CN107573417A - Mycoplasma pneumoniae chimeric antigen, the antigen detecting agent and both preparation methods - Google Patents
Mycoplasma pneumoniae chimeric antigen, the antigen detecting agent and both preparation methods Download PDFInfo
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Abstract
The invention provides mycoplasma pneumoniae chimeric antigen amino acid sequence, includes such as SEQ ID NO:Amino acid sequence shown in 1, and the complete genome sequence of the mycoplasma pneumoniae chimeric antigen of full genome synthesis, include such as SEQ ID NO:Amino acid sequence shown in 2.Present invention also offers the method for structure above-mentioned two gene order.Present invention also offers preparation method, the mycoplasma pneumoniae detection reagent and the preparation method of the above-mentioned mycoplasma pneumoniae chimeric antigen comprising full genome synthesis.Present invention selection Mp recombinates chimeric antigen as marker material, and is applied to gold-marking immunity tomographic system, and the detection architecture is direct mark capturing, sensitivity has raising by a relatively large margin, and antigen its specificity improves, and its antigen is easy to culture purified, more cost-effective；For the clinical new method for providing a kind of both quick and can and accurately detecting mycoplasma pneumoniae IgG, there are good market prospects.
Description
Technical field
In-vitro diagnosis field of immunodetection of the present invention, more particularly to mycoplasma pneumoniae, and the inosculating antibody of mycoplasma pneumoniae
The expression of the structure, chimeric recombinant antigens of former expression vector, prepare, further relate to the kit containing the antigen or antigen composition
And application.
Background technology
Mycoplasma pneumoniae（mycoplasma pneuoniae, MP）It is a kind of hyperfiltration between bacterium and virus
Venereal disease pathogenic microorganism, mainly propagated by the respiratory tract spittle in the form of aerosol particles.1962 first from human primary
SARS（primary atypical pneumonia, PAP）Separate and cultivate in the sputum of patient.
Since the 1990s, with the transition of pneumonia aetology, MP has turned into the important pathogen body of infantile pneumonia, MP
Infection not only causes pulmonary lesion, and can invade other organs such as the heart, brain, liver kidney, causes a variety of Pulmonary hypofuntions.MP or community
Acquired pneumonia（CAP）Important pathogen, especially more than 5 years old children, MP accounts for CAP cause of diseases 10 ~ 20% in non-popular year.Because of MP
Slow-growing, incubation period and carriagable time are grown, and form the epidemic characteristic that interval morbidity is propagated slowly, over a long time, popular reachable number
The moon to the several years.In the last few years, influenza crowd ratio increased year by year, not only invades teenager and children, infant morbidity are also in
Increase trend, and because MP infection often shows without specific clinical, easily mutually obscure with general virus flu.Accordingly, it is capable to
It is enough to make a definite diagnosis which kind of cause of disease is caused a disease in time, accomplish early discovery early treatment, reducing children acute pneumonia, sb.'s illness took a turn for the worse, therefore early diagnoses pole
To be important.
Mycoplasma pneumoniae antibody is divided into two kinds of IgG antibody and IgM antibody, because the incubation period of mycoplasma pneumoniae infection is 2
In week in week -3, when patient symptom occurs and gone to a doctor, IgM antibody has reached at a relatively high level, therefore the IgM antibody positive can conduct
The diagnosis index of acute HIV infection.It can not negate mycoplasma pneumoniae infection such as IgM antibody feminine gender, also need to detect IgG antibody.
There is evening compared with IgM in IgG, needs dynamic to observe.The measure of mycoplasma pneumoniae IgG antibody, in combination with mycoplasma pneumoniae IgM antibody
Detection, available for diseases such as auxiliary diagnosis Eaton agent pneumonias.
Special anti-MP IgG and IgM in indirect method detection human serum, at present using more.Advantage be it is convenient and swift,
Technical operation is simple, requires low to experimental facilities, is adapted to extensive detection.But the country there is no gratifying immunochromatography so far
Detection kit.The immunochromatographytest test kit of development high quality, it is most important that resist from antigenicity is strong, specificity is good
It is former.The MP antigens that China uses at present are the MP extracts of culture mostly, other protein products are often mixed with, so antigentic specificity is not
By force, false positive is easily produced.It is a kind of both economical, practical that high-purity recombinant antigen is largely prepared using technique for gene engineering
Method.As Protocols in Molecular Biology continues to develop, increasing MP major antigen genes are cloned in succession.These genes
The recombinant protein of expression, antigen coat can be used as after expression and purification, for anti-MP antibody tests.But to filter out antigenicity
Good, the higher antigen for being easy to purifying of expression quantity, there is certain difficulty.It can be overcome as Virus monitory antigen by the use of restructuring chimeric antigen
Multiple gene recombinant antigens prepare the shortcomings that complicated, non-specificity is strong and is difficult to purifying, inexpensive, safe, quick and reproducible.
External research confirms that MP P1 albumen and P30 albumen have stronger antigenicity, in the serum of MP the infected
Middle Antibody positive rate is higher, but also has two shortcomings, and one is that false positive is higher, and another is exactly preparation process complexity.Largely
Research has shown that B cell epitope belongs to space conformation specificity epitope corresponding to P1, and albumen belongs to linear epitope.We construct MP
The chimeric antigen of more epitope sequences, the chimeric antigen of epidemic disease chromatography detection display structure have good antigen active, and it is examined
The performance of the product of the sensitivity and specificity of survey more on the market all improves a lot.
The content of the invention
Present invention technical problem to be solved first is to provide a kind of mycoplasma pneumoniae chimeric antigen amino acid sequence, wraps
Containing such as SEQ ID NO:Amino acid sequence shown in 1.
Secondly technical problem to be solved is to provide a kind of mycoplasma pneumoniae chimeric antigen of full genome synthesis to the present invention
Complete genome sequence, include such as SEQ ID NO:Amino acid sequence shown in 2.
It is structure mycoplasma pneumoniae chimeric antigen ammonia first that the present invention also technical problems to be solved, which are to provide a kind of structure,
The method of base acid sequence, step are as follows：S101 utilizes whole amino acid sequences of computer software analysis mycoplasma pneumoniae albumen,
Filter out and antigenic protein sequence P1 is included in mycoplasma pneumoniae albumen：Residues 1177-1535, P30 albumen antigen
Epitope：Residues 170-254 and MPN456 albumen epitope：Residues 765-846, and it is mutual with flexible polypeptide
Connection.
On this basis, present invention also offers a kind of full base for the mycoplasma pneumoniae chimeric antigen for building full genome synthesis
Because of the method for sequence, step also has the following steps on the basis of above-mentioned S101：
According to mycoplasma pneumoniae gene, using full genome synthetic method, CATATG and AAGCTT is added respectively at its 5 ' and 3 ' end
To obtain restriction endonuclease Nde I and Hind III digestion recognition site CATATG and AAGCTT respectively, the bp of sequence length 1668 will
Rare codon is substituted for bacterium preference codon and is connected to pGEM-T Vector in bacterium（Promega Products）
On carrier, the complete genome sequence of mycoplasma pneumoniae chimeric antigen is obtained.
