CN103059109B - Mycoplasma pneumonia antigen, preparation method and immunodetection kit - Google Patents
Mycoplasma pneumonia antigen, preparation method and immunodetection kit Download PDFInfo
- Publication number
- CN103059109B CN103059109B CN201310015513.5A CN201310015513A CN103059109B CN 103059109 B CN103059109 B CN 103059109B CN 201310015513 A CN201310015513 A CN 201310015513A CN 103059109 B CN103059109 B CN 103059109B
- Authority
- CN
- China
- Prior art keywords
- antigen
- mycoplasma pneumoniae
- gene
- present
- mycoplasma pneumonia
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention provides a mycoplasma pneumonia (MP) antigen. The antigen fragment of the antigen is MP371 or MP661, wherein the locus of MP 371 is at an N terminal 371-480aa of MP adhesion protein P1, and the locus of MP661 is at an N terminal 661-772aa of MP adhesion protein P1. The invention also relates to a kit comprising any antigen or antigen composition and the application of the kit in detection of a mycoplasma pneumonia antibody. The mycoplasma pneumonia antigen is a recombinant antigen cloned and expressed by a codon-optimized gene and has stronger antigen specificity than that of the antigen cultivated and extracted by MP. The kit can be used for specific detection of an anti-MP-IgM (immunoglobulin m) antibody in clinical samples, thus identifying and diagnosing the infection of mycoplasma pneumonia at early stage.
Description
Technical field
The present invention relates to the codon optimized gene of mycoplasma pneumoniae (MP) adhesion protein 1 (P1), its antigen, its antigen composition, also relate to the test kit and the application that contain this antigen or antigen composition.
Background technology
Mycoplasma pneumoniae (Mycoplasma pneumonia, MP) infect and cause clinically atypical pneumonia and bronchitis, severe patient can cause the complication beyond respiratory tract, as acute dispersivity encephalomyelitis, blood system, motor system infringement etc., infect object and mostly be 5 years old following children.The pneumonia that MP causes is similar to the symptoms of pneumonia that other cause of diseases cause, but MP does not have cell wall structure, clinical application mainly adopts to infiltrate intracellular macrolide antibiotics, as Roxithromycin, erythromycin, Azythromycin etc., and the antibiotics that conventional pneumonia treatment is adopted, as penicillin, cephalo etc. (being mainly to suppress the synthetic class medicine of pathogen cells wall), and insensitive.Therefore, the differential diagnosis of MP has good directive significance to clinical rational drug use.
The laboratory of MP is detected and is mainly contained isolated culture, serology detection, detection of nucleic acids at present.Isolated culture is the gold standard method that MP detects, but due to MP poor growth, the cycle is long, and positive rate is low, cannot serve as routine clinical detection means.Detection of nucleic acids is mainly for patient's throat swab, sputum, bronchoalveolar lavage fluid equal samples, after washing, processings such as centrifugal, the template of acquisition is carried out pcr amplification detection, there is short, sensitivity specificity advantages of higher of time, but operator are required high, and sample easily pollutes, plant and instrument costliness, has limited it and has further applied.
The at present clinical main agglutination test that adopts Japan's development, cardinal principle is to utilize the MP antigen combination of gelatine particle and extraction, to detecting of MP specific antibody in patients serum.The method is simple and practical, but cannot distinguish IgM, IgG antibody.In practical work, IgM antibody occurs will be early than IgG antibody, is the important marker of MP acute infection.Although exist for IgM-ELISA test kit at present, due to the further investigation lacking MP epitope, test kit sensitivity poor specificity, is difficult to reach the standard of clinical detection.
