CN101270160B - Monoclone antibody 1F1, uses thereof and hybridoma cell line BCSP31-1F1 excreting the antibody - Google Patents
Monoclone antibody 1F1, uses thereof and hybridoma cell line BCSP31-1F1 excreting the antibody Download PDFInfo
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Abstract
The present invention relates to a monoclonal antibody 1F1, applications thereof and a hybridoma cell line BCSP31-1F1 secreting the antibody, and the accession number of the hybridoma cell line is CCTCC C200806. After being purified, the prokaryotically expressed brucella melitensis BCSP31 antigen immunize a mouse, and the hybridoma cell line BCSP31-1F1 is obtained via cell fusion and filtration. The monoclonal antibody 1F1 is prepared and purified via inoculation in the peritoneal cavity of the mouse. The obtained monoclonal antibody belongs to IgG1 subclass and can be specifically reacted with brucella melitensis BCSP31 proteins. By the genetic engineering method, the heavy chain and light chain variable region genes of the mouse monoclonal antibody of the anti-BCSP31 antigen of the present vention and encoding polypeptides thereof can be constructed and expressed as manifold micromolecular genetic engineering antibodies, such as a Fab antibody, a chimeric antibody, a antibody fusion protein, etc., which can be used for the preparation of preparations or drugs used to diagnose or treat brucellosis.
Description
Technical field
The invention belongs to immune Application Areas; Relate to association areas such as molecular biology, immunology; Be particularly related to the hybridoma cell line BCSP31-1F1 of proteic monoclonal antibody 1F1 of anti-sheep Bacillus brucellae BCSP31 and the said antibody of secretion, and use said monoclonal antibody and be used to detect the application aspect Bacillus brucellae and the diagnosis gibraltar fever detection reagent in preparation.The invention still further relates to heavy chain and the chain variable region gene and the coded polypeptide thereof of anti-Bacillus brucellae BCSP31 monoclonal antibody, and said gene and polypeptide are used for diagnosing or treating the application of Bacillus brucellae and aspect of inflammation medicine thereof in preparation.
Background technology
Bacillus brucellae (Brucella) also once was called Brucella in the past, can infect domestic animals such as sheep, ox, pig, also can infect the mankind, caused human gibraltar fever.Bacillus brucellae can pass through number of ways such as skin, mucous membrane, enteron aisle and respiratory tract to be infected; And invade human multiple tissue and organ, the pathological change and the clinical manifestation of its associated diseases are various and complicated, and unstable; Therefore the gibraltar fever clinical diagnosis is comparatively difficult, also lacks effectively prevention preparation.
Gibraltar fever is to propagate one of animal borne disease the most widely in the global range.According to World Health Organization report, having in the world at present has gibraltar fever to take place between the people, animal of more than 160 countries and regions and popular; The whole world has 500,000 people to suffer from gibraltar fever every year approximately.The Law on the Prevention and Control of Infectious Diseases of China is defined as Category B notifiable disease with gibraltar fever, and is classified as in " domestic animals and fowls epidemic prevention regulations detailed rules for the implementation " first of two types of transmissible diseases.Have gibraltar fever epidemic situation in various degree to exist at present between the people and animals of domestic more than 1200 cities and counties in 25 provinces, district, municipality directly under the Central Government's scope, compromised population reaches more than 300,000,000 people.Particularly over past ten years, rebound significantly appears in the gibraltar fever epidemic situation between Chinese people, and the amplitude of its rise increases year by year, becomes worse.In addition, not only serious harm human health of gibraltar fever, and also huge to the animal husbandry development influence of China, behind the animal infection Bacillus brucellae such as ox, sheep, pig, can cause infertile, the sterile or a large amount of miscarriages of a large amount of domestic animals, stillborn foetus etc.According to statistics, the livestock miscarriage that cause because of gibraltar fever every year in main pastoral area such as China Inner Mongol, Xinjiang, Qinghai, nonpregnant etc. causes few living cub to surpass ten million head, and financial loss is more than 1,000,000,000 yuan.See from the generation of SARS and bird flu in recent years, experience and lesson popular and control in addition, should pay attention to Study on etiology and diagnosis, prevention and treatment work that people and animals (beast) suffer from transmissible disease altogether at present especially especially.
