KR100905066B1 - The recombinant proteins for the diagnosis of diseases infected from Mycoplasma pneumoniae and the diagnostic kits comprising the same - Google Patents

Abstract

The present invention is a recombinant protein that specifically reacts with an antibody against mycoplasma pneumoniae, mycoplasma pneumoniae diagnostic composition comprising the same, mycoplasma pneumoniae diagnostic kit using the composition and myco by using the diagnostic kit The present invention relates to a method for diagnosing plasmatic pneumonia.

Mycoplasma pneumoniae, mycoplasma pneumonia, diagnostic kit

Description

The recombinant proteins for the diagnosis of diseases infected from Mycoplasma pneumoniae and the diagnostic kits comprising the same}

The present invention relates to recombinant proteins that specifically react with antibodies against mycoplasma pneumoniae and methods for rapidly diagnosing mycoplasma pneumoniae infection and mycoplasma pneumonia. The present invention also relates to a fusion cell for producing an antibody against the recombinant protein.

Pneumonia can be classified into viral and mycoplasma depending on the cause. In the case of viral pneumonia, respiratory syncytial virus (RSV), adenovirus (adenovirus), parainfluenza virus (parainfluenza virus) is a common cause, influenza virus (influenza virus) is a common cause.

On the other hand, Mycoplasma pneumoniae is the main cause of respiratory infection among 16 mycoplasma isolates from humans. ) Is known to cause genitourinary tract infections and perinatal infections.

20% of mycoplasma infections are asymptomatic and 75% cause mild respiratory infections such as bronchitis and pharyngitis, and 3-10% cause severe infections such as pneumonia. In children, mycoplasma pneumoniae infections account for 10 to 40% of the causes of pneumonia, although it varies during epidemics. Infections can occur throughout the year, but are most common in late summer and early autumn, and the incidence varies from year to year, but is usually reported every four years.

Mycoplasma pneumoniae is poorly treated with beta-lactam antibiotics because it lacks a cell wall. Therefore, treatment with macrolide, quinolone, or tetracycline, which is an antibiotic that inhibits protein or DNA synthesis. Mycoplasma pneumoniae is one of the most common causes of pediatric respiratory infections, but to date it may be the least diagnosed disease due to difficulty in diagnosis. For this reason, until recently, pneumonia caused by mycoplasma pneumoniae infection has not been properly diagnosed, and it has often caused side effects by overdose with other antibiotics.

Standard diagnostic methods for mycoplasma pneumoniae infections known to date include bacterial identification through culture, serological tests, and genetic testing through polymerase reaction (PCR). In the case of identification of bacteria through cultivation, it takes 7 to 21 days to identify bacteria, and since they are cultured only in 40 to 90%, there is a limit to practical use in the clinical field. In addition, the serological diagnosis of mycoplasma pneumoniae infection is the most widely used method, but the complement test (CF test, complement fixation test) takes a long time and shows low sensitivity. (Eg SeroDia MycoII from Fujirebio) or enzyme immunoassay (ELISA; eg, Platelia Myco IgM / IgG from Bio-Rad) is commonly used. However, these two immunoassays also have a disadvantage in that the method of use is complicated, the test takes a long time as 3 to 4 hours, and expensive equipment is used to perform the test in the field. In addition, the concordance rate between the particle agglomeration method and the enzyme immunoassay method is low, which leads to confusion about which method should be used for the diagnosis. Due to these problems, efforts are being actively made to develop point-of-care testing (POCT) kits that can effectively test antibodies to mycoplasma pneumoniae using blood at home and abroad.

Effective antibody screening for mycoplasma pneumoniae requires high antigenic, homogeneous and evolutionarily well-conserved proteins. Mycoplasma pneumoniae adheres to human respiratory epithelial cells and accompanies diseases caused by these adsorption. These adsorption involves Adhesin P1 (170 kDa) and Adhesin P30 (30 kDa). ), Adhesin P40 (40 kDa), and Adhesin P90 (90 kDa) are known to work in combination. Among them, adhesin P1 is known to show strong antigenicity in humans and animals. However, since the Adhesin P1 protein has a very high molecular weight and contains a large number of 21 UGA codons that are recognized as stop codons in Escherichia coli, it is difficult to produce recombinant protein in Escherichia coli. Since it is difficult to produce recombinant proteins with high antigenicity, currently available diagnostic kits have a problem of low specificity of the product because they use the eluate of cultured mycoplasma pneumoniae as it is.

