CN107446893A - Blec protein monoclonal antibodies and its preparation method and application - Google Patents

Blec protein monoclonal antibodies and its preparation method and application Download PDF

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CN107446893A
CN107446893A CN201710418299.6A CN201710418299A CN107446893A CN 107446893 A CN107446893 A CN 107446893A CN 201710418299 A CN201710418299 A CN 201710418299A CN 107446893 A CN107446893 A CN 107446893A
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blec
albumen
monoclonal antibodies
chicken
cell
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CN107446893B (en
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韩凌霞
廉传江
赵丽丽
张圆圆
陈洪岩
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Harbin Veterinary Research Institute of CAAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/465—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from birds
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4724—Lectins

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses Blec protein monoclonal antibodies and its preparation method and application.The present invention, by immune mouse spleen cell and SP2/0 cell fusions, by repeatedly screening and subclone, obtains the specific monoclonal cell strain blec 3D3 and monoclonal antibody blec 3D3 by its secretion using the Blec albumen of prokaryotic expression as immunogen immune mouse.Flow cytometry is carried out to chicken PBLC using monoclonal antibody blec 3D3, the results showed that blec albumen is mainly expressed in CD3+ and CD4+ cells；SABC testing result shows that monoclonal antibody blec 3D3 can detect specific signals in chicken mucosa-immune linked groups.Blec monoclonal antibodies provided by the invention can be as detection antibody test animal tissue or distribution, expression or the biological function of intracellular Blec albumen.

Description

Blec protein monoclonal antibodies and its preparation method and application
Technical field
The present invention relates to chicken C- type agglutinin receptor protein B lec monoclonal antibodies and secrete the monoclonal antibody Hybridoma cell strain, the invention further relates to them in the Histological distribution of detection blec albumen, expression in vivo and in vitro Application, belong to preparation and the application field of chicken agglutinin receptor protein B lec monoclonal antibodies.
Background technology
Chicken blec genes be located at major histocompatibility complex (Major histocompability complex, MHC nucleus), conservative is stronger between different haplotypes.According to the derivation amino acid sequence analysis of encoding proteins, it is C- type agglutinin structures, may the mediation of non-with the NK of chicken MHC I quasi-molecules cell killing function it is relevant.But So far the structure of blec albumen, the Histological distribution in chicken body are there is not yet clearly report.
Using Blec protein specific monoclonal antibodies, blec albumen in chicken body can be analyzed by immunohistochemistry technology Histological distribution, this for study blec albumen structure, biological function and the Histological distribution in chicken body have Important value.
The content of the invention
First technical problem to be solved by this invention is to provide one plant of stably excreting Blec protein specific monoclonal The hybridoma cell strain of antibody；
Second technical problem to be solved by this invention is the Blec protein monoclonals for secreting the hybridoma cell strain Antibody is applied to the expression or distribution of the Blec albumen in detection chicken body.
In order to solve the above technical problems, the technical solution used in the present invention is：
The present invention is suitable for blec protein functions to obtain the conservative blec protein-specific monoclonal antibodies of MHC haplotypes chicken Research, the present invention derive amino acid sequence result according to all different haplotype chicken Blec gene orders announced on the net, comparison, It was found that 5 variant sites be present, in order to obtain the monoclonal antibody for being applied to surface ligand functional analysis, the present invention is final to determine selection Ala59-Leu176 is expressed in region.The most conservative B21 haplotype chickens of selection are experiment material, and normative reference sequences Design is drawn Thing, blec extracellular regions (Ala59-Leu176) gene is expanded, pET-Blec21 recombinant plasmids are built, to ensure that acquisition type is universal Monoclonal antibody.IPTG induces pET-blec21 prokaryotic expression recombinant bacteriums, ultrasonic disruption, is handled with guanidine hydrochloride, obtains inclusion body；Multiple Property liquid in renaturation, concentration, purifying protein in gel permeation chromatography post, obtain the Blec albumen of purifying.