Bacillus brucellae BCSP31 albumen is a kind of Bacillus brucellae outer membrane face that is present in, and immunogenicity is strong, is soluble protein
[4], its molecular weight is 31kDa.BCSP31 albumen has conservative property in 6 kinds of Bacillus brucellae.Existing preliminary experiment proof BCSP31 is a kind of virulence factor, thereby its effect possibly be to suppress the propagation of body scavenger cell directly or indirectly to reduce the ability that immunity system is killed cells infected.In addition, genetically deficient research shows that BSCP31 does not influence the bacteria attack power and the speed of growth
[7]Having confirmed that in recent years BCSP31 has good immunogenicity, is one of important immune protective antigen, and can induce body to produce with B cellular immunization is master's protective immunological reaction
[6]
BCSP31 has high conservative property, but homology is high in the Bacillus brucellae of multiple infected person, so BCSP31 has the potential vital role in the context of detection of gibraltar fever.The main serology detection method of current gibraltar fever is brave red treadmill test, but the false positive rate that this method records is higher.Zeki etc. point out, assisting down of ELISA detection method, can effectively reduce the red dull and stereotyped experiment false positive rate of tiger.Therefore set up and a kind ofly just seem particularly feasible based on proteic ELISA detection method of Bacillus brucellae BCSP31 or detection kit.
Domestic last century the nineties prepare anti-Bacillus brucellae surface antigen A and M successively (antigen A and M be the Bacillus brucellae O antigen; Be LPS) monoclonal antibody and the monoclonal antibody of several kinds of rough type Bacillus brucellae (not identifying antigen), be mainly used in the not evaluation of isotype.Adopted anti-Bacillus brucellae Monoclonal Antibody in addition idiotype antibody (polyclonal antibody), added immune cattle behind the adjuvant, the result shows that it stimulates the lectin antibody that produces to disappear very soon, and incomplete antibody then can be kept more than 1 year.But said monoclonal antibody is not practical application in diagnosis, prevention and the treatment of gibraltar fever all.Domestic studies on Monoclonal Antibody to BCSP31 does not still have report.
Summary of the invention
The present invention follows the principle of " people and animals take into account ", to infecting with Bacillus brucellae and Ia gene BCSP31 expresses, purifying and evaluation; Adopt hybridoma technology to prepare to the proteic a kind of monoclonal antibody of BCSP31.Said monoclonal antibody can be applied to the detection of Bacillus brucellae and the Clinics and Practices of gibraltar fever.
One object of the present invention be that the anti-proteic monoclonal antibody 1F1 of sheep Bacillus brucellae BCSP31 is provided and provide can this monoclonal antibody of continuous release hybridoma cell line BCSP31-1F1.
Another object of the present invention is to provide heavy chain and the chain variable region gene and the encoded polypeptides thereof of the antigenic monoclonal antibody of anti-BCSP31.
Another purpose of the present invention is with said monoclonal antibody and gene thereof or polypeptide in the application that detects aspect Bacillus brucellae and diagnosis or treatment human or animal's gibraltar fever preparation or the medicine.
The present invention is achieved in that
According to an aspect of the present invention, the proteic monoclonal antibody hybridoma cell line BCSP31-1F1 of anti-sheep Bacillus brucellae BCSP31 is preserved in Chinese typical culture collection center (CCTCC) on March 27th, 2008, and preserving number is CCTCC C200806.Utilize sheep Bacillus brucellae BCSP31 prokaryotic expression carrier, through abduction delivering, affinitive layer purification; Obtain reorganization BCSP31 albumen; The routine immunization mouse is got immune spleen cell and myeloma cell SP2/0 and merges, and obtains hybridoma cell line BCSP31-1F1 through the screening of ELISA indirect method.Continuous passage half a year, the still sustainable secrete monoclonal antibody 1F1 of hybridoma.
