CN104894652A - Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) - Google Patents
Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) Download PDFInfo
- Publication number
- CN104894652A CN104894652A CN201510353199.0A CN201510353199A CN104894652A CN 104894652 A CN104894652 A CN 104894652A CN 201510353199 A CN201510353199 A CN 201510353199A CN 104894652 A CN104894652 A CN 104894652A
- Authority
- CN
- China
- Prior art keywords
- antibody
- chain
- ctni
- chain antibody
- sequence
- 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.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the field of gene engineering, and relates to a method for constructing mutant libraries of single-chain antibodies T of cTnI (cardiac troponin I). The method has the advantages that bacteria pRSF-Autodisplay are used as carriers, the capacity of each library can reach 1.5*10<5>, and the libraries are excellent in diversity as proved by DNA (deoxyribonucleic acid) sequencing; cTnI antigens from the fully humanized single-chain antibody libraries of the cTnI are used as targets by the aid of bacterium display technologies, and the humanized single-chain antibodies of the cTnI can be conveniently acquired after multiple screening cycles; the single-chain antibodies are recombinant antibodies, antibody heavy-chain variable regions and light-chain variable regions are connected with one another by a linker peptide by the aid of a gene engineering process to form each recombinant antibody, the single-chain antibodies are the smallest functional antibody fragments for keeping the antigen affinity activity and the specificity of parent antibodies, can obtained by means of in-vitro expression by the aid of gene engineering technologies and can be economically manufactured in the bacteria on a large scale, accordingly, immune detection antibodies can be easily, conveniently and economically manufactured, the detection reagent expenses can be greatly reduced, and the humanized single-chain antibody libraries are high in sensitivity and specificity, easy and convenient to operate and suitable for screening large quantities of samples, and have extremely broad application prospects.
Description
Technical field
The invention belongs to genetically engineered, relate to structure and the application thereof of a kind of people source cTnI single-chain antibody library.
Background technology
Antibody technique development experience three phases, first on behalf of antiserum(antisera) polyclonal antibody, second utilizes B lymphocyte hybridoma technology to prepare authentic monoclonal antibody (McAb) on behalf of the mid-1970s German scholar Kohler and British scholar Milstein, this discovery is one of milestone of biotech development, it is in diagnosis, treatment, the aspects such as prevention and purify protein have shown important.But because M-band easily causes human anti-mouse antibody (Human anti-mouse antibody, HAMA) to react, and people's hybridoma technology is difficult to break through, and has therefore occurred third generation antibody, genetic engineering antibody.Genetic engineering antibody comprises three kinds, i.e. chimeric antibody, antibody prepared by reshaping antibody and Antibody library.Wherein the above two belong to part-humanised antibody, and antibody prepared by Antibody library belongs to full humanized antibody.
Antibody library not only can the process of simulated animal immuning system generating antibody, also has the advantage of many uniquenesses, makes hybridoma technology be difficult to compare.Antibody library without the need to immunity, theoretically, 10
10storage capacity just may contain all antibody.Utilize antigen directly can filter out specific antibody from nonimmune animal's antibody storehouse, and the antibody for this species autoantigen can be screened.Can be the McAb in people source completely from the antibody library of people, be overcome the obstacle being difficult to obtain people source McAb by hybridoma technology.
Yeast is a kind of desirable eukaryotic expression vector host, the expression regulation of its gene and the post translational processing of protein, mechanism of secretion relatively higher eucaryote, to mammalian proteins matter, especially there is unique superiority to the displaying of the protein of people.But be subject to the restriction that transformation into yeast cell rate is low, the foundation in multiple little library when building Yeast libraries, need be carried out, more multiple little library is carried out and storehouse, make to build storehouse process loaded down with trivial details, complicated.In the multiple display technique of exploitation, the applied research of bacterium surface displaying technology makes remarkable progress in recent years, and this combine with technique fluidic cell screens, and can carry out effective high flux screening.Bacterial cell propagation is fast, and toxigenic capacity is cheap, is beneficial to a large amount of preparation high purity antibody.
Summary of the invention
The object of the invention is to provide a kind of cTnI single-chain antibody library, i.e. bacterium single-chain antibody library, containing human antibody heavy chain variable region VH and variable region of light chain VL, and middle attachment zone, have complete antigen-binding site, with bacterium pRSF-Autodisplay for carrier, storage capacity reaches 2 × 10
5.After DNA sequence analysis, prove that its diversity is good, and infection host (E Turbo) can be passed through increase in a large number and regenerate.Utilize the cTNI albumen of bacterium vivoexpression as antigen, by the method for bacteria display, filter out the single-chain antibody that cTNI is special easily, and then for the detection of cTNI.
