CN108659124A - A kind of single-chain antibody of porcine epidemic diarrhea resisting virus and its application - Google Patents
A kind of single-chain antibody of porcine epidemic diarrhea resisting virus and its application Download PDFInfo
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- CN108659124A CN108659124A CN201810510406.2A CN201810510406A CN108659124A CN 108659124 A CN108659124 A CN 108659124A CN 201810510406 A CN201810510406 A CN 201810510406A CN 108659124 A CN108659124 A CN 108659124A
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- sequence
- variable region
- chain variable
- chain antibody
- epidemic diarrhea
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/12—Antidiarrhoeals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
Abstract
The invention discloses a kind of single-chain antibody of porcine epidemic diarrhea resisting virus and its applications.Single-chain antibody provided by the invention, the polypeptide being made of light chain variable region, connection peptide and heavy chain variable region；The connection peptide is between the light chain variable region and the heavy chain variable region；Sequence 1 is from N-terminal shown in the 1st to 107 amino acids residue in the light chain variable region such as sequence table；Sequence 1 is from N-terminal shown in the 123rd to 240 amino acids residue in the heavy chain variable region such as sequence table.Single-chain antibody provided by the invention can neutralize Porcine epidemic diarrhea virus, have important application value in preventing and/or treating pig epidemic diarrhea.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of single-chain antibody of porcine epidemic diarrhea resisting virus and its answer
With.
Background technology
Pig epidemic diarrhea (Porcine Epidemic Diarrhea, PED) is by Porcine epidemic diarrhea virus
A kind of enteric infectious disease caused by (Porcine Epidemic Diarrhea Virus, PEDV), the disease are usually mainly in cold
Winter-spring season, clinical symptoms are mainly shown as diarrhea, vomit, become thin, being dehydrated, and eventually lead to death.Although current vaccine
Certain effect is played using to the control of the disease, but the epidemic disease still has different degrees of generation and prevalence in some areas.
Phage antibody display technology provides more efficient way for the high frequency zone of genetic engineering antibody and directional transformation
Diameter.With the development of animal immune technology, space will become more and more narrower, will be more next using in-vitro method screening antibodies
More attract attention.Display technique of bacteriophage prepares difference, the technology in terms of screening antibodies with traditional monoclonal antibody
By in phage display, being combined with target antigen, by 96 simple and practicable hole Microdilution plate methods, antigen-antibody knot is utilized
It closes, screening time and step is greatly reduced in " absorption-elution-enrichment ".Antibody may be implemented in Phage antibody display technology
The antiantibody phenomenon of antibody in use is reduced in target animals source.
Currently, pig source phage antibody display platform is less, antibody molecule amount is bigger than normal, is easy to be sealed with antigen binding regions
It closes, and the folding ability of mind of single-chain antibody is limited, and traditional monoclonal antibody is essentially mouse source antibody, easy tos produce in use anti-
Antibody influences its therapeutic effect.Existing pig epidemic diarrhea vaccine can not whole prevention and control diseases outburst, brought to pig breeding industry
Huge economic loss.
Invention content
The object of the present invention is to provide a kind of single-chain antibodies of porcine epidemic diarrhea resisting virus.
Single-chain antibody provided by the present invention, the polypeptide being made of light chain variable region, connection peptide and heavy chain variable region；Institute
Connection peptide is stated between the light chain variable region and the heavy chain variable region；The light chain variable region can be such as sequence in sequence table
1 from N-terminal shown in the 1st to 107 amino acids residue；The heavy chain variable region can as in sequence table sequence 1 from N-terminal
Shown in 123 to 240 amino acids residues.
In above-mentioned single-chain antibody, the connection peptide can be such as the 108th to 122 amino acids from N-terminal of sequence 1 in sequence table
Shown in residue.
The single-chain antibody concretely polypeptide shown in sequence 1 in sequence table.
The present invention also protects the nucleic acid molecules for encoding any of the above-described single-chain antibody.Any of the above-described single-chain antibody
Nucleic acid molecules can by the light chain variable region encoding gene, it is described connection peptide encoding gene and the heavy chain variable region volume
Code gene composition.
The encoding gene of the light chain variable region can be such as the 1st to the 321 nucleotide institute from 5 ' ends of sequence 2 in sequence table
Show.
