CN104130326A - Monoclonal antibody of Chlamydophila abortus macrophage infectivity potentiator and hybridoma cell thereof - Google Patents
Monoclonal antibody of Chlamydophila abortus macrophage infectivity potentiator and hybridoma cell thereof Download PDFInfo
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Abstract
The invention discloses a monoclonal antibody of a Chlamydophila abortus macrophage infectivity potentiator (i.e. MIP protein). Chlamydophila abortus MIP protein is taken as the antigen to immunize mice, and the monoclonal antibody is obtained by secretion, wherein the Chlamydophila abortus MIP protein has the amino acid sequence shown as the sequence table 1. Further, the invention also discloses a hybridoma cell able to secrete the monoclonal antibody of the Chlamydophila abortus MIP protein. The monoclonal antibody disclosed by the invention can effectively detect the Chlamydophila abortus MIP protein, the detection mode has strong specificity and high detection rate, and is suitable for establishment of indirect immunofluorescence technique for detection of Chlamydophila abortus.
Description
Technical field
The present invention relates to a kind of monoclonal antibody of the preferendum chlamydozoan MIP albumen of miscarrying and can secrete the hybridoma of this monoclonal antibody, belonging to field of biological detection.
Background technology
Miscarriage preferendum chlamydozoan (Chlamydophila abortus) is Gram-negative special sexual cell endoparasitism prokaryotic organism, belong to Chlamydiaceae (Chlamydiaceae) preferendum chlamydiaceae (Chlamydophila) member, there is host's preferendum widely, main parasitic is in the reproductive tract mucosal epithelium cell of animal, the many animals such as energy infected cattle, sheep, goat and pig, can cause pregnant female miscarriage, premature labor, stillborn foetus, mummy tire or weak tire, the multiple chronic contagious diseases such as male animal genital disease; And miscarriage preferendum chlamydozoan also can infect the mankind, causes pregnant woman to miscarry, and is a kind of important infecting both domestic animals and human disease pathogen.
In recent years, miscarriage preferendum chlamydozoan generally infects in swinery, cows and the flock of sheep of the each provinces and regions of China, and the abortion ratio causing is thus up to more than 50%, becomes one of the most serious disease pathogen of current livestock industry, has caused huge financial loss.
For effective control miscarriage preferendum chlamydozoan, discovery chlamydozoan in time, accurately, as early as possible to carry out effectively diagnosis be to determine that animal suffers from chlamydial basis.At present for miscarriage preferendum chlamydozoan, main detection method is to adopt major outer membrane albumen (MOMP), many types of outer membrane protein (POMP) and lipopolysaccharides (LPS) etc. as object antigen, adopt the direct immunofluorescent assay technique of specific detection LPS, with MOMP, POMP be the enzyme-linked immunosorbent assay kit of envelope antigen and according to its encoding gene ompA set up PCR and real-time round pcr etc., the product of concrete manifestation moulding is chlamydozoan indirect hemagglutination test (IHA) test kit.
In aforesaid method, adopt that round pcr interval between diagnosis is long, narrow application range, be unfavorable for clinical expansion, the chlamydozoan that cannot be used for the many animals such as ox, sheep, goat and pig of large-scale farming detects diagnosis.
Although IHA test kit has been realized scale production and popularization and application, exist sensitivity low, the shortcoming that specificity is poor, increases although the part of such test kit is improved product sensitivity, and production cost is too high, cannot popularization and application.
Summary of the invention
For the defect of prior art, the invention discloses miscarriage preferendum chlamydozoan Macrophage infection potentiator (
macrophage
infectivity
potentiator, MIP) monoclonal antibody and can be used in secretion and produce the hybridoma of this monoclonal antibody, miscarriage preferendum chlamydozoan MIP protein monoclonal antibody of the present invention can effectively detect for miscarriage preferendum chlamydozoan MIP albumen, there is very strong specificity, can provide efficiently in time, prevent and treat accurately information for detection of miscarriage preferendum chlamydozoan.