Another object of the present invention is to provide a kind of construction process of cTNI single-chain antibody library, is achieved through the following technical solutions:
1. the present invention is by extracting 20 masculinity and femininity Adult Spleens and lymphoglandula mRNA respectively, after merging, its reverse transcription is become cDNA.The primer for heavy chain of antibody and variable region of light chain is utilized to increase respectively VH and VL gene fragment, two kinds of Error prone PCR primer are carried out purifying and measured concentration, then form single-chain antibody encoding sequence together with VH with VL gene fragment being assembled into by junction fragment containing the primer of flexible peptide linker.Single-chain antibody encode fragment ScFv purifying is connected with carrier pPNL6, is transformed by efficient lithium acetate transformation method and enter in EBY100 yeast, build semi-synthetic antibody library.
2. be incorporated into row filter with paramagnetic particle method and flow cytometry screening:
A. external escherichia coli expression cTNI albumen is as antigen;
B. by magnetic bead and cTNI protein binding, be combined with antibodies specific by antigen, take turns " absorption-wash-out-amplification " circulation through 3, realize the enrichment isolation to single-chain antibody.
C.cTNI albumen fluorescein FITC marks, and single-chain antibody can emulatively combine with target antigen, is sorted out by flow cell sorter.
D. after fluidic cell sorting, random picking list bacterium colony carries out the secreting, expressing of single-chain antibody, and ELISA method detects expresses the specificity of antibody, and obtaining can the positive strain of expression specificity single-chain antibody.
3. build the competitive expression vector pRSF-Autodisplay be used in bacterium.
4. the positive strain of preliminary screening carries out PCR, obtains the DNA fragmentation of positive strain pPNL6-ScFv.
5. design is containing the primer of Sfil restriction enzyme site, carries out Erron PCR to this DNA fragmentation, amplification heavy chain of antibody and chain variable region gene.
The structure of 6.cTNI single-chain antibody scFv mutant library: by Sfil double digestion scFv gene fragment, and insert pRSF-Autodisplay construction recombination plasmid, transformed competence colibacillus E.coli Turbo cell, obtain anti-cTnI albumen single-chain antibody mutant library.
7. utilize paramagnetic particle method and flow cytometry screening to combine and enrichment isolation is carried out to single-chain antibody mutant library.
8. random picking list bacterium colony carries out the secreting, expressing of single-chain antibody, and ELISA method detects the specificity expressing antibody, and obtaining can the positive strain of expression specificity single-chain antibody.
9. the diversity analysis of single-chain antibody library: the random multiple mono-clonal bacterium of picking, measure the DNA sequence dna of insert district scFv gene, analyze the similarity of its sequence through ClustalW sequence alignment program, judge the diversity analysis of single-chain antibody library, prove that its diversity is good.
Feature of the present invention is:
(1) people source provided by the invention cTNI single-chain antibody mutant library, with bacterium pRSF-Autodisplay for carrier, storage capacity reaches 2 × 10
5, diversity is good; Single-chain antibody is formed by connecting by connection peptides by variable region of heavy chain VH and variable region of light chain VL, and containing complete antigen-binding site.
(2) technology of structure people source provided by the invention cTNI single-chain antibody mutant library, from the semi-synthetic antibody library of the mankind, with cTnI albumen for antigen, obtain wild-type cTnI antibody, utilize Error prone PCR equimolecular biology techniques in conjunction with bacteria display technology, transforming by repeating electricity, obtaining people source cTNI single-chain antibody mutant library antibody library; Technology is workable, and storage capacity is large.
(3) people source of the present invention cTNI single-chain antibody mutant library, because take bacterium as carrier, increased by ehec infection and single-chain antibody is illustrated in bacterium surface, thus can with specific cTNI related antigen as target, filter out the full human single chain variable fragments antibody special to cTNI easily, these human single chain variable fragments antibodies can be used as detection reagent, for cTNI examination.
Embodiment
The structure in embodiment 1: half Humanized single chain antibody storehouse
Its reverse transcription, by extracting 20 masculinity and femininity Adult Spleens and lymphoglandula mRNA respectively, is become cDNA after merging by the present invention.The primer for heavy chain of antibody and variable region of light chain is utilized to increase respectively VH and VL gene fragment, two kinds of Error prone PCR primer are carried out purifying and measured concentration, then form single-chain antibody encoding sequence together with VH with VL gene fragment being assembled into by junction fragment containing the primer of flexible peptide linker.Single-chain antibody encode fragment ScFv purifying is connected with carrier pPNL6, is transformed by efficient lithium acetate transformation method and enter in EBY100 yeast, build semi-synthetic antibody library.