The encoding gene of the heavy chain variable region can be such as the 367th to 720 nucleotide from 5 ' ends of sequence 2 in sequence table
It is shown.
The encoding gene of the connection peptide can be such as the 322nd to the 366 nucleotide institute from 5 ' ends of sequence 2 in sequence table
Show.
The nucleic acid molecules of any of the above-described single-chain antibody concretely A1) or A2) or A3) or A4) shown in DNA points
Son：
A1) code area is DNA molecular shown in sequence 2 in sequence table；
A2) nucleotide sequence is DNA molecular shown in sequence 2 in sequence table；
A3) and A1) or A2) nucleotide sequence that limits has 75% or 75% or more homogeneity, and encodes any of the above-described
The DNA molecular of the single-chain antibody；
A4) under strict conditions with A1) or A2) limit nucleotide sequence hybridization, and encode it is any of the above-described described single-stranded
The DNA molecular of antibody.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used
To be RNA, such as mRNA or hnRNA.
Wherein, sequence 1 is made of 720 nucleotide in sequence table, in sequence table in the nucleotide coding sequence table of sequence 1
Amino acid sequence shown in sequence 2.
Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side
Method is mutated the nucleotide sequence of the coding single-chain antibody of the present invention.Those by manually modified, have and this
The nucleotide sequence 75% of the single-chain antibody of invention or the nucleotide of higher homogeneity, as long as encoding the single-chain antibody
And porcine epidemic diarrhea resisting virus, it is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
The nucleotide sequence for the protein that amino acid sequence shown in the sequence 2 of bright polynucleotide forms has 75% or higher,
80% or higher 85% or higher 90% or higher 95% or higher homogeneity nucleotide sequence.Homogeneity
It can with the naked eye or computer software is evaluated.Using computer software, homogeneity between two or more sequences can be with
It is indicated with percentage (%), can be used for evaluating the homogeneity between correlated series.
The application of any of the above-described single-chain antibody or any of the above-described nucleic acid molecules also belongs to the protection of the present invention
Range.The application of any of the above-described single-chain antibody or any of the above-described nucleic acid molecules can be B1) or B2) or B3) or
B4)：
B1 the product for neutralizing Porcine epidemic diarrhea virus) is prepared；
B2 Porcine epidemic diarrhea virus) is neutralized；
B3 the product for preventing and/or treating disease caused by Porcine epidemic diarrhea virus) is prepared；
B4) prevent and/or treat disease caused by Porcine epidemic diarrhea virus.
In above application, the product concretely drug.
The present invention also protects a kind of product, contains any of the above-described single-chain antibody；The function of the product can be
C1) or C2)：
C1 Porcine epidemic diarrhea virus) is neutralized；
C2) prevent and/or treat disease caused by Porcine epidemic diarrhea virus.
The product concretely drug.
Any of the above-described Porcine epidemic diarrhea virus concretely Porcine epidemic diarrhea virus S protein.
The concretely pig epidemic diarrhea of disease caused by any of the above-described Porcine epidemic diarrhea virus.
It is demonstrated experimentally that single-chain antibody provided by the invention can neutralize Porcine epidemic diarrhea virus, is preventing and/or treating
There is important application value in pig epidemic diarrhea.
Description of the drawings
Fig. 1 is 1.0% agarose gel electrophoresis result of three pcr amplification products.
Fig. 2 is 1.0% agarose gel electrophoresis result of scFv segments first and scFv segment second.
Fig. 3 is the identification in phage single-chain antibody primary library.
Fig. 4 is that phage antibody combines activity.
Specific implementation mode
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Experiment material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.Quantitative experiment in following embodiment is respectively provided with three repeated experiments, as a result makes even
Mean value.
XLI-Blue competent cells are the product that health is century bio tech ltd.Restriction enzyme SfiI is
The product of NEB companies of the U.S..Trizol reagents, PrimeScriptTMII 1st Strand cDNA Synthesis Kit and
DNA maker DL2000 are the product of Takara companies.M13K07 is the product of Invitrogen companies.Pig peripheral blood drenches
Bar cell separating kit is the product of Beijing Suo Laibao Science and Technology Ltd.PComb3XSS carriers are won grand experiment for Qingdao and are moved
The product of object Co., Ltd.Porcine epidemic diarrhea virus S protein is the product of Qingdao Bo Long genetic engineerings Co., Ltd.
