CN102887944A - Chlamydia pneumonia antigen, method for preparing antigen, fast detection method and reagent for detecting anti-chlamydia pneumonia antibody by utilizing antigen - Google Patents
Chlamydia pneumonia antigen, method for preparing antigen, fast detection method and reagent for detecting anti-chlamydia pneumonia antibody by utilizing antigen Download PDFInfo
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Abstract
The invention relates to a chlamydia pneumonia genetic engineering expression artificial antigen, and a method for preparing the antigen. The method comprises the steps of: cloning and amplifying a chlamydia pneumonia MOMP (major outer membrane protein) antigen gene sequence by PCR (polymerase chain reaction), building a prokaryotic expression vector, expressing chlamydia pneumonia MOMP antigen protein by escherichia coli, and obtaining a reconstructed chlamydia pneumonia MOMP antigen with a three-dimensional structure and immunological competence by a dialysis method, a gradient dilution method and a gel chromatography renaturation inclusion body. A fast detection method for detecting the chlamydia pneumonia antigen antibody is provided. The method comprises the application of the chlamydia pneumonia antigen; and the invention provides a fast detection reagent for detecting the chlamydia pneumonia antibody. The reagent contains the chlamydia pneumonia antigen. According to the invention, the chlamydia pneumonia antigen is provided. The antigen has high specificity. The method for preparing the antigen, the method for fast measuring the chlamydia pneumonia antibody, and the reagent for fast measuring the chlamydia pneumonia antibody are provided by the invention.
Description
Technical field
The present invention relates to a kind of Chlamydia pneumoniae antigen for the diagnosis of pneumonia choamydiae infection, prepare the method for this antigen, with method and the reagent of the anti-chlamydia pneumoniae (cp) of this antigen measuring.
The present invention is significant in the diagnosis of the atypical pneumonia that is caused by Chlamydia pneumoniae.
Background technology
Chlamydozoan is that a kind of Grain-negative, obligatory parasitism are in intracellular parasite.Can in people's pulmonary alveolar macrophage, epithelial cell, endotheliocyte, smooth muscle cell, neutrophil leucocyte, breed.Its growth has unique cycle, is positioned at extracellular chlamydozoan elementary body (EB) and She Qu arrive cell formation inclusion body, then changes into netted corpusculum (RB), and RB has fecundity, but does not possess infection ability.The RB that breeds in cell is converted into EB very soon, wears out inclusion body and cell walls and arrives the extracellular.EB does not have fecundity, but has infection ability.At present, confirmed four kinds of chlamydozoans, i.e. chlamydia trachomatis, chlamydia psittaci, cat heart chlamydozoan and Chlamydia pneumoniae, wherein Chlamydia pneumoniae can infect by airborne transmission human.
Chlamydia pneumoniae (Chlamydia pneumoniae, Cpn) is the important pathogen body of human breathing tract disease, mainly causes people's the respiratory tract disease such as severe acute respiratory syndrome, bronchitis, sinusitis paranasal sinusitis.In addition, epidemiology and Study on etiology think, infection involving chlamydia pneumoniae is relevant with cardiovascular disorder.Cpn propagates through the spittle or respiratory secretions, can mutually infect in family and collective place.Chlamydia pneumoniae is because parasitism and the antigenic characteristic of its uniqueness often cause direct or indirect harm to human body, and Cpn has one of pathogenic micro-organism of great effect to be listed in to Chinese's hygiene and health.
Clinical symptom due to the infection involving chlamydia pneumoniae is lighter, is often ignored by people, often needs specific laboratory to detect and just can be found.Other clinical sings and symptoms according to the patient is difficult to the infection of discriminating bacteria, viral, mycoplasma pneumoniae or Chlamydia pneumoniae, so that has difficulties in clinical application, and the laboratory is detected and helped its differential diagnosis.
The detection that mainly comprises the pathogenic bacteria that infection site etc. is located is detected and to the two kinds of methods of antibody test of pathogenic bacteria in patients serum, the body fluid in the laboratory of infected by microbes disease.The detection of pathogenic bacteria comprises the methods such as pathogenicbacteria separation cultivation, the detection of pathogenic bacteria specific antigen and gene test.Although culture method is Chlamydia pneumoniae laboratory diagnosis " gold standard ", Chlamydia pneumoniae is strict cytozoicus, and culture condition requires harsh, and detection sensitivity is low, and clinical applicability is not strong.Take the polymerase chain reaction (PCR) although have higher specificity and susceptibility as the technique of gene detection of representative, its accuracy is subject to the impact of experiment condition and operator's state of the art.