MP genome is double-stranded cyclic DNA, total length 816394bp, hundreds of albumen of encoding.Sticking is the prerequisite that MP causes a disease and infects, and what cause this process is " adhesion protein complex body " mediation on MP top, wherein adhesion protein P1 except infect at MP and pathogenic course in play an important role, still important immunogen.Stimulating body to produce strong immunne response, have good immunogen and antigenicity, is the important target spot of diagnostic antigen research.P1 albumen is made up of 1627 amino acid, and relative molecular weight is about 176.8kD.Chaudhry studies show that for P1 epitope: the 1160-1521aa that is positioned at C-terminal has good immunoreactivity, can react with 72.7% (24/33) positive serum, with the specificity of 32 parts of negative serums be 100%; And the 60-180aa that is positioned at N-terminal does not have antigenic activity.Epitope for P1 PROTEIN C end carries out Bioinformatics Prediction, screen and expressed 1154-1521aa section antigen, sensitivity and specificity are respectively 55.6% (20/36) and 100% (45/45), but to the epitope situation of P1 albumen n end and middle part unclear.
Be different from other pathogenic agent, MP adopts and is different from unique bias codon system, and general terminator codon UGA is coding colors propylhomoserin in MP, if directly adopt the wild gene of MP can cause the interruption of protein translation, has limited the development of restructuring MP antigen.The clonal expression of restructuring MP antigen is evaded UGA conventionally at present, what obtain mostly is not the fragment antigen containing tryptophane, lack effectively prediction and screening of epitope, or directly adopt the whole cell of deactivation to extract antigen, directly affect the development of later stage MP detection reagent.
Summary of the invention
In order to obtain the restructuring MP antigen with diagnostic significance, the present invention is directed between the less 90-1099aa antigenic region of the report of MP adhesion protein P1 and carry out information biology Antigen Epitope Prediction, obtain the aminoacid sequence that immunoreactive antigen fragment wherein may occur with patients serum.
In order to overcome the problem of wild MP gene codon bias, the present invention adopts the aminoacid sequence of the above-mentioned antigen of intestinal bacteria optimal codon reverse translation, obtain the brand-new MP antigen gene being formed by intestinal bacteria optimal codon, its gene order is as shown in sequence 1-6 in sequence table, and employing annealing extension PCR technology obtains improved MP gene.
Adopt gene recombination technology to carry out clonal expression to above-mentioned MP gene, obtain the MP antigen of high expression level, take this recombinant antigen as Foundation IgM prize law MP-ELISA detection reagent, and evaluate the diagnostic significance in its MP detection.
The object of this invention is to provide the position of the interval epitope of mycoplasma pneumoniae P1 adhesion protein 90-1099aa.The above-mentioned antigen epitope genes that another object of the present invention provides intestinal bacteria optimal codon composition the invention provides the MP recombinant antigen and the antigen composition thereof that obtain on said gene basis.The present invention also provides the mycoplasma pneumoniae P1-IgM setting up on above-mentioned antigen and composition basis thereof antibody assay kit, and this test kit has been realized at mycoplasma pneumoniae infection and carried out in early days the accurately object of check.
Particularly, the invention provides a kind of mycoplasma pneumoniae antigen, it is characterized in that: described antigen fragment is MP371 or MP661, described MP371 antigen is the N-terminal antigen of MP adhesion protein P1, and its site is positioned at 371-480aa; Described MP661 antigen is the N-terminal antigen of MP adhesion protein P1, and its site is positioned at 661-772aa.
Further, described MP371 antigenic structure is shown in SEQID NO:3, and described MP661 antigenic structure is shown in SEQ ID NO:4.
The invention provides a kind of codon optimized gene of mycoplasma pneumoniae antigen, described MP371 antigen nucleotide sequence is as shown in list SEQ ID NO:9; Described MP661 antigen nucleotide sequence is as shown in list SEQ IDNO:10.
The present invention also provides the application of a kind of above-mentioned codon optimized gene in the medicine of preparation detection mycoplasma pneumoniae, and described MP371 antigen and the Combination application of MP661 antigen in preparation detection mycoplasma pneumoniae medicine.