The present inventors cloned some of the genes of Adhesin P1, which are expected to have high mycoplasma pneumoniae antigenicity, to produce a recombinant protein, and to create a fusion cell for generating an antibody thereto. Using antibodies, a mycoplasma pneumonia diagnostic kit having high specificity for mycoplasma pneumoniae was constructed and the present invention was completed.

One object of the present invention is to provide an Adhesin P1 recombinant protein of Mycoplasma pneumoniae.

Another object of the present invention is to provide a fusion cell line for producing a monoclonal antibody against the adhesin pi1 recombinant protein.

Another object of the present invention is to provide a mycoplasma pneumoniae diagnostic composition comprising the antibody produced in the fusion cells and mycoplasma pneumoniae diagnostic kit comprising the composition.

In one embodiment, the present invention provides an Adhesin P1 recombinant protein which is a highly antigenic mycoplasma pneumoniae specific antigen.

Adhesin P1 protein has strong antigenicity in humans and animals, but has a very high molecular weight and contains 21 UGA codons that are recognized as stop codons in E. coli due to the genetic code characteristics. Accordingly, the present inventors 1) does not contain UGA codons, there is no truncation in protein expression, 2) contains a large number of epitopes from the epitope prediction program and shows strong antigenicity, 3) isocharge (pI ), The site where the recombinant protein was soluble in water was selected because the acidity and the average hydrophobicity value were 0 or less. Specifically, 1) at least 20 MHC peptide binding motifs from the HLA Peptide Binding Prediction Program, developed by Harvard University, and 2) the Swiss Institute of Bioinformatics. Molecular weight 38.24 kDa, pI 4.72 using ExPASy system developed by Bioinformatics, Switzerland) and 3) no transmembrane domain from SOSUI system (Nagoya University, Japan) and average hydrophobicity value- A site of 0.535 was selected. The recombinant protein has an amino acid sequence of SEQ ID NO: 1, the specific nucleotide sequence thereof is shown in SEQ ID NO: 2.

In the specific method for preparing the recombinant protein, the amplified gene was amplified using a primer pair represented by SEQ ID NOs: 3 and 4, and the amplified gene was cloned into a pGEM-T Easy vector. Expression of the recombinant protein of Adhesin P1 was determined using the pGEX expression system (GE Healthcare, Uppsala, Sweden). That is, a host cell was produced by subcloning the gene of Adhesin P1 with the pGEX-4T-1 expression vector and transforming Escherichia coli BL21 (DE3) as the recombinant plasmid to produce recombinant Adhesin P1. The transformed Escherichia coli was treated with 1 mM concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) to express a large amount of recombinant Adhesin P1 protein, which was subjected to affinity chromatography using GSH-linked Sepharose 64.0. Pure recombinant Adhesin pi1 protein of kDa was obtained (FIG. 3).

In still another aspect of the present invention, there is provided a fusion cell line for producing a monoclonal antibody against the adhesin pi1 recombinant protein.

In a preferred embodiment of the present application, fusion cell lines were prepared by fusing splenocytes obtained by immunizing Adhesin pi 1 recombinant protein obtained by the above method to mice with myeloma cells. First, the purified Adhesin P1 recombinant protein is injected into the abdominal cavity of 6-8 week old mice and immunized. The spleens are extracted to single cell and then reacted with myeloma cells to form fusion cells. Cloning of the fusion cells thus made was repeated until a stable monoclonal cell line was obtained to prepare a fusion cell line producing monoclonal antibodies. The fusion cell line thus prepared was deposited on November 15, 2007, with the accession number KCTC 11242BP to KCTC (Korean Collection for Type Cultures), 52, Eeun-dong, Yuseong-gu, Daejeon, Korea.

In another aspect, the present invention provides novel monoclonal antibodies specific for Adhesin P1 of Mycoplasma for use in the production of the diagnostic kits described above.

As used herein, the term “monoclonal antibody” refers to a highly specific antibody directed against a single antigenic site as it is known in the art. Typically, unlike polyclonal antibodies that include different antibodies directed against different epitopes, monoclonal antibodies are directed against a single determinant on the antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding and also have another advantage of not being contaminated by other immunoglobulins because they are synthesized by fusion culture. Such monoclonal antibodies are known in the art by hybridoma methods (see Kohler and Mislstein (1976) European Journal of Immunology 6: 511-519) or phage antibody libraries (Clackson et al, Nature, 352: 624). -628, 1991, etc.).