The preparation of embodiment 1, phage antibody display libraries
One, the design and synthesis of primer
According to pig IgG heavy chain (No. GenBank：AM177137, light chain Kappa chains (No. GenBank：AY518084) and light
Chain lambda chains (No. GenBank：AF345512 sequence) separately designs primer.The nucleotide sequence and amplification piece of each primer
Segment length is shown in Table 1.
Table 1
Note：Italicized item indicates that linker, underscore indicate the digestion recognition site of restriction enzyme SfiI.In title
Expression upstream containing " for " contains the expression downstream of " back " in title, and VH is heavy chain, and VL (Kappa) is light chain Kappa chains, VL
(Lambda) it is light chain lambda chains, S is C or G, and Y is C or Y, and K is G or T, and R is A or G, and W is A or T.
Each primer shown in table 1 is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
Two, the extraction of peripheral blood lymphocytes
1, the resistance to morbid pig for crossing pig epidemic diarrhea is taken, the pig epidemic diarrhea vaccine (Chinese Academy of Agricultural Sciences is subcutaneously injected
The product of Harbin veterinary institute) it is 3 times immune.Per injection dosage is 1mL.Each immunization interval number of days is 14d.
2,14d after completion step 1, acquires pig peripheral blood.
3, it by 1 parts by volume 10mL pig peripheral bloods and 1-2 parts by volume whole bloods and tissue dilution mixing, then uses outside pig
All blood lymphocyte separating kits extract peripheral blood lymphocytes.
Three, the preparation in phage single-chain antibody primary library
1, the total serum IgE of the peripheral blood lymphocytes prepared using Trizol reagents extraction step two.
2, the total serum IgE extracted using step 1 is template, according to PrimeScriptTMII 1st Strand cDNA
The operating procedure reverse transcription of Synthesis Kit synthesizes cDNA.
3, primer pair 1 (being made of VH for and VH back), primer is respectively adopted as template in the cDNA synthesized using step 2
To 2 (being made of VL (Kappa) for and VL (Kappa) back) and primer pair 3 (by VL (Lambda) for and VL (Lambda)
Back is formed) PCR amplification is carried out, pcr amplification product 1, pcr amplification product 2 and pcr amplification product 3 are obtained successively.
Reaction condition：95℃5min；95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 35 cycles；72 DEG C of extension 10min.
4, pcr amplification product 1 is subjected to 1.0% agarose gel electrophoresis, recycles the DNA fragmentation 1 of about 400bp.PCR is expanded
Increase production object 2 and carry out 1.0% agarose gel electrophoresis, recycles the DNA fragmentation 2 of about 350bp.Pcr amplification product 3 is carried out 1.0%
Agarose gel electrophoresis recycles the DNA fragmentation 3 of about 350bp.
(M is DNA maker DL2000, swimming to 1.0% agarose gel electrophoresis the result is shown in Figure 1 of three pcr amplification products
Road 1 is pcr amplification product 1, and swimming lane 2 is pcr amplification product 2, and swimming lane 3 is pcr amplification product 3).
5, it takes EP to manage, 1 mass parts DNA fragmentation 1 and 1 mass parts DNA fragmentation 2 is added, mixing obtains hybrid dna first.Take EP
Pipe, is added 1 mass parts DNA fragmentation 1 and 1 mass parts DNA fragmentation 3, and mixing obtains hybrid dna second.
Using hybrid dna first as template, PCR amplification is carried out using the primer pair of VL (Kappa) for and VH back compositions, is obtained
The pcr amplification product arrived is the scFv segment first spliced.The structural formula of scFv segment first is：VL(Kappa)-linker-VH.
Using hybrid dna second as template, PCR amplification is carried out using the primer pair of VL (Lambda) for and VH back compositions,
Obtained pcr amplification product is the scFv segment second spliced.The structural formula of scFv segment second is：VL(Lambda)-linker-
VH。
Reaction condition：95℃5min；95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, totally 35 recycle；72 DEG C of extension 10min.
