CN103739679A - Avian metapneumovirus (aMPV) F protein polypeptide and application thereof - Google Patents
Avian metapneumovirus (aMPV) F protein polypeptide and application thereof Download PDFInfo
- Publication number
- CN103739679A CN103739679A CN201410018287.0A CN201410018287A CN103739679A CN 103739679 A CN103739679 A CN 103739679A CN 201410018287 A CN201410018287 A CN 201410018287A CN 103739679 A CN103739679 A CN 103739679A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- protein
- monoclonal antibody
- immunogen
- freund
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 52
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 38
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 38
- 108010068327 4-hydroxyphenylpyruvate dioxygenase Proteins 0.000 title claims abstract description 21
- 241001519465 Avian metapneumovirus Species 0.000 title abstract 4
- 239000000427 antigen Substances 0.000 claims abstract description 14
- 102000036639 antigens Human genes 0.000 claims abstract description 14
- 108091007433 antigens Proteins 0.000 claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 241000351643 Metapneumovirus Species 0.000 claims description 22
- 230000008878 coupling Effects 0.000 claims description 14
- 238000010168 coupling process Methods 0.000 claims description 14
- 238000005859 coupling reaction Methods 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 13
- 210000004408 hybridoma Anatomy 0.000 claims description 12
- 230000002163 immunogen Effects 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- 238000002965 ELISA Methods 0.000 claims description 8
- 239000002671 adjuvant Substances 0.000 claims description 8
- 210000004027 cell Anatomy 0.000 claims description 8
- 102000014914 Carrier Proteins Human genes 0.000 claims description 7
- 108010078791 Carrier Proteins Proteins 0.000 claims description 7
- 244000309466 calf Species 0.000 claims description 6
- 230000006872 improvement Effects 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 210000002966 serum Anatomy 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 5
- 230000028993 immune response Effects 0.000 claims description 5
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 claims description 4
- 210000001185 bone marrow Anatomy 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 230000003053 immunization Effects 0.000 claims description 4
- 238000002649 immunization Methods 0.000 claims description 4
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 3
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 3
- 238000010612 desalination reaction Methods 0.000 claims description 3
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 3
- 229940005654 nitrite ion Drugs 0.000 claims description 3
- 210000000952 spleen Anatomy 0.000 claims description 3
- 210000004988 splenocyte Anatomy 0.000 claims description 3
- 208000011580 syndromic disease Diseases 0.000 claims description 3
- 210000004881 tumor cell Anatomy 0.000 claims description 3
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 claims description 2
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 claims description 2
- 108010017384 Blood Proteins Proteins 0.000 claims description 2
- 102000004506 Blood Proteins Human genes 0.000 claims description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 2
- 108010058846 Ovalbumin Proteins 0.000 claims description 2
- 229960003896 aminopterin Drugs 0.000 claims description 2
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 108060003552 hemocyanin Proteins 0.000 claims description 2
- 229940092253 ovalbumin Drugs 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 229940104230 thymidine Drugs 0.000 claims description 2
- -1 thyroprotein Proteins 0.000 claims description 2
- 238000003766 bioinformatics method Methods 0.000 abstract 1
- 239000002953 phosphate buffered saline Substances 0.000 description 10
- 241000287828 Gallus gallus Species 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 5
- 230000035945 sensitivity Effects 0.000 description 5
- 235000020183 skimmed milk Nutrition 0.000 description 5
- 238000007865 diluting Methods 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 101150034814 F gene Proteins 0.000 description 3
- 238000003119 immunoblot Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 210000003501 vero cell Anatomy 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 208000003322 Coinfection Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000005723 virus inoculator Substances 0.000 description 2
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000027312 Bursal disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000032163 Emerging Communicable disease Diseases 0.000 description 1
- 240000004859 Gamochaeta purpurea Species 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- 206010066226 Metapneumovirus infection Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000272458 Numididae Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000286209 Phasianidae Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000711902 Pneumovirus Species 0.000 description 1
- 206010051497 Rhinotracheitis Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 239000006180 TBST buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003636 conditioned culture medium Substances 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000005571 horizontal transmission Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000002277 temperature effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000005570 vertical transmission Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an F protein polypeptide and an application thereof. The antigen polypeptide is obtained through bioinformatics analysis and comparison according to F protein gene sequences. The amino acid sequence of the antigen polypeptide is CTNAGSTAYYPNKDD. The antigen polypeptide has high conservative property and strong antigenicity, and monoclonal antibodies aiming at the Avian metapneumovirus (aMPV) F proteins can be generated by utilizing the polypeptide.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of fowl metapneumovirus F protein polypeptide, the invention still further relates to this protein polypeptide in the purposes detecting in the infection of fowl metapneumovirus.