For achieving the above object, the present invention is achieved through the following technical solutions:
Miscarriage preferendum chlamydozoan Macrophage infection potentiator (is MIP albumen, below all adopt this abbreviation) monoclonal antibody, obtained as the secretion of antigen immune mouse by miscarriage preferendum chlamydozoan MIP albumen, described miscarriage preferendum chlamydozoan MIP albumen has aminoacid sequence shown in sequence table 1.
Above-mentioned miscarriage preferendum chlamydozoan MIP albumen both can pass through synthetic, also can express acquisition to the encoding gene of miscarriage preferendum chlamydozoan MIP albumen by prokaryotic organism such as intestinal bacteria.
Preferably, for the ease of miscarriage preferendum chlamydozoan MIP protein purification, be connected with 6 × His label at N-terminal or the C-terminal of miscarriage preferendum chlamydozoan MIP albumen.
For the monoclonal antibody of a large amount of enrichments and the above-mentioned miscarriage preferendum chlamydozoan MIP albumen of production, the invention also discloses a kind of hybridoma, for secreting the monoclonal antibody of miscarriage preferendum chlamydozoan MIP albumen, this hybridoma adopts following method preparation:
1) obtain miscarriage preferendum chlamydozoan MIP albumen by synthetic or prokaryotic expression, it has the aminoacid sequence shown in sequence table 1;
2) injection miscarriage preferendum chlamydozoan MIP protein immunization mouse, detects antibody titer from mouse blood sample collection after immunity completes, and the order of magnitude of selecting to tire is not less than 10
7injected in mice booster immunization;
3) under aseptic condition, get the mouse spleen after above-mentioned immunity, prepare splenocyte and SP2/0 myeloma cell's suspension, under fusogen effect, merge;
4) fused cell is suspended evenly and joined in the cell plate that are covered with feeder cell, at 5%CO with HAT selective medium
2in incubator, cultivate;
5) after fused cell clonal growth completes, all culture supernatant that have clonal growth are detected, repeatedly screening and cloning obtain the hybridoma of secretion miscarriage preferendum chlamydozoan MIP protein monoclonal antibody after cultivating.
Preferably, the N-terminal of described MIP albumen or shuttle base end are connected with 6 × His label.
In above-mentioned, mouse used is BALB/c mouse.
In above-mentioned, feeder cell are mouse primary peritoneal macrophages.
In above-mentioned, step 2) immunization mode is the subcutaneous injection of belly multiple spot or antigen liquid abdominal injection.
Wherein, the dosage, number of times, cycle of injection etc. can be adjusted according to practical situation, normally carry out 5-10 immunization with the gap periods in 1-2 week, injected dose is every the each 100-200 μ of mouse g, adopt the subcutaneous injection of belly multiple spot early stage, the later stage adopts antigen liquid abdominal injection to carry out booster immunization.
In above-mentioned, step 3) splenocyte after mouse immune is 5: 1 with SP2/0 myeloma cell's number ratio, fusogen is PEG4000.
In above-mentioned, the time point detecting is that the cell clone after merging grows into while covering 40%～60% left and right, cell hole bottom, adopt indirect ELISA method to detect all culture supernatant that have clonal growth, wherein detect as the hole of strong positive adopts limiting dilution assay and carry out subclone, detect negative hole and detect once again after 3 days, be still feminine gender and abandon.
Further, the invention also discloses the monoclonal antibody of described miscarriage preferendum chlamydozoan Macrophage infection potentiator in the application detecting in miscarriage preferendum chlamydozoan, mainly to detect miscarriage preferendum chlamydozoan by indirect fluorescent method, taking above-mentioned miscarriage preferendum chlamydozoan MIP protein monoclonal antibody as primary antibodie, taking fluorescently-labeled sheep anti-mouse igg as two anti-, detect miscarriage preferendum chlamydozoan by indirect IF staining method.
For the accuracy that ensures to detect, in above-mentioned detection method, also can add the positive contrast of mouse positive serum, with the negative contrast of McCoy cell of not infecting.
On the basis of above-mentioned detection method, for the ease of commercial applications, can also be made detection kit.