The structure of embodiment 2 cTNI single-chain antibody mutant library
1. screen in conjunction with double Humanized single chain antibody storehouse with paramagnetic particle method and flow cytometry screening:
A. external escherichia coli expression cTNI albumen is as antigen;
B. by magnetic bead and cTNI protein binding, be combined with antibodies specific by antigen, take turns " absorption-wash-out-amplification " circulation through 3, realize the enrichment isolation to single-chain antibody.
C.cTNI albumen fluorescein FITC marks, and single-chain antibody can emulatively combine with target antigen, is sorted out by flow cell sorter.
D. after fluidic cell sorting, random picking list bacterium colony carries out the secreting, expressing of single-chain antibody, and ELISA method detects expresses the specificity of antibody, and obtaining can the positive strain of expression specificity single-chain antibody.
2. build the competitive expression vector pRSF-Autodisplay be used in bacterium.
3. the positive strain of preliminary screening carries out PCR, obtains the DNA fragmentation of positive strain pPNL6-ScFv.
4. design is containing the primer of Sfil restriction enzyme site, carries out Erron PCR to this DNA fragmentation, amplification heavy chain of antibody and chain variable region gene.
The structure of 5.cTNI single-chain antibody scFv mutant library: double digestion scFv gene fragment, and insert pRSF-Autodisplay construction recombination plasmid, transformed competence colibacillus E.coli Turbo cell, obtain anti-cTnI albumen single-chain antibody mutant library.
6. experimental technique: random picking 6 strain mono-clonal bacterium, send Qing Ke bio tech ltd, Beijing to carry out inserting the determined dna sequence in region.
Experimental result: determined dna sequence is analyzed known insertion rate > 99%, 6 strain and all can correctly be readed over, analyzes through ClustalW sequence alignment program, and wherein two sequences are consistent, and sequence alignment the results are shown in Table.
Table .ClustalW sequence alignment analysis result
| Alignment score | Alignment score |
| Sequence(1∶2)：75.5 | Sequence(2∶4)：64.3 |
| Sequence(1∶3)：50.1 | Sequence(2∶5)：79.7 |
| Sequence(1∶4)：51.9 | Sequence(3∶4)：91.7 |
| Sequence(1∶5)：68.6 | Sequence(3∶5)：63.5 |
| Sequence(2∶3)：64 | Sequence(4∶5)：64.2 |
7. the DNA sequence dna between the clone of random picking is different, proves that the diversity of this antibody library is good.
Embodiment 3: screen human single chain variable fragments antibody from bacteriocidin mutant library
Screen with paramagnetic particle method:
1. external escherichia coli expression cTNI albumen is as antigen;
2. by magnetic bead and cTNI protein binding, be combined with antibodies specific by antigen, take turns " absorption-wash-out-amplification " circulation through 3, realize the enrichment isolation to single-chain antibody.
3. random choose last take turns the clone 20 obtained, send Qing Ke bio tech ltd, Beijing to carry out the determined dna sequence in scFv region.And carry out the structural analysis of single chain antibody sequence.
| Bacterium sequence number | Length (bp) | Heavy chain is originated | Light chain is originated |
| 1 | 917 | HumIGHV034 | HumIGLV048 |
| 2 | 910 | HumIGHV072 | HumIGLV125 |
| 3 | 785 | HumIGHV072 | HumIGLV162 |
| 4 | 732 | HumIGHV072 | HumIGLV017 |
| 5 | 917 | HumIGHV072 | HumIGLV167 |
The preparation of embodiment 4 antibody
1. pair positive strain carries out revision test, determines the positive strain that stability is best, and by its called after cTnI-Anti-ScFv 2A-2, extract this plasmid DNA, sequencing obtains its corresponding nucleotide sequence.This single-chain antibody is connected to form in turn by variable region of heavy chain, the small peptide connecting described variable region of heavy chain and variable region of light chain, variable region of light chain.Variable region of heavy chain is amino acid residue sequence as described in the 36 to 162, Fig. 1 N end; Variable region of light chain such as Fig. 1 N holds the 183rd to 248 shown amino acid residue sequences.To encode the gene of described cTnI albumen single-chain antibody, by variable region of heavy chain encoding sequence, connect the encoding sequence of variable region of heavy chain and the encoding sequence of variable region of light chain forms.The encoding sequence of variable region of heavy chain is if Fig. 1 is from 5 ' end the 106 to 486 shown nucleotide sequence, the encoding sequence of variable region of light chain is if Fig. 1 is from 5 ' end the 549 to 744 shown nucleotide sequence (catching up with thereafter 3 encoding gene TGA, as terminator).
