid, transferring into competent E.coli BL21(DE3) plysS according to the conventional technology, coating the bacterial liquid on LB plate containing 50 ug/mL kanamycin after the conversion is finished, and screening expression strains according to the conventional method. A single colony transformed with pET-P6' and having the ability to express a foreign protein was picked and inoculated into 100mL of LB medium and cultured overnight at 37 ℃. After taking out the bacterial liquid, the bacterial liquid is prepared according to the following steps of 1: 100 was inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, 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 P6 protein was found to be present in the bacterial cells in dissolved form. Thin-layer scanning shows that the recombinant protein accounts for more than 30% of the total protein of the bacteria, which indicates that higher expression level is obtained after gene optimization. The sonicated supernatant obtained above was filtered through a 0.45 μm filter and purified by His Trap affinity columns (GE healthcare Co.) according to the method described in the specification. The specific method comprises the following steps:
connecting a chromatography system, wherein the system comprises a sample inlet tube, a peristaltic pump, a chromatography column and an ultraviolet detector (Shanghai Huxi analytical instrument factory, model HD1), the column volume is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable; and (5) correcting T%: adjusting a brightness knob to display 100%; rotate the sensitivity to the appropriate position, typically 0.2A; equilibrating the chromatography system with the loading buffer until the reading stabilizes and then rotating "zero" to show "000"; applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid; washing away unbound protein with a loading buffer, recording the reading during the process until the reading does not change any more, and collecting the eluate; eluting with Buffer A +10mM imidazole, and collecting an elution peak; eluting with Buffer A +20mM imidazole, and collecting an elution peak; eluting with Buffer A +40mM imidazole, and collecting an elution peak; eluting with Buffer A +100mM imidazole, and collecting an elution peak; eluting with Buffer A +150mM imidazole, and collecting an elution peak; taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis; as a result, it was found that the target protein was eluted at 100mM imidazole and had a purity of 90% or more, and the concentration was adjusted to 0.5mg/mL for use after the protein concentration was measured with a bradford kit. Thus, the recombinant human Haemophilus influenzae surface protein P6 is prepared.
(3) Preparation of polyclonal antibody against P6 protein
1) Immunization of New Zealand pure rabbits
The recombinant P6 protein prepared above is mixed with Freund's complete adjuvant, emulsified and used as immunogen to immunize 2 male New Zealand rabbits, the total amount of subcutaneous injection for each rabbit is 2ml, and the total amount of antigen is 2 mg/rabbit. And then immunizing once every two weeks by using an emulsion formed by the recombinant P6 protein and Freund's incomplete adjuvant, wherein the immunization is performed for 5 times in total, and the dosage of the antigen is the same as that of the primary immunization. Large amount of blood is taken 3-5 days after five-immunization, placed at 37 ℃ for 1 hour, then placed in a refrigerator at 4 ℃ overnight, and serum is taken every other day.
2) determination of polyclonal antibody titer
The recombinant P6 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, polyclonal antibodies against the P6 protein were prepared.
EXAMPLE 2 preparation of human Haemophilus influenzae surface protein monoclonal antibody Hi-17#
1) immunization of animals
5 BALB/c mice 8 weeks old were immunized with the recombinant human Haemophilus influenzae surface protein P6 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 Haemophilus influenzae thallus (ATCC49247, 1 × 108 thallus/well) is used as coating antigen, and indirect ELISA method is adopted for detection. 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 hominis (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), Streptococcus hominis (ATCC49619, 1 × 108 cells/well), Klebsiella pneumoniae (ATCC 700603, 1 × 108 cells/well), Enterobacter cloacae (ATCC13047, 1 × 108 cells/well), Escherichia coli (ATCC25922, 1 × 108 cells/well), Candida (ATCC1, 1 × 108 cells/well) and other respiratory tract pathogenic bacteria are respectively coated on an enzyme label plate, the specificity of the screened antibody is detected by ELISA, and monoclonal antibodies which have positive reactions with the pathogens are eliminated. In this step, 8 qualified cell lines were selected.
9) Ascites preparation and cell preservation
And performing expanded culture on the finally screened 8 strains of positive monoclonal hybridoma cells. Preparing the monoclonal antibody ascites by a conventional ascites in vivo induction method.
(1) Western blot detection of mAb: and (3) determining the ascites by adopting a conventional Western blot experiment method in the ratio of 1: the results of the specificity at 2000 dilution show that all 8 antibodies only bind to P6 protein with the molecular weight of 16KD in the whole-cell protein of human haemophilus influenzae.
