WO2016086513A1 - 一种人源杀虫基因及其编码杀虫肽与应用 - Google Patents
一种人源杀虫基因及其编码杀虫肽与应用 Download PDFInfo
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- WO2016086513A1 WO2016086513A1 PCT/CN2015/070423 CN2015070423W WO2016086513A1 WO 2016086513 A1 WO2016086513 A1 WO 2016086513A1 CN 2015070423 W CN2015070423 W CN 2015070423W WO 2016086513 A1 WO2016086513 A1 WO 2016086513A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1278—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Bacillus (G)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4233—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-bacterial Ig
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- the invention relates to the field of genetic engineering and biological control, in particular to a human insecticidal gene and an insecticidal peptide thereof and application thereof.
- Bt toxin genes such as Cry1C, Cry1Ab, Cry1B, Cry1F, Cry2Aa, etc.
- Bacillus thuringiensis is an insect pathogenic bacterium, and its Bt toxin has a specific poisoning effect on a variety of agricultural and forest pests. From 1987, Plant Genetic Systems of Belgium reported the success of transgenic Bt insecticide tobacco. Up to now, Bt gene has been transgenic into major crops such as corn, rice, cotton, tomato, potato and tobacco.
- israeltaki HD73 binds to surface proteins in the mouse small intestine
- Vázquez-Padrón et al Biochem Biophys Res Commun, 2000-01
- T cells are positive for T cells in mice when the exotoxin reaches 10 mg/kg and 100 mg/kg.
- the rate, spleen index and macrophage phagocytic function showed obvious inhibition reaction. With the increase of ingestion dose, this inhibition effect became more obvious.
- the experiment also found that when the accumulation coefficient of Bt toxin protein in animals is greater than 6.24.
- Bt toxin protein is also caused by the immune response, and the long-term high dose of Bt toxin protein also causes a significant decrease in the total white blood cell count and hemoglobin content of the animal, which indicates that the Bt toxin protein has obvious immunosuppressive toxicity. Therefore, the development of alternative biological effectors with biological activity of Bt toxin is a hot spot in the field of research and development of biological pesticides.
- Insecticidal peptides are biologically active polypeptides induced by organisms. Some Bt insecticidal crystal proteins can be decomposed into insecticidal peptides in insects to achieve insecticidal effects, thus becoming a substitute for the biological activity of Bt toxins.
- Anti-Id anti-idiotype antibody
- Anti-Id is a kind of polypeptide with biological insecticidal effect. Anti-Id is a pointer to the variable region of antibody molecule. Specific antibodies produced by Idiotype (Id), Bona et al.
- the classified anti-idiotype antibodies into four types ( ⁇ , ⁇ , ⁇ , and ⁇ ) based on the serological response of Id and Anti-Id and the function of AId.
- the ⁇ -type anti-idiotypic antibody has an antigenic function and biological activity because it has an "inner image" effect, that is, has the same antigenic determinant as the (semi)antigen.
- phage display technology is widely used.
- a phage antibody library By constructing a phage antibody library, specific anti-idiotypic antibodies with similar effects of target antigens can be obtained by specific screening.
- the process of screening specific antibodies by phage display technology is called “Panning”. It mainly includes four steps of binding, washing, elution and amplification.
- Raats et al. use anti-cortisol monoclonal anti-coating as a solid phase antigen for direct screening. Before screening, negative monoclonal antibodies of the same species are used. Negative screening was performed to avoid screening for recombinant antibody fragments that bind to the constant region of the antibody, and finally the anti-Id against cortisol was successfully screened; Goletz et al.
- Anti-Id ScFvs anti-Bt toxin type anti-idiotype single-chain antibodies
- An insecticidal peptide encoded by a human insecticidal gene according to the present invention the amino acid sequence of which is shown in SEQ ID NO.
- a prokaryotic vector containing the human insecticidal gene of the present invention is provided.
- insecticidal peptide encoded by the human insecticidal gene for the control of crop pests.
- An insecticide comprising an insecticidal peptide encoded by a human insecticidal gene according to the present invention.
- the present invention screens and obtains a " ⁇ " type anti-Cry2Aa toxin unique type single-chain antibody (insecticidal peptide) having insecticidal activity from the disclosed human gene library, and performs amino acid and nucleotide sequencing on the insecticidal peptide.