7 week old no-special pathogen BALB/c female mices are taken, are immunized with the blec albumen of purifying, 50 μ g/ mouse, Intraperitoneal injection.Immune every 2 weeks once to gather immunized mice blood the 3rd immune latter week, 37 DEG C are placed 1h, 4 DEG C of centrifugation 10min Separate serum.By serum doubling dilution, serum titer is detected with indirect ELISA, selects serum titer highest mouse in fusion Preceding 3 days booster immunizations.By immune mouse spleen cell and SP2/0 cell fusions, by 3 screenings and 3 subclones, specificity is obtained By force, the good monoclonal cell strain blec-3D3 of stability.
The present invention carries out subgroup identification to monoclonal cell strain blec-3D3, and qualification result is shown, blec-3D3 heavy chain It is IgM, light chain is Kappa chains.
Western blot qualification results show, blec-3D3 can specific detection to Ji Tinei mucosa-immunes linked groups In Blec albumen.
The hybridoma cell strain blec-3D3 of secrete monoclonal antibody is submitted the mechanism of patent accreditation to be protected by the present invention Hide, its microbial preservation numbering is：CGMCC No.12998；Classification And Nomenclature is：BALB/c mouse hybridoma.Preservation list Position：China Committee for Culture Collection of Microorganisms's common micro-organisms center；The preservation time is on October 26th, 2016；Preservation Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Flow cytometry result shows, can be used for surface table in detection chicken peripheral blood using the Blec monoclonal antibodies of the present invention Up to the cell category of blec albumen.
SABC testing result shows, the group of blec albumen in detection chicken body is can be used for using the Blec monoclonal antibodies of the present invention Knit credit cloth.
Brief description of the drawings
Fig. 1 is chicken blec genes extracellular region and people and the amino acid sequence of the type (CLEC2) of mouse C- type agglutinin receptors family 2 Row tetraploid rice result.
Fig. 2 is BlecHiload 16/60SuperdexTM75pre grade [global] purification result.
Fig. 3 is Western blot qualification result of the blec-3D3 cell conditioned mediums to prokaryotic expression product：1：PET-30a is carried Body transformed bacteria；2：PET-Blec recombinant plasmid transformed bacterium.
Fig. 4 is to use Flow cytometry chicken PBLC surface blec albumen using Blec monoclonal antibodies of the present invention Expression of results.
Fig. 5 is that application Blec monoclonal antibodies of the present invention carry out the result that SABC detects the distribution of chicken blec albumen tissues.
Embodiment
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and It is apparent.It should be understood that the embodiment is only exemplary, any restrictions are not formed to the scope of the present invention.This area Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and Form is modified or replaced, but these modifications or substitutions each fall within protection scope of the present invention.
The structure of the MHC-B haplotype chicken C- type agglutinin receptor albumen blec monoclonal antibody cell lines of embodiment 1 and identification
1. the selection of immunogene blec protein type conservative regions
To obtain the conservative blec protein-specific monoclonal antibodies of MHC haplotypes chicken, and it is suitable for the research of blec protein functions, this Invention compares according to all different haplotype chicken Blec gene orders announced on the net and derives amino acid sequence result, there is 5 Variant sites (overstriking underscore represents in following table).The most conservative B21 haplotype chickens of selection are experiment material, and reference sequences design Primer, blec genes are expanded, pET-Blec21 recombinant plasmids are built, to ensure the universal monoclonal antibody of acquisition type.
The chicken difference haplotype Blec genes of table 1 derive the mutational site of amino acid
MHC B haplotype amino acid sites
It has been solved with structure is shown to the evolutionary analysis result of Blec protein extracellulars c-type lectin-like domain sequence The people of analysis and subgroup (CLEC2) family similarity of mouse C- types lectin-like domain the 2nd are higher (Fig. 1).Due to mammal CLEC2 there is NK (NK cells) part, thus it is speculated that chicken blec albumen is also likely to be matching somebody with somebody for chicken NK cells Body.In order to obtain the monoclonal antibody for being applied to surface ligand functional analysis, present invention selection Ala59-Leu176 is expressed in region.