Antibody test is one of the comparatively ideal laboratory of infection involving chlamydia pneumoniae detection method.The micro-indirect fluorescence experiments (micro-IF) that Wang and Graystong set up as far back as 1971, micro-IF experiment needs the chlamydozoan EB of purifying, and requires form or the structural integrity of EB, can not use the EB of form or structural modification or destruction.Because EB has infectivity and toxicity, use complete EB as antigen potential infectivity to be arranged, add again the IF experimental procedure complicated, micro-IF experiment seldom use in Clinical Laboratory.The chlamydia pneumoniae (cp) enzyme-linked immunosorbent assay (ELISA) of setting up subsequently has the characteristics such as testing process is simple, high throughput testing, and most of reagent all use complete chlamydozoan EB to make antigen, but chlamydozoan EB antigen can cause nonspecific reaction.
Chlamydial representative genus-specific antigen is lipopolysaccharides (LPS), it and some entero-bacte Re mutant LPS have common antigen, being the morbid substance of Chlamydia pneumoniae, is a kind of cell wall constituent---intracellular toxin, and its toxic action can be neutralized by specific antibody.
The species specific antigen of chlamydozoan is its major outer membrane albumen (MOMP), account for 60% of MOMP-E, molecular weight is about 39.5kDa, closely related with stability, growth and anabolism regulation, antigenicity and the virulence of Chlamydia pneumoniae outer membrane structure, be the main target position of human specific humoral immunity reaction.
Other major outer membrane antigens of chlamydozoan are to belong to specificity as main except MOMP, but also there are some species specificity, be that 40kDa D envelope antigen is to belong to specificity such as molecular weight, molecular weight is that the protein of 43kDa, 46kDa and 53kDa is species-specific antigen, and also having a kind of molecular weight is that the high molecular weight protein of 98kDa may be species-specific antigen.
As mentioned above, chlamydial antigenicity is very complicated, and the chlamydozoan that belongs to together also exists obvious difference not of the same race its antigenicity.The chlamydia pneumoniae (cp) that infection has the individuality of Chlamydia pneumoniae to produce shows the diversity consistent with its antigen complicacy equally.The purifying antigen that dimension, application chlamydozoan EB thalline are done detectable antigens or the not high EB of purity source all can produce nonspecific reaction.In addition, although set up the chlamydia pneumoniae (cp) detection method, they can't reach the clinical demand of Chlamydia pneumoniae rapid detection.
Therefore, prepare the method for the high Chlamydia pneumoniae antigen of a kind of high specificity, detection sensitivity and a kind of simple, High-efficient Production Chlamydia pneumoniae antigen, be conducive to set up more efficient Chlamydia pneumoniae diagnostic method, improve the Chlamydia pneumoniae diagnostic level.
Summary of the invention
The first purpose of the present invention provides a kind of Chlamydia pneumoniae species-specific antigen, and this antigen has species specificity and the immunoreactivity of height.
The second purpose of the present invention provides a kind of method for preparing Chlamydia pneumoniae antigen, and the Chlamydia pneumoniae antigen of the method preparation has species specificity and immunoreactivity.
The 3rd purpose of the present invention provides a kind of method of measuring anti-chlamydia pneumoniae (cp), the method is quick " a step detection method ", has highly sensitive, high specificity, detection speed is fast, easy and simple to handle, need not Special Equipment, be applicable to the multiple occasions such as clinical detection, epidemiology survey and place quarantine.
The 4th purpose of the present invention provides a kind of chlamydia pneumoniae (cp) quick detection reagent, and it is a kind of high specificity, highly sensitive, quick, easy " single stage method " chlamydia pneumoniae (cp) detection reagent.
Main contents of the present invention:
(1) Chlamydia pneumoniae antigen, it is a kind of employing genetic engineering technique artificial antigen.
(2) Chlamydia pneumoniae antigen in (1) above, non-specific cross-reaction does not occur in it and chlamydia trachomatis antibody and Chlamydia psittaci antibody.
(3) a kind of method for preparing Chlamydia pneumoniae antigen, comprise PCR clonal expansion Chlamydia pneumoniae MOMP antigen gene sequences, make up prokaryotic expression carrier, escherichia coli expression Chlamydia pneumoniae MOMP antigen protein, adopt dialysis method, gradient dilution method and gel chromatography renaturation inclusion body, obtain to have three-dimensional structure and immunocompetent restructuring Chlamydia pneumoniae MOMP antigen.
(4) a kind of chlamydia pneumoniae (cp) method for quick comprises the Chlamydia pneumoniae antigen that plant (1)-(2) above using.