According to another aspect of the present invention, by preserving number be the hybridoma cell line excretory monoclonal antibody 1F1 of CCTCC C200806.Get above-mentioned hybridoma through mouse peritoneal inoculation preparation ascites, n-caprylic acid-sulfuric acid ammonia process purifying obtains monoclonal antibody.But said monoclonal antibody combines with sheep Bacillus brucellae BCSP31 albumen specificity.
According to a further aspect of the invention, the weight of monoclonal antibody 1F1, chain variable region gene are from through extracting the RNA of said hybridoma BCSP31-1F1 cell, obtain the heavy chain and the chain variable region gene of this monoclonal antibody through the RT-PCR method.Through sequencing with in NCBI after the BLAST comparative analysis, the nucleotide sequence of variable region of heavy chain of confirming said antibody is shown in the sequence table < 400>1, and its amino acid sequence coded is shown in the sequence table < 400>3; The chain variable region gene nucleotide sequence of said antibody is shown in the sequence table < 400>2, and its amino acid sequence coded is shown in the sequence table < 400>4.
Heavy, the chain variable region gene of the antibody of successfully cloning for the present invention; Use known antibodies geneseq database (IMGT) professional on the Internet respectively and (NCBI) institute's calling sequence is carried out the homology comparison and show with its germline gene source analysis; The gene order that is obtained is really from the mouse germline gene, and is all not quite identical with the various antibody gene sequences of existing report.
According to a further aspect of the invention, weight, chain variable region gene and the encoded polypeptides thereof that the invention still further relates to said antibody and antibody preparation be used for that Bacillus brucellae detects and gibraltar fever diagnosis and treatment preparation or medicine aspect application.
Is core reagent with monoclonal antibody of the present invention with reorganization BCSP31 albumen, sets up the ELISA sandwich assay, can be used for the enzyme linked immunosorbent detection of gibraltar fever.
The present invention through dna recombinant expression and purifying BCSP31 albumen; Immune mouse has prepared anti-BCSP 31 monoclonal antibody specific 1F1; Be the preparation of Bacillus brucellae detection from now on and gibraltar fever diagnostic reagent, also the mechanism of causing a disease for the further investigation Bacillus brucellae lays the foundation.The present invention adopts the RT-PCR method successfully to clone the weight of said monoclonal antibody, chain variable region gene (VH, VL).Based on above-mentioned weight, chain variable region gene, can adopt gene engineering method, make up and be expressed as the small molecules genetic engineering antibody of various ways, like ScFv antibody, Fab antibody, F (ab)
2Antibody, antibody fusion protein etc., preparation is used to diagnose and treat the medicine of Bacillus brucellae and gibraltar fever.
Description of drawings
Fig. 1 is a purifying BCSP31 albumen Western-blot detected result.
Fig. 2 is the antigen-specific detected result of monoclonal antibody.
Fig. 3 is ELISA indirect sandwich assay sensitivity Detection result.
Embodiment
Sheep Bacillus brucellae BCSP31 albumen pronucleus expression, purifying and evaluation
BCSP31-pGEX-4T1 is reorganization BCSP31 albumen pronucleus expression carrier, and its concrete construction process is referring to auspicious document (the Amphixenosis's magazine 2005,21 (11): 961-964) that waits of department.Get this expression vector transformed into escherichia coli DH5 α, the IPTG abduction delivering 4h of 10mmol/L, collection is resuspended in PBS behind the bacterium, ultrasonicly splits bacterium, uses the purification kit purifying of Amersham company, carries out to specifications.Last appearance back PBS flush away foreign protein mouthful is slowly squeezed into the zymoplasm 1ml of 100U/ml with syringe appearance from the GSTrap affinity column, the sealing upper and lower opening, and 4 ℃ of enzymes were cut 20 hours.The binding buffer flow is washed, the fraction collection flowing lotion, and it is 1.5mg/mL that ultraviolet spectrophotometer is measured protein content.The Westernblot detected result shows, the BCSP31 albumen that is obtained can with Bacillus brucellae positive serum specific reaction (referring to Fig. 1).