The amplification of 2.blec genes and prokaryotic expression
MHC-B21 haplotype chicken blec sequences (the NCBI acc.No announced according to NCBI:AM950195) design a pair and draw Thing：
Upstream：5′-CCATATGGCGCAGTGCCCGTTCGATTGGATTG-3 ',
Downstream：5′-CCCTCGAGTTACAACGCGGGTTTGGTGCAAACC-3 ', with no-special pathogen (Specific Pathogen free, SPF) B21 haplotype chicken PBLC total serum IgEs are template, it is extracellular that RT-PCR reactions obtain blec Area (Ala59-Leu176) cDNA, it is cloned into coli expression carrier pET-30a, Transformed E .coli BL21 (DE3) competence Cell, successfully construct prokaryotic expression recombinant plasmid pET-blec21；Expressed amino acid sequence is： MAQCPFDWIGFGGKCYYFSEDESNWTSSQNNCSALGASLAVFDSAEDLSFTMRHKGSSPHWVGLSREGKEHPWEWVN RSPLSHLFQVQGDGLCAYLGDAGLSSSHCSARRNWVCTKPAL(SEQ ID No.1)；
3. the screening of immunogene form
To obtain the monoclonal antibody that adhesion is strong, specificity is good, immunogene is prepared using 2 kinds of approach, according to the blood for being exempted from mouse Clear antibody titer, select potency is high to be exempted from mouse and further prepare monoclonal antibody.
3.1 PAGE gel bars are immunized
Mass propgation, induced expression pET-blec21 recombinant bacteriums, according to the extracting mode of inclusion body, carry out ultrasonic wave and break Broken, centrifugation, after mixing, boil with 5 × sample-loading buffer, carry out SDS-PAGE electrophoresis.The upper batten of broach is could be used without, on Sample.After electrophoresis terminates, conventional Coomassie brilliant blue dye liquor dyes 30min, and boiling water bath 30min decolourizes, and during which repeatedly changes water, until the back of the body Scape Clear colourless, purpose band are clear.According to standard molecular weight, the blade sterilized with 75% ethanol, purpose is accurately cut Band.Pulverize as far as possible after, it is resuspended with PBS, as immunogene.
3.2 Blec protein purification products are immunized
IPTG induces pET-blec21 prokaryotic expression recombinant bacteriums, and thalline 6000r/min centrifugations 20min takes precipitation.Ultrasonic wave It is broken, handled with guanidine hydrochloride solution, obtain inclusion body.In 100mM Tris pH8.0,400mM L-ArgHCl and 2mM EDTA Renaturation 8h in renaturation solution (adding redox couple 5mM GSH/1mM GSSG), concentration (concentration buffer1L:20mMTris/ 50mMNaCl), purifying protein in gel permeation chromatography post.From SUPREDEX75BG, 20mM Tris/50mM NaCl pH8.5 Condition purifies.
SDS-PAGE experimental results (Fig. 2) show that Blec albumen is expressed with inclusion bodies.In Fig. 2, SDS-PAGE results The colour developing band of upper part back swimming lane corresponds to small peak before gel permeation chromatography post figure, the colour developing bar of figure lower front edge swimming lane Peak with corresponding gel permeation chromatography post figure, peak value are higher than 2000mAU.Peak figure result shows that purity of protein is higher, and concentration is 15mg/ml.Appropriate dilution, with 50 μ g/ mouse, is immunized.
The BALB/c female mices of 7 week old are taken, are injected intraperitoneally respectively with above-mentioned 3.1 and 3.2 immunogenes prepared immune small Mouse, it is each immune 3.Exempt from again after 7 days, 2 exempt from latter week collection, separating immune mouse serum.With final concentration of 200ng/100 μ L's Blec purifying protein coated elisa plates, OD450 values are detected with indirect elisa method.As a result the OD450 value average out to of glue immunized mice is cut 0.343, the OD450 values of purifying protein immunized mice are 0.458, blank control wells 0.017.Show to be exempted from using purifying protein Epidemic disease, the antiserum titre of acquisition are immunized better than cutting glue.All it is immunized using purifying protein, was immunized once every 2 weeks afterwards, then After immune 2 times, routine techniques fusion, monoclonal antibody is prepared.