(5) chlamydia pneumoniae (cp) method for quick in (4) above, comprise (1)-the Chlamydia pneumoniae antigen that plants (2) is fixed to a kind of nitrocellulose filter, a kind of traget antibody (two is anti-) is fixed on the solid phase carrier, testing sample contacts with traget antibody (two is anti-), if there is chlamydia pneumoniae (cp) in the testing sample, then chlamydia pneumoniae (cp) and mark two anti-reactions form antibody---mark two anti-mixtures, " mixture " moves horizontally at nitrocellulose filter, run into the Chlamydia pneumoniae antigen that is fixed on the film and produce reaction, " mixture " with marker just is fixed on the antigen specifically, the position of immobilized antigen just has color reaction: if testing sample does not contain chlamydia pneumoniae (cp), then " mixture " can not be fixed on the position of antigen, just there is not color reaction herein, can whether color reaction be arranged according to immobilized antigen position on the nitrocellulose filter, judge detected result, whether chlamydia pneumoniae (cp) is namely arranged in the testing sample.
(6) (5) detect the chlamydia pneumoniae (cp) detection method above, and wherein testing sample is people's serum, blood plasma or whole blood.
(7) measure the method that chlamydia pneumoniae (cp) detects above in (5) or (6), traget antibody wherein (two is anti-) is anti-human IgG antibody or the anti-human IgM antibody of mark.
(8) traget antibody is the antibody of Radioactive colloidal gold or colored latex mark in above-mentioned (5)-(7).
(9) a kind of reagent of rapid detection chlamydia pneumoniae (cp), its used antigen are the Chlamydia pneumoniae antigen in above-mentioned (1)-(2).
(10) be used for the reagent of rapid detection chlamydia pneumoniae (cp) in above-mentioned (9), traget antibody wherein is colloidal gold labeled monoclonal antibody or colored latex traget antibody.
Below the present invention will be described in detail.
Chlamydia pneumoniae antigen of the present invention is a kind of restructuring Chlamydia pneumoniae major outer membrane albumen (MOMP) that obtains by genetic engineering technique.
Chlamydia pneumoniae antigen of the present invention, it is main by Chlamydia pneumoniae major outer membrane albumen (MOMP) is gene constructed to prokaryotic expression carrier PET, MOMP gene-pet sheet reaches carrier and transforms intestinal bacteria, screen positive recombinant bacterium, IPTG induces recombinant bacterium to express Chlamydia pneumoniae MOMP albumen, obtains through steps such as bacteria lysis, solubilization of inclusion bodies and recombinant protein structure renaturation and purifying.
The below will describe the method for preparing Chlamydia pneumoniae antigen among the present invention in detail.
Obtain Chlamydia pneumoniae MOMP gene order from gene BANK, according to the known design of Chlamydia pneumoniae MOMP antigen gene primer, PCR clonal expansion or synthetic Chlamydia pneumoniae MOMP antigen gene sequences.Goal gene is connected with the prokaryotic expression carrier of crossing through same double digestion behind double digestion, transformed competence colibacillus cell (intestinal bacteria), screens positive recombinant bacterium.The positive colony bacterium is extracted plasmid, double digestion is identified and order-checking is identified, proves that insertion gene goal gene is correct.Recombinant bacterium is through IPTG abduction delivering, centrifugal abduction delivering recombinant bacterium, and thalline is resuspended in ultrasonic degradation behind the sample-loading buffer, and the albumen precipitation of centrifugal acquisition inclusion body form is dissolved in the sex change expressing protein of urea soln after washing.Metaprotein need to could obtain the immune response originality of native protein and reach the application standard that chlamydia pneumoniae (cp) detects through structure renaturation and purifying.
For Chlamydia pneumoniae MOMP recombinant protein renaturing inclusion bodies method, can use dialysis method, gradient dilution method, gel chromatography etc., the method that preferred dialysis method and gradient dilution method combine, because they are easy to control, the multiple rate of albumen is high.
For Chlamydia pneumoniae MOMP method for purifying proteins behind the renaturing inclusion bodies, can use gel chromatography, affinity chromatography etc., preferred gel chromatography, because it is easy to control most, yield is high.
The Chlamydia pneumoniae MOMP antigen that obtains by gene recombination has higher species specificity and susceptibility.
For the method for Fast Measurement antibody, what enumerate here is preferred method, as uses the immunochromatographic measurement of various traget antibodies, uses the immunity percolation method of various traget antibodies etc.
The below describes the immunochromatographic measurement method in detail.