Meanwhile present invention also offers it is above-mentioned comprising full genome synthesis mycoplasma pneumoniae chimeric antigen preparation method,
Comprise the following steps：
S301 utilizes whole amino acid sequences of computer software analysis mycoplasma pneumoniae albumen, and the computer software can select
ANTHEWIN etc., filter out and antigenic protein sequence P1 is included in mycoplasma pneumoniae albumen：residues 1177-1535、
The epitope of P30 albumen：Residues 170-254 and MPN456 albumen epitope：Residues 765-846, and
Interconnected with flexible polypeptide, above-mentioned protein sequence is the stronger protein sequence of antigenicity；
S302 is according to mycoplasma pneumoniae gene, using full genome synthetic method, at its 5 ' and 3 ' end respectively plus CATATG and
AAGCTT to obtain restriction endonuclease Nde I and Hind III digestion recognition site CATATG and AAGCTT, sequence length respectively
1668 bp, rare codon it will be substituted for bacterium preference codon in bacterium and be connected to pGEM-T Vector（Promega
Products）On carrier, the complete genome sequence of mycoplasma pneumoniae chimeric antigen is obtained；
The μ g of the carrier of gene plasmid containing mycoplasma pneumoniae 1 III/Nde of restriction enzyme Hind I that S303 obtains above-mentioned steps
Carry out double digestion, digestion products and the 1 μ g carriers pET28a with same digestion（+）, 1% agarose electrophoresis, and glue reclaim purpose
Fragment and carrier segments, the amount of 1% agarose electrophoresis detection recovery fragment, by average molecular number purpose fragment：Carrier segments=4:1
Mixing, add 5 μ gT416 °C of connections of ligase overnight, take 5 μ l connection products to convert the prefabricated DH5a competence of 100 μ l thin
Born of the same parents, resistance LB flat boards are coated with, 37 °C of inversion overnight incubations, the clone containing positive plasmid is selected, contains pneumonia branch by what is obtained
The clone designation of the recombinant expression carrier pair of substance is pET28a-MP.
Further, in addition to step：PET28a-MP containing recombinant expression carrier to be identified is cloned into a small amount of cultures, alkaline process
Rapid extraction plasmid, double digestion is carried out with III/Nde of restriction enzyme Hind I, digestion products carry out agarose electrophoresis inspection
Survey, the mycoplasma pneumoniae gene that a size is 1668bp can be measured.
The full genome synthetic method synthesizes the sequence of mosaic gene, gives expression to restructuring chimeric antigen, solves existing immune
The problem of chromatography detection antigentic specificity is not strong, and sensitivity is not high.
Further, the induced expression process of the recombinant expression carrier of the mycoplasma pneumoniae and expansion incubation are as follows,
Induced expression：
Recombinant expression carrier pET28a-MP is transferred to e. coli bl21 (DE3) competent cell, in the LB trainings containing kanamycins
Support and cultivated on flat board, picking individual colonies are inoculated into the positive restructuring bacterium for screening to obtain in 5mlLB culture mediums and carry out induced expression, production
Thing carries out SDS-PAGE analyses；
Expand culture：
Inoculation identifies that the engineering bacteria single bacterium of purposeful protein expression falls on LBs tri- of the 200ml containing kanamycin by SDS-PAGE
Angle bottle, 200rpm, 37 °C of concussion and cultivates are stayed overnight, next day, by 5% inoculum concentration, are inoculated into 1L triangles of the 400mlLB containing kanamycin
In bottle, 37 °C, 240rpm concussion and cultivates to OD600=0.5, put ice-water bath and be cooled to 22 °C, add IPTG induced pneumonitis mycoplasmas
Gene expression, wherein, IPTG concentration is 0.01 mM, and induction time is 6 hours, and concussion speed is 240rpm/min.
Of the invention to provide a kind of mycoplasma pneumoniae detection reagent bar again in addition, the reagent strip includes PVC board and stickup
NC films, colloidal-gold strip, glass and blotting paper in PVC board, the PVC board head and the tail both ends are sample pad and adsorptive pads, its feature
It is that the T posts of the NC films are coated with the mycoplasma pneumoniae chimeric antigen recombinated as previously described, C posts are coated with as previously described
The monoclonal antibody of the mycoplasma pneumoniae inosculating antibody culture of restructuring.
Finally, the invention provides a kind of preparation method of mycoplasma pneumoniae detection reagent, it comprises the following steps：Colloid
The preparation of gold；
It is prepared by gold mark：
1st, antigen diluent：
A certain amount of mycoplasma pneumoniae recombination antigen as described in claim 2 or 4 is taken, after dialysis, centrifugal treating, uses pH
8.00.1M PB is diluted to 1mg/ml, and 4 DEG C save backup；
2nd, antigen is coupled；
3rd, gold mark working solution is prepared；
4th, colloidal-gold strip is prepared；
5th, the assembling of reagent strip：
NC films, colloidal-gold strip, glass and the blotting paper handled well are pasted in PVC board respectively, the PVC board head and the tail both ends are sample
Product pad and adsorptive pads.
Further, the step is in detail：
The preparation of S801 collaurums：
Take the gold chlorides of 1ml 1%（HAuCl4）Solution, it is added in 100ml water, is heated to boiling, adds the citric acids of 1.5ml 1%
Trisodium, mixing is boiled 5 minutes, until color no longer changes.Now obtained colloid gold particle is 30nm；
It is prepared by S802 gold marks：
1st, antigen diluent：
Take a certain amount of mycoplasma pneumoniae recombination antigen（MP-Ag）, it is dilute with pH 8.00.1M PB after dialysis, centrifugal treating
Release to 1mg/ml, 4 DEG C save backup；
2nd, antigen is coupled：
The 30nm collaurums that 250ml is prepared are taken, pH to 9.0 is adjusted with 1M NaOH, then adds above-mentioned antigen 1 ml, magnetic force stirs
Mix and stirred 1 hour on device, then add a certain amount of BSA to final concentration 1%, continue stirring 0.5 hour；
3rd, the preparation of gold mark working solution
The above-mentioned colloidal gold solution for being coupled antigen is centrifuged（4000rpm, 15 minutes）, precipitation is discarded, retains solution.So
After carry out secondary centrifuging（12000rpm, 30 minutes）, abandoning supernatant, retain precipitation.Precipitation is dilute with the gold mark containing 20% sucrose
Release liquid（pH8.0 0.02M Tris+1%BSA）Redissolve to 10mL, be transferred in brown bottle, 4 DEG C save backup；
4th, the preparation of colloidal-gold strip：
Above-mentioned gold mark working solution 10ml is taken, is loaded to metal spraying machine, it is 1.5 μ l/cm that setup parameter, which chooses discharge rate,.Air pump is opened, is treated
After stable gas pressure, start metal spraying machine, gold mark working solution is uniformly sprayed onto in 30cm*6.5cm gold standard pad.By the good gold mark work of spray
The gold standard pad of liquid is put into drying in 37 DEG C of insulating boxs and taken out after 12 hours, is cut into the wide bars of 8mm and is put into valve bag, then encloses
The aluminium foil bag of built-in drier, 20 DEG C save backup；
The coating of S803 NC films：
1st, antibody and how anti-dilution
By a certain amount of mouse anti-human igg monoclonal antibody（IgG-mab）, it is dilute with pH7.4 0.01M PBS after dialysis, centrifugal treating
Release to final concentration 1.0mg/ml, 4 DEG C save backup.