6, scFv segment first is taken, 1.0% agarose gel electrophoresis, recycling are carried out；Then restriction enzyme SfiI is used
Digestion carries out 1.0% agarose gel electrophoresis, recycles again, obtain DNA fragmentation first.
According to above-mentioned steps, scFv segment first is replaced with into scFv segment second, other steps are constant, obtain DNA fragmentation
Second.
The result that scFv segments first and scFv segment second carry out 1.0% agarose gel electrophoresis is shown in that (M is DNA maker to Fig. 2
DL2000, swimming lane 1 are scFv segment first, and swimming lane 2 is scFv segments second).
7, using restriction enzyme SfiI digestion pComb3XSS carriers, the carrier framework of about 3800bp is recycled.
8, DNA fragmentation first is connected with carrier framework, obtains connection product first.
DNA fragmentation second is connected with carrier framework, obtains connection product second.
9,200 μ L connection products first and 200 μ L connection product second point are 10 times electroporated thin to XLI-Blue competence
Born of the same parents, the bacterium solution that 10 times are obtained mix, and are named as mixed bacteria liquid.
Electroporated step is every time：80 μ L XLI-Blue competent cells are taken, 20 μ L connection products first and 20 are added
μ L connection product second carries out electroporated (shock parameters 2.5KV, 800 Ω), then 1mL SOC culture mediums is used to be resuspended, transfer
To centrifuge tube (specification 50mL), 37 DEG C, 220r/min shaken cultivation 1h obtain bacterium solution.
100 μ L mixed bacteria liquids are spread evenly across on the LB solid plates containing 100 μ g/mL ammonia benzyl mycins, recombination fraction is detected.
Testing result shows that the recombination fraction of mixed bacteria liquid is 90%.
10, the remaining mixed bacteria liquid of step 9 is taken, ammonia benzyl mycin and glucose is added, obtains cultivating system；Cultivating system
In, a concentration of 100 μ g/mL of ammonia benzyl mycin, a concentration of 0.02M of glucose.The cultivating system is placed in 37 DEG C of cultures extremely
OD600nmIt is 0.5,20 μ L M13K07,37 DEG C of infection 30min is first added；Then 37 DEG C, 220r/min shaken cultivation 1h, abandon culture
Base, the isometric SOC culture mediums containing 100 μ g/mL ammonia benzyls mycins, 50 μ g/mL kanamycins, 0.1mM/mL IPTG of addition, 37
DEG C, 220r/min shaken cultivations 6h.
11, after completing step 10, supernatant is collected in centrifugation；Then 1 parts by volume supernatant and 5 parts by volume PEG8000 are mixed
It closes, 4 DEG C overnight；Centrifugation is collected precipitation and is resuspended with the PBS buffer solution of 1.3mL pH7.4,10mM, it is anti-to obtain phage single-chain
Body primary library.
Four, the identification in phage single-chain antibody primary library
1 μ L phage single-chain antibody primary library is taken, primer pair 1 (being made of VH for and VH back), primer is respectively adopted
To 2 (being made of VL (Kappa) for and VL (Kappa) back), primer pair 3 (by VL (Lambda) for and VL (Lambda)
Back is formed), universal primer pair 1 (being made of VL (Kappa) for and VH back) and universal primer pair 2 be (by VL (Lambda)
For and VH back compositions) PCR amplification is carried out, obtain pcr amplification product.
Reaction condition：95℃5min；95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 40s, 35 cycles；72 DEG C of extension 10min.
Each pcr amplification product is subjected to 1.0% agarose gel electrophoresis.As a result see that (M is DNA maker to Fig. 3
DL2000, swimming lane 1 are primer pair 1, and swimming lane 2 is primer pair 2, and swimming lane 3 is primer pair 3, and swimming lane 4 is primer pair 4, and swimming lane 5 is to draw
Object pair is 5).
Five, the measurement of phage single-chain antibody primary library titre
1 μ L phage single-chain antibody primary library is taken, the PBS buffer solution of 999 μ L pH7.4,10mM is added, mixing obtains dilute
Release liquid 1.10 μ L dilutions 1 are taken, the PBS buffer solution of 990 μ L pH7.4,10mM is added, obtains dilution 2；Then 10 multiple proportions are dilute
It releases, dilutes 4~5 gradients.