Anti-chlamydia pneumoniae (cp) immunochromatographic measurement method comprises the steps: that Chlamydia pneumoniae antigen is fixed on the solid phase carrier 1, traget antibody (two is anti-) is fixed on the solid phase 2, testing sample contacts with traget antibody (two is anti-), if there is chlamydia pneumoniae (cp) in the testing sample, then chlamydia pneumoniae (cp) and mark two anti-reactions form antibody---mark two anti-mixtures, " mixture " moves horizontally at solid phase carrier 1, run into the Chlamydia pneumoniae antigen that is fixed on the carrier 1 and produce reaction, " mixture " with marker just is fixed on the antigen specifically, and the position of immobilized antigen just has color reaction on the solid phase carrier; If testing sample does not contain chlamydia pneumoniae (cp), then " mixture " can not be fixed on the position of antigen, just do not have color reaction herein, can whether color reaction be arranged according to immobilized antigen position on the solid phase carrier, judge detected result, whether chlamydia pneumoniae (cp) is namely arranged in the testing sample.
For stationary phase carrier 1, but application examples such as nitrocellulose filter (NC film), pvdf membrane etc.Preferred nitrocellulose filter.
For solid phase carrier 2, but application examples such as fiberglass fiber element film, any form such as non-woven fabrics.The preferred glass fibers element is made binding substances and is discharged pad, because it is easy to control most.
For the antibody of mark, refer to the anti-antibody with different marker marks here, refer to here and resist into IgG antibody and resist the antibody into IgM, or its partially decomposed product, such as F (ab ')
2, Fab etc. can suitably use according to the kind of detected antibody.
Behind the known infection involving chlamydia pneumoniae, IgM occurs after 1～2 week, evening appears in IgG antibody, but can keep the long period.Therefore, in the antibody of mark and step that antibody in the above-mentioned sample contacts, the antibody of the mark that is preferably obtained by the different types of antibody of mark reacts separately in mensuration, the antibody of the mark that obtains for mark different sorts antibody, here refer to the anti-IgM of mark, the anti-IgG antibody of mark etc.
Traget antibody marker applicatory can be used colloid gold and silver, selenium and colored latex etc.
For reagent of the present invention, preferred respectively with the immunochromatographiassay assay reagent of colloid gold label anti-IgM and anti-IgG antibody.
Reagent of the present invention can need to unite other component and make up according to the characteristics of immunochromatographiassay assay reagent.For example, be provided with application of sample end water accepting layer, detection layers and suction side water accepting layer at the PVC liner plate, between detection layers and application of sample end water accepting layer, be provided with golden labeling antibody layer; Be coated with the detection line of antigen formation and the control line that is formed by dynamics in detection layers.
Description of drawings
Fig. 1 has shown the surface structure schematic diagram of invention reagent-check-out console
1-check-out console 2-well 3-peep window
Fig. 2 has shown invention reagent---the external structure schematic diagram of detector bar
4-application of sample end water accepting layer 5-gold labeling antibody layer 6-control line 7-detection line
8-detection layers 9-suction side water accepting layer 10-liner plate
Embodiment
1) recombinant expressed, the structure renaturation of restructuring Chlamydia pneumoniae MOMP antigen and purifying
Mycoplasma pneumoniae MOMP gene carries out full gene with reference to GenBank sequence M64064 and synthesizes, and has removed signal peptide sequence when synthetic and has carried out the optimization of intestinal bacteria rare codon, introduces restriction enzyme site BamHI/XhoI.Synthetic goal gene is 1101bp (366aa) altogether.