In a preferred embodiment of the present invention, the fusion cell line obtained by the above method is immunized by injection into the abdominal cavity of mice 6-8 weeks old, and the ascites is harvested. From this, monoclonal antibodies against mycoplasma pneumoniae were purified. Briefly, ammonium sulfate was added to the collected ascites, centrifuged to separate the antibody, dialyzed, and purified using a column. The monoclonal antibody prepared by the above method is monoclonal antibody 2G6D7.

The antibody purified by the above method is used for the manufacture of a diagnostic kit. Specifically, the strip portion of the kit includes a test line (T) for confirming the result of the sample and a control line (C) portion for confirming the validity of the test, and the test line includes Protein A (Protein A). And, at the control position, dispense each of the above purified Adhesin pi monoclonal antibodies and allow the antibodies to completely immobilize on the nitrocellulose membrane.

The purified monoclonal antibodies described above can be applied on the other hand to directly detect antigens of mycoplasma pneumoniae.

Accordingly, the present invention provides a mycoplasma pneumoniae diagnostic composition comprising the monoclonal antibody.

As another aspect of the present invention, a mycoplasma pneumoniae diagnostic kit is provided using a monoclonal antibody produced in the above-described fusion cells.

In the diagnostic kit of the present invention, the antigen-antibody complex is detected by color particle binding, and the color particles include, for example, colloidal gold particles or color glass or plastic (e.g., polystyrene, polypropylene, latex, etc.). bead). In the present invention, gold colloids are preferably used.

In addition, in the diagnostic kit of the present invention, the capture agent that binds to the antigen-antibody complex includes, in addition to protein A, protein G or anti-human immunoglobulin. . In a specific embodiment of the present invention, Protein A was used as the capture body.

Assay systems for use in the diagnostic kits of the present invention include, but are not limited to, ELISA plates, dip-stick devices, immunochromatography and radiation split immunoassay devices, flow-through devices, and the like. Include. Preferably, a diagnostic kit in the form of a strip or a device using immunochromatography may be used. In the mycoplasma diagnostic kit using the immunochromatography method of the present invention, more specifically, a glass fiber pad in which a nitrocellulose membrane and a recombinant protein-gold condensate with two invisible lines on the surface thereof are stored in a dry state. It consists of two main parts. Two different proteins are immobilized on the invisible line on the nitrocellulose membrane. Specifically, the test line (T) at the bottom has a protein A (Protein A), and the control line (C) at the top has an antiadhesin P1 monoclonal antibody immobilized. In the glass fiber pad, the antigen-gold condensate obtained by conjugating the recombinant adhesin P1 protein to the gold particles is absorbed by the glass fiber pad and dried. When the sample is added to the sample drop of the kit, the antigen-gold condensate stored in the glass fiber pad is hydrated and binds to the antibody in the sample, and at the same time through the micropores in the nitrocellulose membrane by capillary action. Move. Subsequently, protein A (Protein A), which is immobilized on the invisible line, reacts with the antibody bound to the antigen-gold condensate, resulting in a visible red line due to the red color of the antigen-gold condensate. In addition, the antiadhesin P1 monoclonal antibody immobilized on the upper line may appear with each test since it may react with antigen-gold condensate passing through the test line without reacting. You can check it. That is, if there is an antibody against mycoplasma, the test line and control line of the kit appear at the same time, and if there is no antibody, the test line does not appear and only the control line appears.

In one specific embodiment, the present invention can detect and distinguish between human immunoglobulin M (human IgM) and human immunoglobulin G (human IgG) against mycoplasma. More specifically, test line 1 is immobilized with anti human immunoglobulin G, test line 2 with anti human immunoglobulin M, and control line with anti-adhesin pi1 monoclonal antibody. Subsequently, when the sample is developed, the anti-adhesin pi1 antibody in the sample binds to the antigen-gold condensate on the gold condensation pad. When the antibody is IgG, the antibody reacts with test line 1, and the antibody is tested by IgM. React with line 2. As a result, a band of red color is formed in test line 1 when IgG for anti-adhesin blood 1 is present in the sample, and a band is formed in test line 2 when IgM for anti-adhesin blood 1 is present in the sample.

The sensitivity and specificity of the mycoplasma pneumonia diagnosis using the diagnostic kit of the present invention is 90% or higher as compared to a commercially available diagnostic kit (eg, SeroDia Myco II kit (Fujirebio, Japan), etc.). And quick and easy diagnosis compared to the conventional diagnostic kit (see Example 7). Therefore, the diagnostic kit of the present invention will be simple and easy to use for the diagnosis of pneumoconiosis.

In another aspect, the present invention provides a method for diagnosing mycoplasma using the above diagnostic kit.