20 μ L are taken from the solution of each dilution, and 180 μ L OD are added600nm15min is infected in the bacterium solution that value is 0.5, so
It is taken out 20 μ L afterwards to be coated on the LB solid plates containing 100 μ g/mL ammonia benzyl mycins, calculates phage single-chain antibody primary library
Titre.
The result shows that the titre in phage single-chain antibody primary library is 9.5 × 109pfu/mL。
Six, the preparation of phage antibody display libraries
Porcine epidemic diarrhea virus S protein (i.e. antigen) is taken, it is dilute with the sodium carbonate-bicarbonate buffer solution of pH9.6,0.1M
It releases, obtains the antigenic dilution of a concentration of 100 μ g/ μ L.
Utilize antigen-antibody combination screening antibodies library.3 wheel " absorption-elution-have been carried out to phage single-chain antibody primary library
The elutriation of enrichment ", and titer determination is carried out to the bacteriophage that outputs and inputs of each round.By elutriation, antibody library (i.e. bacteriophage
Antibody display libraries) titre it is higher than phage single-chain antibody primary library 80 times (being shown in Table 2), be effectively enriched specificity compared with
Good bacteriophage.It is as follows：
1, the 1st polling is washed in a pan
(1) 96 orifice plates are taken, 100 μ L antigenic dilutions are added per hole, 4 DEG C overnight.
(2) after completing step (1), 96 orifice plate is taken, 100 μ L confining liquids are added per hole and (are dissolved in 0.05g BSA
100mL pH7.4,10mM PBS buffer solution obtain), 37 DEG C standing 2h.
(3) after completing step (2), 96 orifice plate is taken, the 100 μ L phage single-chain antibody primary libraries of addition per hole, 37 DEG C
Stand 1h.
(4) after completing step (3), 96 orifice plate is taken, with PBST buffer solutions (by 8.0g NaCl, 0.2g KCl, 1.15g
Na2HPO4With 0.2g KH2PO4It is dissolved in deionized water, is then settled to 1000mL with deionized water；It is eventually adding 100 μ L tweens
20) it washs 3~4 times, finally (0.5g BSA and 0.75g glycine is dissolved in 100mL ddH with Elution Buffer2O, so
It uses 1M salt acid for adjusting pH to 2.2) eluting afterwards, obtains eluent 1.
20 μ L eluents 1 are taken, titre is measured.
2, the 2nd wheel and the 3rd polling are washed in a pan
(1) remaining eluent 1 is taken, it is electroporated to XLI-Blue competent cells, obtain bacterium solution.
(2) according to (1) in step 1 to (4), phage single-chain antibody primary library is replaced with into the bacterium that step (1) obtains
Liquid, other steps are constant, obtain eluent 2.
(3) 20 μ L eluents 2 are taken, titre is measured.
(4) remaining eluent 2 is taken, it is electroporated to XLI-Blue competent cells, obtain bacterium solution.
(5) according to (1) in step 1 to (4), phage single-chain antibody primary library is replaced with into the bacterium that step (4) obtains
Liquid, other steps are constant, obtain eluent 3.20 μ L eluents 3 are taken, titre is measured.
Eluent 3 is the phage antibody display libraries prepared.
Table 2
| Number is washed in a pan in choosing | Input bacteriophage (pfu) | Export bacteriophage (pfu) | Input/output |
| 1 | 1.0×1011 | 3.0×104 | 3.0×10-7 |
| 2 | 5.0×1010 | 3.7×104 | 7.4×10-7 |
| 3 | 5.0×1010 | 1.2×106 | 2.4×10-5 |
Embodiment 2, the combination activity for detecting phage antibody display libraries
The combination activity of phage antibody display libraries is detected using ELISA method.
Porcine epidemic diarrhea virus S protein (i.e. antigen) is taken, it is dilute with the sodium carbonate-bicarbonate buffer solution of pH9.6,0.1M
It releases, obtains the antigenic dilution of a concentration of 100 μ g/ μ L.
1,96 orifice plates are taken, 100 μ L antigenic dilutions are added per hole, 4 DEG C overnight.
2, after completing step 1,96 orifice plate is taken, 100 μ L confining liquids are added per hole and (0.05g BSA are dissolved in 100mL
The PBS buffer solution of pH7.4,10mM obtain), 37 DEG C of standing 2h.
