CN105274062A - Streptococcus agalactiae monoclonal antibody and preparation method and application thereof - Google Patents
Streptococcus agalactiae monoclonal antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a streptococcus agalactiae monoclonal antibody. The streptococcus agalactiae monoclonal antibody is secreted by a cell strain preserved with a preservation number being CCTCC NO: C2015177 in China Center for Type Culture Collection. The invention further discloses a preparation method and application of the streptococcus agalactiae monoclonal antibody. Rapid colloidal gold test paper prepared by the streptococcus agalactiae monoclonal antibody is high in sensitivity, good in specificity and convenient and rapid to operate, the problem of existing streptococcus agalactiae detection is solved, and application prospect is promising.
Description
Technical field
The present invention relates to a kind of streptococcus agalactiae monoclonal antibody and its production and use.
Background technology
Tilapia is the tropic fishes originating in Africa, and the seventies and eighties China of the upper world repeatedly from overseas introduction, and selects the kind that growing way is fast, output is high, disease resistance is strong, after this successfully promotes cultivation rapidly.At present, China is not only tilapia breeding production state maximum in the world, is also maximum tilapia export State.Domestic tilapia cultivation concentrates on the southern areas such as Guangdong, Hainan, Guangxi, Fujian.Within 2009, start successively to break out tilapia " exophthalmos " in tilapia cultivation compact district, this disease breaks with tremendous force, and sickness rate is up to 35%, and morbidity fish mortality ratio is close to 100%, and the disease time length is long, and medical treatment is difficult to symptom management, and raiser loses bitterness.
Discovery promptly and accurately and diagnosis cause of disease, be successfully the prerequisite of prevention and therapy tilapia streptococcicosis.At present, for the diagnosis of streptococcus agalactiae, main employing (1) molecular biological method, as PCR method is diagnosed, these class methods depend on valuable plant and instrument and technical professional, reach 4-6 hour detection time, are unfavorable for very much promoting the use in basic unit.(2) conventional isolated culture, the microscope form depending on professional operator judges, easily causes erroneous judgement, and the incubation time of these class methods reaches 18-24 hour, is not also suitable for the rapid detection of streptococcus agalactiae.
Summary of the invention
In order to solve foregoing problems, the invention provides a kind of new streptococcus agalactiae monoclonal antibody and preparation method thereof.
The present invention provide firstly a kind of hybridoma cell strain producing streptococcus agalactiae monoclonal antibody, and it is by the preserving number of China typical culture collection center (CCTCC) preservation: the cell strain of CCTCCNO:C2015177.
The present invention expresses the cell strain (Classification And Nomenclature is hybridoma cell strain TWDB-WR-2) of monoclonal antibody CE12, China typical culture collection center (CCTCC) is deposited on October 21st, 2015, its address is: China. Wuhan. and Wuhan University, its preserving number is: CCTCCNO:C2015177.
The invention provides the method for aforementioned hybridoma cells strain, it comprises the steps:
(1) for antigen, BALB/c mouse is inoculated, separating mouse splenocyte with aminoacid sequence recombinant protein as shown in SEQIDNO.1;
(2) get mouse boosting cell, merge with medullary cell, screening.
The invention provides a kind of streptococcus agalactiae monoclonal antibody, it is the monoclonal antibody of being secreted by aforementioned cells strain.
The invention provides the method for aforementioned monoclonal antibody, it comprises the steps:
1) get aforementioned hybridoma cells strain, be injected in BALB/c mouse ascites and breed;
2) collect ascites, use ProteinGSepharose affinity chromatography column purification.
The invention provides aforementioned streptococcus agalactiae monoclonal antibody and detect the purposes in streptococcus agalactiae.
The invention provides the purposes of aforementioned streptococcus agalactiae monoclonal antibody in the reagent of preparation detection streptococcus agalactiae.
The invention provides the purposes of aforementioned streptococcus agalactiae monoclonal antibody in the colloidal gold fast detecting test paper of preparation detection streptococcus agalactiae.