Background technology
Fowl metapneumovirus (aMPV) infects as the emerging infectious disease that harm poultry husbandry develops in a healthy way at present, and its cause of disease aMPV belongs to Paramyxoviridae Pneumovirinae Pneumovirus, without blood clotting and neuraminidase characteristic.AMPV can extensively cause chicken, turkey, pheasant, guinea fowl morbidity, antibody also can be detected in wild birds.Sick fowl infects after aMPV, often has gentleness or serious respiratory symptom, and food consumption and egg productivity decline.The enlargement of many chicken heads, aMPV is the main pathogen of bringing out the swollen syndrome (SHS) of chicken; And turkey main manifestations is acute respiratory symptom-turkey rhinotracheitis (TRT).
AMPV can infect the chicken at each age, and broiler chicken main infection age in days is 4-7 week, and peak is in 5-6 week, and planting chicken is 24-25 age in week.And the inventor detects in breeder flock, some meat kind chicken just had 50% antibody positive rate 10 week age.AMPV has high infectivity, and its pathogenicity rate is high, and velocity of propagation is fast, mortality ratio because of cultivating condition, individual nutrition level and concurrent, secondary infection state difference is widely different.Circulation way is horizontal transmission, mainly passes through the mechanical inoculation of sick fowl direct contact infection and keeper, utensil, feed, can not get rid of the possibility of vertical transmission completely.AMPV can propagate between different birds, even directly or indirectly infected mice.In the situation that chicken infectious bronchitis, infectious bursal disease exist, aMPV can cause serious consequence to chicken group, and after secondary intestinal bacteria, laying rate declines, and death rate obviously raises, and mortality ratio 1-20% not etc., does not depend on the level of control of secondary infection.
The monoclonal antibody of fowl metapneumovirus has to report it is the monoclonal antibody of utilizing C subgroup virus to be prepared at present, obtains altogether 6 strains, is to be all prepared from for virus N albumen, and the monoclonal antibody of other albumen is not reported.
Summary of the invention
First object of the present invention is to provide a kind of F protein polypeptide;
Second object of the present invention is to provide a kind of monoclonal antibody for fowl metapneumovirus F albumen;
The 3rd object of the present invention is to provide a kind of detection kit for fowl metapneumovirus monitoring of infection.
For achieving the above object, the present invention adopts following technical scheme:
A kind of fowl metapneumovirus F protein polypeptide, its aminoacid sequence is CTNAGSTAYYPNKDD.This polypeptide can adopt directly synthetic the obtaining of method of synthetic.
Further, based on this polypeptide, by by itself and carrier protein couplet, can obtain and have immunogenic antigen, described carrier proteins includes but not limited to serum protein, ovalbumin, thyroprotein, hemocyanin and scleroproein.
Further, the invention provides a kind of monoclonal antibody, it is to be prepared as immunogen by described antigen.Can prepare monoclonal antibody according to method well known in the art, by by F protein polypeptide of the present invention and carrier protein couplet, carry out animal immune and obtain sensitization bone-marrow-derived lymphocyte, then itself and homology myeloma cell fusion are obtained to hybridoma, by selectivity, cultivate the hybridoma that screening is merged, and then further screen hybridoma positive colony, finally the positive colony filtering out is carried out to enlarged culturing secretion and obtain monoclonal antibody.
In embodiments of the present invention, adopt and prepare with the following method the protein monoclonal antibody for fowl metapneumovirus F:
1) polypeptide coupling and animal immune
By above-mentioned F protein polypeptide and key hole worm relative keyhole limpet hemocyanin and bovine serum albumin coupling, after desalination, the polypeptide of key hole worm relative keyhole limpet hemocyanin coupling mixes with Freund's complete adjuvant or the freund 's incomplete adjuvant of equivalent, as immunogen, with the immunogen immune mouse mixing with Freund's complete adjuvant, then in subsequently several days, use the repeatedly booster immunization of immunogen mixing with freund 's incomplete adjuvant, enzyme linked immunosorbent assay detects immune response.