The prokaryotic expression miscarriage preferendum chlamydozoan MIP recombinant protein that the present invention obtains by purifying is as antigen immune mouse, by indirect ELISA method screening positive clone, obtain the hybridoma of the monoclonal antibody of secretion miscarriage preferendum chlamydozoan MIP recombinant protein, to hybridoma chromosome number, biological characteristics after continuous passage is cultivated and be frozen, and the type of monoclonal antibody, subclass and the aspect such as tire, obtained monoclonal antibody biological characteristics has been carried out to system identification, result shows that the ability of obtained hybridoma secretory antibody is strong and stable, antibody titer is high.Indirect fluorescent test experience result shows that obtained monoclonal antibody all can detect for the chlamydial MIP albumen of miscarriage preferendum, has very strong specificity and fluorescent characteristic.
Embodiment
Various raw material reagent used all can be from market purchasing in the following embodiments, and main raw material comprises as follows:
Miscarriage preferendum chlamydozoan bacterial strain, market is bought; BALB/c mouse is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.
The rabbit anti-mouse igg of HAT substratum, HT substratum, foetal calf serum, DMEM high glucose medium, RPMI1640 substratum, horseradish peroxidase (HRP) or HRP mark, fluorescein (FITC) mark sheep anti-mouse igg are all purchased from Sigma company.
PET-30a (+) carrier, purchased from Novagen company, expresses bacterium BL21 (DE3) purchased from Beijing Quanshijin Biotechnology Co., Ltd.
The primer is synthetic by Shanghai Sheng Gong biotechnology company limited, restriction enzyme Ndel, Xhol, and T4 DNA ligase and 2 × Power Taq PCR MasterMix are all purchased from the precious biological company limited in Dalian; IPTG, SDS (sodium laurylsulfonate), nucleic acid Marker, Agarose DNA extraction kit, DNA fast purifying reclaim test kit, plasmid rapid extraction test kit all purchased from Dalian Bao Bio-Engineering Company; It is synthetic purchased from Shanghai Sheng Gong biotechnology company limited that bacterial genomes is extracted test kit; Agarose is purchased from Invitrogen company; Dye in advance albumen Marker purchased from Fermentas company.
Embodiment 1: the preparation of miscarriage preferendum chlamydozoan MIP albumen
(1) the coding gene sequence amplification of miscarriage preferendum chlamydozoan MIP albumen
Picking C.abortus strain culture is (for ease of following the trail of, for it is numbered SX5, this bacterial strain is bought from common bacterial classification company), utilize gram negative bacterium genome to extract test kit and extract the genomic dna of SX5, amplify each goal gene taking the genomic dna of SX5 as PCR masterplate.
The amplimer sequence of PCR process is as follows:
Forward primer (line part is Ndel restriction enzyme site): 5 '-AAC
cATATGaAAAAACAATGGTATTTAA-3 ';
Reverse primer (line part is Xhol restriction enzyme site): 5 '-GGA
cTCGAGtGA AGCTGTGTTTTTGTC-3 '.
PCR reaction system comprises: 2 × Power Taq PCR MasterMix of 25 μ L, and the corresponding each 2 μ L of upstream and downstream primer, the DNA masterplate of 2 μ L, deionized water is supplied 50 μ L.
PCR product reclaims test kit and reclaims the encoding gene of the preferendum chlamydozoan MIP albumen that obtains miscarrying with DNA glue.
(2) expression and purification of miscarriage preferendum chlamydozoan MIP albumen
The product of pcr amplification is cut through Ndel and Xhol enzyme, and pET-30 (+) expression vector of cutting with same enzyme connects acquisition recombinant expression vector pET-MIP.Recombinant expression vector proceeds to e. coli bl21 (DE3) (such as BL21, BL21 (DE3) PLySs of other intestinal bacteria etc. also can), under IPTG induction, expresses recombinant protein.