2. bacterial expression antibody protein.
1). with BL21 (DE3) for Host Strains, transformed by cTnI-Anti-ScFv 2A-2, picking list bacterium colony spends the night in 3ml LB culture medium culturing;
2). with 1: 500 ratio, bacterium liquid is forwarded to 400ml LB substratum, constant temperature is at 37 DEG C, and 230rpm is cultured to OD600=0.4-0.6;
3). add IPTG induction, IPTG induces final concentration to be 0.5mM, expresses after 8 hours and receive bacterium under keeping 25 DEG C of conditions.
4) purifying of .cTnI-Anti-ScFv 2A-2 albumen:
A. get 400ml nutrient solution 3000rpm centrifugation and collect thalline after 10 minutes, thalline is resuspended in (50mM Na-PO in 10ml protein extract buffer
4, pH8.0,500mM NaCl, 10mMImidazle), 15MPa is high crushes bacterium, 12,000rpm, 4 DEG C of centrifugal 10min, and supernatant liquor and Ni-NTA hatch 1 hour on ice;
B. cross Ni-NTA affinity column, with protein extraction buffer washing, finally use 250mM Imidazle wash-out, collect elutriant.
C. protein eluate is crossed G50 post and is removed Imidazle, can obtain the cTnI-Anti-ScFv 2A-2 albumen of more than 90% purity like this.
D. collect above-mentioned purified product, carry out 12%SDS-PAGE electrophoresis.
3.cTnI-Anti-ScFv 2A-2 determining the protein quantity:
Accompanying drawing explanation
Fig. 1, bacterial expression vector pRSF-Autodisplay build collection of illustrative plates
The DNA fragmentation of Fig. 2, pcr amplification pPNL6-ScFv
The DNA fragmentation of Fig. 3, erron PCR bis-amplification pPNL6-ScFv.
Claims (3)
1. a cTNI single-chain antibody library, it is characterized in that: this antibody library contains a complete set of human antibody heavy chain variable region VH and variable region of light chain VL, and middle attachment zone linker sequence, there is complete antigen-binding site, be carrier with pRSF-Autodisplay, storage capacity reaches 2 × 10
5.
2. the construction process of a kind of cTNI single-chain antibody library according to claim 1, is characterized in that being realized by following steps:
(1) the present invention is by extracting 20 masculinity and femininity Adult Spleens and lymphoglandula mRNA respectively, after merging, its reverse transcription is become cDNA.The primer for heavy chain of antibody and variable region of light chain is utilized to increase respectively VH and VL gene fragment, two kinds of Error prone PCR primer are carried out purifying and measured concentration, then form single-chain antibody encoding sequence together with VH with VL gene fragment being assembled into by junction fragment containing the primer of flexible peptide linker.Single-chain antibody encode fragment ScFv purifying is connected with carrier pPNL6, is transformed by efficient lithium acetate transformation method and enter in EBY100 yeast, build semi-synthetic antibody library.
(2) using external escherichia coli expression cTNI albumen as antigen, paramagnetic particle method and flow cytometry screening are incorporated into row filter, and obtaining can the positive strain of expression specificity single-chain antibody.
(3) the competitive expression vector pRSF-Autodisplay be used in bacterium is built.
(4) positive strain of preliminary screening carries out PCR, obtains the DNA fragmentation of positive strain pPNL6-ScFv.
(5) design is containing the primer of Sfil restriction enzyme site, carries out Erron PCR to this DNA fragmentation, amplification heavy chain of antibody and chain variable region gene.
(6) Sfil double digestion scFv gene fragment, and insert pRSF-Autodisplay construction recombination plasmid, transformed competence colibacillus E.coli Turbo cell, obtain anti-cTnl albumen single-chain antibody mutant library.