Preferably, sample to be checked is aquaculture water or fish body.
The present invention is adopted to prepare streptococcus agalactiae Sip monoclonal antibody CE12, the high specificity of itself, only be combined with streptococcus agalactiae, and with other bacterium no cross reactions, titre is high, and binding ability is strong, the streptococcus agalactiae Test paper adopting this albumen to prepare is highly sensitive, high specificity, reproducible, accurately can detect actual sample, effectively can solve existing streptococcus agalactiae and detect existence complexity, inaccurate problem, application prospect is good.
The test paper prepared at this antibody there is no the report of the quick detection test paper for streptococcus agalactiae before successfully developing in the world.From existing document, the biomaterial developed required for this test paper is very difficult to preparation and obtains.Therefore, successfully develop the test paper prepared by this antibody and still belong to the first time in the world, be in world lead level.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
The purification result of Fig. 1 monoclonal antibody of the present invention;
Fig. 2 aquatic products common bacteria immunoblot results.1-A first antibody: NC7b; 1-B first antibody: CE12;
The structure of Fig. 3 test paper, 1:PVC base plate; 2: nitrocellulose filter; 3: colloidal gold pad; 4: absorbent pad; 5: detection line; 6: nature controlling line; 7: sample pad;
Fig. 4 sensitivity technique result.1:6 × 10
10cFU/ml; 2:6 × 10
9cFU/ml; 3:6 × 10
8cFU/ml; 4:6 × 10
7cFU/ml; 5:6 × 10
6cFU/ml; CLine: nature controlling line; TLine: detection line;
The specificity test of Fig. 5 test strip;
Fig. 6 falls ill the detected result of fish tissues and water body.
Embodiment
The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to test kit specification sheets.
Experiment material:
Bacterium: streptococcus agalactiae TW3 is used for clone and prepares recombinant antigen, streptococcus agalactiae (ATCC51487), streptococcus agalactiae C918 (Niu Yuan), streptococcus agalactiae TW7 (source of fish), streptococcus agalactiae TW10 (source of fish), Channel-catfish tarda, enterococcus faecalis, Streptococcus iniae, Aeromonas caviae, Salmonella typhimurium, streptococcus aureus, vibrio cholerae, Vibrio parahaemolyticus, Aeromonas hydrophila, subtilis, vibrio alginolyticus, enterobacter cloacae, Shewanella putrefaciens, Klebsiella pneumonia, helicobacter pylori, germ oligotrophy unit cell, Proteus mirabilis, Oidium tropicale, salmonella typhi, salmonella paratyphi, serratia marcescens, colon bacillus, Candida albicans, citrobacter freundii, Pseudomonas aeruginosa, staphylococcus haemolyticus, Acinetobacter bauamnnii and gonorrhoea a kind of apple plucked instrument Salmonella are preserved by Tong Wei share Instituut Voor Veehouderij En Diergezondheid (Id-Dlo).
The preparation of embodiment 1 streptococcus agalactiae Sip of the present invention monoclonal antibody NC7b
1, hybridoma preparation
The preparation of 1.1 recombinant antigens
1) gene clone.The genomic dna of streptococcus agalactiae TW3 is extracted by the DNA extraction kit of Promega company, according to streptococcus agalactiae sip gene (tilapia source, HQ878436.1, Genbank), design primer as shown in table 1, upstream and downstream primer comprises BamHI and SalI restriction endonuclease sites respectively.Adopt the primer pair amplifies sip gene of design, amplification condition is as follows: after DNA94 DEG C of denaturation 5min, setting program 94 DEG C, 30s, 55 DEG C, 35s and 72 DEG C, 78s, totally 30 circulations; Then 72 DEG C extend 10min.
