CN110540597B

CN110540597B - Preparation method of latex microsphere immunochromatographic test paper based on haemophilus influenzae surface protein

Info

Publication number: CN110540597B
Application number: CN201811566005.5A
Authority: CN
Inventors: 杨波; 陶冶; 胡征; 王毅
Original assignee: Hubei University of Technology
Current assignee: Hubei University of Technology
Priority date: 2018-12-20
Filing date: 2018-12-20
Publication date: 2021-04-30
Anticipated expiration: 2038-12-20
Also published as: CN110540597A

Abstract

The invention relates to a preparation method of a latex immunochromatographic test strip for rapidly detecting haemophilus influenzae, the test strip consists of a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a PVC (polyvinyl chloride) plate, wherein a colorful latex microsphere coupled anti-haemophilus influenzae surface protein polyclonal antibody is sprayed on the combination pad, and the nitrocellulose membrane is coated with a detection line of the anti-haemophilus influenzae surface protein polyclonal antibody and a quality control line of goat anti-rabbit IgG antibody. When the added sample contains haemophilus influenzae, the haemophilus influenzae firstly forms a complex with the latex-rabbit anti-haemophilus influenzae surface protein polyclonal antibody, and is captured when the complex migrates to a detection line coated with the haemophilus influenzae surface protein polyclonal antibody under the capillary action, and the detection line is correspondingly colored, so that whether the sample contains haemophilus influenzae or not can be detected. The test strip has the advantages of rapidness, simplicity, high sensitivity and good specificity.

Description

Preparation method of latex microsphere immunochromatographic test paper based on haemophilus influenzae surface protein

Technical Field

The invention belongs to the field of biological detection, and particularly relates to a preparation method of latex microsphere immunochromatographic test paper based on surface protein of haemophilus influenzae.

Background

Haemophilus influenzae (Hi) is a gram-negative bacterium without motility. It was discovered in 1892 by the fuverbo in epidemic cold. It is generally an aerobic organism, but can grow into a facultative anaerobic organism. Haemophilus influenzae was originally mistaken for the cause of influenza, but until 1933 this misunderstanding was not eliminated when the viral etiology of influenza was discovered. 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. Haemophilus influenzae without a capsule can cause ear infections (e.g. otitis media), eye infections (conjunctivitis) and sinusitis in children, and associated pneumonia. Such bacteria have strict nutrient requirements for culture and tend to parasitize in the upper airway, so that false negatives and false positives often occur in culture.

The existing method for detecting the pathogen in the respiratory tract mainly adopts the 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. Among them, immune latex test paper and other immunological detection techniques based on antibody have become an indispensable important means for detecting pathogenic microorganisms due to their characteristics of simplicity, rapidness, sensitivity, accuracy and practicality. Therefore, research and development of antibodies against pathogenic microorganisms having proprietary intellectual property rights are the basis for development of ELISA detection methods, latex microsphere labeling detection methods, and the like having proprietary intellectual property rights.

The choice of antigenic components is critical to the production of highly specific antibodies. The Haemophilus influenzae Pe, D15, PCP and PilA proteins are important molecules located on the cell surface, and the protein is high in conservation, specificity, antigenicity and surface exposure, and is an ideal detection target. In the research, surface proteins Pe, D15, PCP, PilA and the like with interspecific specificity are selected as antigens, gene sequences with high similarity with other bacteria are removed, the cross property of the antigens with other bacteria is reduced, meanwhile, polyclonal antibodies with good specificity are prepared by technical means of gene optimization, fusion expression and the like, and the polyclonal antibodies are applied to the preparation of a haemophilus influenzae latex microsphere immunochromatography detection test strip.

Disclosure of Invention

The invention aims to develop a latex microsphere immunochromatographic assay test strip for rapidly and quickly detecting haemophilus influenzae, which is simple to operate, low in cost and quick, by using an immune latex labeling technology on the basis of a polyclonal antibody.

The purpose of the invention is realized by the following technical scheme:

a preparation method of latex microsphere immunochromatographic test paper based on surface protein of Haemophilus influenzae is characterized by comprising the following steps: the method comprises the following steps:

1) haemophilus influenzae surface protein (D15+ PCP) antibody preparation:

respectively obtaining peptide segments with the most abundant antigenic epitopes in the haemophilus influenzae surface protein D15 and the surface protein PCP extracellular domain, finding out the gene coding sequence of the peptide segments, optimizing the gene coding sequence of the peptide segments, and connecting the optimized gene coding sequence of the peptide segments by using the coding sequence of flexible connecting peptide to form a fusion gene; the accession numbers of the haemophilus influenzae surface protein D15 and the surface protein PCP in the NCBI protein database are AAX87955 and AAX88288 respectively; the sequence of the flexible connecting peptide is ggsggsggsggs; simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as d15 pep;

the complete sequence of the d15 pep gene is:

the protein sequence encoded by the d15 pep gene is:

MQRVTDNDVANIVRSLFVSGRFDDVKAHQEGDVLVVSVVAKSIISDVKIKGNSIIPTEALKQNLDANGFKVGDVLIREKLNEFAKSVKEHYASVGRYNATVEPIVNTLPNNRAEILIQINEDDKAKLASLTFKGNESVSSSTLQEQMELQPDSWWKLWGNKFEGAQFEKDLQSIRDYYLNNGYAKGGSGGSGGSGGSRPVKIQADNQGVIGTLGGGALGGIAGSAIGGGRGQVIAAVVGAIGGAVAGSKIEEKVSQVNGAELVIKKDDGQEIVVVQKADSSFVAGRRVRIVGGGSNLNVSVL；

the protein sequence coded by the D75PCP gene is 50-233aa of the surface protein D15 of the haemophilus influenzae and 50-154aa of the surface protein PCP; the middle of the two protein sequences is connected by flexible connecting peptide; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni²⁺Purifying the recombinant His-D15PCP protein by affinity chromatography; taking the recombinant protein as an immune antigen, mixing the immune antigen with Freund's adjuvant, then repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood for titer determination, separating high-titer recombinant protein antibodies and purifying to finally obtain a haemophilus influenzae surface protein (D15+ PCP) antibody;

2) preparation of haemophilus influenzae surface protein (Pe + pilA) antibody:

respectively obtaining peptide segments with the most abundant antigenic epitopes in the extracellular domains of the surface protein Pe and the surface protein pilA of the haemophilus influenzae, finding out the gene coding sequence of the peptide segments, optimizing the gene coding sequence of the peptide segments, and connecting the two segments of sequences by using the coding sequence of rigid connecting peptide to form a fusion gene; the accession numbers of the surface protein Pe and the surface protein pilA of the haemophilus influenzae in the NCBI protein database are AGT37361 and AAX12377 respectively; the sequence of the rigid linker peptide is eaaakaaaak; simultaneously, introducing a restriction enzyme site NdeI at the 5 'end and introducing a termination signal TAA and a restriction enzyme site BamHI at the 3' end of the fusion gene, and then chemically synthesizing a whole gene sequence which is marked as pepil;

the complete sequence of the pepil gene is:

the protein sequence encoded by the pepil is:

MLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHEAAAAKEAAAAKLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKG；

the protein sequence coded by the pepil gene is 43-130aa of the surface protein Pe of the haemophilus influenzae and 17-133aa of the surface protein pilA, and the two protein sequences are connected by rigid connecting peptide; cloning the gene fragment into a prokaryotic expression vector pET-28a (+) according to a conventional method, inducing recombinant escherichia coli to express by IPTG, and purifying the recombinant His-Pepil protein by Ni2+ affinity chromatography; taking the recombinant protein as an immune antigen, mixing the immune antigen with Freund's adjuvant, then repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood for titer determination, separating high-titer recombinant protein antibodies and purifying to finally obtain a haemophilus influenzae surface protein (Pe + pilA) antibody;

3) preparation of latex microsphere markers for Haemophilus influenzae surface protein (D15+ PCP) antibodies:

3.1) activation of the latex microspheres

Taking 1mL of colored carboxylated polystyrene latex microsphere solution with the concentration of 10%, adding 9mL of MES (2- (N-morpholinyl) ethanesulfonic acid) buffer solution, mixing uniformly, adding NHS (N-hydroxysuccinimide) and EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) until the final concentration of the two is 1mg/mL, slowly mixing uniformly at room temperature for 30 minutes, centrifuging 19000g for 20 minutes after the incubation is finished, removing the supernatant, and precipitating by using 10mL of borax buffer solutionResuspending, oscillating and ultrasonically processing to obtain activated latex microspheres; the MES buffer solution comprises the following components in percentage by weight: 0.1mol/L MES, wherein the pH of the MES buffer is 8.5; the grain size of the colored carboxylated polystyrene latex microspheres is 100 nm; the content of the components in the borax buffer solution is 0.1mol/L Na₂B₄O₇The pH value of the borax buffer solution is 8.5;

3.2) preparation of latex microsphere markers

Diluting the haemophilus influenzae surface protein (D15+ PCP) antibody obtained in the step 1) to 1mg/mL by using a borax buffer solution; adding 10mL of haemophilus influenzae surface protein (D15+ PCP) antibody into 10mL of activated latex microspheres, slowly mixing uniformly for 30 minutes, centrifuging at 19000g for 10 minutes, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, repeating centrifugation for 1 time after ultrasonic pulverization, and removing supernatant; resuspending the precipitate by the same method, repeatedly centrifuging for 1 time after ultrasonic crushing and oscillation, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, namely a latex microsphere marker of Haemophilus influenzae surface protein (D15+ PCP) antibody; the content of the components in the borax buffer solution is 0.1mol/L Na₂B₄O₇The pH value of the borax buffer solution is 8.5;

4) preparation of the bonding pad:

spraying the latex microsphere marker of the haemophilus influenzae surface protein (D15+ PCP) antibody obtained in the step 3) on a bonding pad made of a polyester fiber material, wherein the spraying amount of each square centimeter of polyester fiber film is 10 mu L of the latex microsphere marker; drying at 37 deg.C in environment with relative humidity not more than 30%, sealing at 25 deg.C, drying and storing;

5) preparation of antibody solid-phase nitrocellulose membrane:

diluting the Haemophilus influenzae surface protein (Pe + pilA) antibody obtained in the step 2) to 1.5mg/mL by using a borax buffer solution, and then coating the antibody on a detection line position on a nitrocellulose membrane by using a membrane spraying instrument as a detection line to capture the antibody, wherein the coating parameter is 1 mu L/cm; spraying goat anti-rabbit IgG on a quality control line position on a nitrocellulose membrane as a control line to capture an antibody, wherein the concentration is 1mg/mL, and the coating parameter is 1 mu L/cm; coating the cellulose nitrateDrying the membrane at 37 deg.C in an environment with relative humidity not more than 30%, sealing at 25 deg.C, drying and storing; the content of the components in the borax buffer solution is 0.1mol/L Na₂B₄O₇The pH value of the borax buffer solution is 8.5;

6) preparation of sample pad

Taking a glass cellulose membrane, soaking the glass cellulose membrane in a sample pad treatment solution for at least 3h, placing the sample pad treatment solution in a biological safety cabinet for ventilation drying at 37 ℃, cutting the sample pad treatment solution into required specifications, and sealing, drying and storing the sample pad treatment solution at 25 ℃; thus, a sample pad was prepared;

the sample pad treatment solution comprises the following components in percentage by weight: 0.01mol/L Na₂B₄O₇2g/L sodium chloride, 20g/L casein, 10ml/L Tween-20 and 10ml/L antifoaming agent S-17; the pH of the sample pad treatment solution was 8.5;

7) assembly of test strips

Respectively sticking a water absorption pad, an antibody solid-phase nitrocellulose membrane, a combination pad and a sample pad which are made of water absorption filter paper materials on a PVC (polyvinyl chloride) base plate in sequence, wherein a quality control line on the nitrocellulose membrane is close to the end of the water absorption pad, a detection line is close to the end of the sample pad, cutting the nitrocellulose membrane into test strips with certain width, sealing and packaging the test strips, and drying the test strips for low-temperature storage; thus preparing the Haemophilus influenzae latex microsphere immunochromatography test strip.

The invention has the advantages that:

(1) the invention adopts structural analysis, gene optimization and other modes to construct two brand new fusion genes, and the soluble recombinant D15/PCP fusion protein and Pe/pilA fusion protein are successfully obtained for the first time through soluble over-expression. The two fusion proteins have high expression amount, low preparation cost, good protein solubility, strong antigenicity, high antibody titer and low cost.

(2) The antibody prepared by utilizing the four protein exposed regions on the surface of the haemophilus influenzae for the first time has high titer, many targeted antigen sites, strong capture capacity, no site competition problem and high test strip sensitivity. The detection sensitivity of the kit to a haemophilus influenzae standard strain ATCC49247 reaches 2 x 10⁴CFU/mL is obviously higher than that of the traditional detection method of microorganismsAnd has the advantages of high speed, high efficiency and the like.

(3) The test strip has good specificity, and the test strip has good specificity and stability by using 6 strains of haemophilus influenzae and 18 strains of non-haemophilus influenzae standard strains (containing most common respiratory pathogens), can detect all tested haemophilus influenzae, has no cross reaction with all non-haemophilus influenzae standard strains, and is very suitable for clinical non-diagnostic application.

(4) The test strip can be preserved for two years at normal temperature, effectively prolongs the shelf life and reduces the storage condition; non-professional persons can finish the whole-process detection by using the detection test paper, the operation is simple, and the popularization of the method is facilitated; the whole detection process can be finished within 10min at the fastest speed, and is more suitable for bedside detection.

Drawings

FIG. 1 is a schematic diagram of an explosion structure of a latex microsphere immunochromatographic test paper based on a surface protein of Haemophilus influenzae provided by the invention;

FIG. 2 is a schematic structural diagram of a latex microsphere immunochromatographic test paper based on a surface protein of Haemophilus influenzae provided by the present invention;

wherein:

1-sample pad; 2-a conjugate pad; 3-NC film; 4-absorbent pad; 5-PVC sheet.

Detailed Description

The present invention is further specifically described by the following examples.

Sources of the various materials used or employed in the present invention

1. Latex microspheres: the latex microspheres used in the invention are carboxylated modified polystyrene latex microspheres, are products of Shanghai Yan Biotech Co., Ltd, have the size of 100nm and the color of red, have the tolerance of the average diameter of the products within 10 percent, are in the form of 10 percent solid aqueous suspension, and have the product code of MSI-CAR100 NM.