3. the diversity analysis of single-chain antibody library: the random multiple mono-clonal bacterium of picking, measure the DNA sequence dna of insert district scFv gene, analyze the similarity of its sequence through ClustalW sequence alignment program, judge the diversity analysis of single-chain antibody library, prove that its diversity is good.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510353199.0A CN104894652A (en) | 2015-06-25 | 2015-06-25 | Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510353199.0A CN104894652A (en) | 2015-06-25 | 2015-06-25 | Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104894652A true CN104894652A (en) | 2015-09-09 |
Family
ID=54027594
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510353199.0A Pending CN104894652A (en) | 2015-06-25 | 2015-06-25 | Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104894652A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164399A (en) * | 2017-07-07 | 2017-09-15 | 江苏省疾病预防控制中心 | A kind of high flux expresses the linear expression cassette of monoclonal antibody |
| CN109097382A (en) * | 2018-08-28 | 2018-12-28 | 东北农业大学 | A method of for screening specific antibody variable region |
| CN109972209A (en) * | 2019-01-18 | 2019-07-05 | 南开大学 | The method that single-chain antibody library is rearranged to full length antibody library based on the one-step method of emulsion-based PCR |
| CN111018978A (en) * | 2018-10-10 | 2020-04-17 | 东莞市朋志生物科技有限公司 | Anti-human cardiac troponin I antibody and application thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314363A (en) * | 2000-03-21 | 2001-09-26 | 南京医科大学第一附属医院 | Purification of human myocardial troponin I and proparation of monoclonal anti-body thereof and its quick detecting method |
| CN1408874A (en) * | 2001-09-25 | 2003-04-09 | 韩泽广 | Procaryotic cell in vivo antibody library construction screening and optimizing of antibody and use |
| US20050037339A1 (en) * | 1998-04-24 | 2005-02-17 | The Regents Of The University Of California | Methods of selecting internalizing antibodies |
| CN1644685A (en) * | 2004-12-23 | 2005-07-27 | 复旦大学 | Monoclone antibody against human cardiac troponin I and its use |
| CN1982337A (en) * | 2005-12-14 | 2007-06-20 | 中国医学科学院放射医学研究所 | Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use |
| CN101294308A (en) * | 2007-10-22 | 2008-10-29 | 侯明 | Method for humanizing recombinant phages antibody library |
| CN101948534A (en) * | 2010-08-19 | 2011-01-19 | 中国科学院生物物理研究所 | Method for screening antibodies |
| CN102978713A (en) * | 2012-11-23 | 2013-03-20 | 浙江大学 | Leukemia single-chain antibody library, as well as construction method and application thereof |
| WO2013085368A1 (en) * | 2011-12-09 | 2013-06-13 | Kyungpook National University Industry-Academic Cooperation Foundation | Fusion polypeptide comprising protein scaffold and method for screening the peptide library specific for target protein using the same |
| CN103282560A (en) * | 2010-07-16 | 2013-09-04 | 阿迪马布有限责任公司 | Antibody libraries |
-
2015
- 2015-06-25 CN CN201510353199.0A patent/CN104894652A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050037339A1 (en) * | 1998-04-24 | 2005-02-17 | The Regents Of The University Of California | Methods of selecting internalizing antibodies |
| CN1314363A (en) * | 2000-03-21 | 2001-09-26 | 南京医科大学第一附属医院 | Purification of human myocardial troponin I and proparation of monoclonal anti-body thereof and its quick detecting method |
| CN1408874A (en) * | 2001-09-25 | 2003-04-09 | 韩泽广 | Procaryotic cell in vivo antibody library construction screening and optimizing of antibody and use |
| CN1644685A (en) * | 2004-12-23 | 2005-07-27 | 复旦大学 | Monoclone antibody against human cardiac troponin I and its use |
| CN1982337A (en) * | 2005-12-14 | 2007-06-20 | 中国医学科学院放射医学研究所 | Synthesis of polypeptide antibody from anti-human myocardial troponin I, its production and use |
| CN101294308A (en) * | 2007-10-22 | 2008-10-29 | 侯明 | Method for humanizing recombinant phages antibody library |
| CN103282560A (en) * | 2010-07-16 | 2013-09-04 | 阿迪马布有限责任公司 | Antibody libraries |
| CN101948534A (en) * | 2010-08-19 | 2011-01-19 | 中国科学院生物物理研究所 | Method for screening antibodies |
| WO2013085368A1 (en) * | 2011-12-09 | 2013-06-13 | Kyungpook National University Industry-Academic Cooperation Foundation | Fusion polypeptide comprising protein scaffold and method for screening the peptide library specific for target protein using the same |