(2) Antibody purification and potency determination: the ascites fluid was purified by Protein a affinity chromatography, the procedure was as follows:
The antibodies were purified using a GE-HiTrap Protein A HP pre-packed column as described, in the following manner:
a. 5mL of hybridoma supernatant was added with 0.5mL of 1M Tris (pH8.0) to adjust to pH8.0, and the mixture was centrifuged at 20000g for 20min to remove the precipitate.
b. After loading, the column was washed with 10 column volumes of buffer A (100mM Tris-Cl, pH8.0) and then with 10 column volumes of buffer B (10mM Tris-Cl, pH 8.0).
c. IgG was eluted with approximately three column volumes of IgG elution buffer (100mM glycine, pH 3.0). (0.1 mL IgG-neutralizing buffer (1M Tris-Cl, pH8.0) was preloaded into the collection tube, 0.9mL of eluent was 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 gold-labeled antibodies 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 as Hi-17# is preferably determined to be a gold-labeled antibody. This antibody was designated as monoclonal antibody # Hi-17.
Two hybridoma cell strains secreting the antibody are sent to the China center for type culture Collection for preservation in 2018, 10 months and 15 days, and are classified and named: hybridoma cell strain Hi-17#, preservation number CCTCC NO: c2017214; address: wuhan university in Wuhan, China.
Example 3 human Haemophilus influenzae immunochromatographic assay test strip
The human haemophilus influenzae 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 substrate 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 Hi-17# (hybridoma cell strain Hi-17#, preservation number CCTCC NO: C2017214 secretion) marked by colloidal gold 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 an anti-P6 protein polyclonal antibody and a goat anti-mouse antibody.
When the detected sample contains human haemophilus influenzae, the human haemophilus influenzae can be respectively combined with the gold-labeled human haemophilus influenzae surface protein monoclonal antibody, and then combined with the antibody on the corresponding detection line under the chromatography action to form a macroscopic color band after aggregation, so that the judgment is carried out according to the color development result.
example 4 preparation method of human Haemophilus influenzae immunochromatographic assay test strip
The preparation method of the human haemophilus influenzae immunochromatographic detection test strip 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-P6 protein polyclonal antibody to a final concentration of 2.0mg/ml, diluting a goat anti-mouse IgG (purchased from Beijing Saichi biological science Co., Ltd.) antibody to 0.3mg/ml, and filling the diluted anti-P6 protein polyclonal antibody into a striping machine (Shanghai gold marking biological science Co., Ltd., XYZ three-dimensional striping gold spraying machine, model HM3030) spray head 3 by using PBS buffer (8.5g/L NaCl, 1.4g/L Na2HPO4, 0.2g/L NaH2PO4, pH7.4) and spraying the diluted anti-P6 protein polyclonal antibody on 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 Hi-17#
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 Hi-17# 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 1 milligram 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 Hi-17#
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 human Haemophilus influenzae
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 red appears on both the quality control line and the detection line, the test line is positive for the human haemophilus influenzae, namely the sample contains the human haemophilus influenzae, and the detection line does not appear red and is negative for the human haemophilus influenzae, namely the sample does not contain the human haemophilus influenzae. If the quality control line does not have a red line, the test paper is invalid.
Example 6 determination of specificity and sensitivity of the human Haemophilus influenzae Immunochromatographic test strip
1) Specific assay
In order to verify the specificity of the test strip for immunochromatography detection of human haemophilus influenzae of the present invention, 7 strains of human haemophilus influenzae and 17 strains of non-human haemophilus influenzae standard strains were tested according to the composition and use method of the test strips described in examples 3 and 5, and are shown in table 1. The result shows that the detection results of the kit are positive for all 7 strains of human haemophilus influenzae strains, and the detection results of the kit are negative for other 17 strains of common respiratory pathogenic microorganisms. The kit showed good specificity.