2. Glass cellulose membrane: the thickness is 0.45-0.55mm, the water absorption capacity is 800mg/m²The glass fiber has a diameter of 0.6-3 μm, has good hydrophilicity, and is available in Shanghai gold-labeled biological familyTechnology corporation (model number BT 50).

3. Polyester fiber film: has a thickness of 0.25-0.35mm, a climbing speed of 15-40mm/60s, excellent hydrophilicity, and is used for preparing a bonding pad, and is available from Shanghai gold-labeled Biotech Co., Ltd (model number VL 98).

4. Cellulose nitrate membrane: model number Millipore Corp SHF135, with liner plates, was purchased from Millipore corporation.

5. Water-absorbing filter paper: the thickness is 0.95mm, the water absorption speed is 60s/4cm, and the water absorption capacity is 700mg/cm²Has good water absorption and is used as a material for manufacturing the water absorption pad. Purchased from Shanghai gold Biotech, Inc. (model CH 37K).

6. A bottom plate: is made of high-whiteness PVC material, and is coated with a single-layer high-polymer pressure-sensitive adhesive SM31 purchased from Shanghai gold-labeled Biotech Co.

7. The microorganism samples used in the present invention were purchased from the American Type Culture Collection (ATCC).

8. pET28a (+): e.coli expression vectors, introduced from Novagen, USA.

9. Escherichia coli (e. coli) BL21(DE 3): purchased from northern Biotechnology, Inc., Shanghai.

10. Goat anti-rabbit IgG: is product of bioscience, Dr. Germany, product No. BA1039, and has a concentration of 1 mg/ml.

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

Example 1

Preparation of haemophilus influenzae surface protein (D15+ PCP) antibody:

1.1) cloning of the fusion Gene of Haemophilus influenzae d15pcp

The peptide segment with the most abundant antigenic epitopes in the extracellular domains of the haemophilus influenzae surface protein D15 and PCP (the accession numbers in the NCBI protein database are AAX87955 and AAX88288 respectively) is obtained, the gene coding sequence is found, the gene coding sequence is optimized, and the two sequences are connected by the coding sequence of flexible connecting peptide (ggsgggsgggs) to form the fusion gene. Meanwhile, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as d15 pep. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, protein sequences coded by the D15 pep gene are 50-233aa of a surface protein D15 of haemophilus influenzae and 50-154aa of a surface protein PCP, and flexible connecting peptides (ggsggsggs) are connected between the two protein sequences. The gene sequence is delivered to Nanjing Jinslei Biotech, Inc. for complete gene chemical synthesis, and the artificially synthesized gene fragment is connected to vector pUC57 when delivered. The vector pUC57 containing the artificially synthesized DNA fragment was digested with NdeI and BamHI, and the desired fragment was recovered by a conventional method and used. And carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and BamHI, connecting the d15 pep gene obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional molecular biological method, and transforming Escherichia coli TOP10 to construct a pET-d15 pep expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses a recombinant D15PCP fusion protein.

1.2) expression and purification of Haemophilus influenzae D15PCP fusion protein

Culturing the correctly identified positive clone bacteria, extracting plasmids, transferring into competent E.coli BL21(DE3) according to a conventional technology, coating the bacterial liquid on an LB flat plate containing 50 mu g/mL kanamycin after the conversion is finished, and screening expression strains according to a conventional method. Individual colonies transformed with pET-d15 pep and having the ability to express foreign proteins were picked and inoculated into 100mL of LB medium and cultured overnight at 37 ℃. After taking out the bacterial liquid, the bacterial liquid is prepared according to the following steps of 1: 100 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 0.5mmol/L, and cultured with shaking at 37 ℃ to induce expression of the fusion protein. After induction for 4h, the thalli are collected by centrifugation for 10min at 8000 r/min. The resulting mixture was diluted with 50mL Buffer A (50mM Na)₃PO₄0.5M NaCl; pH7.4) was washed 3 times and 50mL of loading buffer (50mM Na)₃PO₄0.5M NaCl; 5mM imidazole, pH7.4) followed by resuspension, sonication, operating under the following conditions: the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30 min.After the ultrasonic treatment is finished, the mixture is centrifuged at 12000g for 15min, and then the precipitate and the supernatant are respectively collected for electrophoresis detection. The recombinant D15PCP fusion protein was found to be present in the bacterial cells in solubilized form. Thin-layer scanning showed that the recombinant protein accounted for more than 25% of the total bacterial protein. On the other hand, the wild type d15 pep which is not gene-optimized is expressed in the same manner as described above, and as a result, the expression product is found to account for only 5% of the total protein, indicating that the gene optimization effect is outstanding. 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:

1.2.1) connecting a chromatography system, wherein the system comprises a sample inlet pipe, a peristaltic pump (Shanghai Huxi analytical instrument factory, model DHL-A), a chromatography column (GE healthcare product, product name His trade affinity columns) 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;

1.2.2) proofreading T%: adjusting a brightness knob to display 100%;

1.2.3) rotational sensitivity to the appropriate position, typically 0.2A;

1.2.4) equilibrating the chromatography system with the above buffer until the reading is stable and then rotating "zero" to show "000";

1.2.5) applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid;

1.2.6) washing away unbound protein with loading buffer, recording the reading during the process until the reading does not change any more, and collecting the eluate;

1.2.7) eluting with Buffer A +10mM imidazole, and collecting the elution peak;

1.2.8) eluting with Buffer A +20mM imidazole, and collecting the elution peak;

1.2.9) eluting with Buffer A +40mM imidazole, and collecting the elution peak;

1.2.10) eluting with Buffer A +100mM imidazole, and collecting the elution peak;

1.2.11) eluting with Buffer A +150mM imidazole, and collecting the elution peak;

1.2.12) taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis;

1.2.13) was eluted at 100mM imidazole, and the target protein was found to have a purity of 90% or more, and was adjusted to 0.2mg/mL for use after measuring the protein concentration with the bradford kit. Thus, Haemophilus influenzae D15PCP fusion protein was prepared.

1.3) preparation of Haemophilus influenzae surface protein (D15+ PCP) antibody

1.3.1) mixing the Haemophilus influenzae D15PCP fusion protein prepared in the step (1.2) with Freund's complete adjuvant, emulsifying to obtain immunogen for immunizing 2 male New Zealand rabbits, wherein the total amount of subcutaneous injection for each rabbit is 2ml, and the total amount of antigen is 2 mg/rabbit. And then, the emulsion formed by the FhuOmp fusion protein and Freund's incomplete adjuvant is used for immunization once every two weeks, the immunization is carried out for 5 times totally, 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.

1.3.2) determination of the potency of the polyclonal antibody

The D15PCP fusion protein is used as a coating antigen, the coating concentration is 5 mu g/ml, each hole is coated with 100 mu l, and the level of the serum antibody is detected by an indirect ELISA method. The serum dilution times of the experimental groups are as follows: 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.

1.3.3) extraction of polyclonal antibodies

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

1.3.3.1) 5mL of antiserum was taken, 0.5mL of 1M Tris (pH8.0) was added to adjust to pH8.0, and 20,000 g was centrifuged for 20min to remove the precipitate.