2) hybridoma is produced
From the mouse that immune titre is the highest, extract spleen with it, isolate splenocyte, and utilize PEG4000 and rat bone marrow tumour cell SP2/0 to merge in 4:1 ratio, after washing, the new hybridoma merging uses improvement Eagle ' the s culture medium culturing that contains 20% foetal calf serum and 2% xanthoglobulin, aminopterin-induced syndrome and thymidine after 3 days, assign in 96 orifice plates by improvement Eagle ' the s culture medium culturing that contains 20% foetal calf serum and 2%HAT 14 days, with ELISA, detect and filter out positive colony cell, then by improvement Eagle ' the s substratum enlarged culturing that contains 20% foetal calf serum.
Experiment shows, monoclonal anti physical efficiency of the present invention and F albumen test, produce good positive signal, and sensitivity, up to 0.1ng, can be infected and detect for fowl metapneumovirus.
Further, the present invention also provides the detection kit that comprises said monoclonal antibody, the detection of infecting for fowl metapneumovirus, and it also can further comprise one or more in ELIAS secondary antibody, standard substance, substrate nitrite ion, stop buffer etc.
The present invention is according to fowl metapneumovirus F gene protein sequence, and the antigen small peptide conservative property of design is high, antigenicity is strong.Utilize the highly sensitive and stable of monoclonal antibody prepared by this antigen small peptide.
Accompanying drawing explanation
Fig. 1 is the immune response result of small peptide, and it is corresponding to the data of table 1, and the 6th row and the 12nd are classified contrast as.
Fig. 2 is the selection result after hybridoma cell clone, corresponding to the data in table 2, is 1～7 96 orifice plate from left to right from the top down.
Fig. 3 is for Peptide polypeptide specific ELISA the selection result for the second time, corresponding to the data in table 3.
Fig. 4 is for Peptide polypeptide sensitivity detected result, corresponding to the data in table 4.
Fig. 5 is 5 strain monoclonal antibody strain hypotype qualification results, corresponding to the data in table 5.
Fig. 6 is the IFA detected results of 5 strain monoclonal antibodies for F protein expression.
Fig. 7 is that monoclonal antibody detects IFA(Fig. 7 A that fowl metapneumovirus infects) and WB(Fig. 7 B) result.
Embodiment
Following examples are used for further illustrating of the present invention, but should not be construed limitation of the present invention.Do not deviating under the prerequisite of the present invention's spirit and essence, the modification that the present invention is done or replacement, all belong to category of the present invention.
Determining of embodiment 1 fowl metapneumovirus F proteantigen small peptide
According to fowl metapneumovirus F gene protein sequence, carry out bioinformatic analysis comparison, determine that following polypeptide: CTNAGSTAYYPNKDD is as antigen small peptide, called after F protein polypeptide.This antigen small peptide conservative property is high, antigenicity is strong, and synthetic by gill biochemical corp, Shanghai, during coupling, according to maleimide theory, is about to cysteine residues in peptide sequence and the amino coupled in carrier proteins.Therefore, at be everlasting during polypeptide antigen its N end or C end of design, add a cysteine residues and be beneficial to the coupling of polypeptide and carrier proteins.
The preparation of embodiment 2 monoclonal antibodies
1) polypeptide coupling and animal immune
Polypeptide and KLH(Pierce Cat#77611) and BSA(AMRESCO code0332) coupling.After desalination, the polypeptide of KLH coupling mixes with the CFA of equivalent or IFA, and as immunogen, 4 degree are preserved.3 BALB/c mouse were used the immunogen immune of mixing with CFA at the 0th day, and then in subsequently several days, used the repeatedly booster immunization of immunogen mixing with IFA.At the 14th day, extract the tail blood of mouse, the coated not polypeptide of coupling and the polypeptide of carrier proteins BSA coupling, detect corresponding titre to observe the immune response for antigen with ELISA.