The present invention also provides a kind of mycoplasma pneumoniae antigen composition, contains above-mentioned MP371 antigen fragment and MP661 antigen fragment in said composition.
Further, described MP371 antigens c end is connected with described MP661 antigen N-terminal.
The present invention also provides a kind of test kit that detects mycoplasma pneumoniae, above-mentioned any optimized gene of described test kit or above-mentioned composition.
Further, described detection is to utilize IgM antibodycapture to carry out.
The present invention also provides a kind of mentioned reagent box in the application detecting in mycoplasma pneumoniae IgM antibody.
Compared with prior art, the present invention has following characteristics:
1. invention is to set up on the basis of the interval Characterization of antigenic epitopes of mycoplasma pneumoniae P1 (90-1099aa): P1 is that MP mediates the important albumen that sticks and infect, also be the important antigen that can cause organism immune response, but focus mostly at its C-terminal for the research of P1 epi-position at present, the epitope distribution situation of other regions (90-1099aa) is also unclear.This research adopts information biology to carry out systems analysis to above-mentioned interval epi-position and activity thereof, and utilizes the activity of MP hepatitis serum checking screening antigen, thereby obtains the neoantigen epi-position wherein with diagnostic significance.
2. invention is to set up on the basis of the mycoplasma pneumoniae recombination antigen that utilizes codon optimized technology acquisition: mycoplasma pneumoniae detection reagent adopts two class antigens altogether at present, one class is the antigen extracting from the MP whole cell of deactivation, contain multiple film antigenic component, highly sensitive, but poor specificity, and in cause of disease inactivation process, biological safety is poor; Another kind of is to obtain mycoplasma pneumoniae recombination antigen by conventional genetic engineering technique, what adopt due to the mycoplasma pneumoniae of wild-type is bias codon, according to said method obtain recombinant antigen and all interrupt at tryptophane place, be difficult to obtain complete antigen, cause antigen sensitivity low; The present invention utilizes codon optimized technology, obtains the MP optimized gene that does not contain bias codon, realizes the recombinant expressed of the complete antigen of MP, overcomes the defect of at present conventional antigen.
3. the present invention realizes the clinical diagnosis to EV71 on IgM prize law basis: the MP antigen of report is owing to being subject to the restriction of extracting mode, expression amount and follow-up antigenic mark process at present, all to adopt MP antigen coated, the indirect ELISA detection technique of anti-IgM mark horseradish enzyme colour developing, this research will utilize anti-μ chain monoclonal antibody to be coated with elisa plate, with horseradish enzyme labelling restructuring VP1 antigen, set up IgM prize law MP-ELISA detection technique, compared with indirect ELISA technology, there is higher specificity.
Accompanying drawing explanation
Fig. 1 is the prediction of MP-P1 epitope;
Fig. 2 is the acquisition of the gene of MP-P1 Main Antigenic.
Embodiment
Below in conjunction with accompanying drawing, principle of the present invention and feature are described, example, only for explaining the present invention, is not intended to limit scope of the present invention.
For achieving the above object, the present invention, according to mycoplasma pneumoniae P1 aminoacid sequence, adopts position and the corresponding sequence of the interval epitope of bioinformatics technique prediction P1 (90-1099aa); Adopt intestinal bacteria optimal codon reverse translation to become nucleotide sequence; Adopt annealing extension PCR technology, obtain improved P1 antigen epitope genes, after gene engineering expression, large-scale purification preparation restructuring P1 epitope antigen, and carry out horseradish enzyme labelling, preparation ELIAS secondary antibody; Adopt IgM prize law to realize the clinical detection to EV71-IgM.
The present invention is achieved by the following technical programs:
Utilize the Antigen Epitope Prediction function of information biology BIOSUN software, the prediction interval Main Antigenic of P1 (90-1099aa) and corresponding aminoacid sequence (as 1-6 in sequence table), and adopt intestinal bacteria optimal codon reverse translation to become nucleotide sequence (as the 7-12 in sequence table).