Specifically, after the biological sample is left to be absorbed in contact with the sample dropping portion of the diagnostic kit of the present invention, the sample is estimated to be infected with mycoplasma pneumoniae according to the presence or absence of a test line of the diagnostic kit in which the reaction is completed. After the blood (serum or plasma) is left in contact with the sample dropping portion of the above diagnostic kit, the sample is left to be absorbed.

As used herein, the term "biological sample" refers to serum, plasma, and the like of a mammal including a human whose suspicious or suspected pulmonary pneumoconiosis is to be examined, and may also be referred to as "sample" or "sample" in the present invention. The biological sample may be used with or without dilution before contacting the sample drop of the diagnostic kit of the present invention.

The test time is faster than the conventional mycoplasma pneumoniae diagnostic method, gelatin particle agglomeration method and enzyme immunoassay method, and the usage is simple, so that mycoplasma infection can be effectively diagnosed. In addition, misuse of antibiotics caused by misdiagnosis can be reduced, and improved therapeutic effect can be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Gene Cloning, Expression, and Purification of Adhesin P1 of Mycoplasma Pneumoniae

1-1. Cloning of the Gene of Adhesin P1 of Mycoplasma Pneumoniae

Genomic DNA of Mycoplasma pneumoniae was obtained by purchasing directly from Advanced Biotechnologies Inc. (USA). From this genome DNA, a part of 1053 bp-adhesin pi1 gene shown by SEQ ID NO: 2 was amplified. In other words, the prepared mycoplasma pneumoniae DNA was used as a template, and 1) there was no truncation in protein expression because it did not contain UGA codon, and 2) many epitopes from the epitope prediction program. 3) isoelectric charge (pI) is acidic, and the average hydrophobicity value is 0 or less, so that the recombinant protein is water-soluble. The portion corresponding to the 1053 bp size, which is part of the Adhesin P1 gene, was amplified using two primers represented by SEQ ID NOs: 3 and 4, that is, AP1F (SEQ ID NO: 3) and AP1R (SEQ ID NO: 4). Cloning with pGEM-Easy T vector (Promega, USA). The base sequence of the cloned gene was analyzed by requesting to Bioneer (Chungwon, Korea). As a result, it was confirmed that the sequence was identical to the initial 1053 bp Adhesin blood gene sequence, and the result was compared with Mycoplasma pneumoniae sequence (GeneBank Accession Number: AF290001) in GeneBank. It was confirmed that the gene of Monia's Adhesin P1 protein. This gene was then subcloned into an expression vector pGEX-4T-1 (GE Healthcare, Sweden) capable of expressing protein in E. coli. The restriction enzyme of the gene used was EcoRI in the 5 'direction and XhoI in the 3' direction, and BL21 (DE3) species was used as the E. coli for the expression of the recombinant protein.

1-2. Expression and Purification of Adhesin P1 Recombinant Protein in Escherichia Coli

Escherichia coli BL21 (DE3) transformed with pGEX-4T-1-P1 plasmid was cultured and absorbance was measured at 600 nm. When 0.6 to 0.8, 1 mM concentration of IPTG (isopropyl-β-D-thiogalactopyranoside) was added to the culture. After the treatment, the culture was further performed for 4 to 5 hours to induce mass expression of Adhesin P1 recombinant protein. E. coli expressing the recombinant protein was collected by centrifugation, cells were disrupted using an ultrasonic mill, and then centrifuged to extract only the supernatant. Purification of the protein was performed by affinity chromatography using GSH-bound Sepharose (Sepharose). Specifically, centrifugation supernatant was injected into a column filled with GSH-binding sepharose to bind the GST-adhesin pi1 fusion protein to the filler and the other unbound materials were washed with a sufficient amount of buffer. The GST-adhesin pi1 fusion protein was then eluted from the column using Tris buffer (pH 7.0) containing 100 mM GSH (glutathion). The isolated GST-adhesin pi1 fusion protein was used in the invention after dialysis and concentration without further treatment. However, if necessary, thrombin (GE Healthcare, Sweden) was treated to remove GST, and only Adhesin P1 recombinant protein was isolated and used in the invention.

Example  2: recombination Adsin At 1  About Monoclonal  Construction of Antibodies

2-1. Immunization with Adhesin P1 Recombinant Protein

Six to eight-week-old female mice (BALB / C, prepared) were made of a suspension containing the same volume of complete Freund's adjuvant as a solution containing 100 μg of the Adhesin P1 recombinant protein obtained in Example 1 above. Intraperitoneal injection of Daehan Biolink Co., Ltd., Korea. Two weeks later, incomplete Freund's adjuvant was used to inject once more with the same technique, and again two weeks later with the same technique. Two days after the last immunization, blood was collected from the tail and serum was obtained. The antibody titer was determined by ELISA (enzyme-linked immunosorbent assay) by diluting to 1/1000 in phosphate buffer. If titers were low, they were immunized again after 2 weeks.