By a certain amount of mycoplasma pneumoniae recombination antigen monoclonal antibody（MP-mab）, after dialysis, centrifugal treating, use
PH7.4 0.01M PBS are diluted to final concentration 1.0mg/ml, and 4 DEG C save backup.
2nd, antigen coat
By above-mentioned antigen and it is how anti-be loaded to respectively in T, C post of a film machine, setup parameter selection discharge rate be 1.0 μ l/cm.Adjustment
The position of film head is drawn, makes T, C line in the middle part of NC, and spacing 5mm.Start point film machine, by T, C line solution coating to NC films.Will
The gold standard pad of the good gold mark working solution of spray is put into dried 24 hours in 37 DEG C of insulating boxs after take out, be put into valve bag, then enclose
The aluminium foil bag of built-in drier, 20 DEG C save backup；
The processing of S805 glasses：
Glass fibre element film is cut into 17*300mm bars, is put into after being soaked 1 hour in glass treatment fluid and takes out, be put into 37 DEG C of perseverances
Taken out after being dried 12 hours in incubator, be put into valve bag, then enclose the aluminium foil bag of built-in drier, 20 DEG C save backup；
The assembling of S806 reagent strips：
NC films, colloidal-gold strip, glass and the blotting paper handled well are pasted in PVC board respectively, are then cut into 4.0mm bars,
The PVC board head and the tail both ends are sample pad and adsorptive pads.
The reagent or reagent strip be low testing cost, high sensitivity, the detection range of linearity is wide, reappearance is high, can quantify,
Mycoplasma pneumoniae IgG kits simple to operate, calibration product are detected using gold-marking immunity chromatography, test agent sample,
And performance is carried out to mycoplasma pneumoniae IgG gold-marking immunitys chromatographic assay system（Sensitivity, linear, precision, interference）Comment
Valency.
It is of the invention compared with current technology, there is advantages below：
1st, present invention selection Mp recombinates chimeric antigen as marker material, and is applied to gold-marking immunity tomographic system,
The detection architecture is direct mark capturing, preferably, using single fragment recombinant antigen is examined with market assessment as coated IgG
Survey product to compare, sensitivity has a raising by a relatively large margin, and antigen its specificity is compared with using MP cultures as coated product
It is improved, and its antigen is easy to culture purified, it is more cost-effective；
2nd, it is the clinical new method that a kind of both quick and can is provided and accurately detects mycoplasma pneumoniae IgG, is provided for diagnosis MP infection
Help, there are good market prospects.
Brief description of the drawings
What Fig. 1 was represented is that recombinant expression carrier pET28a-MP digestion products carry out agarose electrophoresis testing result, is as a result shown
Show after recombinant expression carrier pET28a-MP digestions it can be seen that two bands, above one be pET28a, below one be that size is
1668bp mycoplasma pneumoniae gene.
What Fig. 2 was represented is the induced expression SDS-PAGE protein electrophoresis figures for recombinating mycoplasma pneumoniae.
What Fig. 3 was represented is the mycoplasma pneumoniae SDS-PAGE protein electrophoresis figures of purifying.
Fig. 4 is reagent strip schematic diagram of the present invention.
Fig. 5 is the performance schematic diagram of NC films of the present invention.
Embodiment
The present invention can be described in more detail in the following examples, but can not limit the present invention in any form.Also, implement
Involved experimental method, is normal experiment method unless otherwise specified in example.
Embodiment one, the structure of recombinant expression carrier containing mycoplasma pneumoniae gene and the system of restructuring MP chimeric antigens
It is standby：
1st, the screening of MP P1 albumen, p30 albumen and MPN456 Protein Epitopes
Using softwares such as ANTHEWIN, pass through whole amino acid sequences of computer analysis MP albumen（M. pneumonia FH
strain ）, filter out MP albumen and include antigenic protein sequence P1 (residues 1177-1535)；Filter out P30 eggs
White epitope（residues 170–254）With the epitope ((residues 765-846 of MPN456 albumen）, it is used in combination
Flexible polypeptide（GLy4Ser）3Interconnect.Their amino acid sequence is SEQ ID NO:1.
2nd, the acquisition of mycoplasma pneumoniae gene
Different from other pathogens, MP is used different from unique bias cryptography subsystem, and general terminator codon UGA is in MP
Middle codes for amino acid tryptophan, if can directly cause the interruption of protein translation using MP wild gene, limit restructuring MP antigens
Develop.In order to obtain the restructuring MP antigens with diagnostic significance, the present invention for MP adhesion protein 1 (P1), P30 and
MPN456 proteantigens section carries out bioinformatics Antigen Epitope Prediction, and obtain wherein may occur immune response with patients serum
The amino acid sequence of antigen fragment, while employ and do not influence the flexible polypeptide of antigen reactivity by three fragment expressing in series,
Reduce the difficulty of Prepare restructuring antigen.
The problem of in order to overcome wild MP gene codons bias, the present invention is using in Escherichia coli optimal codon reverse translation
The amino acid sequence of antigen is stated, obtains the brand-new MP Chimeric antigen genes being made up of Escherichia coli optimal codon.
According to mycoplasma pneumoniae gene（M. pneumonia FH strain）, full genome synthetic method is taken, and at it
5 ' and 3 ' ends add CATATG and AAGCTT to obtain restriction endonuclease Nde I and Hind III digestion recognition site respectively respectively
CATATG and AAGCTT, the bp of sequence length 1668, bacterium preference codon and company will be substituted for by rare codon in bacterium
It is connected on pGEM-T Vector（Promega Products）On carrier, mycoplasma pneumoniae is obtained（Mp）The full genome of chimeric antigen
Sequence, its gene order such as SEQ ID NO:Shown in 2.