The detection method of ELISA is: 100ng detects in the former every hole that is added to 96 orifice plates (Corning, USA), and 4 ℃ of wrapper sheets spend the night.Phosphate buffered saline buffer (PBS) is washed after twice, and then 5% skim-milk PBS room temperature sealing one hour, adds tail blood (1:500to1:50000) room temperature of diluting with 5% skim-milk PBS 1 hour.PBS washes after twice, and the sheep anti-mouse igg Fc γ-specific bis-that uses of horseradish peroxidase (HRP) coupling of diluting with 5% skim-milk PBS resists (1:2000, Jackson Immune) room temperature 1 hour.PBST washes after five times, adds HRP substrate TMB colour developing.After 10min, read 450nm absorbancy with Microplate reader.
The results are shown in Table 1 and Fig. 1, as can be seen from the results, observe high titre, signal is respectively 1#>2#.Because two of use resists, be mouse IgG Fc γ-specific, high titre shows to have produced a large amount of IgG for small peptide in animal body, and 1# mouse is cooked further fusion.
The immune response result of table 1 small peptide
Note: NC represents blank, does not add primary antibodie.
2) hybridoma is produced
From the mouse that immune titre is the highest, extract spleen with it, isolate splenocyte, and utilize PEG4000 and rat bone marrow tumour cell SP2/0 to merge in 4:1 ratio.After washing, the new hybridoma merging is cultivated with the DMEM that contains 20%FBS and 2%HAT several times, after 3 days, assigns in 96 orifice plates and cultivates 14 days with the DMEM that contains 20%FBS and 2%HAT.After clone, the hybridoma of picking out is more further by the DMEM enlarged culturing that contains 20%FBS.
Collect single crosses oncocyte clone's supernatant and for polypeptide Peptide-BSA, with 96 orifice plates, screen detection for the first time.The results are shown in Table 2 and Fig. 2, in 700 detected mono-clonals, have 474 clones to produce the monoclonal antibody for polypeptide Peptide, select 20 strain OD values higher than 3.00 clone.
The selection result after table 2 hybridoma cell clone
| Clone | OD |
| 6B7 | ****. |
| 6B2 | ****. |
| 5A4 | ****. |
| 2B8 | ****. |
| 2A7 | ****. |
| 4C11 | ****. |
| 5C7 | ****. |
| 2B7 | ****. |
| 7B2 | ****. |
| 3B7 | ****. |
| 7B1 | ****. |
| 1A10 | 3.433 |
| 3A7 | 3.432 |
| 6A1 | 3.431 |
| 3A10 | 3.38 |
| 5D12 | 3.372 |
| 3A12 | 3.334 |
| 1D8 | 3.332 |
| 2D7 | 3.258 |
| 4D9 | 3.257 |
| NC | 0.046 |
| PC | 3.411 |
Positive colony is picked out and enlarged culturing.Then, carry out multiple sieve test, coated Peptide-BSA polypeptide further detects cells and supernatant, and as shown in table 3 and Fig. 3,20 strain clones that primary dcreening operation is selected all show strong positive, further enlarged culturing; And then sieve again for the second time (sensitivity detection), cell conditioned medium detects for polypeptide Peptide-BSA, as shown in table 4 and Fig. 4,3 strain clone sensitivity reach 0.1ng, 11 strain clones reach 1ng, and 6 strain clones reach 10ng, and all 20 strain clones are through being further verified as the positive strain of stablizing, final this 20 strain monoclonal antibody that retains, for further verification.
Table 3 is for the specific ELISA screening for the second time of Peptide polypeptide
Note: NC represents blank, does not add primary antibodie.PC represents positive control, and primary antibodie is mouse polyvalent antibody.
Table 4 detects for the sensitivity of Peptide polypeptide
Note: NC represents blank, does not add primary antibodie.PC represents positive control, and primary antibodie is mouse polyvalent antibody.