3, after completing step 2,96 orifice plate is taken, 100 μ L phage antibody display libraries, 37 DEG C of standings are added per hole
2h。
4, after completing step 3,96 orifice plate is taken, the antibody of the anti-M13 of 100 μ L HRP labels is added per hole (as enzyme
Mark secondary antibody), 37 DEG C of standing 1h finally use OD of the microplate reader detection per hole450nm。
" phage antibody display libraries " in step 3 are replaced with into M13K07, other steps are constant, as negative right
According to.
" phage antibody display libraries " in step 3 are replaced with to the PBS buffer solution of pH7.4,10mM, other steps are equal
It is constant, as blank control.
Experimental result finds one plant and the preferable porcine epidemic diarrhea resisting disease of the affine activity of Porcine epidemic diarrhea virus S protein
The single-chain antibody of poison, OD450nmFor 0.762, (see Fig. 4, Control M13K07, CloneA are that the affine activity of discovery is preferable
Porcine epidemic diarrhea resisting virus single-chain antibody).Through sequencing, the single-chain antibody such as sequence table of porcine epidemic diarrhea resisting virus
Shown in middle sequence 1.In sequence table in sequence 1, from N-terminal the 1st to 107 amino acids residue be light chain variable region, the 108th to
122 amino acids residues are connection peptide, and the 123rd to 240 amino acids residue is heavy chain variable region.
<110>Qingdao Bo Long genetic engineerings Co., Ltd
<120>A kind of single-chain antibody of porcine epidemic diarrhea resisting virus and its application
<160> 2
<170> PatentInversion3.5
<210>1
<211>720
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>1
gccatccaga tgacccagtc tccagcctcc ctggctgcat ctctcggaga cacggtctcc 60
atcacttgcc gggccagtca gagcattagc agttatttag cctggtatca acaacaacca 120
gggacggctc ctaaacgctt gatctatgct gcatccagtt tgcaaagtgg ggtcccatcc 180
cggttcaagg gcagtggatc tggcaccgat ttcaccctca ccatcagtgg cctgcaggct 240
gaagatgttg caacttatta ctgtttgcag aataataatg tacctccgac gttcggccaa 300
ggaaccaagc tggaactcaa acgggctgat gccaagccat ccgtcggtgg ttcctctaga 360
tcttcccagg agaagctggt ggagtctgga ggaggcctgg tgcagcctgg ggggtctctc 420
agactctcct gtgtcggctc tggattcacc ttcagtagta cctggattaa ctgggtccgc 480
caggctccag ggaaggggct ggagtggctg gcaggcattt atagtagtgc aggtagcacc 540
gtccactcag actctgtgaa gggccgattc accgtctcag gagacaactc ccagaacacg 600
gcctatctgc aaatgaacag cctgagaacc gaagacacgg cccgctatta ctgtacaaaa 660
tccaactggt atacgttgga tgtctggggc ccaggcgttg aggtcgtcgt gtcctcaggc 720
<210>2
<211>240
<212>PRT
<213>Artificial sequence
<220>
<223>
<400>2
Ala Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ala Ala Ser Leu Gly
1 5 10 15
Asp Thr Val Ser Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Gln Pro Gly Thr Ala Pro Lys Arg Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Leu Gln Ala
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Leu Gln Asn Asn Asn Val Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Lys
100 105 110
Pro Ser Val Gly Gly Ser Ser Arg Ser Ser Gln Glu Lys Leu Val Glu
115 120 125
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
130 135 140
Val Gly Ser Gly Phe Thr Phe Ser Ser Thr Trp Ile Asn Trp Val Arg
145 150 155 160
Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala Gly Ile Tyr Ser Ser
165 170 175
Ala Gly Ser Thr Val His Ser Asp Ser Val Lys Gly Arg Phe Thr Val
180 185 190
Ser Gly Asp Asn Ser Gln Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu
195 200 205
Arg Thr Glu Asp Thr Ala Arg Tyr Tyr Cys Thr Lys Ser Asn Trp Tyr
210 215 220
Thr Leu Asp Val Trp Gly Pro Gly Val Glu Val Val Val Ser Ser Gly
225 230 235 240
Claims (10)
1. a kind of single-chain antibody, the polypeptide being made of light chain variable region, connection peptide and heavy chain variable region；The connection peptide is located at
Between the light chain variable region and the heavy chain variable region；Sequence 1 is the 1st from N-terminal in the light chain variable region such as sequence table
Shown in 107 amino acids residues；The 123rd to 240 bit amino from N-terminal of sequence 1 in the heavy chain variable region such as sequence table
Shown in sour residue.