The experiment that applicant repeatedly carries out shows, it is 37 DEG C at inducing temperature, when inductor IPTG final concentration is 0.8mM, target protein great expression, and in induction expression amount maximum 5 hours time, target protein size is 27kD (containing the 6 × His label on carrier pET-30a (+)).Miscarriage preferendum chlamydozoan MIP albumen is solubility expression at BL21 (DE3), collects thalline, after ultrasonic degradation, and centrifuging and taking supernatant, supernatant is through Ni-NTA purification column (Invitrogen) purifying target protein.
Embodiment 2: animal immune
As the antigen immune BALB/c mouse in 6 week age, be total to immunity 6 times, every minor tick 1～2 week with purifying miscarriage preferendum chlamydozoan MIP albumen.
After being wherein emulsified into water-in-oil with antigen and equivalent Freund's complete adjuvant for the first time, carry out immunity, immunizing dose is 200 μ g/, and totally 2, immunization route is the subcutaneous injection of belly multiple spot.
After two weeks, carry out immunity for the second time, immunity is for the third time carried out at interval for one week, all uses for twice and antigen equivalent Freund's incomplete adjuvant, and immunizing dose is 100 μ g/, and immunization route is the subcutaneous injection of belly multiple spot.
Then interval is carried out the 4th immunity and the 5th immunity for one week, adopts antigen liquid immunity, and dosage is 100 μ g/, and immunization route is abdominal injection.
After the 5th immunity one week, mouse tail is blood sampling in a small amount, detected antibody titer, selected to tire the high order of magnitude 10
7above mouse, is cooked booster immunization in first three day of fusion, again uses antigen liquid abdominal injection, and dosage is 100 μ g/.
Wherein, the concentration of antigen liquid is not subject to special restriction, and concentration is 5mg/ml conventionally.
Embodiment 3: immune antibody cell and myeloma cell's cytogamy
Merge the SP2/0 myeloma cell that recovery is preserved first 3 days.Under aseptic condition, get the mouse spleen after above-mentioned immunity, prepare splenocyte, draw respectively 1 × 10
8individual splenocyte and 2 × 10
7individual myeloma cell's suspension, under fusogen PEG4000 effect, merges.
Fused cell is selected to cultivate suspension evenly with HAT, and add in the 96 porocyte plates that are covered with feeder cell, every hole 100 μ L, are placed in 5%CO
2in incubator, cultivate.Every day observed and recorded Growth of Cells situation.
Embodiment 4: the screening of hybridoma and subclone
Cell clone after fusion grows into while covering 40%～60% left and right, cell hole bottom, uses the indirect ELISA method establishing to detect all culture supernatant with clonal growth.
Detect as strong positive hole adopts limiting dilution assay and carry out subclone; Negative hole detects once after 3 days again, abandons it if still negative.
Cultivate through the screenings of 3 times and cloning, obtain secretion miscarriage preferendum chlamydozoan MIP protein monoclonal antibody and the high hybridoma of tiring.
Continuous passage stability to hybridoma obtained above detects, hybridoma is added to 0.1mL 1% colchicine, incubated overnight, collecting cell, the centrifugal 10min of 2000r/min, abandons supernatant, in precipitation, add 0.075mol/L KCl 10mL, even with suction pipe pressure-vaccum, put 37 DEG C of water-bath 20min, make cellular swelling.Centrifugal abandoning after supernatant, the fixing 20min of the mixed solution 10mL of 3 parts of methyl alcohol, 1 part, Glacial acetic acid for cell precipitation, with 2000r/min, centrifugal 10min, gets cell precipitation smear after abandoning supernatant, after seasoning by Giemsa dyeing, oily spectroscopy.Hybridoma karyomit(e) is 95～103, and the cultured cells that shows to go down to posterity does not morph.
Embodiment 5: the preparation of odd contradictive hydroperitoneum and Antibody types qualification
By the hybridoma of gained, be inoculated in the healthy BALB/c mouse abdominal cavity of using in advance 10 week age inductor silica gel H (or sterilising liq paraffin, 0.5mL/ only) to process 10d, consumption is that 0.5mL/ is only (containing 5 × 10
5～1 × 10
6individual hybridoma), through 14d, visible mouse web portion obviously increases, and now gathers ascites and by the ascites 3000r/min collecting, and centrifugal 10min is odd contradictive hydroperitoneum after removing grease and precipitation.