The gene composition sequence
>CpnMOMPSequence
GGC
GGATCCTTGCCTGTAGGTAACCCTTCTGATCCAAGCTTATTAATTGATGGTACAATCTGGGAGGGTGCTGCAGGTGATCCTTGCGATCCTTGCGCTACTTGGTGCGACGCTATTAGCTTACGTGCTGGTTTTTACGGTGACTATGTTTTCGACCGTATCTTAAAAGTAGATGCACCTAAAACATTTTCTATGGGTGCCAAGCCTACTGGTTCCGCTGCTGCAAACTATACTACTGCCGTAGATCGTCCTAACCCGGCCTACAATAAGCATTTACACGATGCAGAGTGGTTCACTAATGCAGGCTTCATTGCCTTAAACATTTGGGATCGCTTTGATGTTTTCTGTACTTTAGGTGCTTCTAATGGTTACATTCGTGGTAACTCTACAGCGTTCAATCTCGTTGGTTTATTCGGTGTTAAAGGTACTACTGTAAATGCAAATGAACTCCCAAACGTTTCTTTAAGTAACGGTGTTGTTGAACTTTACACAGACACCTCTTTCTCTTGGAGCGTAGGCGCTCGTGGTGCCTTATGGGAATGCGGTTGTGCAACTTTGGGTGCTGAATTCCAATATGCACAGTCCAAACCTAAAGTTGAAGAACTTAATGTGATCTGTAACGTATCGCAATTCTCTGTAAACAAACCTAAGGGCTATAAAGGCGTTGCTTTCCCTTTGCCAACAGACGCTGGCGTAGCAACAGCTACTGGTACAAAGTCTGCGACCATCAATTATCATGAATGGCAAGTAGGTGCCTCTCTCTCTTACCGTCTCAACTCTTTAGTGCCATACATTGGTGTACAATGGTCTCGCGCAACTTTTGATGCTGATAACATCCGCATTGCTCAGCCAAAACTCCCTACAGCTGTTTTAAACTTAACTGCATGGAACCCTTCTTTACTCGGTAATGCCACAGCATTGTCTACTACTGATTCGTTCTCAGACTTCATGCAAATTGTTTCCTGTCAGATCAACAAGTTTAAATCTCGTAAAGCTTGTGGTGTTACTGTAGGTGCTACTTTAGTTGATGCTGATAAATGGTCACTTACTGCAGAATTTAATTAACGAGCGTGCTGCTCACGTATCTGGTCAGTTCCGTTTCTAA
CTCGAGGGC
The Construction and identification of recombinant expression plasmid.With BamHI and XhoI Chlamydia pneumoniae MOMP goal gene fragment and pET30a plasmid are carried out double digestion respectively, after enzyme is cut the product purification recovery, 16 ℃ of connections are spent the night, and change E.coli DH5a competent cell over to again, and picking conversion bacterium colony extraction plasmid carries out PCR, enzyme is cut and check order evaluation.Identify through PCR and double digestion, all confirm to have the gene fragment insertion vector of big or small approximately 1.1Kb, consistent with expected result.Sequencing result shows that inserting the goal gene fragment is 1101bp, and the Chlamydia pneumoniae MOMP gene order of delivering in nucleotide sequence and the GenBank database is identical, and the expression vector open reading frame of structure is correct, can express recombinant protein.
The abduction delivering of recombinant protein: get 50 μ lpET30a-cpnMOMP/BL21 (DE3) bacterium liquid and join in the LB liquid nutrient medium that 5ml contains 100 μ g/ml kantlex, 37 ℃, 200rpm shakes bacterium and spends the night, and increased bacterium in the LB substratum that contain kantlex in by 1: 50 next day, when being cultured to extinction A600 and being 0.6 left and right sides, add IPTG to concentration be 1mmol/L, carried out abduction delivering 4 hours, centrifugal 30 minutes of 5000rpm, collect thalline, suspend abundant mixing ,-80 ℃ with the TE damping fluid, multigelation 3 times, at power 370W, by ultrasonic 5 minutes, 5 minutes the frequency ice-bath ultrasonic in gap 180 times, 4 ℃, centrifugal 15 minutes of 10000 * g, collecting precipitation (contains 50mmol/L Tris-HCl with the urea of 2M, pH 3.0) wash 2 times, precipitation is dissolved in 8M urea soln (contain 50mmol/L Tris-HCl, pH 8.0), 4 ℃ of preservations.Precipitation SDS-PAGE analyzing proteins expression amount.Deposition condition, concentrated gum concentration are 5%, and resolving gel concentration is 12%.Protein staining is the coomassie brilliant blue staining method, coomassie brilliant blue R_250 staining fluid dyeing 3h, acetic acid-ethanol decolorization liquid decolour colourless to background till.Contain 414 amino acid through the amino acid analysis recombinant protein, molecular weight is 44.6kDa, and waiting a point is 6.43.
Structure renaturation and the purifying of recombinant protein: 10mol/L under the room temperature condition, PH 7.2PBS damping fluid carries out gradient dialysis dilution to the lysate of Chlamydia pneumoniae MOMP recombinant expression protein 8mol/L urea, be respectively 6mol/L, 4mol/L, 3mol/L control dilution gradient by urea concentration in the solution, the time of each gradient dilution is respectively 3～4h.4 ℃ of placements of the lysate that contains 3mol/L urea 24h of dilution refolding will be finished.The centrifugal sedimentable matter of going out of the lysate 10000r/min of dilution refolding, supernatant liquor carries out purifying by the gel chromatography partition method take S-300 as medium, thereby obtains restructuring Chlamydia pneumoniae MOMP antigen.Gained group Chlamydia pneumoniae MOMP antigen component carries out the SDS-polyacrylamide gel electrophoresis, to determine molecular weight and the purity of protein of protein.Deposition condition, concentrated gum concentration answers 5%, and resolving gel concentration is 12%.Protein staining is the coomassie brilliant blue staining method, coomassie brilliant blue R_250 staining fluid dyeing 3h, acetic acid-ethanol decolorization liquid decolour colourless to background till.Main protein band appears at the 44.6kDa place, and purity of protein reaches more than 95%.