| CN102978713A (en) * | 2012-11-23 | 2013-03-20 | 浙江大学 | Leukemia single-chain antibody library, as well as construction method and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| MARTIN FISCHLECHNER等: "Evolution of Enzyme Catalysts Caged in Biomimetic Gel-Shell Beads", 《NATURE CHEMISTRY》 * |
| P.J.CONROY等: "Cardiac Troponin I：a case study in rational antibody design for human diagnostics", 《PROTEIN ENGINEERING，DESIGN ＆ SELECTION》 * |
| 刘亮伟，陈红歌主编: "《基因工程原理与实验指导》", 31 December 2010 * |
| 刘萱: "基于酵母表面展示系统的HIV抗体分离和表达", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 吴梧桐主编: "《生物制药工艺学》", 31 December 2013 * |
| 沈倍奋等主编: "《重组抗体》", 30 December 2005 * |
| 贾娟娟等: "抗人心肌肌钙蛋白I 单克隆抗体的制备及初步鉴定", 《现代免疫学》 * |
| 邓宁主编: "《动物细胞工程》", 31 December 2014 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107164399A (en) * | 2017-07-07 | 2017-09-15 | 江苏省疾病预防控制中心 | A kind of high flux expresses the linear expression cassette of monoclonal antibody |
| CN109097382A (en) * | 2018-08-28 | 2018-12-28 | 东北农业大学 | A method of for screening specific antibody variable region |
| CN111018978A (en) * | 2018-10-10 | 2020-04-17 | 东莞市朋志生物科技有限公司 | Anti-human cardiac troponin I antibody and application thereof |
| CN109972209A (en) * | 2019-01-18 | 2019-07-05 | 南开大学 | The method that single-chain antibody library is rearranged to full length antibody library based on the one-step method of emulsion-based PCR |
| CN109972209B (en) * | 2019-01-18 | 2022-05-03 | 南开大学 | Method for rearranging single-chain antibody library into full-length antibody library by one-step method based on emulsion PCR |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6466397B2 (en) | Protocol for identification and isolation of antigen-specific B cells and production of antibodies against the desired antigen | |
| CN104894652A (en) | Construction and application of humanized single-chain antibody libraries of cTnI (cardiac troponin I) | |
| CN106047857A (en) | Method for mining antibody with specific function | |
| CN113699118A (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and application of hybridoma cell strain | |
| CN109971726B (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and preparation method of antibody | |
| CN105524173B (en) | Nano antibody aiming at humanized antibody Fc fragment and application thereof | |
| CN109777785A (en) | Hybridoma cell strain and its application | |
| CN103603057B (en) | Based on can the antibody screening in infecting virus particle type heavy chain of antibody storehouse/light chain storehouse and preparation method | |
| CN112321709A (en) | Macadamia nut allergen Vicilin specific nano antibody and application thereof | |
| Chen et al. | Generation and characterization of chicken-sourced single-chain variable fragments (scFvs) against porcine interferon-gamma (pIFN-γ) | |
| CN112321708B (en) | Peanut allergen Arah3 specific nano antibody and application thereof | |
| CN110205300B (en) | Bar monoclonal antibody hybridoma cell strain, antibody produced by same and preparation method thereof | |
| CN112342198A (en) | PAT/PAT monoclonal antibody hybridoma cell strain, antibody produced by same and preparation method thereof | |
| CN102094035B (en) | Expression vector for constructing high-quality bacteriophage antibody library | |
| CN113150122B (en) | Preparation method of high-throughput whole rabbit source monoclonal antibody | |
| CN114106167B (en) | Nanobody for specifically recognizing listeria monocytogenes, recombinant vector, host cell and application of nanobody | |
| Orcutt et al. | Yeast display and selections | |
| CN116003606B (en) | TIGIT nano antibody and preparation method and application thereof | |
| CN115785277B (en) | Preparation and application of anti-IL4i1 nano antibody | |
| CN113912729B (en) | Single-domain antibody aiming at sST2, and derivative protein and application thereof | |
| CN117843798A (en) | anti-CHI 3L1 nanobody and application thereof | |
| CN117126277B (en) | Monoclonal antibody and application thereof | |
| CN114044819B (en) | High-flux preparation method of whole sheep-derived monoclonal antibody | |
| CN114773462B (en) | Recombinant single-chain antibody for detecting bovine CRP protein and application thereof | |
| US10088471B1 (en) | Immunoglobulin G mRNA based plasma cell separation and antibody gene cloning |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150909 |
|
| WD01 | Invention patent application deemed withdrawn after publication |