2) conversion, expression and purification.Be connected on pET32a carrier by the sipDNA after amplification, this carrier contains the sequence of coding hexahistine.With pET32a-sip transform E. coli BL21.Transformant 37 DEG C of cultivations in the LB liquid nutrient medium containing 100 μ g/mL kantlex.Add 0.8mM isopropyl-β-D-thiogalactoside(IPTG) (IPTG) and cultivate 5h again in 37 DEG C, to express the restructuring Sip (rSip) of band hexahistine label.Ultrasonic vibration extracts the soluble part containing rSip, then uses IMAC affinity column to carry out purifying and wash-out, and uses bicinchoninic acid detection kit (Sigma-Aldrich) to measure protein concentration.
Table 1 increases son (~ 1302bp) for the primer of expressing sip gene
3) electrophoresis and immunoblotting.Carry out SDS-PAGE electrophoresis (gel strength 12%) to the rSip of purifying, coomassie brilliant blue staining or immunoblotting show protein bars, use resisting for streptococcus agalactiae more simultaneously, to detect the activity of rSip.
Antigen in detecting as the immunogen and ELISA of producing monoclonal antibody with albumen rSip, the aminoacid sequence (SEQIDNO.1) of rSip is as follows:
MEMNKKVLLTSTMAASLLSVASVQAQETDTTWTARTVSEVKADLVKQDNKSSYTVKYGDTLSVISEAMSIDMNVLAKINNIADINLIYPETTLTVTYDQKSHTATSMKIETPATNAAGQTTATVDLKTNQVSVADQKVSLNTISEGMTPEAATTIVSPMKTYSSAPALKSKEVLAQGQAVSQAAANEQVSPAPVKSITSEVPAAKEEVKPTQTSVSQSTTVSPASVAAETPAPVAKVAPVRTVAAPRVASVKVVTPKVETGASPEHVSAPAVPVTTTSTATDSKLQATEVKSVPVAQKAPTATPVAQPASTTNAVAAHPENARLQPHVAAYKEKVASTYGVNEFSTYRAGDPGDHGKGLAVDFIVGKNQALGNEVAQYSTQNMAANNISYVIWQQKFYSNTNSIYGPANTWNAMPDRGGVTANHYDHVHVSFN。
1.2 immune programme for children
With through not formula Freund's complete adjuvant (Sigma-Aldrich) emulsification (emulsification method: by antigen and Freund's complete adjuvant equal-volume mixing, suck in a syringe, take off syringe needle, with another syringe hose connection, band fixes flexible pipe, promote two syringes in turn, mixed solution is flowed between syringe back and forth by flexible pipe, thus reach emulsification object) 25 μ g, 50 μ g, 100 μ g and 150 μ grSip albumen abdominal injection female BAl BIc/c mouse (8 week age) respectively.After 4 and 8 weeks, again inject (emulsification and injected dose the same) mouse by the same antigen through not formula Freund's incomplete adjuvant (Sigma-Aldrich) emulsification.10th week, last abdominal injection rSip albumen (injected dose is the same) separately once.
1.3 hybridomas generations and the monoclonal antibody for Sip albumen are produced
After last booster shots three days, obtain immune mouse spleen cell (separation method: get height and exempt from BALB/c mouse, after drawing neck to put to death, 3 ~ 5min is soaked in 75% alcohol, abdominal cavity is opened in aseptic technique, and the careful spleen that takes out is as in plate, adds a small amount of DMEM basic medium rinsing 2 ~ 3 times, and carefully removes the reticular tissue around spleen.Then in another culture dish, pour serum-free into by copper mesh lid thereon, get spleen on copper mesh, grind gently with grinding rod, after cell release completely, collect splenocyte, the centrifugal 10min of 1000rpm).Method according to Situ and Wu describes: with 50% (w/v) Macrogol 4000 (Sigma-Aldrich), splenocyte and myeloma cell S/P20 are carried out merging (the step of cytogamy: S/P20 cell is so kind as to give by Chengdu University of Traditional Chinese Medicine professor Rao Chaolong with the ratio of 5:1.Fusion steps: the S/P20 cell in vegetative period of taking the logarithm and splenocyte fully mix in proportion, the centrifugal 10min of 1000rpm, abandons supernatant liquor, touches at the bottom of centrifugal bottle with palm, breaks up the cell of precipitation.Centrifugal bottle is placed in 40 DEG C of water-baths, rotates limit add 1mlPEG4000 at 1min inner edge, then add 15ml serum-free RPMI-1640 substratum termination fusion, add in 90s, from slow to fast, front 5s adds 1ml.Room temperature leaves standstill 10min, and the centrifugal 10min of 1000rpm, abandons supernatant, and be added in cytogamy thing by the RPMI-1640 substratum containing HAT and 15% foetal calf serum, suspension cell, is assigned to 96 porocyte culture plates of existing feeder cell, is placed in 37 DEG C, 5%CO
2incubator in cultivate), obtain hybridoma.