Adopt Sigma company mouse monoclonal antibody parting kit, application agar diffusion test is measured, and experimental result shows that the monoclonal antibody of the hybridoma secretion obtaining is IgG1 type.It is 1: 10 that application indirect ELISA method detects in ascites that monoclonal antibody tires
6.
Embodiment 6: monoclonal antibody specificity detects
Mouse ascites monoclonal antibody is carried out to western blot test, the miscarriage preferendum chlamydozoan SX5 strain of purifying boils 10min preparation miscarriage preferendum I (chlamydia) protein sample with 1 × SDS Ioading buffer, prepare expression vector pET-30 (+) oneself expression label protein, carry out SDS-PAGE with the restructuring MIP of purifying simultaneously, and be transferred on pvdf membrane, use successively ascites monoclonal antibody, the sheep anti-mouse igg of HRP mark or mouse-anti 6 × His monoclonal antibody, the sheep anti-mouse igg of HRP mark is hatched, the ECL chemical illuminating reagent mixing is dropped on pvdf membrane, in darkroom, use X-exposure, develop, photographic fixing.
Experimental result shows, the pvdf membrane that the miscarriage preferendum chlamydozoan MIP monoclonal antibody of preparing using the present invention is hatched as primary antibodie, respectively there is a specific band at 27KDa place at the miscarriage preferendum chlamydozoan MIP of purifying albumen and miscarriage preferendum I (chlamydia) protein sample, and in expression vector pET-30 (+) oneself expression label protein sample, there is no band; The pvdf membrane of hatching as primary antibodie using 6 × His monoclonal antibody, in the MIP of purifying albumen and expression vector pET-30 (+) oneself expression label protein sample, all there is specific band, this show prepared monoclonal antibody only with miscarriage preferendum chlamydozoan MIP albumen generation specific reaction, and do not react with expression vector pET-30 (+) oneself expression label protein.
Embodiment 7: miscarriage preferendum chlamydozoan MIP protein monoclonal antibody indirect immunofluorescene assay method
The pathological material of disease of taking doubtful miscarriage preferendum choamydiae infection, cuts appropriate lungs, spleen, uterine mucosa, vaginal mucosa, and 4% paraformaldehyde is fixed, and makes the tissue slice of 4 μ m thickness; Cut equally the above-mentioned tissue of part, after grinding under 4 DEG C of conditions, adding final concentration is Streptomycin sulphate and the kantlex of 1mg/mL, effect 30min, 2000r/min, centrifugal 20min, get supernatant, infect the McCoy cell being grown on cover glass, infect 48h, take out the cover glass infecting, with the fixing 10min of methanol/acetone (1: 1) of precooling.The concentration that drips 50 μ L on the tissue slice preparing and cell climbing sheet is the MIP protein monoclonal antibody of 100 μ g/mL, be placed in wet box, hatch 1h for 37 DEG C, PBST washing 3 times, each 10min, then drip the sheep anti-mouse igg of the FITC mark of the 10 μ g/ml of 50 μ L, be placed in wet box, hatch 1h for 37 DEG C, PBST washing 3 times, each 10min, glycerine mounting, fluorescence microscope.Simultaneously with the positive contrast of mouse positive serum, with the negative contrast of McCoy cell of not infecting.If positive, can observe the light-emitting particles of ellipse, bright green, systematicness, feminine gender does not have.
The miscarriage preferendum chlamydozoan MIP protein monoclonal antibody of preparing using the present invention is as primary antibodie, adopt indirect immunofluorescence to detect 58 parts of miscarriage chlamydozoan pathological material of diseases of clinical censorship, and with real-time PCR detection method (Alexandra Pantchev, Reinhard Sting, Rolf Bauerfeind, Judith Tyczka, Konrad Sachse, New real-time PCR tests for species-specific detection of Chlamydophila psittaci and Chlamydophila abortus from tissue samples, The Veterinary Journal, 181 (2009) 145-150) compare, the positive coincidence rate of result is 96%, with PCR result negative match-rate be 100% (table 1).