2-2. Fusion of Splenocytes and Myeloma Cells

After removing the spleen from the immunized mice in the same manner as in Example 2-1, crushing the spleen with a tissue grinder, the cell suspension was placed in a container and centrifuged to recover the cells. Myeloma cells were recovered in a cell culture flask, suspended in RPMI1640 medium, and the number of cells was counted. Splenocytes also counted the number of cells. 1 × 10 ⁷ myeloma cells and 1 × 10 ⁸ splenocytes were transferred to a 50 ml container, and an appropriate amount of RPMI1640 medium was added, followed by centrifugation at 200 × g for 5 minutes to recover the cells. The recovered cells were suspended and shaken slowly in RPMI1640 medium, and 1 ml of PEG (50% polyethylene glycol) solution was added for 1 minute. After 5 minutes 1 ml of RPMI1640 medium was added slowly over 30 seconds and again 3 ml of RPMI1640 culture was added over 30 seconds. Again, 17 ml of RPMI1640 culture was added over 1 minute, followed by further addition of 20 ml of RPMI1640 culture and allowed to react for 5 minutes. It was centrifuged at 200 x g for 5 minutes and then the medium was removed. After carefully suspending the RPMI1640 culture medium containing 50 ml of 1% HAT (hypoxathine-aminopterin-thymidine), 100 μl / well of the cells were added to a 96-well cell culture plate containing feeder cells. Then incubated in 37 ℃, 5% CO ₂ incubator.

2-3. Antibody Production Using Cloning and Ascites of Fusion Cell Lines

After culturing the cells cultured in the same manner as in Example 2-2 for 10 days, when it is confirmed that the fusion cells grow by forming a colony, about 100 μl of the medium is removed from the 96-well cell culture plate and purified by Eliza method. The wells in which the cells producing the antibody against the Hessin P1 antigen grew were identified, and the cells of the wells were recovered, transferred to a 24-well cell culture vessel, and cultured, and the cloning was continued until a stable monoclonal cell line was obtained. After cloning was completed, the cells were transferred to a T flask, cultured in large quantities, frozen, and stored in liquid nitrogen.

0.5 ml of incomplete Freund's adjuvant was injected into the abdominal cavity of 6-8 week old mice (BALB / C). On the first day of week, the fused cells were suspended and counted in phosphate buffer, 1.5 × 10 ⁶ cells per mouse. The cells were suspended in 0.5 ml phosphate buffer and then injected intraperitoneally. After 1-2 weeks ascites (ascitic fluid) is properly filled, the ascites were collected using a syringe and stored frozen.

2-4. Purification of Monoclonal Antibodies

Ammonium sulfate was added at a concentration of 10% to the ascites collected in the same manner as in Example 2-3, mixed for 30 minutes, centrifuged at 15,000 rpm for 30 minutes, and the supernatant was taken. Again, ammonium sulfate was added to the supernatant at a concentration of 50%, and left at 4 ° C for 30 minutes. After centrifugation at 15,000 rpm for 30 minutes, the supernatant was discarded and the precipitate was suspended in 20 mM phosphate buffer (pH 7.0). After the suspension was dialyzed in 20 mM phosphate buffer solution for 18 hours or more, the dialysate was injected into a Protein G-coupled column equilibrated with 20 mM phosphate buffer solution (pH 7.0). Phosphoric acid buffer solution was used to remove the unadsorbed materials. The antibody bound to the column was eluted with a 10 mM glycine solution (pH 2.8) solution. At this time, 1 M Tris (pH 9.0) solution was added to the 1/10 volume of the eluate to calibrate to pH 7.0 ~ 7.5. The eluted antibody solution was concentrated, dialyzed in 150 mM phosphate buffer solution, and after dialysis, the amount of the antibody was confirmed by Bradford assay and stored frozen until use. The purified monoclonal antibody was named monoclonal antibody 2G6D7.