In order to obtain said gene, the present invention is to 4 pairs of primers of every kind of P1 antigen epitope genes design, point 4 extension PCRs of annealing; To expression vector, introduce BamHI and two restriction enzyme sites of EcoRI at the two ends of connecting arm, to be applicable to expression vector pGEX-4T-2 for the ease of gene clone.After double digestion, in insertion vector pGEX-4T-2, build corresponding expression plasmid.Sequencing proves that each gene fragment has obtained correctly insertion.
After the expression plasmid Transformed E .coliBL21 that contains above-mentioned P1 antigen epitope genes, induce by IPTG, get full bacterium liquid and carry out SDS-PAGE evaluation, prove that this plasmid has all obtained efficient expression, through nickel post and gel-filtration purifying, can obtain electrophoretically pure P1 antigen sterling again.Adopt method of sodium iodide to carry out horseradish enzyme labelling, prepare enzyme-labelled antigen mixture.Adopt IgM prize law to detect IgM in mycoplasma pneumoniae patients serum, and evaluate sensitivity and the specificity of its detection.
The results show, MP93 provided by the present invention, MP236, MP371, MP661, MP874, MP987 recall rate in 20 parts of mycoplasma pneumoniae patients is respectively 15%, 30%, 65%, 55%, 80%, 20%; In 50 parts of Healthy Human Serums, specificity is 100%, 80%, 100%, 100%, 30%, 90%, MP371 and MP661 antigen that wherein the present invention obtains have the specific immunologic competence of MP, the sensitivity that both combine use is 80%, specificity position 100% has certain using value in diagnostic reagent research.
Four aspects that lower mask body the present invention relates to.
1, the prediction of MP-P1 epitope
Utilize the Antigen Epitope Prediction function of information biology BIOSUN software, the interval Main Antigenic of prediction P1 (90-1099aa) lays respectively at 93-199aa, 236-346aa, 371-480aa, 661-772aa, 874-987aa, 987-1099aa, as shown in Figure 1, called after MP93 respectively, MP236, MP371, MP661, MP874, MP987, corresponding aminoacid sequence is shown in the 1-6 in sequence table.
2, the codon optimized and acquisition of MP-P1 the main antigen epitope gene
By bioinformatics software, adopt intestinal bacteria optimal codon that the aminoacid sequence reverse translation of above-mentioned 6 kinds of antigens is become to nucleotide sequence, improved 6 kinds of gene orders are as shown in sequence 7-12 in sequence table.
Consider the synthetic accuracy of current domestic gene primer, the present invention by every kind of gene design F1, R1, F2, R2, F3, R3, F4, R4 totally 8 primers synthesized respectively, the matching sequence of 16 Nucleotide of every primer end tool, every section of synthetic primer sequence is as shown in the 13-60 in sequence table.
Described preparation method for convenience of description, is corresponding gene sequences name, and SEQ ID NO:13 to 20 is called after MP93F4, MP93F3, MP93F2, MP93F1, MP93R1, MP93R2, MP93R3, MP93R4 respectively; SEQ ID NO:21 to 28 is called after MP236F4, MP236F3, MP236F2, MP236F1, MP236R1, MP236R2, MP236R3, MP236R4 respectively; SEQ ID NO:29 to 36 is called after MP371F4, MP371F3, MP371F2, MP371F1, MP371R1, MP371R2, MP371R3, MP371R4 respectively; SEQ ID NO:37 to 44 is called after MP661F4, MP661F3, MP661F2, MP661F1, MP661R1, MP661R2, MP661R3, MP661R4 respectively; SEQ ID NO:45 to 52 is called after MP874F4, MP874F3, MP874F2, MP874F1, MP874R1, MP874R2, MP874R3, MP874R4 respectively; SEQ ID NO:52 to 60 is called after MP987F4, MP987F3, MP987F2, MP987F1, MP987R1, MP987R2, MP987R3, MP987R4 respectively.