4. the screening of positive hybridoma strain
It is lethal that eyeball blood sampling will be plucked by immunized mice, it is sterile to win spleen, with plunger in scattered spleen on 300 mesh copper mesh Cell.With appropriate murine myeloma cell SP2/0 mixing with cells, after centrifugation, the 96 hole cells containing feeder cells are dispensed into Plate culture.After 10-12 days, the cell hole of nutrient solution jaundice is selected to detect one by one.Judge that positive criteria is：(sample well OD450nm- blank control OD450nm)/(negative control hole OD450nm- blank control D450nm)>2.1.
Repeatedly screening and subclone, eliminate the cell that antibody-secreting is unstable, potency is not high, finally obtain satisfactory Monoclonal cell strain blec-3D3.
5. hypotype is identified
UsingRapid ELISA Mouse mAb Isotyping Kit specifications carry out sub- to Blec mAb Class is identified.Use Na2CO3/NaCO3It is coated with Blec albumen, concentration 200ng/ul.After 5% skimmed milk closing 3h, add blec-3D3 Cell conditioned medium, 37 DEG C of secondary antibodies (1 being incubated in 1h, then reagent adding box:800 dilutions), 37 DEG C of incubation 30min.As a result show, Blec-3D3 heavy chain is IgM, and light chain is Kappa chains.
6. the preparation of hybridoma ascites and Western blot identifications
Take 8-12 week old BALB/c mouse intraperitoneal injection paraffin oil 0.5mL/ only, pneumoretroperitoneum injects exponential phase within one week Hybridoma, every mouse about 106Individual cell, routine techniques prepare ascites.Blec prokaryotic expression products and pET-30a are carried Body transformed bacteria carries out SDS-PAGE electrophoresis, carries out half-dried transfer and skimmed milk closing.It is thin that the NC films closed are put into blec-3D3 In born of the same parents' ascites, room temperature effect 1h, PBST washing 3 times, 5min/ times；The mountain sheep anti-mouse igg marked with the HRP enzymes of 8000 times of dilutions For secondary antibody, room temperature effect 1h, PBST washing 3 times, 5min/ times.Infrared ray sweeps the colour developing of film instrument.As a result show that blec-3D3 can be special Property detects Blec albumen (Fig. 3).
Expression of the Blec albumen of experimental example 1 in PBLC
Add 5ml chickens lymphocyte separation medium in 15ml centrifuge tubes, then slow equivalent adds 72 week old SPF chicken wings vein lemons Lemon acid sodium anticoagulation.2000r/min centrifuges 15min, takes more intermediate annular milky buffy coat.2 times are washed with 1 × PBS, every time 5min.The cell being separated to is divided into 3 parts in equal volume, every part of 500ul.Wherein 1 part is not dyed, and does negative control gating, will be another Outer cell is separately added into chicken CD3 monoclonal antibodies, CD4 monoclonal antibodies, CD8 monoclonal antibodies and the B cell monoclonal antibody of appropriate Blec-3D3 ascites and commercialization, In 37 DEG C of lucifuge 30min；1600r/min centrifuges 5min, discards supernatant, is washed 2 times with PBS, each 5min；Cell is resuspended with PBS, The mountain sheep anti-mouse igg (H＆L) of FITC marks is added, 37 DEG C of lucifuge 30min, cell, the filtering of 300 mesh copper mesh is resuspended with 1 × PBS Afterwards, flow cytomery is used.As a result show the cell surface expression blec albumen for having 1%, be distributed in CD3+ and CD4+ cells Surface (Fig. 4)；Testing result shows, can be used for surface expression blec eggs in detection chicken peripheral blood using the Blec monoclonal antibodies of the present invention White cell category.