1.3.3.2) was applied to the column, and then 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).

1.3.3.3) eluted IgG 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)

1.3.3.4) the eluate was dialyzed against 50 volumes of Tris (10mM Tris-Cl, pH 8.0).

1.3.3.5) ultrafiltering and concentrating, adjusting the concentration to 5mg/ml, and storing at-70 ℃ for later use. Thus, a Haemophilus influenzae surface protein (D15+ PCP) antibody was prepared.

Example 2

Preparation of haemophilus influenzae surface protein (Pe + pilA) antibody:

2.1) cloning of the Haemophilus influenzae Pepil fusion Gene

The method comprises the steps of obtaining peptide segments with the most abundant antigenic epitopes in extracellular domains of Haemophilus influenzae surface proteins Pe and pilA (the access numbers in an NCBI protein database are AGT37361 and AAX12377 respectively), finding out gene coding sequences of the peptide segments, optimizing the gene coding sequences of the peptide segments, and connecting the two sequences by using coding sequences of rigid connecting peptides (eaaakaaaak) to form a fusion gene. Meanwhile, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as Pepil. The complete gene sequence and the coded amino acid sequence are shown in a sequence table. Specifically, the protein sequences coded by the Pepil gene are 43-130aa of the surface protein Pe of the haemophilus influenzae and 17-133aa of the surface protein pilA, and the two protein sequences are connected by rigid connecting peptide (eaaakaaaak). The gene sequence is delivered to Nanjing Jinslei Biotech, Inc. for complete gene chemical synthesis, and the artificially synthesized gene fragment is connected to vector pUC57 when delivered. The vector pUC57 containing the artificially synthesized DNA fragment was digested with NdeI and BamHI, and the desired fragment was recovered by a conventional method and used. And simultaneously carrying out double enzyme digestion on the vector pET-28a (+) by NdeI and BamHI, connecting Pepil genes obtained after double enzyme digestion into the pET-28a (+) vector according to a conventional molecular biological method, and transforming Escherichia coli TOP10 to construct a pET-Pepil expression vector. The construction of the expression vector is verified to be correct by enzyme digestion and sequence determination. The vector expresses a recombinant Pepil fusion protein.

2.2) expression and purification of Haemophilus influenzae Pepil fusion proteins

Culturing the correctly identified positive clone bacteria, extracting plasmids, transferring into competent E.coli BL21(DE3) according to a conventional technology, coating the bacterial liquid on an LB flat plate containing 50 mu g/mL kanamycin after the conversion is finished, and screening expression strains according to a conventional method. Individual colonies transformed with pET-Pepil having the ability to express foreign proteins were picked and inoculated into 100mL of LB medium and cultured overnight at 37 ℃. After taking out the bacterial liquid, the bacterial liquid is prepared according to the following steps of 1: 100 was inoculated into 100mL of LB medium containing 50. mu.g/mL of kanamycin, and when the cells were cultured at 37 ℃ until OD600 became 0.6, 1mol/L of IPTG was added to a final concentration of 0.5mmol/L, and the cells were cultured with shaking at 37 ℃ to induce expression of the fusion protein. After induction for 4h, the thalli are collected by centrifugation for 10min at 8000 r/min. The resulting mixture was diluted with 50mL Buffer A (50mM Na)₃PO₄0.5M NaCl; pH7.4) was washed 3 times and 50mL of loading buffer (50mM Na)₃PO₄0.5M NaCl; 5mM imidazole, pH7.4) followed by resuspension, sonication, operating under the following conditions: the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30 min. After the ultrasonic treatment is finished, the mixture is centrifuged at 12000g for 15min, and then the precipitate and the supernatant are respectively collected for electrophoresis detection. The recombinant Pepil fusion protein was found to be present in the bacterial cells in solubilized form. Thin-layer scanning showed that the recombinant protein accounted for more than 20% of the total bacterial protein. And the wild Pepil which is not optimized by the gene is expressed in the same way, and the result shows that the expression product only accounts for 3 percent of the total protein, which indicates that the gene optimization effect is outstanding. 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:

(1) connecting a chromatography system, wherein the system comprises a sample inlet pipe, a peristaltic pump (Shanghai analytical instrument factory, model DHL-A), a chromatography column (product of GE healthcare company, trade name His Trap affinity column) and an ultraviolet detector (Shanghai analytical instrument factory, model HD1), the column volume is 2ml, and the ultraviolet detector is preheated for about 30min until the reading is stable;

(2) and (5) correcting T%: adjusting a brightness knob to display 100%;

(3) rotate the sensitivity to the appropriate position, typically 0.2A;

(4) equilibrating the chromatography system with the loading buffer until the reading stabilizes and then rotating "zero" to show "000";

(5) applying protein sample, controlling the flow rate within 5ml/min, and collecting penetration liquid;

(6) 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;

(7) eluting with Buffer A +10mM imidazole, and collecting an elution peak;

(8) eluting with Buffer A +20mM imidazole, and collecting an elution peak;

(9) eluting with Buffer A +40mM imidazole, and collecting an elution peak;

(10) eluting with Buffer A +60mM imidazole, and collecting an elution peak;

(11) eluting with Buffer A +100mM imidazole, and collecting an elution peak;

(12) eluting with Buffer A +150mM imidazole, and collecting an elution peak;

(13) taking 100ul of each elution peak sample to carry out SDS-PAGE electrophoresis;

(14) as a result, it was found that the target protein was eluted at 60mM imidazole and had a purity of 90% or more, and the concentration was adjusted to 0.2mg/mL for use after the protein concentration was measured with a bradford kit. Thus, Haemophilus influenzae Pepil fusion protein is prepared.

2.3) preparation of Haemophilus influenzae surface protein (Pe + pilA) antibody

2.3.1) mixing the Haemophilus influenzae Pepil fusion protein prepared in the step 2.2) with Freund's complete adjuvant, emulsifying to obtain immunogen for immunizing 2 male New Zealand rabbits, wherein 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 emulsion formed by the Pepil fusion protein and Freund's incomplete adjuvant, wherein the immunization is carried out for 5 times totally, 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.

2.3.2) determination of the potency of the polyclonal antibody

Pepil fusion protein is used as a coating antigen, the coating concentration is 5 mu g/ml, each hole is coated with 100 mu l, and the level of serum antibody is detected by an indirect ELISA method. The serum dilution times of the experimental groups are as follows: 1: 200. 1: 400. 1: 800. 1: 1600. 1: 3200. 1: 6400. 1: 12800. 1: 25600. 1: 51200. 1: 102400, 1: 204800;

2.3.3) extraction of polyclonal antibodies

2.3.3.1) 5mL of antiserum was taken, 0.5mL of 1M Tris (pH8.0) was added to adjust to pH8.0, and 20,000 g was centrifuged for 20min to remove the precipitate.

2.3.3.2) was applied to the column, and then 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).

2.3.3.3) eluting IgG 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)

2.3.3.4) the eluate was dialyzed against 50 volumes of Tris (10mM Tris-Cl, pH 8.0).

2.3.3.5) concentrating by ultrafiltration, adjusting the concentration to 5mg/ml, and storing at-70 deg.C for use. Thus, a Haemophilus influenzae surface protein (Pe + pilA) antibody was prepared.