- the primary culture has the binding activity to the peritrophic membrane-specific receptor BBMV of the rice leaf roller, which is not immunized by the animal, has a short preparation cycle and a small amino acid sequence, and is suitable for large-scale production in vitro.
- the insecticidal peptide explores and expands new insecticidal gene resources with simulated Bt toxin biological activity, reduces various safety risks existing in the widespread use of existing Bt toxins, and may replace Bt in the future.
- the biological control of agricultural pests and the reduction of the use of pesticides have important scientific and practical significance.
- Figure 1 is a schematic diagram showing the results of F2 ELISA.
- Figure 2 is a schematic diagram showing the results of F2 biological assay.
- Fig. 3 is a schematic diagram showing the death of the third instar larvae of Cnaphalocrocis medinalis after feeding F2, CK+ and CK-soaked rice leaves, respectively.
- Fig. 4 is a schematic diagram showing the death of the third instar larvae of Plutella xylostella after being fed with F2, CK+, CK-soaked cabbage tablets, respectively.
- Glucose having a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% was added to a 2 ⁇ TY medium.
- a final concentration of 100 ⁇ g/ml ampicillin and 50 ⁇ g/ml kanamycin was added to the 2 ⁇ TY medium.
- a final concentration of 100 ⁇ g/ml ampicillin, 50 ⁇ g/ml kanamycin, and a mass ratio of 1% glucose were added to the 2 ⁇ TY medium.
- a final concentration of 100 ⁇ g/ml ampicillin and a mass ratio of 1% glucose were added to the TYE solid medium.
- 0.05% PBST was added to a PBS solution at a volume ratio of 0.05% Tween-20.
- PBST 0.1% PBST was added to a PBS solution at a volume ratio of 0.1% Tween-20.
- Citrate buffer (CPBS, substrate buffer, pH 5.5):
- skim milk powder 3 g was weighed, dissolved in 80 ml of PBS solution, and made up to 100 ml with PBS solution.
- the HRP-goat anti-rabbit IgG described in the examples was diluted in 3% MPBS solution.
- BBMV irrelevant anti-idiotype single-chain antibody
- non-" ⁇ " type Anti-Id ScFv cabbage leaf
- third instar larvae of Plutella xylostella were provided by the Key Laboratory of Agricultural Product Quality Safety Control Technology and Standards, Ministry of Agriculture, Jiangsu Academy of Agricultural Sciences ;
- Negative serum collected from 1.5-2.0 kg of pure male New Zealand white rabbits, the collection time is one week before immunization;
- Anti-Cry2Aa polyclonal antibody Cry2A protein standard (Shanghai Youlong Biotechnology Co., Ltd.) was used as the immunogen. Three 1.5-2.0 kg purebred male New Zealand white rabbits were selected as test animals. The specific immunization procedure was as follows: Each 200 ⁇ g immunogen was mixed with an equal volume of Freund's complete adjuvant, emulsified into a water-in-oil structure and then subcutaneously injected into the back (about 40 points). After two weeks, the same dose of immunogen was used with an equal volume of incomplete The adjuvant was boosted and then boosted every two weeks. The last time the immunogen was diluted with an equal volume of normal saline, and the ear was injected directly into the ear.
- the heart was bled, serum was prepared, and a final concentration of 0.01% thiomersal was added.
- the serum was purified by the method described in "Contemporary Immunology Technology and Application” (Bad Year, Beijing Medical University, China Union Medical University, United Press, 1998. 309-322) to obtain an anti-Cry2Aa polyclonal antibody.
- the humanized phage antibody library, TG1 bacteria and helper phage KM13 were purchased from Source BioScience, UK;
- HRP-goat anti-M13-IgG was purchased from Wuhan Boster Company;
- Cry2Aa toxin and Cry1Ab toxin were purchased from Shanghai Youlong Biotechnology Co., Ltd.;
- the third instar larvae of rice leaves and leaf roller are provided by Yangzhou Luyuan Biochemical Co., Ltd.
- the screening method adopts positive and negative screening, negative serum is used as negative screening, anti-Cry2Aa polyclonal antibody is used as positive screening, negative negative and negative cells first.