The SABC of experimental example 2 detects distribution of the blec albumen in chicken body tissue
Spleen, liver, kidney, lungs, heart, the bursa of farbricius, thymus gland and the cecal tonsil of 12 week old SPF chickens are taken, is protected It is stored in the 50mL centrifuge tubes equipped with the formaldehyde fixers of 30mL 10%, it is fixed.With FFPE and cut into slices.Staining procedure reference Immunohistochemical reagents box specification is carried out, and anti-Blec protein-specifics monoclonal antibody Blec-3D3 dilution factors are 1: 100.Dye Observed under an optical microscope after.As a result it is smooth in bursa of farbricius epithelium, the smooth muscle cell of lung tertiary bronchus, kidney, small intestine Stronger specific positive signal is detected in flesh, thymus gland Kazakhstan corpusculum and pulpa lienis matter, and (figure is not detected by heart and liver 5)；Test result indicates that it can be used for the Histological distribution of blec albumen in detection chicken body using the Blec monoclonal antibodies of the present invention.
SEQUENCE LISTING
<110>Harbin Veterinary Medicine Inst., China Academy of Agriculture
<120>Blec protein monoclonal antibodies and its preparation method and application
<130> HLJ-2001-170109A
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 119
<212> PRT
<213> artifical sequence
<400> 1
Met Ala Gln Cys Pro Phe Asp Trp Ile Gly Phe Gly Gly Lys Cys Tyr
1 5 10 15
Tyr Phe Ser Glu Asp Glu Ser Asn Trp Thr Ser Ser Gln Asn Asn Cys
20 25 30
Ser Ala Leu Gly Ala Ser Leu Ala Val Phe Asp Ser Ala Glu Asp Leu
35 40 45
Ser Phe Thr Met Arg His Lys Gly Ser Ser Pro His Trp Val Gly Leu
50 55 60
Ser Arg Glu Gly Lys Glu His Pro Trp Glu Trp Val Asn Arg Ser Pro
65 70 75 80
Leu Ser His Leu Phe Gln Val Gln Gly Asp Gly Leu Cys Ala Tyr Leu
85 90 95
Gly Asp Ala Gly Leu Ser Ser Ser His Cys Ser Ala Arg Arg Asn Trp
100 105 110
Val Cys Thr Lys Pro Ala Leu
115

Claims (6)

1. the hybridoma cell strain of one plant of stably excreting Blec protein monoclonal antibody, it is characterised in that its microbial preservation is compiled Number it is：CGMCC No.12998.
2. the Blec protein monoclonal antibodies that the hybridoma cell strain as described in claim 1 is secreted.
3. hybridoma cell strain described in claim 1 is preparing detection Blec albumen in animal tissue or cell inner expression or tissue Learn the application in distribution agent.
4. blec protein monoclonal antibodies described in claim 2 are preparing detection Blec albumen in animal tissue or cell inner expression Or the application in Histological distribution reagent.
5. detection Blec albumen is expressed in animal tissue or cell or the antibody assay kit of Histological distribution, including detection Antibody, it is characterised in that：Described detection antibody is the Blec protein monoclonal antibodies described in claim 2.
6. detection Blec albumen is expressed in animal tissue or the immunologic combined detection reagent kit of Histological distribution, including detection is anti- Body, it is characterised in that：Described detection antibody is the Blec protein monoclonal antibodies described in claim 2.
CN201710418299.6A 2017-06-06 2017-06-06 Blec protein monoclonal antibody and its preparation method and application Expired - Fee Related CN107446893B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111848779A (en) * 2019-08-07 2020-10-30 北京市农林科学院 Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6339185B1 (en) * 1994-09-02 2002-01-15 Drexel University Plant termination sequence
CN103589690A (en) * 2013-11-15 2014-02-19 中国农业科学院哈尔滨兽医研究所 Chicken interleukin-18 monoclonal antibody as well as preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6339185B1 (en) * 1994-09-02 2002-01-15 Drexel University Plant termination sequence
CN103589690A (en) * 2013-11-15 2014-02-19 中国农业科学院哈尔滨兽医研究所 Chicken interleukin-18 monoclonal antibody as well as preparation method and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111848779A (en) * 2019-08-07 2020-10-30 北京市农林科学院 Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof
CN111848779B (en) * 2019-08-07 2022-02-22 北京市农林科学院 Chicken complement receptor 2(ChCR2) monoclonal antibody and application thereof

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