The hybridoma preparation
1, the preparation of immune spleen cell
Get the BCSP31 albumen and the abundant mixing of Freund's complete adjuvant of above-mentioned purifying, carry out the subcutaneous multiple spot immunity in back according to 100 a μ g/ mouse; After 2～3 weeks, get BCSP 31 albumen and the incomplete abundant mixing of Freund's complete adjuvant of purifying, only carry out the subcutaneous multiple spot immunity in back according to 100 μ g/; Get the BCSP31 albumen of purifying after 2～3 weeks and inoculate according to 100 μ g/ abdominal cavities, immunity is three times altogether.Two weeks adopted the ELISA method to measure antibody titer in the mice serum after the last immunity.Get height tire mouse through the abdominal cavity booster immunization 1 time, originate as splenocyte.
2, cytogamy
Merge to collect the same day and be in that logarithmic phase, growth conditions are good, viable count is higher than 95% murine myeloma cell (Sp2/0) 3 * 10
7Individual/ml, the full nutrient solution washing 2 times (each centrifugal 5min of 1000rpm) of toing many or too much for use.Get and process suspension after above-mentioned immune mouse spleen cell grinds, the same washing of the full nutrient solution that toos many or too much for use 2 times, and adjustment cell count to 2 * 10
8Individual/ml.Myeloma cell and splenocyte are pressed 1.5: 10 mixed together, and the full nutrient solution that in the 50ml plastic centrifuge tube, toos many or too much for use is washed (the centrifugal 8min of 1200rpm) 1 time.Abandon supernatant, the exhaustion residual liquid.Flick at the bottom of the centrifuge tube pipe, make deposition loosening, slowly add 0.7ml PEG at room temperature 60 seconds, left standstill in the resorb transfer pipet 30 seconds, slowly blow out in 30 seconds.Add incomplete 1640 substratum of 25ml in the 5min, the centrifugal 8min of 1000rpm abandons supernatant.The resuspended deposition of 10ml HAT nutrient solution adds 96 orifice plates that are covered with feeder cell, 2/hole.Place 5%CO
2Incubator continues for 37 ℃ to cultivate.
3, select the screening of cultivation and positive colony
Fused cell is used the HAT culture medium culturing, cultivates and changes liquid 1 time after 4～5 days, and 1/2 liquid in sucking-off 96 orifice plates is added 1 * HAT complete culture solution, and 100 μ l/ holes are put 37 ℃ of 5%CO2 incubators and continued to cultivate, every day observation of cell clonal growth state.After selecting nutrient solution to keep to cultivate for 1 week with HAT, use the HT nutrient solution instead, keep again and cultivated 3～4 days.
, hybridoma during 1/10 area (about the 8th～10 day), detects culture supernatant with indirect elisa method: encapsulate BCSP31 albumen with 5 μ g/ml concentration, 100 μ l/ holes at the bottom of being covered with the hole; 4 ℃ are spent the night, and the washing back adds the 3%BSA sealing, 37 ℃ of reaction 2h; The washing back adds cells and supernatant again, 37 ℃ of reaction 1h, and the washing back adds the sheep anti-mouse igg with 1: 3000 HRP mark of diluted; 37 ℃ of reaction 1h behind the thorough washing, add substrate solution (OPD); Color development at room temperature 30min, 2M H2SO4 stopped reaction and mensuration OD value in the 492nm place.Do the positive and negative contrast with Sp2/0 cells and supernatant and immune serum respectively.With limiting dilution assay positive porocyte is carried out repeatedly cloning, set up hybridoma cell line.Obtain strain of hybridoma system, cultured continuously half a year, but this clone stably excreting monoclonal antibody still are with this clone called after: BCSP31-1F1.