TABLE 1
| strain name | Results of the detection | strain name | Results of the detection |
| Human Haemophilus influenzae ATCC49247 | Positive for | Legionella pneumophila ATCC33152 | Negative of |
| Human Haemophilus influenzae ATCC33391 | Positive for | streptococcus pyogenes ATCC19615 | Negative of |
| Human Haemophilus influenzae ATCC10211 | Positive for | Staphylococcus aureus ATCC25923 | Negative of |
| Human haemophilus influenzae ATCC49766 | Positive for | Streptococcus pneumoniae ATCC49619 | Negative of |
| Human haemophilus influenzae ATCC35056 | Positive for | klebsiella pneumoniae ATCC700603 | negative of |
| human haemophilus influenzae ATCC31441 | positive for | Enterobacter cloacae ATCC13047 | Negative of |
| human Haemophilus influenzae ATCC49144 | Positive for | Escherichia coli ATCC25922 | Negative of |
| Mycoplasma pneumoniae ATCC15531 | negative of | Candida ATCC10231 | Negative of |
| Pseudomonas aeruginosa ATCC27853 | Negative of | Influenza A virus ATCCVR-1743 | negative of |
| Moraxella catarrhalis ATCC25240 | Negative of | Influenza B virus ATCCVR-790 | Negative of |
| Acinetobacter baumannii ATCC19606 | Negative of | Respiratory syncytial virus ATCCVR26 | Negative of |
| Haemophilus parainfluenza ATCC7901 | Negative of | adenovirus ATCCVR-3 | Negative of |
2) sensitivity assay
the human haemophilus influenzae ATCC49247 strain is inoculated in a sheep blood chocolate culture medium, after 24 hours of culture at 37 ℃, 10 times of gradient dilution is carried out by normal saline, simultaneously, plates are counted to obtain a thallus solution with the thallus concentration of 108-103CFU/mL, 100 mu L of the thallus solution is taken, and detection is carried out according to the test strip composition and the method 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 haemophilus influenzae surface protein monoclonal antibody and application
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 471
<212> DNA
<213> P6' Gene Sequence (Artificial Sequence)
<400> 1
catatgaaca aattcgttaa atcacttctt gttgctggtt cagttgctgc tcttgctgct 60
tgttcatcat caaacaacga cgctgctggt aacggtgctg ctcaaacttt cggtggttac 120
tcagttgctg accttcaaca acgttacaac actgtttact tcggtttcga caaatacgac 180
atcactggtg aatacgttca aatccttgac gctcacgctg cttaccttaa cgctactcca 240
gctgctaaag ttcttgttga aggtaacact gacgaacgtg gtactccaga atacaacatc 300
gctcttggtc aacgtcgtgc tgacgctgtt aaaggttacc ttgctggtaa aggtgttgac 360
gctggtaaac ttggtactgt ttcatacggt gaagaaaaac cagctgttct tggtcacgac 420
gaagctgctt actcaaaaaa ccgtcgtgct gttcttgctt actaactcga g 471
<210> 2
<211> 153
<212> PRT
<213> P6' protein Sequence (Artificial Sequence)
<400> 2
Met Asn Lys Phe Val Lys Ser Leu Leu Val Ala Gly Ser Val Ala Ala
1 5 10 15
Leu Ala Ala Cys Ser Ser Ser Asn Asn Asp Ala Ala Gly Asn Gly Ala
20 25 30
Ala Gln Thr Phe Gly Gly Tyr Ser Val Ala Asp Leu Gln Gln Arg Tyr
35 40 45
Asn Thr Val Tyr Phe Gly Phe Asp Lys Tyr Asp Ile Thr Gly Glu Tyr
50 55 60
Val Gln Ile Leu Asp Ala His Ala Ala Tyr Leu Asn Ala Thr Pro Ala
65 70 75 80
Ala Lys Val Leu Val Glu Gly Asn Thr Asp Glu Arg Gly Thr Pro Glu
85 90 95
Tyr Asn Ile Ala Leu Gly Gln Arg Arg Ala Asp Ala Val Lys Gly Tyr
100 105 110
Leu Ala Gly Lys Gly Val Asp Ala Gly Lys Leu Gly Thr Val Ser Tyr
115 120 125
Gly Glu Glu Lys Pro Ala Val Leu Gly His Asp Glu Ala Ala Tyr Ser
130 135 140
Lys Asn Arg Arg Ala Val Leu Ala Tyr
145 150
Claims (10)
1. A hybridoma cell strain for generating a human haemophilus influenzae surface protein monoclonal antibody is characterized in that: the hybridoma cell strain for generating the human haemophilus influenzae surface protein monoclonal antibody is a hybridoma cell strain which is preserved by China center for type culture Collection with the preservation number of CCTCC NO: c2017214 hybridoma cell strain Hi-17 #.