Example  3: Rapid Immunochromatography Strip Form Diagnosis Using Recombinant Antigens Of kit  Produce

3-1. Immobilization of Protein A and Anti-adhesin P1 Monoclonal Antibodies

As shown in FIG. 4, the protein A (Protein A) is placed at a specific position of the nitrocellulose membrane attached to the plastic card, that is, the test line (T), and the antiadhesin P1 is placed at the control line (C) position. Monoclonal antibody (anti-adhesin P1 monoclonal antibody) 2G6D7 was each dispensed and dried in a 30 ° C incubator for 2 days to ensure that the protein A and monoclonal antibody completely immobilized on the nitrocellulose membrane.

3-2. Preparation of Gold Condensate and Preparation of Gold Condensation Pads

Adhesin P1 recombinant protein was mixed with colloidal gold particles having a size of about 40 nm, and then reacted in a constant temperature water bath at 37 ° C. for 1 hour to bind to each other. After 1 hour, bovine serum albumin (BSA) and sucrose (sucrose) were added to 3% and 1%, respectively, and then reacted for 1 hour to react with the sites of colloidal gold particles to which the antibody did not bind. It was forever. Thereafter, centrifuged at 10,000 rpm for 20 minutes to recover the recombinant protein-conjugated gold condensate, the absorbance was measured at 540 nm, and then refrigerated. The prepared gold condensate was injected into a plastic microwell plate and dried to prepare a gold conjugate well.

3-3. Assembly of the strip kit

As illustrated in FIG. 4, a strip is formed by attaching an absorbent pad to absorb a sample and a buffer to an upper end of a plastic card to which a protein A and a monoclonal antibody-bound nitrocellulose membrane are attached. The kit was completed.

Example  4: Rapid Immunochromatography Using Recombinant Antigens device  Form diagnosis Of kit  Produce

4-1. Immobilization of Protein A and Anti-adhesin P1 Monoclonal Antibodies

As shown in FIG. 4, the protein A (Protein A) is placed at a specific position of the nitrocellulose membrane attached to the plastic card, that is, the test line (T), and the antiadhesin P1 is placed at the control line (C) position. Monoclonal antibodies (anti-adhesin P1 monoclonal antibody) 2G6D7 were each dispensed and dried in a 30 ° C. incubator for 2 days to ensure that the monoclonal antibody was completely immobilized on the nitrocellulose membrane.

4-2. Preparation of Gold Condensate and Preparation of Gold Condensation Pads

Adhesin P1 recombinant protein was mixed with colloidal gold particles having a size of about 40 nm and then reacted for 1 hour in a 37 ° C. constant temperature water bath to bind to each other. After 1 hour, bovine serum albumin (BSA) and sucrose (sucrose) were added at 3% and 1%, respectively, and then reacted for 1 hour to react with the sites of colloidal gold particles to which the antibody did not bind. It was forever. Thereafter, centrifuged at 10,000 rpm for 20 minutes to recover the recombinant protein-conjugated gold condensate, the absorbance was measured at 540 nm, and then refrigerated. The prepared gold condensate was absorbed into a glass fiber pad and dried to prepare a gold condensation pad.

4-3. Assembly of Device Shape Kits

As illustrated in FIG. 6, an absorption pad capable of absorbing a sample and a buffer is attached to an upper end of a plastic card to which a protein A and a monoclonal antibody-bound nitrocellulose membrane are attached. Sample pads and condensation pads were attached to the specimen. In the case of the device form, the assembled semifinished product was placed in a housing made of plastic as shown in FIG. 6 to complete the kit.

Example  5: strip form Of kit  method of inspection

Add 1 drop of wash solution to the gold condensation well supplied with the kit. Then, add 4 microliters of serum sample using a pipette and mix. After that, the strip-type kit is plugged in so that the sample liquid is absorbed and left for 10 minutes. Put 3 drops of washing solution into the washing well, insert the strip-type kit into which the sample solution is absorbed, and leave it for 10 minutes for washing reaction. After completion of the reaction, the strip is read for antibody to mycoplasma pneumoniae according to the position of the red band on the test line as indicated in FIG. 5.

Example  6: device  shape Of kit  method of inspection

Take out the device kit contained in the aluminum pouch and place it on a flat floor. Serum samples are collected in 4 microliters (μl, microliter) and placed in a sample window as indicated in FIG. 6A to allow the sample to be absorbed. Thereafter, three drops of the assay buffer were dropped and left for 15 minutes. After the reaction was completed, the kit was read for antibodies against mycoplasma pneumoniae according to the position of the red band on the test line as indicated in FIG. 7.