- the culture flask is coated with negative serum, and the positive cell culture flask is coated with anti-Cry2Aa polyclonal antibody, and the elution method uses four rounds of competitive elution:
- the anti-Cry2Aa polyclonal antibody and the negative serum concentration in the second, third and fourth rounds of panning were still 100 ⁇ g/ml, and the phage antibody used was the phage antibody obtained by the previous round of panning, and the panning method still used the first one.
- the positive and negative screening strategy of the round the difference is that the second round of washing the bottle with 0.1% PBST solution 10 times, adding 1ml 10mg / ml trypsin (Trypsin) and competitive elution 1h, the third and fourth times are 0.1% Wash the PBST solution for 20 times, then add 500 ⁇ l of 100 ⁇ g/ml Cry2Aa polyclonal antibody instead. Trypsin competes for elution, the third round of competitive elution time is 1 h, and the fourth round of competitive elution time is 30 min.
- 10 ⁇ l of the phage antibody in the fourth round of panning was infested with 1 ml of TG1 bacteria in logarithmic growth phase, incubated at 37 ° C for 1 h, then coated on TYE-AG solid medium, and cultured overnight at 37 ° C; the next day random pick list Colonies, inoculated into 96-well plates containing 100 ⁇ l/well 2 ⁇ TY-AG liquid medium, cultured overnight at 37° C.; 2 ⁇ l of bacterial solution was aspirated from the wells of the plate the next day and transferred to a new 96-well plate well at 37 ° C.
- the supernatant in step 2 corresponding to the solution is the selected supernatant containing the anti-Cry2Aa toxin unique type single-chain antibody, i.e., insecticidal peptide.
- the nucleotide sequence of the selected insecticidal peptide was determined by Sanger sequencing method as SEQ ID NO. 1 as follows:
- amino acid sequence of the selected insecticidal peptide was determined by Sanger sequencing method as SEQ ID NO. 2, as follows:
- the supernatant containing the insecticidal peptide screened in Example 1 was transferred to 10 ml of 2 ⁇ TY-AG liquid medium at a volume ratio of 1:100, incubated at 37 ° C for 2 h, and 100 ⁇ l of helper phage with a titer of 10 12 was added.
- KM13 was rescued, incubated at 30 ° C for 2 h, centrifuged at 1800 g for 10 min, the supernatant was removed, and the precipitated strain was resuspended in 2 ⁇ TY-AK liquid medium, and cultured overnight at 30 ° C, 250 rpm; the next day, 1800 g was centrifuged for 30 min, on which The supernatant is the supernatant containing the F2 primary culture.
- the experiment was divided into 6 experimental groups and corresponding control groups, and the solutions were prepared according to Table 1.
- F1 in Table 1 is the supernatant of the F2-containing primary culture obtained in Example 2;
- the experiment was divided into experimental group 1, experimental group 2, experimental group 3, positive control group, negative control group 1, negative control group 2, and negative control group 3; the experimental steps were as follows:
- F2 is the supernatant of the F2-containing primary culture obtained in Example 2;
- step b Incubate the 96-well plate after step b for 2 h at room temperature, wash the plate 3 times with 250 ⁇ l PBST per well, then add 100 ⁇ l of 10 ⁇ g/ml anti-Cry2Aa polyclonal antibody per well, and use 250 ⁇ l PBST per well. Wash the plate 3 times, add 100 ⁇ l of 1:5000 diluted HRP-goat anti-rabbit IgG to each well for 1 h at room temperature; wash the plate 3 times with 250 ⁇ l PBST per well, add 100 ⁇ l of substrate color development solution per well, and react for 10-20 min at room temperature. To the appearance of blue, finally add 50 ⁇ l of 2 mol/L H 2 SO 4 per well to terminate the reaction, and then measure the OD 450 value with a microplate reader.
- the experiment was divided into experimental group and control group:
- the experimental group obtained the supernatant (F2) containing the F2 primary culture obtained in Example 2;
- the positive control group used Cry1Ab toxin (CK+) at a concentration of 0.2 g/L;
- the negative control group used anti-Id ScFvs (CK-) other than " ⁇ " type;
- Figure 3 shows the rice leaf roller after feeding the rice leaves soaked in the experimental group (F2), the positive control group (CK+), and the negative control group (CK-).