Screening step 1:
The hybridoma obtained is cultivated in xanthoglobulin-aminopterin-thymidine medium, using the rSip of 5 μ g/ml as envelope antigen, detects the antibody in culture supernatant with ELISA, to screen hybridoma, obtain ELISA and be accredited as positive hybridoma.
Screening step 2:
ELISA is accredited as positive hybridoma to breed in BALB/c mouse ascites.Collect ascites, use ProteinGSepharose affinity column (GEHealthcareLifeSciences) purifying (purification process: draw mouse ascites with syringe puncture, at 4 DEG C, the grumeleuse that under 12000g condition, centrifugal 15min removing is larger.The PBS damping fluid of the ascites 0.02MpH7.0 handled well is diluted 10 times, 0.45 μm of membrane filtration, the protein g affinity chromatography post that the PBS damping fluid that then loading flows through 0.02MpH7.0 balances.Be that elutriant carries out wash-out with the Gly-HCl of 0.1MpH2.7, collect elution peak, and adjust pH extremely about 7.0 with the Tris-HCl of 1.0MpH9.0, last dialysis desalting lyophilize) rSip monoclonal antibody in ascites.
Be combined with Radioactive colloidal gold liquid by the monoclonal antibody (concentration 2mg/ml) of purifying, room temperature (RT) places 5min.After adding 5%PEG, the centrifugal 30min of 8000rpm immediately.Red precipitate is collected in 12ml centrifuge tube, resuspended with Radioactive colloidal gold protective material, and be diluted to working concentration (usual OD
532=30-50).Then solution is placed in 4 DEG C.
With 0.1MPBS, another monoclonal antibody is diluted to 2mg/ml.With 0.1MPBS, sheep anti-mouse igg is diluted to 1.0mg/ml.The former is for wrapping by the detection line on NC film, and the latter is used for nature controlling line.Then film is kept 24h at 37 DEG C.After closing with BB damping fluid (ArtronBioResearchInc), wash film with WB damping fluid, then drying at room temperature.
Test strip is assembled with sample pad, pad, reaction film and absorption layer.With automatic strip-cutting machine, test paper is cut into the little bar of 3mm.
By all combinations-monoclonal antibody and bag quilt-monoclonal antibody matches, prepare for test strip.The sample pad of test strip is inserted in streptococcus agalactiae sample, until the half of NC film is immersed in liquid.Under room temperature, after 3-10min, observe the formation of nature controlling line and detection line.Screening demonstrates the strongest positive signal to streptococcus agalactiae, other test bacteria is demonstrated to the pairing antibody of negative findings.
84 positive cell lines are obtained altogether after cytogamy.All 84 monoclonal antibodies of purifying are also used as the coated antibody/binding antibody of test strip assembling.Through repeatedly matching after test, finding NC7b-CE12 to match and demonstrating and have peak signal to streptococcus agalactiae, to other bacterium no signal.
The hybridoma cell strain of monoclonal antibody NC7b and monoclonal antibody CE12 will be expressed respectively, called after hybridoma cell strain TWDB-WR-1 and hybridoma cell strain TWDB-WR-2 respectively, and being deposited in China typical culture collection center (CCTCC) on October 21st, 2015, preserving number is respectively CCTCCNO:C2015178 and CCTCCNO:C2015177.