2) preparation of chlamydia pneumoniae (cp) gold labeled quick detection reagent
Above-mentioned resulting gene recombination Chlamydia pneumoniae MOMP albumen is made detectable antigens, at the coated detection line of nitrocellulose filter, with reference to Fig. 2, preparation chlamydia pneumoniae (cp) gold labeled quick detection reagent, its moiety comprises: be provided with application of sample end water accepting layer 4, detection layers 8 and water accepting layer 9 at liner plate 10, between detection layers and application of sample end water accepting layer 4, be provided with the anti-chlamydia pneumoniae (cp) antibody layer 5 of gold mark, be coated with detection line 7 and control line 6 in detection layers 8.Wherein application of sample end water accepting layer 4 and suction side water accepting layer 9 are made by multi-layer filter paper; Detection layers 8 is nitrocellulose filter; Gold labeling antibody layer is glass fibre or non-woven fabrics leaching colloidal gold labeled monoclonal antibody.
The detection reagent preparation procedure comprises: prepare control line 6, detection line 7 coated of golden labeling antibody layer 5 and detection layers 8, and then in liner plate 10 combination gold label test strip and test card 1.Paste respectively application of sample end absorbent paper layer 4 and suction side water accepting layer 9 at the two ends of plastic base plate 10; Section is pasted the Mierocrystalline cellulose rete 8 of coated detection line and control line therein, at the handing-over position of application of sample end absorbent paper layer 4 with Mierocrystalline cellulose rete 8, folder pastes the glass fibre that contains golden labeling antibody layer 5, and 4/5 part of glass fibre is in the middle of application of sample end absorbent paper layer 4, and 1/5 part is on Mierocrystalline cellulose rete 8.Then by 4 mm wides, 8 cm long specification slittings.Refer again to Fig. 5, detector bar is assembled in forms test card 1 in the plastics casing.Well 2 is over against the application of sample end absorbent paper layer 4 of test strip on the lid, and vision slit 3 is over against the detection layers 8 of cellulose membrane.
The preparation method of above-mentioned golden labeling antibody layer comprises the steps: the preparation of i Radioactive colloidal gold, getting the 100mg hydrochloro-auric acid is dissolved in the 1000ml tri-distilled water, add 15ml, be 1% trisodium citrate in concentration, boiled 15 minutes, can obtain the colloidal gold solution of 15～50 nanometers; The anti-human IgG monoclonal antibody of ii colloid gold label is got the 100m/ colloidal gold solution, transfers pH8.4 with the 0.2m solution of potassium carbonate, enter the anti-human IgG monoclonal antibody of 6mg, stirred 20 minutes, add again 240mg bovine serum albumin (BSA), continue to stir 5 minutes, 4 ℃ left standstill 2～4 hours; Iii with above-mentioned colloidal gold solution through 2000 rev/mins centrifugal 10～15 minutes, remove throw out, get supernatant liquor; Iv with supernatant liquor through 10000 rev/mins of centrifugal 60 minutes centrifugal throw outs; V is dissolved in 4ml 0.02M PH7.4Tris-Hcl damping fluid with throw out and gets colloidal gold solution, contains 0.25%BSA and 0.02% sodium azide in this damping fluid; Till vi immersed colloidal gold solution glass fibre or non-woven fabrics to liquid and begins to ooze out, 37 ℃ of dryings formed golden labeling antibody layer 5 in 2 hours.
Described detection layers 8 is to be provided with the detection line 7 of restructuring Chlamydia pneumoniae MOMP antigen formation and the nature controlling line 6 that is formed by sheep or rabbit anti-mouse igg antibody at nitrocellulose filter.Its preparation method is to get above-mentioned 1) prepared restructuring Chlamydia pneumoniae MOMP antigen, accent concentration is 1.5mg/ml, adds 2% methyl alcohol, sprays detection line 7 in the cellulose membrane stage casing with Membrane jetter; Getting sheep or rabbit anti-mouse igg accent concentration is 1.5mg/ml again, with Membrane jetter in the cellulose membrane stage casing, apart from detection line 0.5cm place, spray control line 6 arranges spray film amount by 20 μ l/10cm, and the spray film is placed on 37 ℃ of dryings 2 hours, sheet 0.01mlpH7.0 contains the PBS of 10% calf serum again, in 37 ℃ of lower sealings 30 minutes, the PBS rinsing of 0.01mlpH7.0,37 ℃ of dryings.