Example  7: Mycoplasma  Rapid immunochromatography strip form for antibody detection and device  shape Of kit  Efficacy test

7-1. Positive sensitivity efficacy test

The sensitivity performance of the kit for mycoplasma positive clinical specimens was tested using the diagnostic kits prepared in Examples 3-4 above. The test to confirm the sensitivity of the diagnostic kit was performed using 95 positive clinical samples provided from Cheongju St. Mary's Hospital (Cheongju, Chungbuk, Korea). In addition, the same sample was used to perform parallel testing using the SeroDia Myco II kit (Fujirebio, Japan), and compared with the diagnostic kit achieved in the present invention. The results are as shown in Table 1.

7-2. Voice specificity efficacy test

Specificity performance of the kit for mycoplasma antibody negative clinical specimens was tested using the diagnostic kits prepared in Examples 3-4 above. Testing to determine the specificity of the diagnostic kit was performed using 120 negative clinical specimens provided by the Department of Parasitology, Sungkyunkwan University School of Medicine. In addition, the same sample was used to perform parallel testing using the SeroDia Myco II kit (Fujirebio, Japan), and compared with the diagnostic kit achieved in the present invention. The results are shown in Table 2.

7-3. Conclusion through sensitivity and specificity test results

As described above, the diagnostic kit of the present invention was found to be clinically effective due to its high specificity and sensitivity despite the rapid use of the diagnostic kit compared to the conventional diagnostic kit, and quick examination without the help of expensive equipment.

1 is a view showing the form of mycoplasma pneumoniae according to the present invention.

Figure 2 is a flow chart showing a method for producing a recombinant protein of Mycoplasma pneumoniae Adhesin P1 (Adhesin P1) according to the present invention.

Figure 3 is a view showing the result of separation and purification of recombinant adhesin pi 1 according to the present invention. In this figure, M is the protein molecular weight marker, lane 1 is the cell eluate after protein expression, lane 2 is the cell eluate before protein expression, and lane 3 is the purified recombinant Adhesin P1. SD-polyacrylamide gel electrophoresis (SDS-PAGE) was performed.

Figure 4 is a schematic diagram showing the configuration of a rapid immunochromatography strip form for diagnosing antibodies to Mycoplasma pneumoniae from an infected patient according to the present invention.

Figure 5 is a view showing a photograph of the test results in the form of a rapid immunochromatography strip for diagnosing the antibody against mycoplasma pneumoniae according to the present invention.

Fig. 6 shows a schematic diagram of a configuration of a rapid immunochromatography device for diagnosing antibodies against Mycoplasma pneumoniae from an infected patient according to the present invention.

Figure 7 is a view showing a photograph of the test results in the form of a rapid immunochromatography device for diagnosing the antibody against mycoplasma pneumoniae according to the present invention.