2, monoclonal antibody preparation
Get hybridoma cell strain TWDB-WR-1 and hybridoma cell strain TWDB-WR-2 and be prepared as follows monoclonal antibody NC7b and monoclonal antibody CE12 respectively:
Get aforementioned hybridoma cells strain to breed in BALB/c mouse ascites, specifically by the BALB/c mouse in 8 week age of sterilising liq paraffin abdominal injection, 0.5ml/ only.After 7 days, aforementioned two strain of hybridoma strains are hanged with serum-free RPMI-1640 substratum respectively, the centrifugal 5min of 1000rpm, abandon supernatant, wash away the serum in substratum, again by appropriate serum-free RPMI-1640 substratum re-suspended cell precipitation, be injected into mouse peritoneal respectively, every 0.5ml is (containing about 10
6individual hybridoma.Collect ascites (taking belly obviously to heave the ascites of mouse after 7 days), use ProteinGSepharose affinity column (GEHealthcareLifeSciences) purifying (concrete steps of purifying: draw mouse ascites with syringe puncture, at 4 DEG C, the grumeleuse that under 12000g condition, centrifugal 15min removing is larger.The PBS damping fluid of the ascites 0.02MpH7.0 handled well is diluted 10 times, 0.45 μm of membrane filtration, the protein g affinity chromatography post that the PBS damping fluid that then loading flows through 0.02MpH7.0 balances.Be that elutriant carries out wash-out with the Gly-HCl of 0.1MpH2.7, collect elution peak, and adjust pH extremely about 7.0 with the Tris-HCl of 1.0MpH9.0, last dialysis desalting lyophilize) rSip monoclonal antibody in ascites.
Purification result as shown in Figure 1, can be found out, present invention obtains monoclonal antibody NC7b and the monoclonal antibody CE12 of sterling.
3, the qualification of monoclonal antibody
3.1 immunoblotting
Get sterling monoclonal antibody NC7b and monoclonal antibody CE12rSip monoclonal antibody that step 2 prepares respectively, carry out immunoblot experiment respectively: in immunoblot experiment, test streptococcus agalactiae and other aquatic products common bacteria.Use the anti-streptococcus agalactiae monoclonal antibody of three strains as the first antibody of immunoblotting respectively.
As shown in Figure 2, monoclonal antibody NC7b and CE12 can specific recognition streptococcus agalactiae for result, and with other test bacteria no cross reaction.
3.2 bioactivity
Bioactivity result please be provided:
The present invention prepares the hybridoma cell strain TWDB-WR-2 expressing streptococcus agalactiae Sip monoclonal antibody CE12, and has prepared sterling monoclonal antibody CE12, its high specificity, only be combined with streptococcus agalactiae, and with other bacterium no cross reactions, simultaneously, titre is high, and binding ability is strong.
Embodiment 2 adopts streptococcus agalactiae Sip monoclonal antibody NC7b of the present invention to prepare the application of Test paper
1, the preparation of Test paper
Test strip of the present invention is double antibody sandwich method test strip, shown in test paper structure figure Fig. 3:
Prepared by Radioactive colloidal gold: the main trisodium citrate reduction method that adopts prepares Radioactive colloidal gold.In 10mL system chlorauric acid solution, add 150 μ l Trisodium Citrates, visual inspection colloid gold particle color is homogeneous, and in burgundy, have obvious halation, in the same size through electron microscopic observation colloid gold particle, particle diameter is at about 20nm.Through UV scanning gold grain, its peak width is narrower, shows colloidal gold solution favorable dispersity.
The preparation method of gold labeling antibody: get monoclonal antibody CE12 of the present invention (prepared by embodiment 1, concentration 2mg/ml), be combined with Radioactive colloidal gold liquid, room temperature (RT) places 5min.After adding 5%PEG, the centrifugal 30min of 8000rpm immediately.Red precipitate is collected in 12ml centrifuge tube, resuspended with Radioactive colloidal gold protective material, and be diluted to working concentration (OD
532=30-50), 4 DEG C save backup).