3) mensuration of chlamydia pneumoniae (cp)
Above-mentioned sample diluting liquid can be selected 0.01ml pH7.0PBS and 0.9%NaCl, preferred 0.01ml pH7.0PBS, and the result is stable because it develops the color.
4) to the susceptibility of human serum and special
Prepare chlamydia pneumoniae (cp) (IgG) gold mark detection reagent with antigen of the present invention and detection method, detect the 36 parts of chlamydia pneumoniae (cp) positive (IgG) patients serums and 84 parts of chlamydia pneumoniae (cp) feminine genders (IgG) serum sample carries out specificity and sensitivity test.Simultaneously, in order to verify effect of the present invention, chlamydia pneumoniae (cp) (IgG) the ELISA test kit that the gold mark detection reagent of carrying out with Chlamydia pneumoniae antigen of the present invention and Ou Meng company (EUROIMMUN Medizinische Labordiagnostika AG) are produced carries out correlation analysis, the results are shown in Table 1, table 2.
Table 1: the specificity of antigen of the present invention and sensitivity Detection result
Table 1 result demonstration, the susceptibility of antigen of the present invention reaches 94.44%, and specificity reaches 97.61%.
Table 2: the detection reagent and the ELISA of the Ou Meng company reagent that carry out with antigen of the present invention carry out the correlation analysis result
Table 2 result shows, the positive coincidence rate of detection method of the present invention and the ELISA of Ou Meng company reagent is 89.19%, negative match-rate is 97.59, total coincidence rate is 95.00%, illustrates that detected result of the present invention and Ou Meng company have preferably dependency with chlamydia pneumoniae (cp) (IgG) ELISA reagent.Therefore, the present invention has preferably clinical value.
Claims (10)
1. Chlamydia pneumoniae antigen, it is a kind of employing genetic engineering technique artificial antigen.
2. the Chlamydia pneumoniae antigen of claim 1, non-specific cross-reaction does not occur in it and chlamydia trachomatis and Chlamydia psittaci antibody.
3. method for preparing Chlamydia pneumoniae antigen, comprise PCR clonal expansion Chlamydia pneumoniae MOMP antigen gene sequences, make up prokaryotic expression carrier, escherichia coli expression Chlamydia pneumoniae MOMP antigen protein, adopt dialysis method, gradient dilution method and gel chromatography renaturation inclusion body, obtain to have three-dimensional structure and immunocompetent restructuring Chlamydia pneumoniae MOMP antigen.
4. a chlamydia pneumoniae (cp) method for quick comprises that application rights requires the Chlamydia pneumoniae antigen of arbitrary claim among the 1-3.
5. chlamydia pneumoniae (cp) method for quick in the claim 4, comprise that the Chlamydia pneumoniae antigen among the claim 1-3 is fixed to a kind of nitrocellulose filter, a kind of traget antibody (two is anti-) is fixed on the solid phase carrier, testing sample contacts with traget antibody (two is anti-), if there is chlamydia pneumoniae (cp) in the testing sample, then chlamydia pneumoniae (cp) and mark two anti-reactions form antibody one mark two anti-mixtures, " mixture " moves horizontally at nitrocellulose filter, run into the Chlamydia pneumoniae antigen that is fixed on the film and produce reaction, " mixture " with marker just is fixed on the antigen specifically, the position of immobilized antigen just has color reaction, if testing sample does not contain chlamydia pneumoniae (cp), then " mixture " can not be fixed on the position of antigen, just there is not color reaction herein, can whether color reaction be arranged according to immobilized antigen position on the nitrocellulose filter, judge detected result, whether chlamydia pneumoniae (cp) is namely arranged in the testing sample.
6. claim 5 detects the chlamydia pneumoniae (cp) detection method, and wherein testing sample is people's serum, blood plasma or whole blood.
7. measure the method that chlamydia pneumoniae (cp) detects in the top claim 5 or 6, traget antibody wherein (two is anti-) is anti-human IgG antibody or the anti-human IgM antibody of mark.
8. traget antibody is the antibody of Radioactive colloidal gold or colored latex mark among the claim 5-7.
9. the reagent of a rapid detection chlamydia pneumoniae (cp), its used antigen is the Chlamydia pneumoniae antigen among the claim 1-3.