Example 3

Preparation of latex microsphere markers for Haemophilus influenzae surface protein (D15+ PCP) antibodies:

3.1) activation of the latex microspheres

Taking 1mL of a 10% red carboxylated polystyrene latex microsphere (100nm) solution, adding 9mL of 2- (N-morpholinyl) ethanesulfonic acid (MES) buffer solution (0.1mol/L MES, pH8.5), and mixing uniformly; preparing 10mg/mL of N-hydroxysuccinimide (NHS) and 10mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution by using MES buffer solution;

adding 1mL NHS solution and 1mL EDC solution into polystyrene latex microsphere (100nm) solution in sequence, slowly mixing at room temperature for 30min, centrifuging 19000g after incubation for 20min, removing supernatant, precipitating with 10mL borax buffer (0.1mol/L Na)₂B₄O₇Ph8.5), resuspension, shaking, sonication (sonicator model: YJ92-IIDN, the power is 50W, the working time is 2s, the interval time is 3s, the alarm temperature is 60 ℃, and the total time is 30min) to obtain the activated latex microspheres.

3.2) preparation of latex microsphere markers

Using borax buffer (0.1mol/L Na)₂B₄O₇pH8.5) the antibody against the surface protein of Haemophilus influenzae (D15+ PCP) obtained in step 1 was diluted to 1 mg/mL. 10mL of Haemophilus influenzae surface protein (D15+ PCP) antibody is added into 10mL of activated latex microspheres, slowly and uniformly mixed for 30 minutes, then 19000g is centrifuged for 10 minutes, and the supernatant is removed. The precipitate was resuspended in 10mL of borax buffer containing 1% casein, and after ultrasonication (model: YJ92-IIDN, power 50W, working time 2s, interval time 3s, alarm temperature 60 ℃ C., total time 30min), 19000g was centrifuged repeatedly 1 time (10 min) to remove the supernatant. The pellet was resuspended in the same way, sonicated and centrifuged again at 19000g for 1 time (10 min) and the supernatant removed. The precipitate was resuspended in 10mL borax buffer containing 1% casein, which was the latex microsphere marker for Haemophilus influenzae surface protein (D15+ PCP) antibody.

Example 4

Preparation of the bonding pad:

the polyester fiber film was cut into pieces of 4cm × 0.8 cm/piece, and 32 μ L of the latex microsphere marker prepared in example 3 was dropped onto the cut pieces of the film. After spraying, drying at 37 ℃ for 12h in an environment with the relative humidity of 20%. Sealing, drying and storing at 25 ℃.

Example 5

Preparation of antibody solid-phase nitrocellulose membrane:

the nitrocellulose membrane was cut to a size of 4cm by 2.3 cm. Using borax buffer solution (0.1mol/L Na)₂B₄O₇pH8.5) the Haemophilus influenzae surface protein (Pe + pilA) antibody obtained in example 2 and goat anti-rabbit IgG were diluted to 1.5mg/mL and 1mg/mL, respectively; filling the diluted haemophilus influenzae surface protein (Pe + pilA) antibody into a nozzle 1 of a BIODOT membrane scribing instrument, and spraying the diluted haemophilus influenzae surface protein (Pe + pilA) antibody on a nitrocellulose membrane in an amount of 1.0 mu l/cm to form a detection line, wherein the edge distance between the detection line and the nitrocellulose membrane is 0.8 cm; and (3) filling the diluted goat anti-rabbit IgG into a nozzle 2 of a BIODOT membrane scribing instrument, setting the volume of 1.0 mu l/cm, spraying the diluted goat anti-rabbit IgG on a nitrocellulose membrane to be used as a quality control line, and setting the distance between the diluted goat anti-rabbit IgG and the detection line to be 0.7 cm. After coating, the nitrocellulose membrane is put in an environment with the relative humidity of 20 percent, dried for 12 hours at 37 ℃, sealed, dried and stored at 25 ℃.

Example 6

Preparation of sample pad:

preparing sample pad treating fluid with different formulas, observing the release effect of the latex microsphere labeled antibody, and optimizing through multiple orthogonal tests to obtain the optimal formula (0.01mol/L Na) of the sample pad treating fluid₂B₄O₇2g/L sodium chloride, 20g/L casein, 10ml/L Tween-20, 10ml/L antifoam S-17, pH 8.5). Taking a glass cellulose membrane, soaking the glass cellulose membrane in the sample pad treatment solution for 3h, then placing the sample pad in a biological safety cabinet for 12h at 37 ℃ for ventilation drying, cutting the sample pad into strips with the specification of 4cm multiplied by 3cm, sealing and drying at 25 ℃ for storage, and obtaining the sample pad. Thus, a sample pad was prepared. Tests prove that the use of the sample pad greatly improves the release rate of the latex microsphere labeled antibody on the bonding pad, and achieves better application effect.

EXAMPLE 7 absorbent pad cutting

The water-absorbing filter paper purchased from Shanghai gold-labeled Biotechnology Co., Ltd, model number CH37K, was cut into pieces of 4 cm. times.3 cm/strip for use.

EXAMPLE 8 tailoring of PVC sheets

A high-whiteness PVC sheet purchased from Shanghai gold-labeled Biotechnology Co., Ltd, model number SM31, was cut into 4cm by 8.5cm strips for use.

EXAMPLE 9 Assembly of the test strips

Referring to fig. 1 and 2, an NC film 3, a bonding pad 2, a water absorbent pad 4 and a sample pad 1 are sequentially adhered to a single-sided PVC plate 5, wherein the bonding pad 2 and the water absorbent pad 4 are laminated on the NC film 3, and are respectively overlapped with the NC film 3 by about 2mm, and the sample pad 1 is laminated on the bonding pad 2, and are overlapped with the bonding pad 2 by about 2 mm. The NC membrane 3 is marked with a detection line T and a quality control line C. And cutting the adhered test paper board into test paper strips with the width of 4mm by using a cutting machine, and putting the prepared test paper strips and the drying agent into an aluminum foil bag for sealing and storing.

Example 10

The use method of the test strip comprises the following steps:

10.1) treatment of the sample to be examined

The throat swab of the subject was obtained by a conventional method, and inserted into a 500. mu.L sample treatment solution (0.01mol/L Na)₂B₄O₇2g/L sodium chloride, 20ml/L Tween-20), the wall of the plastic tube is pressed to fully dissolve the sample on the swab.

10.2) adding the sample to be detected, and judging the result

Adding 100 mu L of sample into a randomly extracted and assembled test strip, binding Haemophilus influenzae in a sample solution with an antibody marked by latex microspheres on a binding pad and binding with an antibody of a detection line (T line) under the action of chromatography, and acting for 10min at room temperature to obtain two red lines, namely a detection line T line and a quality control line C line, as a positive result; if the test sample does not contain the haemophilus influenzae, a negative result only shows that a red line appears on the quality control line C, which shows that the sample does not contain the haemophilus influenzae.