Described method is as follows:
Synthetic said gene needs 3 to take turns the reaction of annealing extension PCR altogether, first round PCR reaction system is the hundred 2 × PCR of Imtech reaction solution, 25 μ l, distilled water 23 μ l, XF1 and the each 1 μ l of XR1 primer, interim X represents MP93, MP236, MP371, MP661, MP874, MP987, PCR reaction conditions is 94 ℃ after 1 minute, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 point, 5 circulations; Follow-up n wheel PCR reaction system is the hundred 2 × PCR of Imtech reaction solution, 25 μ l, distilled water 22 μ l, XFn and the each 1 μ l of XRn primer, n-1 wheel PCR product 1 μ l, and n represents 2,3,4; Reaction conditions is that PCR reaction conditions is 94 ℃ after 3 minutes, 94 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1 minute, after 30 circulations again 72 ℃ extend 5 minutes, obtain MP93, MP236, MP371, MP661, MP874, six kinds of genes of MP987, as shown in Figure 2.
3, the clone of MP-P1 antigen epitope genes, expression and mark
1.MP-P1 the structure of the main antigen epitope gene antigen presentation plasmid
1.1PCR product and expression vector pGEX-4T-2 double digestion
Getting the each 30 μ l of above 6 kinds of gene products and pGEX-4T-2 expression vector is put in respectively in Eeppendorf centrifuge tube, respectively add 10 × buffer (D), 4 μ l, BamHI (10u/ μ l) and EcoRI (12u/ μ is each 1 μ l l), add sterile purified water to 40 μ l, put 37 ℃ of water-bath enzymes and cut and spend the night.
Enzyme is cut agarose gel electrophoresis purifying and the recovery of product: PCR product and vector pGEX-4T-2, after double digestion, carry out purifying with 1.2% sepharose, and concrete grammar is undertaken by the method for " molecular cloning " (Science Press, second edition).The a small amount of glue that purified genes is produced with Shanghai Hua Shun biotechnology company limited again reclaims test kit and reclaims: under ultraviolet lamp, cut respectively the agarose containing plasmid and goal gene; be put in Eeppendorf centrifuge tube; respectively add S1 liquid; put 55 ℃ of water-baths and within 10 minutes, make peptization solution; add equivalent Virahol, mix, 55 ℃ of temperature are bathed 1 minute; then move into respectively after adsorption column, carry out purifying by test kit specification sheets.
1.2. connect: in sterilizing Eeppendorf centrifuge tube, add carrier after above-mentioned enzyme is cut and the each 1 μ l of goal gene, 10 × T4DNA Ligasebuffer1 μ l, T4DNA Li gase (12u/ul) 1 μ l, add sterile purified water to 10 μ l, put 16 ℃ and spend the night.
1.3 transform: in Bechtop, (competent cell is by " molecular cloning " (Science Press to get 100 μ l competent cells with aseptic suction nozzle, the second edition) method carry out) suspension is in Eppendorf, add above-mentioned connector 5 μ l, rotation mixes gently, 30 points of ice baths.Transfer to immediately in 42 ℃ of water-baths and place 2 minutes, every pipe adds 0.5ml LB substratum (not added with antibiotic), 30 ℃ of shaking baths were cultivated after 60 minutes, respectively get 0.2ml and be applied to respectively (containing microbiotic) on LB agar culture plate, after room temperature is dried, put 37 ℃ of thermostat containers and be inverted overnight incubation.Select several bacterium colonies, be inoculated in respectively (5ml/ pipe) in LB, overnight incubation, respectively get 0.1ml next day and transfer to (2ml/ pipe) in LB, cultivate 3 hours 1mM IPTG inducing culture 8h for 32 ℃, receive bacterium, with SDS-PAGE evaluation, select goal gene to obtain high expression level bacterial strain, and order-checking is identified.