The golden labeling antibody marked is coated on glass fibre membrane, makes colloidal gold pad: get golden labeling antibody solution and join in the shower nozzle storage bottle of spraying machine, sprayed glass tunica fibrosa, dry, be colloidal gold pad.
Test paper assembling (structure as shown in Figure 3): the upper nitrocellulose filter (2) pasted of PVC base plate (1), sample pad (7) is connected with nitrocellulose filter (2) one end by colloidal gold pad (3), nitrocellulose filter (2) the other end is overlapped with absorbent pad (4), the upper side near colloidal gold pad (3) of described nitrocellulose filter (2) is fixed with NC7b antibody as detection zone (5), is fixed with sheep anti-mouse igg as quality control region (6) near the side of absorbent pad (4).
The method of sessile antibody in colloidal gold pad: get antibody, make antibody-solutions, joins in the shower nozzle storage bottle of spraying machine, and spraying test paper, dries.
Adopt the testing method of test paper of the present invention: infiltrate measuring samples by the sample pad of Test paper of the present invention, whether observation detection zone and quality control region develop the color.Wherein, if the detection zone of test paper 5 and quality control region 6 position all occur red stripes, then measuring samples is positive for detecting; If only there is red stripes on quality control region 6 position, and redfree band on position, detection zone 5, then measuring samples is negative for detecting; If red stripes does not appear to the quality control region of test strip 6 in position, no matter whether detection line position occurs red stripes, detected result is all invalid.
2, nature examination
2.1 test strip sensitivity tests
Be 6 × 10 by streptococcus agalactiae doubling dilution
10cFU/ml to 6 × 10
6cFU/ml, then tests by test strip to assess its minimum detection quantity.
2.2 test strip specificity tests
Streptococcus agalactiae (ATCC51487) is tested by test strip, streptococcus agalactiae C918 (Niu Yuan), Channel-catfish tarda, enterococcus faecalis, Streptococcus iniae, Aeromonas caviae, Salmonella typhimurium, streptococcus aureus, vibrio cholerae, Vibrio parahaemolyticus, Aeromonas hydrophila, subtilis, vibrio alginolyticus, enterobacter cloacae, Shewanella putrefaciens, Klebsiella pneumonia, helicobacter pylori, germ oligotrophy unit cell, Proteus mirabilis, Oidium tropicale, salmonella typhi, salmonella paratyphi, serratia marcescens, colon bacillus, Candida albicans, citrobacter freundii, Pseudomonas aeruginosa, staphylococcus haemolyticus, Acinetobacter bauamnnii and gonorrhoea a kind of apple plucked instrument Salmonella.
2.3 test strip reperformance tests
Three streptococcus agalactiae samples and three negative samples are tested, each sample repeated test ten times respectively by test strip.
2.4 actual sample tests
Gather ill tilapia, dissect and get pisiformis tissue to be checked (liver or brain) in 1.5mlEp pipe, after smashing to pieces, add 1ml physiological saline, test after mixing, aseptic technique is simultaneously separated the bacterium in liver, uses PCR system to detect, compare the detected result of the two after purifying.
3 detected results
3.1 sensitivity test results
Streptococcus agalactiae nutrient solution 10 is doubly diluted to 6 × 10
10cFU/ml ~ 6 × 10
6cFU/ml, with the sensitivity of test paper bar.When concentration is 6 × 10
7during CFU/ml, T line faint as seen, but when concentration continues to be down to 6 × 10
6during CFU/ml, T line there will not be (Fig. 4).The detection that Fig. 4 shows this pairing antibody is limited to 6 × 10
7cFU/ml.