10. be used for the reagent of rapid detection chlamydia pneumoniae (cp) in the claims 9, traget antibody wherein is colloidal gold labeled monoclonal antibody or colored latex traget antibody.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104131020A (en) * | 2014-08-01 | 2014-11-05 | 中国农业科学院兰州兽医研究所 | Coexpression vector of Chlamydophila abortus and Chlamydophila psittaci protective antigen MOMP and MIP, construction and expression method thereof |
| CN104198710A (en) * | 2014-08-18 | 2014-12-10 | 湖北工业大学 | Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously |
| CN104280543A (en) * | 2013-07-11 | 2015-01-14 | 国家纳米科学中心 | Test paper and method for detecting IgG (immunoglobulin G) and IgM (immunoglobulin m) in liquid sample simultaneously |
| CN105319360A (en) * | 2014-08-18 | 2016-02-10 | 董俊 | Chlamydia pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1133192A (en) * | 1994-08-03 | 1996-10-16 | 日立化成工业株式会社 | Monoclonal antibody with idiocrasy to pneumonia chlamydia and preparation process |
| CN1184532A (en) * | 1995-03-28 | 1998-06-10 | 日立化成工业株式会社 | Chlamydia pneumoniae antigen, process for producing the same, method for assaying anti-chlamydia pneumoniae antibody by using the same, and reagent for assaying anti-chlamydia pneumoniae antibody |
| CN1263599A (en) * | 1998-04-14 | 2000-08-16 | 大塚制药株式会社 | Method for assaying antibody and device for assaying antibody |
| CN101699290A (en) * | 2009-04-03 | 2010-04-28 | 李克生 | Method for testing mycoplasma pneumoniae antibody, gold-labeled quick test card and preparation method |
-
2012
- 2012-09-06 CN CN2012103263264A patent/CN102887944A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1133192A (en) * | 1994-08-03 | 1996-10-16 | 日立化成工业株式会社 | Monoclonal antibody with idiocrasy to pneumonia chlamydia and preparation process |
| CN1184532A (en) * | 1995-03-28 | 1998-06-10 | 日立化成工业株式会社 | Chlamydia pneumoniae antigen, process for producing the same, method for assaying anti-chlamydia pneumoniae antibody by using the same, and reagent for assaying anti-chlamydia pneumoniae antibody |
| CN1263599A (en) * | 1998-04-14 | 2000-08-16 | 大塚制药株式会社 | Method for assaying antibody and device for assaying antibody |
| CN101699290A (en) * | 2009-04-03 | 2010-04-28 | 李克生 | Method for testing mycoplasma pneumoniae antibody, gold-labeled quick test card and preparation method |
Non-Patent Citations (4)
| Title |
|---|
| 姚菊明等: "桑蚕丝素-RGD融合蛋白的固态结构及其细胞粘附性分析", 《第十届陈维稷优秀论文奖论文汇编》 * |
| 王文勇主编: "《免疫细胞（组织）化学和分子病理学技术》", 30 November 2010, 西安：第四军医大学出版社 * |
| 陈学勤等: "肺炎衣原体主要外膜蛋白重组抗原制备与应用的研究", 《中国卫生检验杂志》 * |
| 陈学勤等: "肺炎衣原体主要外膜蛋白重组抗原制备与应用的研究", 《中国卫生检验杂志》, vol. 18, no. 1, 31 January 2008 (2008-01-31), pages 36 - 39 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104280543A (en) * | 2013-07-11 | 2015-01-14 | 国家纳米科学中心 | Test paper and method for detecting IgG (immunoglobulin G) and IgM (immunoglobulin m) in liquid sample simultaneously |
| CN104280543B (en) * | 2013-07-11 | 2016-08-24 | 国家纳米科学中心 | A kind of method of IgG and IgM in reagent paper and a kind of fluid sample of detection simultaneously |
| CN104131020A (en) * | 2014-08-01 | 2014-11-05 | 中国农业科学院兰州兽医研究所 | Coexpression vector of Chlamydophila abortus and Chlamydophila psittaci protective antigen MOMP and MIP, construction and expression method thereof |
| CN104198710A (en) * | 2014-08-18 | 2014-12-10 | 湖北工业大学 | Magnetic separation and multicolor quantum dot marking-based method and kit for quickly detecting anti-human chlamydia pneumoniae IgM and IgG antibodies simultaneously |
| CN104198710B (en) * | 2014-08-18 | 2016-01-06 | 湖北工业大学 | Based on the anti-human Chlamydia pneumoniae IgM of magnetic resolution and color quantum point mark, the IgG antibody method of altogether inspection and kit fast |
| CN105319360A (en) * | 2014-08-18 | 2016-02-10 | 董俊 | Chlamydia pneumoniae quantum dot immunochromatographic assay detection card and preparing method and application thereof |
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