Example 11

And (3) testing the performance of the test strip:

11.1) specific analysis

In order to verify the specificity of the latex microsphere immunochromatographic assay test strip for detecting haemophilus influenzae, the compositions and the use methods of the test strips in the embodiments 9 and 10 are 2 × 10⁵6 strains of Haemophilus influenzae and 17 non-Haemophilus influenzae of CFU/mL were tested and shown in Table 1. The results show that the test strip of the invention can treat all 6 strainsThe detection results of the haemophilus influenzae strains are positive, and the detection results of other 17 strains of respiratory common pathogenic microorganisms are negative. The test strip shows good specificity.

TABLE 1

Strain name	Results of the detection
		Human Haemophilus influenzae ATCC49247	Positive for
Human Haemophilus influenzae ATCC 33391	Positive for
		Human Haemophilus influenzae ATCC 10211	Positive for
Human haemophilus influenzae ATCC 49766	Positive for
		Human Haemophilus influenzae ATCC 35056	Positive for
Human haemophilus influenzae ATCC 31441	Positive for
		Mycoplasma pneumoniae ATCC 15531	Negative of
Pseudomonas aeruginosa ATCC 27853	Negative of
		Acinetobacter baumannii ATCC 19606	Negative of
Moraxella catarrhalis ATCC 46327	Negative of
		Haemophilus parainfluenza ATCC 7901	Negative of
Legionella pneumophila ATCC 33152	Negative of
		Streptococcus pyogenes ATCC 19615	Negative of
Staphylococcus aureus ATCC 25923	Negative of
		Streptococcus pneumoniae ATCC 49619	Negative of
Klebsiella pneumoniae ATCC 700603	Negative of
		Enterobacter cloacae ATCC 13047	Negative of
Escherichia coli ATCC 25922	Negative of
		Candida ATCC 10231	Negative of
Influenza A virus ATCC VR-1743	Negative of
		Influenza B virus ATCC VR-790	Negative of
Respiratory syncytial virus ATCC VR26	Negative of
		Adenovirus ATCC VR-3	Negative of

At the same time, the concentration is 2 multiplied by 10⁵The test strip is used for detecting 121-strain clinical isolates of the human haemophilus influenzae in CFU/mL, the results are positive, and the high detection coverage of the test strip on the clinically isolated human haemophilus influenzae is shown.

11.2) sensitivity determination

Inoculating human Haemophilus influenzae ATCC49247 strain to goat blood chocolate culture medium, culturing at 35 deg.C for 36 hr, diluting with 10 times of normal saline, and counting to obtain 10-fold thallus concentration⁸-10³CFU/mL of the bacterial solution, 100. mu.L of the bacterial solution was dropped on the sample pad, and the test was performed according to the composition and the method of use of the test strips described in examples 7 and 8. The result shows that the detection sensitivity of the kit is 2 multiplied by 10⁴CFU/mL。

11.3) stability test

The test strips were dried and sealed, and then subjected to 4 ℃, 25 ℃, 37 ℃, and the physiological saline dilutions thereof for detecting Haemophilus influenzae ATCC49247 after 6 months, 12 months, 18 months, 21 months, and 24 months, respectively, and the results were observed.

After the test strip is dried and sealed, the test strip can still detect strong positive results after being respectively placed at 4 ℃ and 25 ℃ for 6-24 months; positive results were also detected after 6-18 months at 37 ℃, but were reduced after 21-24 months of storage. The test strip can be stored at 4 ℃ or 25 ℃ for at least 2 years.

Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Sequence listing

<110> Hubei university of industry

<120> preparation method of latex microsphere immunochromatographic test paper based on haemophilus influenzae surface protein

<160> 4

<170> SIPOSequenceListing 1.0

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<212> DNA

<213> d15 pep Gene sequence (d15 pep)

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catatgcagc gcgtaaccga caacgacgtt gcaaacatcg tacgttctct gtttgtgtcc 60

ggccgtttcg atgacgttaa agctcaccag gaaggtgatg ttctggttgt ttctgttgtt 120

gcaaaatcca tcatcagtga cgttaaaatc aagggcaact ctatcatccc aactgaagct 180

ctgaagcaga acctggacgc taacggtttt aaagtaggtg atgttctgat ccgtgaaaag 240

ctgaacgaat tcgctaaatc cgttaaggag cattatgcga gtgttggtcg ttacaacgct 300

accgtagaac cgattgttaa cactctgccg aacaaccgcg cggaaatcct gatccagatc 360

aacgaagatg ataaagcaaa actggcttct ctgaccttca aaggtaacga atctgtatcc 420

tcatccactc ttcaggaaca gatggaactg cagccggact cttggtggaa gctgtggggc 480

aacaaattcg aaggcgcgca gttcgaaaaa gacctgcaat ctattcgtga ttattacctg 540

aacaacggtt atgcaaaagg tggttctggt ggttctggtg gttctggtgg ttctcgcccg 600

gttaaaatcc aggctgataa ccagggtgta atcggcaccc tgggcggcgg cgctctgggc 660

ggtatcgcag gttctgctat cggcggcggt cgcggccagg ttattgccgc tgtagtaggc 720

gctatcggtg gtgcggtggc cggttctaaa atcgaagaaa aggtttccca ggtaaacggt 780

gctgagctgg taatcaaaaa agacgatggc caggaaatag ttgttgtaca gaaagccgac 840

tcttcctttg tagcaggtcg tcgcgtgcgc atcgttggtg gtgggtctaa cctgaacgtc 900

tctgttctgt aaggatcc 918

<210> 2

<211> 302

<212> PRT

<213> d15 pep protein sequence (d15 pep)

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Met Gln Arg Val Thr Asp Asn Asp Val Ala Asn Ile Val Arg Ser Leu

1 5 10 15

Phe Val Ser Gly Arg Phe Asp Asp Val Lys Ala His Gln Glu Gly Asp

20 25 30

Val Leu Val Val Ser Val Val Ala Lys Ser Ile Ile Ser Asp Val Lys

35 40 45

Ile Lys Gly Asn Ser Ile Ile Pro Thr Glu Ala Leu Lys Gln Asn Leu

50 55 60

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

65 70 75 80

Asn Glu Phe Ala Lys Ser Val Lys Glu His Tyr Ala Ser Val Gly Arg

85 90 95

Tyr Asn Ala Thr Val Glu Pro Ile Val Asn Thr Leu Pro Asn Asn Arg

100 105 110

Ala Glu Ile Leu Ile Gln Ile Asn Glu Asp Asp Lys Ala Lys Leu Ala

115 120 125

Ser Leu Thr Phe Lys Gly Asn Glu Ser Val Ser Ser Ser Thr Leu Gln

130 135 140

Glu Gln Met Glu Leu Gln Pro Asp Ser Trp Trp Lys Leu Trp Gly Asn

145 150 155 160

Lys Phe Glu Gly Ala Gln Phe Glu Lys Asp Leu Gln Ser Ile Arg Asp

165 170 175

Tyr Tyr Leu Asn Asn Gly Tyr Ala Lys Gly Gly Ser Gly Gly Ser Gly

180 185 190

Gly Ser Gly Gly Ser Arg Pro Val Lys Ile Gln Ala Asp Asn Gln Gly

195 200 205

Val Ile Gly Thr Leu Gly Gly Gly Ala Leu Gly Gly Ile Ala Gly Ser

210 215 220

Ala Ile Gly Gly Gly Arg Gly Gln Val Ile Ala Ala Val Val Gly Ala

225 230 235 240

Ile Gly Gly Ala Val Ala Gly Ser Lys Ile Glu Glu Lys Val Ser Gln

245 250 255

Val Asn Gly Ala Glu Leu Val Ile Lys Lys Asp Asp Gly Gln Glu Ile

260 265 270

Val Val Val Gln Lys Ala Asp Ser Ser Phe Val Ala Gly Arg Arg Val

275 280 285

Arg Ile Val Gly Gly Gly Ser Asn Leu Asn Val Ser Val Leu

290 295 300

<210> 3

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<212> DNA

<213> pepil Gene sequence (pepil)