3rd, recombinant expression carrier is built
The μ g of the carrier of gene plasmid containing mycoplasma pneumoniae 1 that above-mentioned steps obtain are carried out with III/Nde of restriction enzyme Hind I
Double digestion, digestion products and the 1 μ g carriers pET28a with same digestion（+）, 1% agarose electrophoresis, and glue reclaim purpose fragment
And carrier segments, the amount of 1% agarose electrophoresis detection recovery fragment, by average molecular number purpose fragment：Carrier segments=4:1 is mixed
Close, add 5 μ gT416 °C of connections of ligase are overnight.5 μ l connection products are taken to convert the prefabricated DH5a competent cells of 100 μ l,
It is coated with resistance LB flat boards, 37 °C of inversion overnight incubations.The clone containing positive plasmid is selected, contains mycoplasma pneumoniae by what is obtained
The clone designation of recombinant expression carrier pair be pET28a-MP.
4th, the digestion identification of recombinant expression carrier
PET28a-MP containing recombinant expression carrier to be identified is cloned into a small amount of cultures, alkaline process Rapid extraction plasmid, uses restriction enzyme
III/Nde of enzyme Hind I carry out double digestion, and digestion products carry out agarose electrophoresis detection, as a result as shown in Figure 1.Wherein swimming lane 1
For the agarose gel electrophoresis result of recombinant expression carrier pET28a-MP digestion products, M is DNA molecular amount standard.Specification is attached
What Fig. 1 was represented is that recombinant expression carrier pET28a-MP digestion products carry out agarose electrophoresis testing result, as a result shows restructuring table
It can be seen that two bands after up to carrier pET28a-MP digestions, above one be pET28a, below one be that size is 1668bp
Mycoplasma pneumoniae gene.
Embodiment two, the induced expression of mycoplasma pneumoniae and expansion culture
Example one obtain recombinant expression carrier pET28a-MP be transferred to e. coli bl21 (DE3) competent cell, containing
Have and cultivated on the LB culture plates of kanamycins, picking individual colonies are inoculated into the positive restructuring for screening to obtain in 5mlLB culture mediums
Bacterium carries out induced expression, and product carries out SDS-PAGE analyses, and is carried out pair with the bacterium that compares containing expression vector pET28a (+)
Than as a result as shown in Fig. 2 the expression of Figure of description 2 is the induced expression SDS-PAGE protein electrophoresises for recombinating mycoplasma pneumoniae
Figure, wherein foreign protein is more.
With the bacteria break supernatant of the control bacterium without target gene compared with total protein (swimming lane 3,4 in Fig. 2), positive restructuring
Can be observed in the bacteria break supernatant and total protein of bacterium recombinant protein band that molecular weight is about 60 kDa (swimming lane 1 in Fig. 2,
2), the molecular weight of the recombinant protein is consistent with recombinating the molecular weight of mycoplasma pneumoniae, and the preliminary judgement band is MP.It is although broken
Destination protein (swimming lane 1 in Fig. 2) in bacterium supernatant is high without destination protein (swimming lane 2 in Fig. 2) content in total protein,
But its proportion is suitable with destination protein proportion in total protein, illustrate that MP is largely expressed with soluble form, Neng Goufang
Just purified by way of nickel affinity chromatography.
Inoculation identifies that the engineering bacteria single bacterium of purposeful protein expression falls on 200ml containing kanamycin's by SDS-PAGE
LB triangular flasks, 200rpm, 37 °C of concussion and cultivates are stayed overnight, and next day, by 5% inoculum concentration, are inoculated into 1Ls of the 400mlLB containing kanamycin
In triangular flask, 37 °C, 240rpm concussion and cultivates to OD600=0.5, put ice-water bath and be cooled to 22 °C, add IPTG induced pneumonitis branch
Chlamydia genes are expressed, wherein, IPTG concentration is 0.01 mM, and induction time is 6 hours, and concussion speed is 240rpm/min.
Embodiment three, mycoplasma pneumoniae isolate and purify
Fusion tag is carried on the recombinant expression carrier pET28a-MP built due to above-described embodiment 1（His·Tag）Gene
Sequence, so with HisTag labels on the destination protein given expression to.Therefore, passed through using the agarose for being complexed Ni affine
Chromatography can carry out the purifying of destination protein, and affinity column is purchased from GE Healthcare companies.
After method according to above-described embodiment 2 carries out induced expression with IPTG, centrifuge 5 min with 10000 rpm/min and receive
Obtain bacterial sediment；By bacterial sediment with Buffer A (20 mM Tris, 0.5 M NaCl pH 8.0)（60ml Buffer
A/L thalline）It is resuspended, 250 W ultrasonic degradations 90 times, each 5s, interval 10s, 12000 rpm/min centrifugations 20 under the conditions of 4 °C
Min collects supernatant and prepares loading.
Specific purification step is as follows：
1）It is constant to OD280 readings with Buffer A balance Ni posts；
2）The sample filtration supernatant upper prop of collection；
3）Post elution, which is crossed, with the Buffer A containing 40 mM imidazoles removes foreign protein；
4）Post is crossed with the Buffer A containing 300 mM imidazoles are slow, the destination protein of purifying is collected into centrifuge tube；
5）Destination protein after purification is taken to carry out SDS-PAGE, observation, the purification effect of destination protein；
As a result as shown in figure 3, the expression of Figure of description 3 is the mycoplasma pneumoniae SDS-PAGE protein electrophoresis figures purified；Wherein,
Swimming lane 1 is the SDS-PAGE testing results after Sample Purification on Single, and swimming lane 2M is the kDa of 14.0kda~116.0 protein molecular weight
Standard.
Example IV, mycoplasma pneumoniae detection reagent prepare and detection
First, the preparation of collaurum
Take the gold chlorides of 1ml 1%（HAuCl4）Solution, it is added in 100ml water, is heated to boiling, adds the citric acids of 1.5ml 1%
Trisodium, mixing is boiled 5 minutes, until color no longer changes.Now obtained colloid gold particle is 30nm.
2nd, prepared by gold mark
1st, antigen diluent
Take a certain amount of mycoplasma pneumoniae recombination antigen（MP-Ag）, it is dilute with pH 8.00.1M PB after dialysis, centrifugal treating
Release to 1mg/ml, 4 DEG C save backup.
2nd, antigen is coupled
The 30nm collaurums that 250ml is prepared are taken, pH to 9.0 is adjusted with 1M NaOH, then adds above-mentioned antigen 1 ml, magnetic force stirs
Mix and stirred 1 hour on device, then add a certain amount of BSA to final concentration 1%, continue stirring 0.5 hour.
3rd, the preparation of gold mark working solution
The above-mentioned colloidal gold solution for being coupled antigen is centrifuged（4000rpm, 15 minutes）, precipitation is discarded, retains solution.So
After carry out secondary centrifuging（12000rpm, 30 minutes）, abandoning supernatant, retain precipitation.Precipitation is dilute with the gold mark containing 20% sucrose
Release liquid（pH8.0 0.02M Tris+1%BSA）Redissolve to 10mL, be transferred in brown bottle, 4 DEG C save backup.