- the death of the third instar larvae Fig. 4 shows the death of the third instar larvae of Plutella xylostella after feeding the cabbage tablets soaked in the experimental group (F2), the positive control group (CK+) and the negative control group (CK-), respectively.
- the experimental group insecticidal peptide F2 has a good insecticidal effect.
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Abstract
Description
Claims (5)
- 一种人源杀虫基因,其核苷酸序列如SEQ ID NO.1所示。
- 如权利要求1所述人源杀虫基因编码的杀虫肽,其氨基酸序列如SEQ ID NO.2所示。
- 一种含有权利要求1所述人源杀虫基因的原核载体。
- 如权利要求2所述杀虫肽在农作物害虫防治方面的应用。
- 一种含有权利要2所述杀虫肽的杀虫剂。
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JP2016535244A JP6211702B2 (ja) | 2014-12-05 | 2015-01-09 | ヒト殺虫遺伝子およびそれによりコードされた殺虫ペプチドならびに用途 |
EP15851632.8A EP3067423B1 (en) | 2014-12-05 | 2015-01-09 | Human insecticidal gene and insecticidal peptide encoded thereby, and use thereof |
US15/103,515 US9725521B2 (en) | 2014-12-05 | 2015-01-09 | Human-derived insecticidal gene and insecticidal peptide encoded thereby and application thereof |
BR112016014689-1A BR112016014689B1 (pt) | 2014-12-05 | 2015-01-09 | Gene inseticida derivado de ser humano e peptídeo inseticida codificado pelo mesmo e aplicação dos mesmos |
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CN105085683B (zh) * | 2015-09-16 | 2018-11-27 | 江苏省农业科学院 | 一种具有杀虫活性的猪源化改型抗体及其制备方法与应用 |
CN105131123B (zh) * | 2015-09-16 | 2018-11-27 | 江苏省农业科学院 | 一种具有杀虫活性的牛源化改型抗体及其制备方法与应用 |
WO2017100632A1 (en) * | 2015-12-11 | 2017-06-15 | Arizona Board Of Regents On Behalf Of Arizona State University | Human alzheimer's disease and traumatic brain injury associated tau variants as biomarkers and methods of use thereof |
CN111732661A (zh) * | 2020-06-11 | 2020-10-02 | 军事科学院军事医学研究院军事兽医研究所 | 抗h5n1病毒入胞抗体ptd-7b及其应用 |
CN113150161B (zh) * | 2021-05-26 | 2022-06-17 | 江苏省农业科学院 | 苏云金芽孢杆菌Cry2Aa毒素的抗独特型单克隆抗体及应用 |
CN113512115B (zh) * | 2021-08-09 | 2023-09-15 | 江苏省农业科学院 | 一种广谱识别小菜蛾中肠受体的抗Cry2Aa独特型单域抗体及其应用 |
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CN103571853A (zh) * | 2012-08-08 | 2014-02-12 | 中国科学院亚热带农业生态研究所 | 密码子优化的Cry2Aa基因与重组载体以及改变作物抗性的方法 |
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EP1263281B1 (en) * | 2000-02-29 | 2012-04-11 | Auburn University | Multiple gene expression for engineering novel pathways and hyperexpression of foreign proteins in plants |
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CN103773776B (zh) * | 2014-01-26 | 2015-11-18 | 江苏省农业科学院 | 一种人源抗虫基因及其编码的抗Cry1C毒素独特型单链抗体与应用 |
CN103773774B (zh) * | 2014-01-26 | 2015-11-18 | 江苏省农业科学院 | 一种人源抗虫基因及其编码的抗Cry1Ab毒素独特型单链抗体与应用 |
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2014
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Also Published As
Publication number | Publication date |
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JP6211702B2 (ja) | 2017-10-11 |
US20170088633A1 (en) | 2017-03-30 |
BR112016014689A2 (pt) | 2017-12-12 |
BR112016014689B1 (pt) | 2022-06-07 |
EP3067423B1 (en) | 2020-07-15 |
EP3067423A1 (en) | 2016-09-14 |
CN104498501A (zh) | 2015-04-08 |
EP3067423A4 (en) | 2017-04-26 |
CN104498501B (zh) | 2017-07-18 |
US9725521B2 (en) | 2017-08-08 |
JP2017502655A (ja) | 2017-01-26 |
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