2. the expression and purification of antigen
The cultivation of 2.1 expression strains: the expression strain 20 μ l that get-70 ℃ of preservations are inoculated in (100ml LB/500ml triangular flask) in LB substratum, 30 ℃ of air table overnight incubation, next day, 30 ℃ of air tables were cultivated approximately 3 hours, in the time that OD600 value reaches 0.7 in 5% ratio transferred species in LB substratum (the same), add 1mM IPTG, inducing culture 8h, merges bacterium liquid centrifugal 20 minutes of 6000rpm, abandon supernatant, collecting precipitation part.
2.2 extract inclusion body: will precipitate and claim weight in wet base, will precipitate and hang with the 20mmol/L pH8.0TE damping fluid of 10 times of volumes, and add N,O-Diacetylmuramidase (1mg/ml suspension), at room temperature magnetic agitation 10 minutes.Ultrasonic disruption bacterium in ice bath surpassed for 30 seconds at every turn, in 30 seconds of interval, surpassed altogether 10 times.8 ℃, 1,2000rpm, centrifugal 20 minutes, abandon supernatant, 1mol/L NaCl for precipitation (preparing with TE) washes once, then washes 2 times collecting precipitation with TE.8M urea for precipitation (preparing with PH8.0TE) dissolving, adds 1% beta-mercaptoethanol.In 20 ℃, 1,2000rpm, centrifugal 10 minutes, goes precipitation to get supernatant again.
2.3 purifying: the inclusion body solution of above-mentioned dissolving is crossed to nickel ion post, with adsorption-buffering liquid (pH8.0,20mmol/L TE is containing 6mol/L urea, 0.1% beta-mercaptoethanol, 5mM imidazoles, 0.25M NaCl) balance cleans after loading, with elution buffer (pH8.0,20mmol/L TE is containing 6mol/L urea, 0.1% beta-mercaptoethanol, 200mM imidazoles) wash-out, collect the first elution peak.After Sephardex G-50 gel-filtration column, damping fluid adopts pH8.0, and 20mmol/L TE, containing 0.1%SDS, collects the first elution peak.Wash-out antigen adopts SDS-PAGE to carry out Purification.
3. horseradish enzyme (HRP) mark MP-P1 epitope antigen
Get HRP5mg and be dissolved in 0.2mol/L PH5.6 acetate buffer 0.5ml; Add freshly prepared 0.1mol/L Na IO40.25ml; Mix.(now solution colour should be become from yellowish brown blackish green)
4 ℃ of 30min; Add 2.5% hexylene glycol 0.5ml, mix.Room temperature is put 30min.(now solution should revert to yellow)
Add antibody 5～10mg to be marked, with 1.0mol/L PH9.5CBS tune PH to 9.0, mix.4 ℃ are spent the night; Add NaHB40.1ml (0.5mg), mix.
4 ℃ after 2 hours to 0.01mol/L PH7.4PBS dialysis, 4 ℃ are spent the night.Add packing in a small amount after appropriate neutral glycerine.
4, foundation and the application thereof of MP-IgM antibody test (prize law) technology based on restructuring P1 epitope antigen
1. set up MP-IgM antibody test (prize law) technology based on restructuring MP-P1 epitope antigen
Anti-IgM-μ chain monoclonal antibody is coated with elisa plate (PBS is diluted to 2 μ g/ml), and every hole 100 μ l abandon liquid after spending the night for 4 ℃, and distilled water flushing 3 times, pats dry, and every hole adds 1%BSA100 μ l, and room temperature 2h, abandons liquid.Every hole adds after 100 μ l sample thin liquids, add respectively the sample to be tested serum of 10 μ l, 37 ℃ of 30min, abandon liquid, after washing plate 5 times with washings, abandon liquid, pat dry, add MP-P1 antigen (1: 1000) the 100 μ l of horseradish peroxidase-labeled, 37 ℃ of temperature are bathed 20min, abandon liquid and wash plate and pat dry for 5 times.Add the each 50 μ l of TMB nitrite ion: A and B liquid, 37 ℃ of lucifuges colour developing 10min, every hole adds stop buffer 50 μ l, reads 450nm light absorption value OD by microplate reader.OD > 0.1 is judged as the positive.