4th, the preparation of colloidal-gold strip
Above-mentioned gold mark working solution 10ml is taken, is loaded to metal spraying machine, it is 1.5 μ l/cm that setup parameter, which chooses discharge rate,.Air pump is opened, is treated
After stable gas pressure, start metal spraying machine, gold mark working solution is uniformly sprayed onto in 30cm*6.5cm gold standard pad.By the good gold mark work of spray
The gold standard pad of liquid is put into drying in 37 DEG C of insulating boxs and taken out after 12 hours, is cut into the wide bars of 8mm and is put into valve bag, then encloses
The aluminium foil bag of built-in drier, 20 DEG C save backup.
3rd, the selection of NC films
Choose the NC films of three higher companies of in the market utilization rate：PALL Vivid 170、Millipore 135、
Sartorius CN 140, specification 2.5*30cm, tape backing, aperture are that 8um is contrasted, it is desirable to the width of film each point and thickness
Spend homogeneous；Performance of metalling run out meets the requirements：4cm time is chromatographed in 130s-140s, and without inclination and blank；Performance test meets
It is required that：Three kinds of NC films are coated with 1.0mg/mL mouse anti-human igg respectively and match corresponding colloidal-gold strip, a negative sample of test
Originally, a critical negative sample（It is required that 3-5 minutes develop the color）And a critical positive sample（It is required that developed the color within 5 minutes）
As shown in figure 5, interpretation of result, we select lifes of the Millipore 135 as mycoplasma pneumiae anti-body detection reagent box
NC films are used in production.
4th, the coating of NC films
1st, antibody and how anti-dilution
By a certain amount of mouse anti-human igg monoclonal antibody（IgG-mab）, it is dilute with pH7.4 0.01M PBS after dialysis, centrifugal treating
Release to final concentration 1.0mg/ml, 4 DEG C save backup.
By a certain amount of mycoplasma pneumoniae recombination antigen monoclonal antibody（MP-mab）, after dialysis, centrifugal treating, use
PH7.4 0.01M PBS are diluted to final concentration 1.0mg/ml, and 4 DEG C save backup.
2nd, antigen coat
By above-mentioned antigen and it is how anti-be loaded to respectively in T, C post of a film machine, setup parameter selection discharge rate be 1.0 μ l/cm.Adjustment
The position of film head is drawn, makes T, C line in the middle part of NC, and spacing 5mm.Start point film machine, by T, C line solution coating to NC films.Will
The gold standard pad of the good gold mark working solution of spray is put into dried 24 hours in 37 DEG C of insulating boxs after take out, be put into valve bag, then enclose
The aluminium foil bag of built-in drier, 20 DEG C save backup.
5th, the processing of glass
Glass fibre element film is cut into 17*300mm bars, is put into after being soaked 1 hour in glass treatment fluid and takes out, be put into 37 DEG C of perseverances
Taken out after being dried 12 hours in incubator, be put into valve bag, then enclose the aluminium foil bag of built-in drier, 20 DEG C save backup.
Glass prescription for the treatment of liquid
6th, the assembling of reagent strip
The NC films 2 handled well, colloidal-gold strip 4, glass and blotting paper are pasted in PVC board 1 respectively, are then cut into 4.0mm
Bar, as shown in figure 4, the PVC board head and the tail both ends are sample pad 5 and adsorptive pads 3.
7th, with restructuring MP chimeric antigens detection MP IgG antibodies
The mycoplasma pneumiae anti-body detection reagent box assembled is applied to the detection of mycoplasma pneumoniae antibody.As a result show, it is sensitive
Degree and specificity have more significant raising compared with having listed other Products.And with containing HBsAg antibody, HIV
Cross reaction does not occur for antibody, HCV antibody, TP antibody, HAV antibody samples and HTLV antibody samples.
In the case where not conflicting, the feature in embodiments of the invention and embodiment can be mutually combined.It is described above
Only it is the exemplary embodiment of the present invention, not for limiting the scope of the invention, protection scope of the present invention is by weighing
Profit requires to determine.
SEQUENCE LISTING
<110>Hangzhou Long Ji Bioisystech Co., Ltd
<120>Mycoplasma pneumoniae chimeric antigen, the antigen detecting agent and both preparation methods
<130> 201708
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 556
<212> PRT
<213> Mycoplasma pneumoniae
<400> 1
Gly Ala His Ile Val Arg Thr Leu Leu Leu Ala Ala Ser Gln Lys Asp
1 5 10 15
Val His Ser Lys Phe Thr Gln Lys Ile Asp Gln Gln Asn Thr Ala Ser
20 25 30
Thr Thr Ser Asp Val Thr Val Lys Lys Ala Asp Ser Ser Gln Asp Ser
35 40 45
Ser Lys Ser Asn Thr Glu Glu Glu Lys Trp Asp Asp Val Thr Ser Ala
50 55 60
Asp Leu Phe Lys Asp Asp Pro Tyr Val Leu Lys Asn Phe Gly Asp Ala
65 70 75 80
Lys Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
85 90 95
Ser Arg Thr Gly Phe Pro Pro Gln Pro Gly Met Ala Pro Arg Pro Gly
100 105 110
Met Pro Pro His Pro Gly Met Ala Pro Arg Ser Gly Phe Pro Pro Gln
115 120 125
Pro Gly Met Ala Pro Arg Pro Gly Met Pro Pro His Pro Gly Met Ala
130 135 140
Pro Arg Pro Gly Phe Pro Pro Gln Pro Gly Met Ala Pro Arg Pro Gly
145 150 155 160
Met Pro Pro His Pro Gly Met Ala Pro Arg Pro Gly Phe Pro Pro Gln
165 170 175
Pro Gly Met Ala Pro Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
180 185 190
Gly Gly Gly Gly Ser Ala Ala Phe Arg Gly Ser Trp Val Asn Arg Leu
195 200 205
Gly Arg Val Glu Ser Val Trp Asp Leu Lys Gly Val Trp Ala Asp Gln
210 215 220