<400> 3

catatgctgg taaaaaacgt taactactac atcgactctg aatccatctg ggttgataac 60

caggaaccac agatcgtaca cttcgacgct gttgttaacc tcgataaagg cctgtacgtt 120

tacccggaac caaagcgtta cgctcgttct gttcgtcagt acaaaatcct taactgtgcg 180

aactaccatc tgacccaggt acgtaccgac ttttacgacg aattctgggg tcagggcctg 240

cgcgcagctc cgaaaaaaca gaaaaaacac gaagctgctg ctgctaaaga agctgctgct 300

gctaaactga ttgaactgat gatcgtgatc gcgatcatcg caattctggc gaccatcgca 360

atcccgtctt accagaacta cactaaaaaa gctgctgtct ccgaactgct gcaagcatct 420

gctccgtaca aagctgacgt tgaactctgc gtttactcta cgaacgagac caccaactgc 480

actggtggca aaaacggtat tgcggctgac atcaccactg ctaagggtta cgtaaaatct 540

gttaccacct ctaacggcgc aatcactgtg aaaggggacg gcaccctggc taacatggaa 600

tacattctgc aggctaccgg taacgcagcg accggcgtga cctggactac cacctgcaaa 660

ggctaaggat cc 672

<210> 4

<211> 220

<212> PRT

<213> pepil protein sequence (pepil)

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Met Leu Val Lys Asn Val Asn Tyr Tyr Ile Asp Ser Glu Ser Ile Trp

1 5 10 15

Val Asp Asn Gln Glu Pro Gln Ile Val His Phe Asp Ala Val Val Asn

20 25 30

Leu Asp Lys Gly Leu Tyr Val Tyr Pro Glu Pro Lys Arg Tyr Ala Arg

35 40 45

Ser Val Arg Gln Tyr Lys Ile Leu Asn Cys Ala Asn Tyr His Leu Thr

50 55 60

Gln Val Arg Thr Asp Phe Tyr Asp Glu Phe Trp Gly Gln Gly Leu Arg

65 70 75 80

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

85 90 95

Ala Ala Ala Ala Lys Leu Ile Glu Leu Met Ile Val Ile Ala Ile Ile

100 105 110

Ala Ile Leu Ala Thr Ile Ala Ile Pro Ser Tyr Gln Asn Tyr Thr Lys

115 120 125

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

130 135 140

Asp Val Glu Leu Cys Val Tyr Ser Thr Asn Glu Thr Thr Asn Cys Thr

145 150 155 160

Gly Gly Lys Asn Gly Ile Ala Ala Asp Ile Thr Thr Ala Lys Gly Tyr

165 170 175

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

180 185 190

Gly Thr Leu Ala Asn Met Glu Tyr Ile Leu Gln Ala Thr Gly Asn Ala

195 200 205

Ala Thr Gly Val Thr Trp Thr Thr Thr Cys Lys Gly

210 215 220

Claims

1. A Haemophilus influenzae surface protein D15+ PCP, characterized by: the amino acid sequence of the haemophilus influenzae surface protein D15+ PCP is as follows:

the complete sequence of the nucleic acid gene used for encoding the haemophilus influenzae surface protein D15+ PCP is:

CATATGCAGCGCGTAACCGACAACGACGTTGCAAACATCGTACGTTCTCTGTTTGTGTCCGGCCGTTTCGATGACGTTAAAGCTCACCAGGAAGGTGATGTTCTGGTTGTTTCTGTTGTTGCAAAATCCATCATCAGTGACGTTAAAATCAAGGGCAACTCTATCATCCCAACTGAAGCTCTGAAGCAGAACCTGGACGCTAACGGTTTTAAAGTAGGTGATGTTCTGATCCGTGAAAAGCTGAACGAATTCGCTAAATCCGTTAAGGAGCATTATGCGAGTGTTGGTCGTTACAACGCTACCGTAGAACCGATTGTTAACACTCTGCCGAACAACCGCGCGGAAATCCTGATCCAGATCAACGAAGATGATAAAGCAAAACTGGCTTCTCTGACCTTCAAAGGTAACGAATCTGTATCCTCATCCACTCTTCAGGAACAGATGGAACTGCAGCCGGACTCTTGGTGGAAGCTGTGGGGCAACAAATTCGAAGGCGCGCAGTTCGAAAAAGACCTGCAATCTATTCGTGATTATTACCTGAACAACGGTTATGCAAAAGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTCGCCCGGTTAAAATCCAGGCTGATAACCAGGGTGTAATCGGCACCCTGGGCGGCGGCGCTCTGGGCGGTATCGCAGGTTCTGCTATCGGCGGCGGTCGCGGCCAGGTTATTGCCGCTGTAGTAGGCGCTATCGGTGGTGCGGTGGCCGGTTCTAAAATCGAAGAAAAGGTTTCCCAGGTAAACGGTGCTGAGCTGGTAATCAAAAAAGACGATGGCCAGGAAATAGTTGTTGTACAGAAAGCCGACTCTTCCTTTGTAGCAGGTCGTCGCGTGCGCATCGTTGGTGGTGGGTCTAACCTGAACGTCTCTGTTCTGTAAGGATCC。

2. a preparation method of Haemophilus influenzae surface protein D15+ PCP is characterized in that: the preparation method comprises the following steps: respectively obtaining peptide segments with the most abundant antigenic epitopes in the haemophilus influenzae surface protein D15 and the surface protein PCP extracellular domain, finding out the gene coding sequence of the peptide segments, optimizing the gene coding sequence of the peptide segments, and connecting the optimized gene coding sequence of the peptide segments by using the coding sequence of flexible connecting peptide to form a fusion gene;

the accession numbers of the haemophilus influenzae surface protein D15 and the surface protein PCP in the NCBI protein database are AAX87955 and AAX88288 respectively;

the sequence of the flexible connecting peptide is ggsggsggsggs;

simultaneously, enzyme cutting site NdeI is introduced into the 5 'end of the fusion gene, and termination signal TAA and enzyme cutting site BamHI are introduced into the 3' end of the fusion gene, and then a complete gene sequence is chemically synthesized and is marked as d15 pep;

the complete sequence of the d15 pep gene is:

CATATGCAGCGCGTAACCGACAACGACGTTGCAAACATCGTACGTTCTCTGTTTGTGTCCGGCCGTTTCGATGACGTTAAAGCTCACCAGGAAGGTGATGTTCTGGTTGTTTCTGTTGTTGCAAAATCCATCATCAGTGACGTTAAAATCAAGGGCAACTCTATCATCCCAACTGAAGCTCTGAAGCAGAACCTGGACGCTAACGGTTTTAAAGTAGGTGATGTTCTGATCCGTGAAAAGCTGAACGAATTCGCTAAATCCGTTAAGGAGCATTATGCGAGTGTTGGTCGTTACAACGCTACCGTAGAACCGATTGTTAACACTCTGCCGAACAACCGCGCGGAAATCCTGATCCAGATCAACGAAGATGATAAAGCAAAACTGGCTTCTCTGACCTTCAAAGGTAACGAATCTGTATCCTCATCCACTCTTCAGGAACAGATGGAACTGCAGCCGGACTCTTGGTGGAAGCTGTGGGGCAACAAATTCGAAGGCGCGCAGTTCGAAAAAGACCTGCAATCTATTCGTGATTATTACCTGAACAACGGTTATGCAAAAGGTGGTTCTGGTGGTTCTGGTGGTTCTGGTGGTTCTCGCCCGGTTAAAATCCAGGCTGATAACCAGGGTGTAATCGGCACCCTGGGCGGCGGCGCTCTGGGCGGTATCGCAGGTTCTGCTATCGGCGGCGGTCGCGGCCAGGTTATTGCCGCTGTAGTAGGCGCTATCGGTGGTGCGGTGGCCGGTTCTAAAATCGAAGAAAAGGTTTCCCAGGTAAACGGTGCTGAGCTGGTAATCAAAAAAGACGATGGCCAGGAAATAGTTGTTGTACAGAAAGCCGACTCTTCCTTTGTAGCAGGTCGTCGCGTGCGCATCGTTGGTGGTGGGTCTAACCTGAACGTCTCTGTTCTGTAAGGATCC；

the protein sequence encoded by the d15 pep gene is:

the protein sequence coded by the D15PCP gene is formed by connecting two sections of protein sequences, namely 50-233aa of the Haemophilus influenzae surface protein D15 and 50-154aa of the surface protein PCP, by flexible connecting peptide; cloning the gene fragment into prokaryotic expression vector pET-28a (+) according to conventional method, inducing recombinant Escherichia coli expression by IPTG, and using Ni²⁺And (3) purifying the recombinant D15PCP protein by an affinity chromatography method.

3. A method for preparing a haemophilus influenzae surface protein D15+ PCP antibody, characterized in that: the method comprises the steps of taking the recombinant D15PCP protein as an immune antigen as claimed in claim 2, mixing the immune antigen with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, performing titer measurement by blood drawing, separating high-titer recombinant protein antibodies and purifying to finally obtain the H.influenzae surface protein D15+ PCP antibodies.

4. A latex microsphere immunochromatographic test paper based on surface protein of Haemophilus influenzae is characterized in that: the latex microsphere immunochromatographic test paper based on the surface protein of the haemophilus influenzae comprises a latex microsphere marker coated with an antibody of the surface protein D15+ PCP of the haemophilus influenzae prepared by the method of claim 3 and a nitrocellulose membrane coated with an antibody of the surface protein Pe + pilA of the haemophilus influenzae; the haemophilus influenzae surface protein Pe + pilA antibody is prepared by mixing recombinant Pepil protein serving as an immune antigen with Freund's adjuvant, repeatedly and artificially immunizing healthy New Zealand white rabbits, drawing blood to perform titer determination, separating high-titer recombinant protein antibodies and purifying to finally obtain the haemophilus influenzae surface protein Pe + pilA antibody;

the amino acid sequence of the recombinant Pepil protein is:

MLVKNVNYYIDSESIWVDNQEPQIVHFDAVVNLDKGLYVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPKKQKKHEAAAAKEAAAAKLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVELCVYSTNETTNCTGGKNGIAADITTAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWTTTCKG。

5. a method for preparing the latex microsphere immunochromatographic test paper based on the surface protein of Haemophilus influenzae according to claim 4, characterized in that: the method comprises the following steps:

1) preparing an antibody of Haemophilus influenzae surface protein D15+ PCP and an antibody of Haemophilus influenzae surface protein Pe + pilA;

2) preparing a latex microsphere marker of the haemophilus influenzae surface protein D15+ PCP antibody:

2.1) activation of the latex microspheres

Taking 1mL of colored carboxylated polystyrene latex microsphere solution with the concentration of 10%, adding 9mL of MES buffer solution, uniformly mixing, adding NHS and EDC until the final concentration of the two is 1mg/mL, slowly and uniformly mixing for 30 minutes at room temperature, centrifuging for 20 minutes at 19000g after the incubation is finished, removing supernatant, re-suspending the precipitate with 10mL of borax buffer solution, oscillating, and performing ultrasonic treatment to obtain activated latex microspheres; the MES buffer solution comprises the following components in percentage by weight: 0.1mol/L MES, wherein the pH of the MES buffer is 8.5; the grain size of the colored carboxylated polystyrene latex microspheres is 100 nm; the components and the content of the borax buffer solution are 0.1mol/L Na₂B₄O₇The pH value of the borax buffer solution is 8.5;

2.2) preparation of latex microsphere markers

Diluting the haemophilus influenzae surface protein D15+ PCP antibody obtained in the step 1) to 1mg/mL by using a borax buffer solution; adding 10mL of haemophilus influenzae surface protein D15+ PCP antibody into 10mL of activated latex microspheres, slowly mixing uniformly for 30 minutes, centrifuging at 19000g for 10 minutes, and removing supernatant; resuspending the precipitate with 10mL borax buffer solution containing 1% casein, repeating centrifugation for 1 time after ultrasonic pulverization, and removing supernatant; resuspending the precipitate by the same method, repeatedly centrifuging for 1 time after ultrasonic crushing and oscillation, and removing supernatant; the precipitate was resuspended in 10mL borax buffer containing 1% casein,namely a latex microsphere marker of the haemophilus influenzae surface protein D15+ PCP antibody; the components and the content of the borax buffer solution are 0.1mol/L Na₂B₄O₇The pH value of the borax buffer solution is 8.5;

3) preparation of the bonding pad:

spraying the latex microsphere marker of the haemophilus influenzae surface protein D15+ PCP antibody obtained in the step 2) on a bonding pad made of a polyester fiber material, wherein the spraying amount of a polyester fiber film per square centimeter is 10 mu L of the latex microsphere marker; drying at 37 deg.C in environment with relative humidity not more than 30%, sealing at 25 deg.C, drying and storing;

4) preparation of antibody solid-phase nitrocellulose membrane:

diluting the Haemophilus influenzae surface protein Pe + pilA antibody obtained in the step 1) to 1.5mg/mL by using a borax buffer solution, and then coating the antibody on a detection line position on a nitrocellulose membrane by using a membrane spraying instrument as a detection line to capture the antibody, wherein the coating parameter is 1 mu L/cm; spraying goat anti-rabbit IgG on a quality control line position on a nitrocellulose membrane as a control line to capture an antibody, wherein the concentration is 1mg/mL, and the coating parameter is 1 mu L/cm; after coating, putting the nitrocellulose membrane in an environment with the relative humidity not more than 30%, drying at 37 ℃, sealing at 25 ℃, drying and storing; the components and the content of the borax buffer solution are 0.1mol/L Na₂B₄O₇The pH value of the borax buffer solution is 8.5;

5) preparation of sample pad

6) assembly of test strips