Table 1: miscarriage chlamydozoan pathological material of disease detected result
| Pathological material of disease | Indirect immunofluorescence | PCR | Coincidence rate |
| Positive | 41 | 43 | 95％ |
| Negative | 17 | 15 | 100％ |
In sum, the miscarriage preferendum chlamydozoan MIP protein monoclonal antibody that prepared by the present invention can be used for quick diagnosis miscarriage preferendum chlamydiosis.
Claims (5)
1. the monoclonal antibody of miscarriage preferendum chlamydozoan Macrophage infection potentiator, is characterized in that being obtained as the secretion of antigen immune mouse by miscarriage preferendum chlamydozoan MIP albumen, and the preferendum of wherein miscarrying chlamydozoan MIP albumen has aminoacid sequence shown in sequence table 1.
2. a hybridoma for the secretion miscarriage preferendum chlamydozoan MIP protein monoclonal antibody obtaining with miscarriage preferendum chlamydozoan MIP protein immunization mouse, is characterized in that adopting following method preparation: 1) obtain miscarriage preferendum chlamydozoan MIP albumen by synthetic or prokaryotic expression; 2) injection miscarriage preferendum chlamydozoan MIP protein immunization mouse, detects antibody titer from mouse blood sample collection after immunity completes, and the order of magnitude of selecting to tire is not less than 10
7injected in mice booster immunization; 3) under aseptic condition, get the spleen of mouse after above-mentioned immunity and prepare splenocyte and SP2/0 myeloma cell's suspension, under fusogen effect, merge; 4) fused cell is suspended evenly and joined in the cell plate that are covered with feeder cell, at 5%CO with HAT selective medium
2in incubator, cultivate; 5) fused cell clonal growth detects all culture supernatant that have clonal growth after completing, and repeatedly screening and cloning obtain the hybridoma of secretion miscarriage preferendum chlamydozoan MIP protein monoclonal antibody after cultivating.
3. hybridoma according to claim 2, is characterized in that step 2) mode of immunization is the subcutaneous injection of belly multiple spot or antigen liquid abdominal injection.
4. hybridoma according to claim 2, is characterized in that step 3) splenocyte after mouse immune is 5: 1 with SP2/0 myeloma cell's number ratio, fusogen is PEG4000.
5. the monoclonal antibody of the miscarriage preferendum chlamydozoan Macrophage infection potentiator of claim 1 is in the application detecting in miscarriage preferendum chlamydozoan.
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Cited By (3)
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| CN108486066A (en) * | 2018-02-27 | 2018-09-04 | 中国农业大学 | A kind of monoclonal antibody of the anti-current of hybridoma cell strain and its secretion production Chlamydia |
| CN110129278A (en) * | 2019-05-31 | 2019-08-16 | 湖北省农业科学院畜牧兽医研究所 | Hybridoma cell strain CMOMP-5D7 and its monoclonal antibody and application of secretion |
| CN111662854A (en) * | 2020-07-27 | 2020-09-15 | 中国农业科学院兰州兽医研究所 | Cell culture method and application of Chlamydia abortus |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108486066A (en) * | 2018-02-27 | 2018-09-04 | 中国农业大学 | A kind of monoclonal antibody of the anti-current of hybridoma cell strain and its secretion production Chlamydia |
| CN108486066B (en) * | 2018-02-27 | 2021-07-27 | 中国农业大学 | Hybridoma cell strain and monoclonal antibody secreted by hybridoma cell strain and resisting chlamydia abortus |
| CN110129278A (en) * | 2019-05-31 | 2019-08-16 | 湖北省农业科学院畜牧兽医研究所 | Hybridoma cell strain CMOMP-5D7 and its monoclonal antibody and application of secretion |
| CN111662854A (en) * | 2020-07-27 | 2020-09-15 | 中国农业科学院兰州兽医研究所 | Cell culture method and application of Chlamydia abortus |
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Application publication date: 20141105 |