<110> Bioland Ltd. <120> The recombinant proteins for the diagnosis of diseases infected          from Mycoplasma pneumoniae and the diagnostic kits comprising the          same <130> PA070684 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 357 <212> PRT <213> Mycoplasma pneumoniae <220> <221> ACT_SITE (222) (1) .. (357) <223> adhesin P1 <400> 1 Phe Arg Gly Ser Trp Val Asn Arg Leu Gly Arg Val Glu Ser Val Trp   1 5 10 15 Asp Leu Lys Gly Val Trp Ala Asp Gln Ala Gln Ser Asp Ser Gln Gly              20 25 30 Ser Thr Thr Thr Ala Thr Arg Asp Ala Leu Pro Glu His Pro Asn Ala          35 40 45 Leu Ala Phe Gln Val Ser Val Val Glu Ala Ser Ala Tyr Lys Pro Asn      50 55 60 Thr Ser Ser Gly Gln Thr Gln Ser Thr Asn Ser Ser Pro Tyr Leu His  65 70 75 80 Leu Val Lys Pro Lys Lys Val Ile Gln Ser Asp Lys Leu Asp Asp Asp                  85 90 95 Leu Lys Asn Leu Leu Asp Pro Asn Gln Val Arg Thr Lys Leu Arg Gln             100 105 110 Ser Phe Gly Thr Asp His Ser Thr Gln Pro Gln Pro Gln Ser Leu Lys         115 120 125 Thr Thr Thr Pro Val Phe Gly Thr Ser Ser Gly Asn Leu Ser Ser Val     130 135 140 Leu Ser Gly Gly Gly Ala Gly Gly Gly Ser Ser Gly Ser Gly Gln Ser 145 150 155 160 Gly Val Asp Leu Ser Pro Val Glu Lys Val Ser Gly Trp Leu Val Gly                 165 170 175 Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser Thr Asn Asn Leu             180 185 190 Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly Val Gly Arg Leu         195 200 205 Ser Glu Ser Asn Ala Ala Lys Met Glu Thr Asn Asp Asp Val Asp Gly     210 215 220 Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly Glu Gly Gln Thr 225 230 235 240 Ala Asp Thr Gly Pro Gln Ser Val Lys Phe Lys Ser Pro Asp Gln Ile                 245 250 255 Asp Phe Asn Arg Leu Phe Thr His Pro Val Thr Asp Leu Phe Asp Pro             260 265 270 Val Thr Met Glu Thr Leu Val Tyr Asp Gln Tyr Ile Pro Leu Phe Ile         275 280 285 Asp Ile Pro Ala Ser Val Asn Pro Lys Met Glu Thr Val Arg Leu Lys     290 295 300 Val Leu Ser Phe Asp Thr Asn Glu Gln Ser Leu Gly Leu Arg Leu Glu 305 310 315 320 Phe Phe Lys Pro Asp Gln Asp Thr Gln Pro Asn Asn Asn Val Gln Val                 325 330 335 Asn Pro Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr Ala Ser Ser Gln             340 345 350 Gly Pro Gln Thr Leu         355 <210> 2 <211> 1053 <212> DNA <213> Mycoplasma pneumoniae <220> <221> gene (222) (1) .. (1053) <223> adhesin P1 <400> 2 tttcgtggca gttgggtcaa ccggttgggc cgcgtggaga gtgtgtggga tttgaagggg 60 gtgtgggcgg atcaagctca gtccgactcg caaggatcta ccaccaccgc aacaagggac 120 gccttaccgg agcacccgaa tgctttggcc tttcaggtga gtgtggtgga agcgagtgct 180 tacaagccaa acacgagctc cggccaaacc caatccacta acagttcccc ctacctgcac 240 ttggtgaagc ctaagaaagt tatccaatcc gacaagttag acgacgatct taaaaacctg 300 ttggacccca accaggttcg caccaagctg cgccaaagct ttggtacaga ccattccacc 360 cagccccagc cccaatcgct caaaacaacg acaccggtat ttgggacgag tagtggtaac 420 ctcagtagtg tgcttagtgg tgggggtgct ggagggggtt cttcaggctc aggtcaatct 480 ggcgtggatc tctcccccgt tgaaaaagtg agtgggtggc ttgtggggca gttaccaagc 540 acgagtgacg gaaacacctc ctccaccaac aacctcgcgc ctaatactaa tacggggaat 600 gatgtggtgg gggttggtcg actttctgaa agcaacgccg caaagatgaa cgacgatgtt 660 gatggtattg tacgcacccc actcgctgaa ctgttagatg gggaaggaca aacagctgac 720 actggtccac aaagcgtgaa gttcaagtct cctgaccaaa ttgacttcaa ccgcttgttt 780 acccacccag tcaccgatct gtttgatccg gtaactatgt tggtgtatga ccagtacata 840 ccgctgttta ttgatatccc agcaagtgtg aaccctaaaa tggttcgttt aaaggtcttg 900 agctttgaca ccaacgaaca gagcttaggt ctccgcttag agttctttaa acctgatcaa 960 gatacccaac caaacaacaa cgttcaggtc aatccgaata acggtgactt cttaccactg 1020 ttaacggcct ccagtcaagg tccccaaacc ttg 1053 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer (APIF) <400> 3 actatggatt ctttcgtggc agttgggtc 29 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer (APIR) <400> 4 ggttctcgag caaggtttgg ggacct 26  

Claims (10)

Mycoplasma pneumoniae diagnostic composition comprising a monoclonal antibody specific for the mycoplasma pneumoniae adhesin pi 1 recombinant protein having the amino acid sequence of SEQ ID NO: 1. delete The diagnostic composition of claim 1, wherein the monoclonal antibody specific for the Mycoplasma pneumoniae adhesin pi 1 recombinant protein is monoclonal antibody 2G6D7. The diagnostic composition according to claim 3, wherein the monoclonal antibody 2G6D7 is produced by accession number KCTC 11242BP fusion cells. Mycoplasma pneumoniae diagnostic kit comprising the composition of any one of claims 1, 3 or 4. The diagnostic kit of the strip type according to claim 5, wherein the immunochromatography method is used. 6. A diagnostic kit of device type according to claim 5, using immunochromatography. The diagnostic kit according to claim 5, wherein the antigen-antibody complex is detected by color particle binding. Antibody 2G6D7 produced in a fusion cell line of Accession No. KCTC 11242BP. Fusion cell of Accession No. KCTC 11242BP.

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