2. A preparation method of hybridoma cell strain for generating monoclonal antibody of human haemophilus influenzae surface protein is characterized in that: the preparation method comprises the following steps:
1) Preparation of recombinant human haemophilus influenzae P6 protein:
Bioinformatics analysis is carried out on a human haemophilus influenzae surface protein P6 gene, wherein the accession number in an NCBI protein database of the human haemophilus influenzae surface protein P6 is WP _005652235, the DNA coding sequence of the human haemophilus influenzae surface protein P6 is optimized by combining GC content, codon preference, mRNA secondary structure, RNA unstable motif and mRNA free energy stability, meanwhile, a complete gene sequence is chemically synthesized after enzyme cutting site NdeI is introduced into 5 ' of the DNA coding sequence of the human haemophilus influenzae surface protein P6, and a termination signal TAA and enzyme cutting site XhoI are introduced into 3 ' end of the DNA coding sequence of the human haemophilus influenzae surface protein P6, and the chemically synthesized complete gene sequence is connected to a pUC57 and is marked as P6 '; cloning P6' into 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 haemophilus influenzae P6 protein by Ni2+ affinity chromatography;
2) screening anti-human haemophilus influenzae surface protein positive hybridoma cell strains:
a) Preparing immune spleen cells: immunizing 5 BALB/c mice with the age of 8 weeks by taking the recombinant human Haemophilus influenzae P6 protein prepared in the step 1) as an antigen, and setting 2 mice without being 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 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;
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.
3. A human haemophilus influenzae surface protein monoclonal antibody is characterized in that: the human haemophilus influenzae surface protein monoclonal antibody is a monoclonal antibody secreted by the hybridoma cell strain Hi-17# of claim 1.
4. A method of preparing a human haemophilus influenzae surface protein monoclonal antibody as claimed in claim 3, characterized in that: the method comprises the following steps:
1) Carrying out expanded culture on a hybridoma cell strain capable of producing a human haemophilus influenzae surface protein monoclonal antibody to obtain a 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 of human, pseudomonas aeruginosa, moraxella catarrhalis, acinetobacter baumannii, haemophilus parainfluenza, legionella pneumophila, streptococcus pyogenes, staphylococcus aureus, streptococcus pneumoniae of human, 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 haemophilus influenzae surface Protein monoclonal antibody.
5. the use of the human haemophilus influenzae surface protein monoclonal antibody according to claim 3 in the detection of human haemophilus influenzae.
6. the use of the human haemophilus influenzae surface protein monoclonal antibody of claim 3 in the preparation of a reagent for detecting human haemophilus influenzae.
7. The use of the human haemophilus influenzae surface protein monoclonal antibody of claim 3 in the preparation of an immunochromatographic test strip for the detection of human haemophilus influenzae.
8. The application of the human haemophilus influenzae surface protein monoclonal antibody in preparing an immunochromatography detection test strip for detecting human haemophilus influenzae according to claim 3, wherein the human haemophilus influenzae surface protein monoclonal antibody secreted by the hybridoma cell strain Hi-17# is a labeled monoclonal antibody.
9. An immunochromatography test strip for detecting human haemophilus influenzae is characterized in that: the immunochromatographic detection test strip for detecting the human haemophilus influenzae comprises a nitrocellulose membrane detection layer and a combination pad; the binding pad is sprayed with a colloidal gold marker of a human haemophilus influenzae surface protein monoclonal antibody secreted by a hybridoma cell strain Hi-17 #; and a detection line for the polyclonal antibody against the P6 protein is sprayed on the nitrocellulose membrane.
10. A preparation method of an immunochromatographic detection test strip for detecting human haemophilus influenzae 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-P6 protein antibody:
a) Immunizing a new Zealand pure rabbit by adopting recombinant human Haemophilus influenzae P6 protein, and detecting the level of a serum antibody by an indirect ELISA method;
b) Purifying and extracting the polyclonal antibody by using a GE-HiTrap Protein A HP pre-packed column to prepare an anti-P6 Protein polyclonal antibody;
1.2) coating:
Diluting the anti-P6 protein polyclonal antibody to 2.0mg/ml, diluting the goat anti-mouse IgG antibody to 0.3mg/ml, filling the diluted anti-P6 protein polyclonal antibody into a sprayer of a film scratching instrument, and spraying the diluted anti-P6 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 haemophilus influenzae surface protein monoclonal antibody secreted by the hybridoma cell strain Hi-17# by colloidal gold labeling;
2.3) marking the load of the monoclonal antibody of the surface protein of the human haemophilus influenzae secreted by the hybridoma cell strain Hi-17# with 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.
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