Ala Gln Ser Asp Ser Gln Gly Ser Thr Thr Thr Ala Thr Arg Asp Ala
225 230 235 240
Leu Pro Glu His Pro Asn Ala Leu Ala Phe Gln Val Ser Val Val Glu
245 250 255
Ala Ser Ala Tyr Lys Pro Asn Thr Ser Ser Gly Gln Thr Gln Ser Thr
260 265 270
Asn Ser Ser Pro Tyr Leu His Leu Val Lys Pro Lys Lys Val Ile Gln
275 280 285
Ser Asp Lys Leu Asp Asp Asp Leu Lys Asn Leu Leu Asp Pro Asn Gln
290 295 300
Val Arg Thr Lys Leu Arg Gln Ser Phe Gly Thr Asp His Ser Thr Gln
305 310 315 320
Pro Gln Pro Gln Ser Leu Lys Thr Thr Thr Pro Val Phe Gly Thr Ser
325 330 335
Ser Gly Asn Leu Ser Ser Val Leu Ser Gly Gly Gly Ala Gly Gly Gly
340 345 350
Ser Ser Gly Ser Gly Gln Ser Gly Val Asp Leu Ser Pro Val Glu Lys
355 360 365
Val Ser Gly Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn
370 375 380
Thr Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp
385 390 395 400
Val Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn
405 410 415
Asp Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp
420 425 430
Gly Glu Gly Gln Thr Ala Asp Thr Gly Pro Gln Ser Val Lys Phe Lys
435 440 445
Ser Pro Asp Gln Ile Asp Phe Asn Arg Leu Phe Thr His Pro Val Thr
450 455 460
Asp Leu Phe Asp Pro Val Thr Met Leu Val Tyr Asp Gln Tyr Ile Pro
465 470 475 480
Leu Phe Ile Asp Ile Pro Ala Ser Val Asn Pro Lys Met Val Arg Leu
485 490 495
Lys Val Leu Ser Phe Asp Thr Asn Glu Gln Ser Leu Gly Leu Arg Leu
500 505 510
Glu Phe Phe Lys Pro Asp Gln Asp Thr Gln Pro Asn Asn Asn Val Gln
515 520 525
Val Asn Pro Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr Ala Ser Ser
530 535 540
Gln Gly Pro Gln Thr Leu Phe Ser Pro Phe Asn Gln
545 550 555
SEQUENCE LISTING
<110>Hangzhou Long Ji Bioisystech Co., Ltd
<120>Mycoplasma pneumoniae chimeric antigen, the antigen detecting agent and both preparation methods
<130> 201708
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 0
<212> PRT
<213> Mycoplasma pneumoniae
<400> 1
000
<210> 2
<211> 1668
<212> DNA
<213> Mycoplasma pneumoniae
<400> 2
ggcgcgcata ttgtgcgcac cctgctgctg gcggcgagcc agaaagatgt gcatagcaaa 60
tttacccaga aaattgatca gcagaacacc gcgagcacca ccagcgatgt gaccgtgaaa 120
aaagcggata gcagccagga tagcagcaaa agcaacaccg aagaagaaaa atgggatgat 180
gtgaccagcg cggatctgtt taaagatgat ccgtatgtgc tgaaaaactt tggcgatgcg 240
aaagcgggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag ccgcaccggc 300
tttccgccgc agccgggcat ggcgccgcgc ccgggcatgc cgccgcatcc gggcatggcg 360
ccgcgcagcg gctttccgcc gcagccgggc atggcgccgc gcccgggcat gccgccgcat 420
ccgggcatgg cgccgcgccc gggctttccg ccgcagccgg gcatggcgcc gcgcccgggc 480
atgccgccgc atccgggcat ggcgccgcgc ccgggctttc cgccgcagcc gggcatggcg 540
ccgcgcggcg gcggcggcag cggcggcggc ggcagcggcg gcggcggcag cgcggcgttt 600
cgcggcagct gggtgaaccg cctgggccgc gtggaaagcg tgtgggatct gaaaggcgtg 660
tgggcggatc aggcgcagag cgatagccag ggcagcacca ccaccgcgac ccgcgatgcg 720
ctgccggaac atccgaacgc gctggcgttt caggtgagcg tggtggaagc gagcgcgtat 780
aaaccgaaca ccagcagcgg ccagacccag agcaccaaca gcagcccgta tctgcatctg 840
gtgaaaccga aaaaagtgat tcagagcgat aaactggatg atgatctgaa aaacctgctg 900
gatccgaacc aggtgcgcac caaactgcgc cagagctttg gcaccgatca tagcacccag 960
ccgcagccgc agagcctgaa aaccaccacc ccggtgtttg gcaccagcag cggcaacctg 1020
agcagcgtgc tgagcggcgg cggcgcgggc ggcggcagca gcggcagcgg ccagagcggc 1080
gtggatctga gcccggtgga aaaagtgagc ggctggctgg tgggccagct gccgagcacc 1140
agcgatggca acaccagcag caccaacaac ctggcgccga acaccaacac cggcaacgat 1200
gtggtgggcg tgggccgcct gagcgaaagc aacgcggcga aaatgaacga tgatgtggat 1260
ggcattgtgc gcaccccgct ggcggaactg ctggatggcg aaggccagac cgcggatacc 1320
ggcccgcaga gcgtgaaatt taaaagcccg gatcagattg attttaaccg cctgtttacc 1380
catccggtga ccgatctgtt tgatccggtg accatgctgg tgtatgatca gtatattccg 1440
ctgtttattg atattccggc gagcgtgaac ccgaaaatgg tgcgcctgaa agtgctgagc 1500
tttgatacca acgaacagag cctgggcctg cgcctggaat tttttaaacc ggatcaggat 1560
acccagccga acaacaacgt gcaggtgaac ccgaacaacg gcgattttct gccgctgctg 1620
accgcgagca gccagggccc gcagaccctg tttagcccgt ttaaccag 1668
Claims (9)
1. a kind of mycoplasma pneumoniae chimeric antigen amino acid sequence, it is characterised in that include such as SEQ ID NO:Amino shown in 1
Acid sequence.
2. a kind of complete genome sequence of the mycoplasma pneumoniae chimeric antigen of full genome synthesis, it is characterised in that include such as SEQ ID
NO:Amino acid sequence shown in 2.
A kind of 3. method for building mycoplasma pneumoniae chimeric antigen amino acid sequence as claimed in claim 1, it is characterised in that
It comprises the following steps：
S101, which is filtered out, includes antigenic protein sequence P1 in mycoplasma pneumoniae albumen：Residues 1177-1535, P30 eggs
White epitope：Residues 170-254 and MPN456 albumen epitope：Residues 765-846, and with flexibility
Polypeptide interconnects.
4. a kind of method for the mycoplasma pneumoniae chimeric antigen for building full genome synthesis as claimed in claim 2, its feature exist
Comprise the following steps in it：
S201, which is filtered out, includes antigenic protein sequence P1 in mycoplasma pneumoniae albumen：Residues 1177-1535, P30 eggs
White epitope：Residues 170-254 and MPN456 albumen epitope：Residues 765-846, and with flexibility
Polypeptide interconnects；
S202 is according to mycoplasma pneumoniae gene, using full genome synthetic method, at its 5 ' and 3 ' end respectively plus CATATG and
AAGCTT to obtain restriction endonuclease Nde I and Hind III digestion recognition site CATATG and AAGCTT, sequence length respectively
1668 bp, rare codon it will be substituted for bacterium preference codon in bacterium and be connected on pGEM-T Vector carriers,
Obtain the complete genome sequence of mycoplasma pneumoniae chimeric antigen.