The detectability of table 2 streptococcus agalactiae test strip
| Streptococcus agalactiae | Serotype | Separation source | Detectability |
| TW3 | Ia | Fish | 6×10 7CFU/ml |
| TW6 | Ia | Fish | 1.1×10 7CFU/ml |
| TW7 | Ia | Fish | 1.3×10 7CFU/ml |
| TW9 | Ia | Fish | 1.1×10 6CFU/ml |
| TW10 | Ia | Fish | 1×10 7CFU/ml |
| TW31 | II | Niu Yuan | Negative |
3.2 specificity test results
The Rapid detection test strip of two pairings has high degree of specificity to streptococcus agalactiae, with other bacteria tested no cross reaction (Fig. 5 and table 3).
The specificity test of table 3 streptococcus agalactiae test strip
3.3 reperformance test results
Yin and yang attribute coincidence rate is 100% (table 4).
Table 4 reperformance test result
The detection of 3.4 actual samples
The water body of the streptococcus agalactiae of research and development trial-production to ill tilapia and morbidity pond is used to detect, result shows, containing a large amount of streptococcus agalactiaes in morbidity fish body, and streptococcus agalactiae (Fig. 6) in water body, do not detected, consistent with the detected result of PCR, illustrate that the inventive method detected result is accurate.
Test-results illustrates, the streptococcus agalactiae Test paper adopting monoclonal antibody CE12 of the present invention to prepare is highly sensitive, high specificity, reproducible, accurately can detect actual sample.
To sum up, the present invention is adopted to prepare streptococcus agalactiae Sip monoclonal antibody CE12, the high specificity of itself, only be combined with streptococcus agalactiae, and with other bacterium no cross reactions, titre is high, binding ability is strong, the streptococcus agalactiae Test paper adopting this albumen to prepare is highly sensitive, high specificity, reproducible, accurately can detect actual sample, application prospect is good.
Claims (9)
1. produce a hybridoma cell strain for streptococcus agalactiae monoclonal antibody, it is characterized in that: it is by the cell strain of the preserving number of China typical culture collection center preservation: CCTCCNO:C2015177.
2. prepare a method for hybridoma cell strain described in claim 1, it is characterized in that: it comprises the steps:
(1) for antigen, BALB/c mouse is inoculated, separating mouse splenocyte with aminoacid sequence recombinant protein as shown in SEQIDNO.1;
(2) get mouse boosting cell, merge with medullary cell, screening.
3. a streptococcus agalactiae monoclonal antibody, is characterized in that: it is the monoclonal antibody of being secreted by hybridoma cell strain described in claim 1.
4. prepare a method for monoclonal antibody described in claim 3, it is characterized in that: it comprises the steps:
1) get the hybridoma cell strain described in claim 1, be injected in BALB/c mouse ascites and breed;
2) ascites is collected, purifying.
5. method according to claim 4, is characterized in that: step 2) in, described purifying adopts ProteinGSepharose affinity chromatography column purification.
6. streptococcus agalactiae monoclonal antibody according to claim 3 is detecting the purposes in streptococcus agalactiae.
7. streptococcus agalactiae monoclonal antibody according to claim 3 detects the purposes in the reagent of streptococcus agalactiae in preparation.
8. streptococcus agalactiae monoclonal antibody according to claim 3 detects the purposes in the colloidal gold fast detecting test paper of streptococcus agalactiae in preparation.
9. according to the purposes described in claim 6 ~ 8, it is characterized in that: sample to be checked is aquaculture water or fish body.
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| WO2017075937A1 (en) * | 2015-11-06 | 2017-05-11 | 通威股份有限公司 | Colloidal-gold rapid detection test paper for streptococcus agalactiae |
| CN115572716A (en) * | 2022-09-30 | 2023-01-06 | 云南沃森生物技术股份有限公司 | Monoclonal antibody for resisting group III streptococcus group B capsular polysaccharide and hybridoma cell strain |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017075937A1 (en) * | 2015-11-06 | 2017-05-11 | 通威股份有限公司 | Colloidal-gold rapid detection test paper for streptococcus agalactiae |
| CN115572716A (en) * | 2022-09-30 | 2023-01-06 | 云南沃森生物技术股份有限公司 | Monoclonal antibody for resisting group III streptococcus group B capsular polysaccharide and hybridoma cell strain |
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