Purification of Monoclonal Antibodies
Hybridoma is used the serum-free medium mixing, and cell count is transferred to 1～2 * 10
6Individual/ml, every BALB/c mouse is through abdominal injection 0.5ml.Behind the inoculation hybridoma about 10～15 days, visible mouse web portion obviously expanded, and extracts ascites, and the centrifugal 15min of 3000rpm collects supernatant.Adopt n-caprylic acid-saturated ammonium sulphate method that ascites is carried out purifying.With the acetate buffer solution dilution of ascites, with 1mol/L sodium hydroxide adjust pH to 4.5 with 4 times of volumes; The amount that adds 25 μ l by sample after every milliliter of dilution adds n-caprylic acid, and the limit edged stirs, and slowly adds.After placing 30min, the centrifugal 30min of 10000 * g.(9 duplicate samples add 1 part 10 * PBS), regulate pH to 7.4 to add 10 times of spissated PBS.Supernatant is cooled to 4 ℃.The ammonium sulfate precipitation supernatant that adds 45% saturation ratio stirs 30min, the centrifugal 15min of 5000 * g.Use a small amount of PBS dissolution precipitation again, under 4 ℃, with the PBS dialysed overnight of 50-100 times of volume.The concentration of measuring mAb is 2.0mg/mL.The SDS-PAGE electrophoresis is accredited as the monoclonal antibody of purifying, and purity is 97%.This monoclonal antibody can be used for preparing the reagent that detects Bacillus brucellae antibody.
The antigen-specific detected result of monoclonal antibody
The BCSP31 albumen of getting above-mentioned purifying carries out the SDS-PAGE electrophoresis, is transferred to NC film or pvdf membrane, and with the monoclonal antibody reactive of above-mentioned purifying, its concentration is 1～50ug/mL, with the HRP mark rabbit anti-mouse igg detection of dilution in 1: 1000～1: 10000, DAB colour developing.The result show said monoclonal antibody can with BCSP31 protein-specific reaction (referring to Fig. 2).
Confirming of monoclonal antibody subclass
Adopt Sigma company mouse monoclonal antibody subclass test kit to detect prepared monoclonal antibody subclass.The said monoclonal antibody subclass is IgG1.
The application of monoclonal antibody in diagnosis sheep gibraltar fever
1, the ELISA indirect sandwich assay detects Bacillus brucellae antibody
Monoclonal antibody (1～10 μ g/ml) is in elisa plate after encapsulating purifying, 100 μ l/ holes, 4 ℃ of incubated overnight.Elisa plate is with the BCSP31 albumen (10～50 μ g/ml) that adds purifying behind the PBS/T washing 3mi n * 3 times, 100 μ l/ holes, 37 ℃ of reaction 1h.After the washing, add sheep blood serum to be checked (1: 100), 100 μ l/ holes, 37 ℃ of reaction 1h.After the PBS/T washing, add the anti-sheep of rabbit (goat or the sheep) IgG (Zhongshan Bioengineering Co., Ltd.) of the HRP mark of 1: 2000～50000 dilutions, 100 μ l/ holes, 37 ℃ of reaction 1h.After elisa plate washs 3min * 3 time with PBS/T, with O-Phenylene Diamine (O-phenyl-enediamine, OPD) or TMB (3,3 ', 5,5 '-tetramethyl-benzidine, TMB) be the substrate colour developing, 100 μ l/ holes, 37 ℃ of reaction 15min.Stop buffer 25 μ l/ hole (2 M H
2SO4) stopped reaction and measure the OD value in the 492nm place.With reorganization BCSP31 albumen sheep immune serum or the positive contrast of gibraltar fever human serum, with normal sheep or the negative contrast of human serum.Result of determination: P/N value=sample OD value/negative control OD value; The P/N value of positive control must be more than or equal to 2.1, and the P/N value of testing sample is judged as positive findings more than or equal to 2.1, less than 2.1 negative results or suspicious; Need to detect once again, still be lower than at 2.1 o'clock and think negative findings.
The anti-sheep IgG of the rabbit of the HRP mark that in said ELISA indirect sandwich assay, relates to serves as according to selecting for use with serum kind to be checked source.For example, when detecting the serum in goat source, need with the anti-goat IgG of the rabbit of HRP mark; When detecting the serum in sheep source, need with the anti-sheep IgG of the rabbit of HRP mark.Detect other species like need and infect serum, during like animals such as deer, pigs, can adopt the antibody of the anti-corresponding species IgG of HRP mark.Like this, this method can be widely used in the diagnosis of gibraltar fever.
2, the susceptibility of above-mentioned ELISA indirect sandwich assay
Use the sheep Bacillus brucellae serum that above-mentioned ELISA method detects serial dilution, serum dilution is from stoste to 10
-7The result shows that its this method susceptibility is 5 * 10
-4(Fig. 3).