5. the preparation method of a kind of mycoplasma pneumoniae chimeric antigen as described in claim 2 or 4, it is characterised in that it is included such as
Lower step：
S301, which is filtered out, includes antigenic protein sequence P1 in mycoplasma pneumoniae albumen：Residues 1177-1535, P30 eggs
White epitope：Residues 170-254 and MPN456 albumen epitope：Residues 765-846, and with flexibility
Polypeptide interconnects；
S302 is according to mycoplasma pneumoniae gene, using full genome synthetic method, at its 5 ' and 3 ' end respectively plus CATATG and
AAGCTT to obtain restriction endonuclease Nde I and Hind III digestion recognition site CATATG and AAGCTT, sequence length respectively
1668 bp, rare codon it will be substituted for bacterium preference codon in bacterium and be connected on pGEM-T Vector carriers,
Obtain the complete genome sequence of mycoplasma pneumoniae chimeric antigen；
The μ g of the carrier of gene plasmid containing mycoplasma pneumoniae 1 III/Nde of restriction enzyme Hind I that S303 obtains above-mentioned steps
Carry out double digestion, digestion products and the 1 μ g carriers pET28a with same digestion（+）, 1% agarose electrophoresis, and glue reclaim purpose
Fragment and carrier segments, the amount of 1% agarose electrophoresis detection recovery fragment, by average molecular number purpose fragment：Carrier segments=4:1
Mixing, add 5 μ gT416 °C of connections of ligase overnight, take 5 μ l connection products to convert the prefabricated DH5a competence of 100 μ l thin
Born of the same parents, resistance LB flat boards are coated with, 37 °C of inversion overnight incubations, the clone containing positive plasmid is selected, contains pneumonia branch by what is obtained
The clone designation of the recombinant expression carrier pair of substance is pET28a-MP.
6. the preparation method of mycoplasma pneumoniae chimeric antigen as claimed in claim 5, it is characterised in that after step S303 also
Digestion authentication step including following recombinant expression carrier：
PET28a-MP containing recombinant expression carrier to be identified is cloned into a small amount of cultures, alkaline process Rapid extraction plasmid, uses restriction enzyme
III/Nde of enzyme Hind I carry out double digestion, and digestion products carry out agarose electrophoresis detection, and it is 1668bp's that can measure a size
Mycoplasma pneumoniae gene.
7. a kind of mycoplasma pneumoniae detection reagent bar, the reagent strip includes PVC board and the NC films, the Jin Biao that are pasted onto in PVC board
Bar, glass and blotting paper, the PVC board head and the tail both ends are sample pad and adsorptive pads, it is characterised in that the T posts coating of the NC films
Just like the mycoplasma pneumoniae chimeric antigen of the restructuring of claim 2 or 4, C posts are coated with just like the restructuring of claim 2 or 4
Mycoplasma pneumoniae inosculating antibody culture monoclonal antibody.
8. a kind of preparation method of mycoplasma pneumoniae detection reagent, it is characterised in that it comprises the following steps：
The preparation of collaurum；
It is prepared by gold mark：
1st, antigen diluent：
A certain amount of mycoplasma pneumoniae recombination antigen as described in claim 2 or 4 is taken, after dialysis, centrifugal treating, uses pH
8.00.1M PB is diluted to 1mg/ml, and 4 DEG C save backup；
2nd, antigen is coupled；
3rd, gold mark working solution is prepared；
4th, colloidal-gold strip is prepared；
5th, the assembling of reagent strip：
NC films, colloidal-gold strip, glass and the blotting paper handled well are pasted in PVC board respectively, the PVC board head and the tail both ends are sample
Product pad and adsorptive pads.
A kind of 9. preparation method of mycoplasma pneumoniae detection reagent as claimed in claim 8, it is characterised in that this method it is detailed
Carefully step is：
The preparation of S801 collaurums：
The chlorauric acid solutions of 1ml 1% are taken, are added in 100ml water, are heated to boiling, add the trisodium citrates of 1.5ml 1%, are mixed
Boil 5 minutes, until color no longer changes, now obtained colloid gold particle is 30nm；
It is prepared by S802 gold marks：
1st, antigen diluent：
A certain amount of mycoplasma pneumoniae recombination antigen as described in claim 2 or 4 is taken, after dialysis, centrifugal treating, uses pH
8.00.1M PB is diluted to 1mg/ml, and 4 DEG C save backup；
2nd, antigen is coupled：
The 30nm collaurums that 250ml is prepared are taken, pH to 9.0 is adjusted with 1M NaOH, then adds above-mentioned antigen 1 ml, magnetic force stirs
Mix and stirred 1 hour on device, then add a certain amount of BSA to final concentration 1%, continue stirring 0.5 hour；
3rd, the preparation of gold mark working solution
The above-mentioned colloidal gold solution for being coupled antigen is centrifuged, discards precipitation, retains solution；Then secondary centrifuging is carried out,
Abandoning supernatant, retain precipitation；Precipitation is redissolved to 10mL with the gold mark dilution containing 20% sucrose, is transferred in brown bottle, 4 DEG C
Save backup；
4th, the preparation of colloidal-gold strip：
Above-mentioned gold mark working solution 10ml is taken, is loaded to metal spraying machine, it is 1.5 μ l/cm that setup parameter, which chooses discharge rate, opens air pump, treats
After stable gas pressure, start metal spraying machine, gold mark working solution is uniformly sprayed onto in 30cm*6.5cm gold standard pad；By the good gold mark work of spray
The gold standard pad of liquid is put into drying in 37 DEG C of insulating boxs and taken out after 12 hours, is cut into the wide bars of 8mm and is put into valve bag, then encloses
The aluminium foil bag of built-in drier, 20 DEG C save backup；
The coating of S803 NC films：
1st, antibody and how anti-dilution
By a certain amount of mouse anti-human igg monoclonal antibody, after dialysis, centrifugal treating, final concentration is diluted to pH7.4 0.01M PBS
1.0mg/ml, 4 DEG C save backup；
By a certain amount of mycoplasma pneumoniae recombination antigen monoclonal antibody, after dialysis, centrifugal treating, with pH7.4 0.01M
PBS is diluted to final concentration 1.0mg/ml, and 4 DEG C save backup；
2nd, antigen coat