3) hypotype is identified
Frozen multiple receipts of further carrying out monoclonal antibody strain, finally obtains 2B7,2B8,3A10,5C7 and the 5D12 stable positive monoclonal antibody strain of totally 5 Expression of Plant Heights.Use the Southern Biotech monoclonal antibody hypotype identification kit SBA Clonotyping System/HRP of company, this 5 strain monoclonal antibody is carried out to hypotype evaluation, the results are shown in Table 5 and Fig. 5, wherein the heavy chain hypotype of 5C7 is IgG1, the heavy chain hypotype of 3A10 is IgG2a, the heavy chain hypotype of 2B7 and 2B8 is IgG2b, and the heavy chain hypotype of 5D12 is IgG3, and the light chain of this 5 strain monoclonal antibody is κ hypotype.
Table 55 strain monoclonal antibody hypotype is identified
| Clone | 2B7 | 2B8 | 3A10 | 5C7 | 5D12 | NC |
| IgG1 | 0.127 | 0.131 | 0.145 | 1.621 | 0.994 | 0.08 |
| IgG2a | 0.329 | 0.334 | 1.736 | 0.099 | 0.841 | 0.08 |
| IgG2b | 1.598 | 1.591 | 0.193 | 0.389 | 0.736 | 0.065 |
| IgG3IgA | 0.1060.1 | 0.1060.103 | 0.1120.0890.1050.071 | 1.7420.634 | 0.0750.064 |
| IgM | 0.203 | 0.197 | 0.1350.103 | 0.734 | 0.07 |
| λ | 0.107 | 0.114 | 0.1220.1 | 0.707 | 0.076 |
| κ | 1.135 | 1.188 | 1.3531.549 | 1.516 | 0.08 |
4) 5 strain monoclonal antibodies are for the detection of F protein expression
Fowl metapneumovirus F gene clone builds pCMV-HA-F recombinant vectors in carrier for expression of eukaryon pCMV-HA, and transfection Vero cell fixedly carries out indirect immunization method (IFA) and detects after 24 hours, and primary antibodie is this 5 strain monoclonal antibody, the results are shown in Figure 6.This 5 strain monoclonal antibody all can with F albumen test, produce good positive signal.
The detection that IFA method prepared by embodiment 3 monoclonal antibodies and immunoblotting (WB) method infect for fowl metapneumovirus
The monoclonal antibody that monoclonal antibody strain 2B8 produces is used for detecting the infection conditions of fowl metapneumovirus on Vero cell, uses indirect immunofluorescence (IFA) and immunoblotting (WB) method to detect.IFA detection method is as follows: in 96 porocyte plates, grow up to the Vero cell of individual layer, with phosphate buffered saline buffer (PBS) washed cell 2 times, then inoculate fowl metapneumovirus, containing 37 ℃ of cultivations in 5%CO2 incubator; Given time point after virus inoculation, fixes 30 minutes by 4% paraformaldehyde room temperature; After washing, 37 ℃ of the anti-fowl metapneumovirus F protein monoclonal antibodies (diluent is 3%BSA-PBS) that doubly dilute with 1:30 of cell hole are hatched 90 minutes; After washing, the fluorescein-labelled mouse IgG(diluent that doubly dilutes with 1:200 of cell hole is 3%BSA-PBS) 37 ℃ hatch 60 minutes; Fluorescence microscope photograph after washing.WB detection method is as follows: the cell extract of different virus infection time point is gathered to the gel electrophoresis of propionyl ammonia and then transfer to nitrocellulose filter (NC); After transferring film, with sealing damping fluid (5% skimmed milk-TBST) room temperature sealing 2h; After washing, add with the anti-fowl metapneumovirus F protein monoclonal antibody doubly diluting containing the TBST1:500 of 1% skimmed milk, hatch 2h for 37 ℃; After washing, add with the horseradish peroxidase-labeled goat anti-mouse IgG room temperature effect 1.5h doubly diluting containing the TBST1:1000 of 1% skim-milk; After washing, add enhancement type O-phthalic ammonia substrate nitrite ion, lucifuge colour developing when there is object band (5-20min), takes out while there is clearly band on NC film.The results are shown in Figure 7A and 7B.
IFA method detected result shows that 24h, 48h, 72h, 96h, 120h all detect fluorescent reaction after virus inoculation, and control test less than, repeatedly reproducible results is identical, similarly result exists too on immunoblotting detects, and shows that thus the monoclonal antibody that the present invention makes as core using F protein polypeptide can be used for fowl metapneumovirus infection detection.