3, monoclonal antibody detects the application in the sheep Bacillus brucellae
The said monoclonal antibody of this patent can be used for the detection of Bacillus brucellae: the said monoclonal antibody of this patent can directly be used for the detection of Bacillus brucellae.Through the separation and Culture to sample, with the bacterium and the monoclonal antibody reactive of gained, with enzyme labelling two anti-or fluorescent mark two anti-reactions, the two anti-antibody with above-mentioned HRP mark here are consistent again, and the mark of antibody can be HRP, AP or FITC.Can be used for Bacillus brucellae identifies.
The said monoclonal antibody of this patent in addition can be directly with marks such as FITC or HRP, AP, directly be used for the detection of Bacillus brucellae or its antigen BCSP31.
The amplification of monoclonal antibody heavy chain and chain variable region gene, clone and sequencing
Extract the total RNA of hybridoma 1F1 with Trizol (Gibco).It is synthetic to carry out cDNA with the cDNA first chain synthetic agent box of U.S. Promega company.Use specific variable region of heavy chain (VH) primer (table 1) and variable region of light chain (VL) primer (table 2) to increase.The pcr amplification condition is: 94 ℃ of 5min, and 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, after 30 circulations, 72 ℃ of 10min.Be cloned in the T easy Vector carrier of U.S. Promega company, measure dna sequence dna in AudioCodes Bioisystech Co., Ltd.Wherein said monoclonal antibody heavy chain variable region gene has the sequence of sequence table < 400>1, and chain variable region gene has the sequence of sequence table < 400>3.
Amplification primers: according to Luo Wen etc. (structure and the sequential analysis of mouse source monoclonal antibody 3G1 single-chain antibody gene. the journal .2003 of Xi'an Joint Univ.; 6 (2): 25-8) document is synthetic.
Table 1: heavy chain H variable region primer
* be the degeneracy sign indicating number in the primer sequence bracket
* be the degeneracy sign indicating number in the primer sequence bracket
To the antigenic monoclonal anti body weight of the anti-BCSP31 that is cloned, that chain variable region gene carries out sequential analysis is following
Use following two DBs respectively:
http://imgt.cines.fr
http://www.ncbi.nlm.nih.gov/blast
Institute's calling sequence and existing other various antibody genes of having reported are carried out homology relatively, and analyze its germline gene source, the result is following:
(1) germline gene of 1F1 heavy chain < 400>1 source:
V-GENE：BN000872 IGHV1-62-2*01，or AC163348 IGHV1-71*01
D-GENE：J00434 IGHD1-1*01
J-GENE：V00762 IGHJ1*01，or X63164 IGHJ1*03
Analyze demonstration through FR-IMGT and CDR-IMGT:
CDR1：ggc tac att ttc act gag tat att
CDR2：ttt tcc cct gga agt ggt agt a ta
CDR3：gca aga cac gaa gcc tac ggt agt acc ctt tac tgg tac ttc gatgtc
The homology comparative result shows among the NCBI:
RID：XJEJ7N2R016
Database：All GenBank+EMBL+DDBJ+PDB sequences(but no EST，STS，GSS，environmental samples or phase 0，1 or 2 HTGS sequences)
6,540,228 sequences；23，165，110，747 total lettersQuery＝Leng th＝384
Database：All GenBank+EMBL+DDBJ+PDB sequences(but no EST，STS，GSS，environmental samples or phase 0，1 or 2 HTGS sequences)
2,655,401 sequences；12，045，272，748 total lettersgi|84487350|dbj|AB159968.1|Mus musculus mRNA for immunoglobulinH-chain V-region，partial cds，clone：0S_b-01
Length＝357
Score＝558 bits(618)，Expect＝6e-156
Identities＝344/368(93％)，Gaps＝12/368(3％)
Strand＝Plus/Plus
(2) germline gene of 1F1 light chain < 400>3 source:
V-GENE：Y15976 IGKV6-15*01
J-GENE：V00777 IGKJ4*01
Analyze demonstration through FR-IMGT and CDR-IMGT:
CDR1：cag aat gtg ggt act tat
CDR2：tcg gca tcc
CDR3：cag caa tat aac agc tat cca ttc acg
The homology comparative result shows among the NCBI:
RID：XJAVVTUD014
Query＝Length＝4 50
Database：All GenBank+EMBL+DDBJ+PDB sequences(but no EST，STS，GSS，environmental samples or phase 0，1 or 2 HTGS sequences)
6,540,228 sequences；23，165，110，747 total lettersgi|5853231|gb|AF178624.1|AF178624 Mus musculus 23-7 immunoglobulinlight chain variable region
mRNA，partial cds
Length＝321
Score＝513bits(568)，Expect＝3e-142
Identities＝304/317(95％)，Gaps＝0/317(0％)
Strand＝Plus/Plus
Above-mentioned analytical results shows: the monoclonal antibody 1F1 gene order that is obtained is all not quite identical from various other known antibodies genes of mouse germline gene and existing report really, is to belong to a kind of new antibody gene to BCSP31 that function is arranged.