2. single-chain antibody as described in claim 1, it is characterised in that：Sequence 1 is from N-terminal in the connection peptide such as sequence table
Shown in 108th to 122 amino acids residue.
3. single-chain antibody as claimed in claim 1 or 2, it is characterised in that：The single-chain antibody is shown in sequence 1 in sequence table
Polypeptide.
4. encode any single-chain antibody of claims 1 to 3 nucleic acid molecules, by the encoding gene of the light chain variable region,
The encoding gene of the connection peptide and the encoding gene composition of the heavy chain variable region.
5. nucleic acid molecules as claimed in claim 4, it is characterised in that：In the encoding gene of the light chain variable region such as sequence table
Sequence 2 is from 5 ' ends shown in the 1st to 321 nucleotide；In the encoding gene of the heavy chain variable region such as sequence table sequence 2 from
It rises shown in the 367th to 720 nucleotide 5 ' ends.
6. nucleic acid molecules as claimed in claim 4, it is characterised in that：Sequence in the encoding gene of the connection peptide such as sequence table
2 from 5 ' ends shown in the 322nd to 366 nucleotide.
7. the nucleic acid molecules as described in claim 4 to 6 is any, it is characterised in that：The nucleic acid molecules be A1) A2) or A3)
Or A4) shown in DNA molecular：
A1) code area is DNA molecular shown in sequence 2 in sequence table；
A2) nucleotide sequence is DNA molecular shown in sequence 2 in sequence table；
A3) and A1) or A2) nucleotide sequence that limits has 75% or 75% or more homogeneity, and encodes claims 1 to 3
The DNA molecular of any single-chain antibody；
A4) under strict conditions with A1) or the A2) nucleotide sequence hybridization that limits, and it is any described to encode claims 1 to 3
The DNA molecular of single-chain antibody.
8. the application of any single-chain antibody or any nucleic acid molecules of claim 4 to 7 of claims 1 to 3, is
B1) or B2) or B3) or B4)：
B1 the product for neutralizing Porcine epidemic diarrhea virus) is prepared；
B2 Porcine epidemic diarrhea virus) is neutralized；
B3 the product for preventing and/or treating disease caused by Porcine epidemic diarrhea virus) is prepared；
B4) prevent and/or treat disease caused by Porcine epidemic diarrhea virus.
9. a kind of product contains any single-chain antibody of claims 1 to 3；The function of the product is C1) or C2)：
C1 Porcine epidemic diarrhea virus) is neutralized；
C2) prevent and/or treat disease caused by Porcine epidemic diarrhea virus.
10. the product described in application as claimed in claim 8 or claim 9, it is characterised in that：The product is drug.
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| CN111848785A (en) * | 2020-07-10 | 2020-10-30 | 青岛博隆基因工程有限公司 | Feline herpesvirus antibody sequences, tetrapeptide chain molecules, immunoglobulin molecules |
| CN111909259A (en) * | 2020-07-10 | 2020-11-10 | 青岛博隆基因工程有限公司 | Feline parvovirus antibody sequence, tetrapeptide chain molecule, globulin molecule and application |
| TWI829223B (en) * | 2021-07-08 | 2024-01-11 | 中央研究院 | Recombinant antibody and uses thereof |
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| CN111848785A (en) * | 2020-07-10 | 2020-10-30 | 青岛博隆基因工程有限公司 | Feline herpesvirus antibody sequences, tetrapeptide chain molecules, immunoglobulin molecules |
| CN111909259A (en) * | 2020-07-10 | 2020-11-10 | 青岛博隆基因工程有限公司 | Feline parvovirus antibody sequence, tetrapeptide chain molecule, globulin molecule and application |
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| TWI829223B (en) * | 2021-07-08 | 2024-01-11 | 中央研究院 | Recombinant antibody and uses thereof |
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