Claims (9)
1. a F protein polypeptide, is characterized in that, the aminoacid sequence of this polypeptide is CTNAGSTAYYPNKDD.
2. an antigen, it is to be obtained by F protein polypeptide and carrier protein couplet described in claim 1.
3. according to right, will remove the antigen described in 2, it is characterized in that, described carrier proteins is selected from: serum protein, ovalbumin, thyroprotein, hemocyanin or scleroproein.
4. a monoclonal antibody, it is to be prepared as immunogen by the antigen described in claim 2 or 3.
5. a detection kit, it contains monoclonal antibody claimed in claim 4.
6. detection kit according to claim 5, it also comprises and is selected from one or more in ELIAS secondary antibody, standard substance, substrate nitrite ion and stop buffer.
7. contain the biological products of F protein polypeptide claimed in claim 1.
8. F protein polypeptide claimed in claim 1 is being prepared for the application on fowl metapneumovirus F protein monoclonal antibody.
9. application according to claim 8, is characterized in that, comprises the steps:
1) polypeptide coupling and animal immune
Polypeptide claimed in claim 1 and key hole worm relative keyhole limpet hemocyanin and bovine serum albumin coupling, after desalination, the polypeptide of key hole worm relative keyhole limpet hemocyanin coupling mixes with Freund's complete adjuvant or the freund 's incomplete adjuvant of equivalent, as immunogen, with the immunogen immune mouse mixing with Freund's complete adjuvant, then in subsequently several days, use the repeatedly booster immunization of immunogen mixing with freund 's incomplete adjuvant, enzyme linked immunosorbent assay detects immune response;
2) hybridoma is produced
From the mouse that immune titre is the highest, extract spleen with it, isolate splenocyte, and utilize PEG4000 and rat bone marrow tumour cell SP2/0 to merge in 4:1 ratio, after washing, the new hybridoma merging uses improvement Eagle ' the s culture medium culturing that contains 20% foetal calf serum and 2% xanthoglobulin, aminopterin-induced syndrome and thymidine after 3 days, assign in 96 orifice plates by improvement Eagle ' the s culture medium culturing that contains 20% foetal calf serum and 2%HAT 14 days, with ELISA, detect and filter out positive colony cell, then by improvement Eagle ' the s substratum enlarged culturing that contains 20% foetal calf serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410018287.0A CN103739679B (en) | 2014-01-15 | 2014-01-15 | Avian metapneumovirus (aMPV) F protein polypeptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410018287.0A CN103739679B (en) | 2014-01-15 | 2014-01-15 | Avian metapneumovirus (aMPV) F protein polypeptide and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103739679A true CN103739679A (en) | 2014-04-23 |
| CN103739679B CN103739679B (en) | 2015-07-22 |
Family
ID=50496763
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410018287.0A Expired - Fee Related CN103739679B (en) | 2014-01-15 | 2014-01-15 | Avian metapneumovirus (aMPV) F protein polypeptide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103739679B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296439A (en) * | 2015-09-08 | 2016-02-03 | 北京市农林科学院 | Chicken C-type acian metapneumovirus strain(aMPV-JCX) and application thereof |
| CN105693829A (en) * | 2016-04-14 | 2016-06-22 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus F protein |
| CN105770884A (en) * | 2016-04-14 | 2016-07-20 | 青岛易邦生物工程有限公司 | Type B acian metapneumovirus subunit vaccine |
| CN105861349A (en) * | 2016-04-14 | 2016-08-17 | 青岛易邦生物工程有限公司 | Recombinant strain for producing B-type acian metapneumovirus F protein |
-
2014
- 2014-01-15 CN CN201410018287.0A patent/CN103739679B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| COSWIG LT ET AL.: "Production of Monoclonal Antibodies for Avian Metapneumovirus (SHS-BR-121) Isolated in Brazil.", 《BRAZILIAN JOURNAL OF POULTRY SCIENCE》, 31 December 2007 (2007-12-31), pages 255 - 258 * |
| MARIO H. SKIADOPOULOS ET AL.: "Individual contributions of the human metapneumovirus F, G, and SH surface glycoproteins to the induction of neutralizing antibodies and protective immunity.", 《VIROLOGY》, 31 December 2006 (2006-12-31), pages 492 - 501 * |