The application of the antigenic monoclonal anti body weight of anti-BCSP31, chain variable region gene and encoded polypeptides product thereof
It is the Bacillus brucellae cell surface protein that bibliographical information BCSP31 albumen is arranged, and has high conservative property, is a kind of Bacillus brucellae immunodominance antigen and protective antigen, can stimulate body to produce protective immunological reaction.
The weight of monoclonal antibody according to the invention, chain variable region gene can reconstruct and be expressed as the protein drug of various ways, can directly be used for the diagnosis and the treatment of anti-SARS-Coy inflammation.For example: small molecular antibody.Mainly contain by VH-CH1 and VL-C1 form Fab antibody, with a polypeptide (GLy4Ser)
3The single domain antibody that joint connects single-chain antibody that VH gene and VL gene form, be made up of with non covalent bond be combined into Fv fragment antibody, by VH or functional domain of VL VH and VL, the atom that constitutes by single CDR etc.; The multivalence miniantibody.Mainly contain double-stranded antibody, (ScFv)
2, Flex miniantibody, LD miniantibody, F (ab ')
2, F (ab ')
3, (ScFv)
4Deng.Because the polyvalent antigen binding site arranged, avidity is high, and molecular size is moderate, can penetrate tissue, and also slower characteristics of the clearance rate in kidney and have high clinical value; Bi-specific antibody.Be one type of antibody, claim bifunctional antibody again with dual specificity and dual-use function; Recombinant antibody fusion proteins.Gene fragments such as Fab or Fv, with having that other protein genes such as the toxin of non-antibody or enzyme are connected to form a kind of recombinant protein of specific biological activity guiding target site; Recombinant immunotoxin.The recombination of encoding antibody and toxin is produced, and characteristics are efficient, and non-special toxicity is low, and the body internal stability is good; Humanized's whole antibody mainly contains by VH-CH and VL-C1 and forms complete antibody.