The clinical detection of 2.MP-IgM antibody test (prize law) technology
MP93 provided by the present invention, MP236, MP371, MP661, MP874, MP987 recall rate in 40 parts of mycoplasma pneumoniae patients is respectively 17.5% (7/40), 32.5% (13/40), 65% (26/40), 55% (22/40), 77.5% (31/40), 17.5% (7/40); In 50 parts of Healthy Human Serums, specificity is 100% (50/50), 80% (40/50), 100% (50/50), 100% (50/50), 30% (15/50), 90% (45/50), MP371 and MP661 antigen that wherein the present invention obtains have the specific immunologic competence of MP, both combine the specificity 100% (50/50) of use, sensitivity is 85% (34/40), is significantly higher than single antigen and uses (X
2=8.628; P < 0.05); The sensitivity that cold agglutination (Sai Lediya-Mai can II) detects and specificity are respectively 75% (15/20) and 98% (49/50), two kind of detection method and have good consistence (kappa=0.800; P < 0.01) in diagnostic reagent research, there is certain using value.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (5)
1. a mycoplasma pneumoniae antigen composition, is the combination of MP371 antigen and MP661 antigen, and described MP371 antigen is the N-terminal antigen of mycoplasma pneumoniae adhesion protein P1, and its site is positioned at 371-480aa, and concrete structure is shown in SEQ ID NO:3; Described MP661 antigen is the N-terminal antigen of mycoplasma pneumoniae adhesion protein P1, and its site is positioned at 661-772aa, and concrete structure is shown in SEQ ID NO:4.
2. mycoplasma pneumoniae antigen composition described in claim 1, is characterized in that: the Nucleotide of coding MP371 antigen is as shown in SEQ ID NO:9; The nucleotide sequence of coding MP661 antigen is as shown in SEQ ID NO:10.
3. described in claim 1, mycoplasma pneumoniae antigen composition detects the application in mycoplasma pneumoniae reagent in preparation.
4. a test kit that detects mycoplasma pneumoniae, is characterized in that, described test kit contains the composition described in claim 1.
5. test kit according to claim 4, is characterized in that, described detection is to utilize IgM antibodycapture to carry out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310015513.5A CN103059109B (en) | 2013-01-16 | 2013-01-16 | Mycoplasma pneumonia antigen, preparation method and immunodetection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310015513.5A CN103059109B (en) | 2013-01-16 | 2013-01-16 | Mycoplasma pneumonia antigen, preparation method and immunodetection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103059109A CN103059109A (en) | 2013-04-24 |
| CN103059109B true CN103059109B (en) | 2014-05-14 |
Family
ID=48102034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310015513.5A Active CN103059109B (en) | 2013-01-16 | 2013-01-16 | Mycoplasma pneumonia antigen, preparation method and immunodetection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103059109B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834668B (en) * | 2014-03-17 | 2016-08-17 | 英诺特（唐山）生物技术有限公司 | A kind of restructuring mycoplasma pneumoniae albumen and application thereof |
| CN105203768B (en) * | 2014-08-18 | 2017-01-18 | 董俊 | Method and kit for fast detection of human chlamydia pneumoniae antigen based on magnetic resolution and quantum dot labelling |
| JP6616332B2 (en) * | 2015-01-29 | 2019-12-04 | 株式会社タウンズ | Immunological detection method and kit for Mycoplasma pneumoniae |
| CN104678097B (en) * | 2015-03-10 | 2017-04-05 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of mycobacterium tuberculosis combined antigen for diagnosis of pulmonary tuberculosis |