By above-mentioned antigen and it is how anti-be loaded to respectively in T, C post of a film machine, setup parameter selection discharge rate be 1.0 μ l/cm, adjust
The position of film head is drawn, makes T, C line in the middle part of NC, and spacing 5mm, start point film machine, T, C line solution are coated with onto NC films, will
The gold standard pad of the good gold mark working solution of spray is put into dried 24 hours in 37 DEG C of insulating boxs after take out, be put into valve bag, then enclose
The aluminium foil bag of built-in drier, 20 DEG C save backup；
The processing of S804 glasses：
Glass fibre element film is cut into 17*300mm bars, is put into after being soaked 1 hour in glass treatment fluid and takes out, be put into 37 DEG C of perseverances
Taken out after being dried 12 hours in incubator, be put into valve bag, then enclose the aluminium foil bag of built-in drier, 20 DEG C save backup；
The assembling of S805 reagent strips：
NC films, colloidal-gold strip, glass and the blotting paper handled well are pasted in PVC board respectively, are then cut into 4.0mm bars,
The PVC board head and the tail both ends are sample pad and adsorptive pads.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110759977A (en) * | 2018-12-29 | 2020-02-07 | 南京拂晓生物科技有限公司 | Preparation and application of mycoplasma pneumoniae recombinant antigen |
| CN110964089A (en) * | 2019-11-06 | 2020-04-07 | 南京诺唯赞医疗科技有限公司 | Mycoplasma pneumoniae antigen and application thereof in simultaneous quantitative detection of content of mycoplasma pneumoniae IgG and IgM in peripheral blood |
| CN111087453A (en) * | 2019-12-31 | 2020-05-01 | 南京拂晓生物科技有限公司 | Preparation method and application method of chlamydia pneumoniae recombinant antigen |
| CN111548423A (en) * | 2020-06-04 | 2020-08-18 | 珠海丽珠试剂股份有限公司 | Mycoplasma pneumoniae fusion antigen and preparation method and application thereof |
| CN112979769A (en) * | 2021-03-08 | 2021-06-18 | 珠海丽禾医疗诊断产品有限公司 | Amino acid sequence, protein, preparation method and application thereof |
| CN113024642A (en) * | 2021-03-08 | 2021-06-25 | 珠海丽禾医疗诊断产品有限公司 | Protein based on mycoplasma pneumoniae and preparation method and application thereof |
| CN115028716A (en) * | 2022-06-10 | 2022-09-09 | 青岛汉德森生物科技有限公司 | Monoclonal antibody and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820394A (en) * | 2013-12-31 | 2014-05-28 | 杭州隆基生物技术有限公司 | Anti-morphine monoclonal antibody, cell strain for generating anti-morphine monoclonal antibody, morphine detection kit and manufacturing method thereof |
| CN103834668A (en) * | 2014-03-17 | 2014-06-04 | 英诺特（唐山）生物技术有限公司 | Recombination mycoplasma pneumoniae protein and application thereof |
| CN105063065A (en) * | 2015-08-25 | 2015-11-18 | 四川农业大学 | Codon optimization type 1 type duck hepatitis A virus VP1 gene and application of recombinant protein of codon optimization type 1 type duck hepatitis A virus VP1 gene |
| CN105859843A (en) * | 2016-04-29 | 2016-08-17 | 兰州雅华生物技术有限公司 | Preparation method of respiratory syncytial virus antigen and rapid respiratory syncytial virus antibody detection kit prepared from antigen |
| CN105859845A (en) * | 2016-04-14 | 2016-08-17 | 石河子大学 | Mycoplasma ovipneumoniae multi-epitope fusion antigen MO-meAg5 and preparing method and application thereof |
-
2017
- 2017-08-17 CN CN201710708246.8A patent/CN107573417A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103820394A (en) * | 2013-12-31 | 2014-05-28 | 杭州隆基生物技术有限公司 | Anti-morphine monoclonal antibody, cell strain for generating anti-morphine monoclonal antibody, morphine detection kit and manufacturing method thereof |
| CN103834668A (en) * | 2014-03-17 | 2014-06-04 | 英诺特（唐山）生物技术有限公司 | Recombination mycoplasma pneumoniae protein and application thereof |
| CN105063065A (en) * | 2015-08-25 | 2015-11-18 | 四川农业大学 | Codon optimization type 1 type duck hepatitis A virus VP1 gene and application of recombinant protein of codon optimization type 1 type duck hepatitis A virus VP1 gene |
| CN105859845A (en) * | 2016-04-14 | 2016-08-17 | 石河子大学 | Mycoplasma ovipneumoniae multi-epitope fusion antigen MO-meAg5 and preparing method and application thereof |
| CN105859843A (en) * | 2016-04-29 | 2016-08-17 | 兰州雅华生物技术有限公司 | Preparation method of respiratory syncytial virus antigen and rapid respiratory syncytial virus antibody detection kit prepared from antigen |
Non-Patent Citations (6)
| Title |
|---|
| ELISA BEGHETTO 等: "Discovery of new Mycoplasma pneumoniae antigens by use of a whole-genome lambda display library", 《MICROBES AND INFECTION》 * |
| F. MONTAGNANI 等: "Use of recombinan t chimeric antigens for the serodiagnosis of Mycoplasma pneumoniae infection", 《EUR J CLIN MICROBIOL INFECT DIS》 * |
| NCBI: "adhersin-related protein P30 [Mycoplasma pneumoniae]", 《GENBANK DATABASE》 * |
| NCBI: "adhesin P1 [Mycoplasma pneumoniae]", 《GENBANK DATABASE》 * |
| NCBI: "RecName: Full=Uncharacterized lipoprotein MG321 homolog; Flags: Precursor", 《GENBANK DATABASE》 * |
| 王丽琼 等: "肺炎支原体主要黏附蛋白的研究进展", 《现代生物医学进展 》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110759977A (en) * | 2018-12-29 | 2020-02-07 | 南京拂晓生物科技有限公司 | Preparation and application of mycoplasma pneumoniae recombinant antigen |
| CN110964089A (en) * | 2019-11-06 | 2020-04-07 | 南京诺唯赞医疗科技有限公司 | Mycoplasma pneumoniae antigen and application thereof in simultaneous quantitative detection of content of mycoplasma pneumoniae IgG and IgM in peripheral blood |
| CN111087453A (en) * | 2019-12-31 | 2020-05-01 | 南京拂晓生物科技有限公司 | Preparation method and application method of chlamydia pneumoniae recombinant antigen |
| CN111548423A (en) * | 2020-06-04 | 2020-08-18 | 珠海丽珠试剂股份有限公司 | Mycoplasma pneumoniae fusion antigen and preparation method and application thereof |
| CN111548423B (en) * | 2020-06-04 | 2021-11-30 | 珠海丽珠试剂股份有限公司 | Mycoplasma pneumoniae fusion antigen and preparation method and application thereof |
| CN112979769A (en) * | 2021-03-08 | 2021-06-18 | 珠海丽禾医疗诊断产品有限公司 | Amino acid sequence, protein, preparation method and application thereof |
| CN113024642A (en) * | 2021-03-08 | 2021-06-25 | 珠海丽禾医疗诊断产品有限公司 | Protein based on mycoplasma pneumoniae and preparation method and application thereof |
| CN115028716A (en) * | 2022-06-10 | 2022-09-09 | 青岛汉德森生物科技有限公司 | Monoclonal antibody and application thereof |
| CN115028716B (en) * | 2022-06-10 | 2023-02-03 | 青岛汉德森生物科技有限公司 | Monoclonal antibody and application thereof |
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