Sequence table
< 110>The Fourth Military Medical University of P.L.A
< 120>a kind of monoclonal antibody 1F1 and application thereof and the hybridoma cell line of secreting this antibody
BCSP31-1F1
<160>4
<210>1
<211>384
<212>DNA
< 213>mouse
<220>
<221>V-region
<222>(7)...(376)
<400>1
atggccgagg cccagctgca gcagtctgga gctgagctgg tgaaacccgg ggcatcagtg 60
aagctgtcct gcaaggcttc tggctacatt ttcactgagt atattataca ctggataaag 120
cagaggtctg gacagggtct tgagtggatt gggtggtttt cccctggaag tggtagtata 180
aagtacaatg agaaatttaa ggacaaggcc acattgactg cggacaaatc ctccagcaca 240
gtctatatgg agcttagtag attgacatct gaagactctg cggtctattt ctgtgcaaga 300
cacgaagcct acggtagtac cctttactgg tacttcgatg tctggggcca agggaccacg 360
gtcaccgtct cctcaaatca ctag 384
<210>2
<211>123
<212>PRT
< 213>mouse
<220>
<221>V-segment
<222>(1)...(123)
<400>2
Glu Ala Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly
1 5 10 15
Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr
20 25 30
Glu Tyr Ile Ile His Trp Ile Lys Gln Arg Ser Gly Gln Gly Leu
35 40 45
Glu Trp Ile Gly Trp Phe Ser Pro Gly Ser Gly Ser Ile Lys Tyr
50 55 60
Asn Glu Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser
65 70 75
Ser Ser Thr Val Tyr Met Glu Leu Ser Arg Leu Thr Ser Glu Asp
80 85 90
Ser Ala Val Tyr Phe Cys Ala Arg His Glu Ala Tyr Gly Ser Thr
95 100 105
Leu Tyr Trp Tyr Phe Asp Val Trp Gly Glu Gly Thr Thr Val Thr
110 115 120
Val Ser Ser
123
<210>3
<211>450
<212>DNA
< 213>mouse
<220>
<221>V-region
<222>(130)...(450)
<400>3
atgaccatga ttacgccaag ctatttaggt gacactatag aatactcaag ctatgcatcc 60
aacgcgttgg gagctctccc atatggtcga cctgcaggcg gccgcgaatt cactagtgat 120
tggctgcagg atattcagct gacacaagct caaaaattca tgtccacatc agtaggagac 180
agggtcagcg tcacctgcaa ggccagtcag aatgtgggta cttatgtagc ctggtatcaa 240
cagaaaccag ggcaatctcc taaatcactg atttactcgg catcctaccg gtacagtgga 300
gtccctgatc gcttcacagg cagtggatct gggacagatt tcactctcac catcagcaat 360
gtacagtctg aagacttggc agagtatttc tgtcagcaat ataacagcta tccattcacg 420
ttcggctcgg ggaccaagct ggagatctaa 450
<210>4
<211>106
<212>PRT
< 213>mouse
<220>
<221>V_segment
<222>(1)...(106)
<400>4
Asp Ile Gln Leu Thr Gln Ala Gln Lys Phe Met Ser Thr Ser Val
1 5 10 15
Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly
20 25 30
Thr Tyr Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
35 40 45
Ser Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp
50 55 60
Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75
Ser Asn Val Gln Ser Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln
80 85 90
Tyr Asn Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
95 100 105
Ile
106
Claims (7)
1. the proteic mouse monoclonal antibody 1F1 of anti-sheep Bacillus brucellae BCSP31 is to be the hybridoma cell line secretion generation of CCTCC C200806 by deposit number.
2. can secrete the hybridoma cell line BCSP31-1F1 of monoclonal antibody according to claim 1 for one kind, its deposit number is: CCTCC C200806.
3. by the described a kind of monoclonal antibody 1F1 of claim 1, it is characterized in that: its heavy chain variable region gene has the sequence of sequence table < 400>1, and chain variable region gene has the sequence of sequence table < 400>3.
4. by the described monoclonal antibody 1F1 of claim 3, it is characterized in that: its heavy chain variable region gene encoded polypeptides has the sequence of sequence table < 400>2, and its chain variable region gene encoded polypeptides has < 400>4 sequence.
5. be used for detecting the application of Bacillus brucellae and diagnosis human or animal gibraltar fever detection reagent in preparation by the described monoclonal antibody 1F1 of claim 1.
6. be used for detecting the application of Bacillus brucellae and diagnosis or treatment human or animal's gibraltar fever reagent and medicine in preparation by the heavy chain of the described monoclonal antibody 1F1 of claim 3 and chain variable region gene.
7. be used for detecting the application of Bacillus brucellae and diagnosis or treatment human or animal's gibraltar fever reagent and medicine in preparation by the heavy chain of the described monoclonal antibody 1F1 of claim 4 and chain variable region gene encoded polypeptides.
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| CN2008100181302A CN101270160B (en) | 2008-05-05 | 2008-05-05 | Monoclone antibody 1F1, uses thereof and hybridoma cell line BCSP31-1F1 excreting the antibody |
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| CN101270160B true CN101270160B (en) | 2012-05-02 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101020064A (en) * | 2007-03-27 | 2007-08-22 | 北京大学 | Brucellosis nucleic acid vaccine |
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