| RENE ALVAREZ, M. ET AL.: "Development of a Nucleoprotein-Based Using a Synthetic Peptide Antigen for Antibodies in Turkey Sera.", 《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》, 31 December 2004 (2004-12-31), pages 245 - 249 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296439A (en) * | 2015-09-08 | 2016-02-03 | 北京市农林科学院 | Chicken C-type acian metapneumovirus strain(aMPV-JCX) and application thereof |
| CN105296439B (en) * | 2015-09-08 | 2019-01-04 | 北京市农林科学院 | One breeder c-type fowl metapneumovirus strain aMPV/C-JCX and its application |
| CN105693829A (en) * | 2016-04-14 | 2016-06-22 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus F protein |
| CN105770884A (en) * | 2016-04-14 | 2016-07-20 | 青岛易邦生物工程有限公司 | Type B acian metapneumovirus subunit vaccine |
| CN105861349A (en) * | 2016-04-14 | 2016-08-17 | 青岛易邦生物工程有限公司 | Recombinant strain for producing B-type acian metapneumovirus F protein |
| CN105770884B (en) * | 2016-04-14 | 2020-11-27 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus subunit vaccine |
| CN105693829B (en) * | 2016-04-14 | 2022-03-15 | 青岛易邦生物工程有限公司 | B-type avian metapneumovirus F protein |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103739679B (en) | 2015-07-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dey et al. | Infectious bursal disease virus in chickens: prevalence, impact, and management strategies | |
| Jackwood et al. | A proposed nomenclature for infectious bursal disease virus isolates | |
| Cavanagh et al. | Location of the amino acid differences in the S1 spike glycoprotein subunit of closely related serotypes of infectious bronchitis virus | |
| Webster | Antigenic hybrids of influenza A viruses with surface antigens to order | |
| CN102636644B (en) | Double-antibody sandwich ELISA (enzyme linked immunosorbent assay) kit used for detecting avian leukosis group specific antigen | |
| CN103739679B (en) | Avian metapneumovirus (aMPV) F protein polypeptide and application thereof | |
| US8790657B2 (en) | Astrovirus | |
| JP4953256B2 (en) | Epitope of HN protein recognized by avian immune system and Newcastle disease virus containing said epitope mutation | |
| Czifra et al. | Detection of PMV‐1 specific antibodies with a monoclonal antibody blocking enzyme‐linked immunosorbent assay | |
| Ingrao et al. | Characterization of two recombinant HVT-IBD vaccines by VP2 insert detection and cell-mediated immunity after vaccination of specific pathogen-free chickens | |
| CN102031245B (en) | Monoclonal antibody of avian reticuloendothelial hyperplasia protein gp90 and application thereof | |
| CN105949321A (en) | B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof | |
| CN101395175A (en) | Chicken virus vaccine and diagnostic | |
| CN103923881A (en) | Hepatitis A virus monoclonal antibody and its application | |
| CN104619337A (en) | Vaccine for preventing viral hemorrhagic sepsis of flatfishes | |
| Nayak et al. | Evaluation of the genetic diversity of avian paramyxovirus type 4 | |
| Lardinois et al. | A model for the transfer of passive immunity against Newcastle disease and avian influenza in specific pathogen free chickens | |
| Kefford et al. | Serological identification of avian adenoviruses isolated from cases of inclusion body hepatitis in Victoria, Australia | |
| CN103923882A (en) | Hepatitis A virus monoclonal antibody and its application | |
| CN106749647A (en) | Salmon trout IHNV monoclonal antibodies and detection kit | |
| CN110161246A (en) | Distinguish the indirect competitive ELISA method and kit of newcastle disease virus sample particle vaccines immune serum and wild virus infection serum | |
| CN110452883A (en) | Avian leukosis P27 protein monoclonal antibody and its preparation method and application | |
| CN115894671B (en) | Coronavirus detection kit | |
| CN104292310B (en) | Duck plague virus UL15 gene exonI recombinant proteins and its preparation method and application | |
| CN115925894B (en) | Coronavirus monoclonal antibody and detection reagent prepared from same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150722 |