| CN105585635B (en) * | 2016-03-08 | 2019-01-11 | 湖北工业大学 | The immune chromatography reagent kit of anti-human mycoplasma pneumoniae p1 protein antibody and the application antibody |
| CN108314710B (en) * | 2018-01-31 | 2020-09-11 | 广东唯实生物技术有限公司 | Mycoplasma pneumoniae recombinant antigen and application thereof |
| CN110964089B (en) * | 2019-11-06 | 2021-02-19 | 南京诺唯赞医疗科技有限公司 | Mycoplasma pneumoniae antigen |
-
2013
- 2013-01-16 CN CN201310015513.5A patent/CN103059109B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103059109A (en) | 2013-04-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103059109B (en) | Mycoplasma pneumonia antigen, preparation method and immunodetection kit | |
| CN105384803B (en) | A kind of Schistosoma japonicum recombinant protein SjSAPLP4 and its encoding gene and application | |
| CN103293306B (en) | Preparation method for African swine fever virus antibody detection colloidal gold immunochromatography test paper strip | |
| CN108614121B (en) | Bovine viral diarrhea virus E2 protein antigen multi-epitope fusion peptide and preparation and application thereof | |
| CN107176977B (en) | Application of mycoplasma bovis Mbovp730 protein in natural infection and vaccine immune identification | |
| CN107118262A (en) | A kind of Mycoplasma bovis MbovP579 albumen and its application | |
| CN106442981B (en) | A kind of 1 type antibody indirect ELISA diagnostic kit of human bocavirus | |
| CN106279378B (en) | Varicellazoster virus gE antigens and its purposes in varicella zoster virus antibody is detected | |
| CN101363859B (en) | Test paper strip for rapidly detecting brucellosis antibody | |
| CN115873079A (en) | Canine infectious hepatitis virus hexon protein antigen, truncation and application thereof | |
| CN109239341B (en) | Indirect ELISA kit for detecting bovine haemolytic mannheimia antibody and application thereof | |
| CN105061602A (en) | Fusion protein for detecting anti-ETEC (enterotoxigenic escherichia coil) antibody of pigs, as well as preparation method and application thereof | |
| CN111621506B (en) | Mycoplasma bovis secretory protein Mbovp0145 and application thereof | |
| CN110483625A (en) | A kind of Mycoplasma bovis imagination albumen MbovP732 and its application | |
| CN105254732B (en) | A kind of Schistosoma japonicum recombinant protein SjSAPLP5 and its encoding gene and application | |
| CN104610443A (en) | High-stability recombinant procalcitonin and preparation method and application thereof | |
| CN109679970B (en) | Preparation method for rapidly detecting feline herpes type I virus | |
| CN111978410A (en) | Fusion protein of brucella outer membrane protein OMP25 and periplasmic protein BP26 as well as expression and application thereof | |
| CN109111507B (en) | Virus recombinant glycoprotein and eukaryotic cell high-efficiency expression method and application thereof | |
| CN109824766A (en) | A kind of indirect ELISA testing kit using 4 type antibody of Fiber2 Protein Detection aviadenovirus | |
| CN111537732B (en) | Application of salmonella gallinarum SifA protein in preparation of ELISA antibody detection kit for detecting salmonella gallinarum antibody | |
| CN102520166A (en) | ELISA (Enzyme-Linked Immunosorbent Assay) kit for detecting swine chlamydophila abortus antibody | |
| CN110540599B (en) | Klebsiella pneumoniae Elisa detection kit based on Klebsiella pneumoniae surface protein antibody and preparation method thereof | |
| CN110554187B (en) | Expression protein for detecting bovine viral diarrhea virus antibody, ELISA kit, preparation method and application thereof | |
| CN101979406B (en) | Multi-epitope protein for South Africa type II foot-and-mouth disease, and preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |