CN112266926A - Synthetic antibody library and construction method and application thereof - Google Patents

Synthetic antibody library and construction method and application thereof Download PDF

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CN112266926A
CN112266926A CN202011117094.2A CN202011117094A CN112266926A CN 112266926 A CN112266926 A CN 112266926A CN 202011117094 A CN202011117094 A CN 202011117094A CN 112266926 A CN112266926 A CN 112266926A
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黄海明
庄贤韩
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Shanghai Nawei Biotechnology Co ltd
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Abstract

The invention relates to the field of antibody library construction, in particular to a synthetic antibody library, a construction method and application thereof. The method for constructing the synthetic antibody library comprises the following steps: the nucleotide sequences of the CDR regions of the template antibody were mutated so that the probability of each amino acid at each site in all the antibody CDRs after mutation was as follows: 0.7-1.5% of Met; asn 0.7-1.5%; 0.3-2.1% of Pro; 0.6-1.4% of Trp; 9.8-14% of Gly; 7-14% of Ser; 10-22% of Tyr; 3.1-7.4% of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr and Val respectively; cys is 0. The synthetic antibody library of the invention avoids the use of hybridoma method for screening antibody, does not need to operate animal or animal cell, has simple method and screening process, and can obtain monoclonal antibody with treatment or detection function without humanization.

Description

Synthetic antibody library and construction method and application thereof
Technical Field
The invention relates to the field of antibody library construction, in particular to a synthetic antibody library, a construction method and application thereof.
Background
An antibody is a protein that specifically recognizes its antigen, and conventional antibodies have two polypeptide chains, one Heavy Chain (HC) and one Light Chain (LC) linked together by a disulfide bond to form a heterodimer. The Fc ends of the heavy chains are in turn linked by additional disulfide bonds to form heterotetramers. The antibody recognizes its antigen by 6 Complementarity Determining Regions (CDRs) on the light and heavy chains, 3 on the light chain, CDRL1, CDRL2 and CDRL3, respectively, and correspondingly, 3 on the heavy chain, CDRH1, CDRH2 and CDRH3, respectively. These 6 CDR regions are the main regions for antibodies to recognize and bind to their antigens. It has been found in research that in camelid animals, the antibody has only one heavy chain, which is called a single domain antibody, and the N-terminus of the single domain antibody lacks the CH1 region of a conventional antibody, has only about 100 amino acid residues, is only a few tens of nanometers in diameter, and is therefore also called a nanobody. Similar to conventional antibodies, there are also 3 complementarity determining regions on nanobodies: CDRH1, CDRH2 and CDRH 3.
The invention of hybridoma technology enabled humans to produce high quality monoclonal antibodies for the first time. The technology fuses immune B lymphocytes and myeloma cells into hybridoma cells, and the obtained hybrid cells have the genetic characteristics of amphiphilic cells, can secrete antibodies like B lymphocytes and can proliferate indefinitely like myeloma cells, so that the problem that the survival period of the B lymphocytes in vitro is short is successfully solved. And culturing the hybridoma cell to obtain the monoclonal antibody. Its advantages are high purity of monoclonal antibody, and high productivity of monoclonal antibody. It has the disadvantages that animals need to be raised and operated, and the operation steps are complicated; the monoclonal antibodies produced by the hybridoma technology are mostly of murine origin, and as therapeutic antibodies, human anti-mouse antibody (HAMA) reactions occur, which are rapidly cleared in the human circulatory system, and the like.
The transgenic mouse technology destroys the mouse's own antibody expression system by means of transgenosis, and then introduces the human antibody production system. The transgenic mouse can directly generate fully human antibodies aiming at a certain antigen. However, it also has the disadvantages of cumbersome operation, high cost and inability of high throughput screening.
The single B cell antibody directly carries out single cell level separation, analysis and screening on the B cells, so that the B cells secreting target antibody molecules are accurately and efficiently screened out, and then the target antibody sequence can be obtained by combining a single cell sequencing technology. However, this technique is also costly and technically difficult. Cloning antibody genes from single cells and carrying out in vitro expression verification has large workload and long time, so the method is not suitable for large-throughput screening.
Natural proteins are composed of 20 amino acids, and analysis of large data shows that the distribution of each amino acid in the CDR regions of antibodies is not uniform, for example, the content (or probability of occurrence) of Tyr, Ser, Gly, Asp, Arg is much higher than the average, so that there are reports in the literature that only a few of the 20 natural amino acids are used to construct synthetic antibody libraries. However, this construction strategy neglects the action of other low-content amino acids, which, although low in content, play a non-negligible or even very important role in different situations.
The synthetic antibody is prepared by using a known natural antibody as a template, randomly mutating its hypervariable region (i.e., 6 CDR regions) by genetic engineering means, and forming a large antibody library after mutation, wherein the library molecules are generally displayed on the surface of phage (or other cells, such as yeast and mammalian cells) for screening. At present, synthetic antibody libraries are represented by the HuCal library series of MorphoSys, germany, from which selected IL23 antibodies have been approved by the FDA for the treatment of psoriasis, with more synthetic antibody drug candidate molecules in clinical trials and early development stages. The HuCal library mutation strategy of MorphoSys company is ingenious in design, but is very complex and not beneficial to construction of general researchers, so that more and more researchers develop simple and easy mutation strategies which are effective to construct a synthetic antibody library.
Disclosure of Invention
In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a synthetic antibody library, a method for constructing the same, and use thereof, which solve the problems of the prior art.
To achieve the above and other related objects, the present invention provides a method for constructing a synthetic antibody library, which comprises mutating the nucleotide sequence of the CDRs of a template antibody such that the probability of each amino acid at each position in all the mutated CDRs of the antibody is as follows:
Met 0.7～1.5％；
Asn 0.7～1.5％；
Pro 0.3～2.1％；
Trp 0.6～1.4％；
Gly 9.8～14％；
Ser 7～14％；
Tyr 10～22％；
3.1-7.4% of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr and Val respectively;
cys is not contained.
The present invention also provides a synthetic antibody library obtained by the above method.
The invention also provides polynucleotides or polynucleotide libraries encoding one or more antibodies in the synthetic antibody libraries.
The invention also provides isolated polypeptides encoded by the polynucleotides.
The invention also provides the application of the polynucleotide or the polynucleotide library, the polypeptide and the synthetic antibody library in antibody in vitro screening and antibody discovery.
As described above, the synthetic antibody library of the present invention, the method for constructing the same, and the use thereof, have the following advantageous effects: the method avoids the use of a hybridoma method for screening the antibody, does not need to operate animals or animal cells, has simple method and screening process, and can obtain the monoclonal antibody with the treatment or detection effect without humanization. The library is constructed in vitro, the target molecule is also screened in vitro, expensive instruments and reagents are not needed, and the cost is greatly reduced; high-throughput screening can be performed, and the screening period is short, so that the capital and labor cost is greatly saved.
Drawings
FIG. 1 is a schematic diagram showing a construction process of a synthetic antibody library of the present invention (X represents a polynucleotide of the present invention).
FIG. 2 shows the results of detecting the purity and molecular weight of the positive antibody protein of the present invention by SDS-PAGE.
FIG. 3 is a graph showing the results of Size Exclusion Chromatography (SEC) for detecting the presence or absence of aggregation of the positive antibody protein of the present invention; FIGS. 3-1 to 3-6 show the results of RBD-B7, S1-A11, S-D8, S-D9, S-F12 and S-G6, respectively.
FIG. 4 is a graph showing the results of the affinity assay of the positive antibody proteins of the present invention for their respective antigens using a biofilm interference assay (BLI); FIGS. 4-1 to 4-6 show the results of RBD-B7, S1-A11, S-D8, S-D9, S-F12 and S-G6, respectively.
Detailed Description
The invention provides a construction method of a synthetic antibody library, which comprises the following steps: the nucleotide sequences of the CDR regions of the template antibody were mutated such that the probability of each amino acid at each position in all antibody CDRs is as follows:
Met 0.7～1.5％；
Asn 0.7～1.5％；
Pro 0.3～2.1％；
Trp 0.6～1.4％；
Gly 9.8～14％；
Ser 7～14％；
Tyr 10～22％；
3.1-7.4% of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr and Val respectively;
Cys 0。
the probability refers to the number of occurrences of an amino acid at a particular position in the CDRs of all antibodies in a synthetic antibody library divided by the total number of antibodies, multiplied by 100%. For example: assuming that the total number of all antibodies in the antibody library is 100, Met co-occurs 1 time at the first site of CDRH1 of 100 antibodies, and the probability is 1/100 × 100% ═ 1%, and the probability calculation method for other sites or other CDRs is the same.
In one embodiment, the probability of Met is selected from any of the following ranges: 0.7-0.9%, 0.9-1.1%, 1.1-1.3%, 1.3-1.5%.
In one embodiment, the probability of Asn is selected from any of the following ranges: 0.7-0.9%, 0.9-1.1%, 1.1-1.3%, 1.3-1.5%.
In one embodiment, the probability of Pro is selected from any of the following ranges: 0.3-0.7%, 0.7-1.1%, 1.1-1.5%, 1.5-2.1%.
In one embodiment, the probability of Trp is selected from any of the following ranges: 0.6-0.8%, 0.8-1.0%, 1.0-1.2%, 1.2-1.4%.
In one embodiment, the probability of Gly is selected from any of the following ranges: 9.8-10.5%, 10.5-11.0%, 11.0-11.5%, 11.5-12.0%, 12.0-12.5%, 12.5-13.0%, 13.0-13.5%, 13.5-14.0%.
In one embodiment, the probability of Ser is selected from any of the following ranges: 7-9%, 9-11%, 11-13%, 13-14%.
In one embodiment, the probability of Tyr is selected from any of the following ranges: 10-13%, 13-15%, 15-17%, 17-19%, 19-22%.
In one embodiment, the probability of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr, Val is selected from any of the following ranges: 3.1-4.0%, 4.0-5.0%, 5.0-6.0%, 6.0-7.4%.
The kind of antigen against which the template antibody is directed is not particularly limited. For example, the 1ZVH antibody used in one embodiment of the invention is an anti-lysozyme antibody.
In one embodiment, the template antibody is in a form selected from IgG, scFv, Fab or F (ab')2One or more of them. Correspondingly, the CDR regions of the template antibody include CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH 3.
In one embodiment, the template antibody is in the form of a nanobody. Correspondingly, the CDR regions of the nanobody include CDRH1, CDRH2, and CDRH 3.
The nano antibody can be a natural antibody or a recombinant protein synthesized by genetic engineering. The natural antibody may be derived from a camelid. The camelid mammals include camel, alpaca, vicuna, and guanaco. In one embodiment, the nanobody is selected from the group consisting of nanobodies numbered 1ZVH from the protein structure database (PDB), and 1ZVH is a camelid-derived nanobody.
The CDR length of the antibody obtained by the method for constructing a synthetic antibody library of the present invention is not particularly limited.
In one example, the amino acid lengths of CDRH1, CDRH2, and CDRH3 of the antibodies obtained using the synthetic antibody library construction method are 7, 8, and 14 amino acids, respectively. In other embodiments of the invention, the construction methods may also result in antibodies with CDR lengths different from those exemplified above.
In one embodiment, the mutation is a site-directed mutation. Site-directed mutagenesis techniques can be used to arbitrarily substitute, insert or delete nucleotide fragments of a certain length in a known DNA sequence. It will be appreciated by those skilled in the art that the site-directed mutagenesis may be selected from the group consisting of oligonucleotide primer-mediated site-directed mutagenesis, PCR-mediated site-directed mutagenesis, and cassette mutagenesis, or may be modified over the foregoing methods, such as the Kunkel mutagenesis method.
In certain embodiments of the invention, a Kunkel mutation method is used in which various antibodies having different CDR regions from the template antibody are produced by making the CDR regions of the template antibody have a mutated base sequence by using a primer set containing a mutated base (i.e., a base included in the polynucleotide).
In one embodiment, a library of synthetic antibodies is constructed using the Kunkel mutation method, which comprises the steps of:
1) designing a primer group containing mutant bases, and copying by using uracil-containing single-chain DNA of a template antibody as a template to generate heteroduplex DNA molecules;
2) transforming the heteroduplex DNA molecules of step 1) into E.coli cells to obtain a synthetic antibody library.
The synthesis of uracil-containing single-stranded DNA of a template antibody is known to those skilled in the art. For example, the template antibody can be obtained by cloning the nucleotide sequence of the template antibody into a phage display vector, and then transforming the vector into E.coli for culture.
The primer set containing the mutant base includes a plurality of primers, and the nucleotide sequence of each primer is not particularly limited. In one embodiment, the nucleotide sequence of the primer is as shown in SEQ ID NO. 3-5:
TGTGCAGCAAGTGGAXXXXXXXCTAGGCTGGTTTCGT(SEQ ID NO.3)
GAAGGAGTTGCTGCAXXXXXXXXTACTACGCCGATAGC(SEQ ID NO.4)
TACTATTGTGCGGCCXXXXXXXXXXXXXXAACTACTGGGGCCAA(SEQ ID NO.5)
wherein X represents a nucleotide sequence corresponding to Met, Asn, Pro, Trp, Gly, Ser, Tyr, Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr or Val.
In a preferred embodiment, the probability of each amino acid corresponding to the nucleotide sequence in the primer set containing the mutant base is as follows:
1% of Met; asn 1%; pro 1%; 1% of Trp; gly 10 percent; 10% of Ser; tyr 16 percent; ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr, Val are each 5%; cys is 0.
The inventor researches and discovers that: met is easy to oxidize, so the probability is not too high; asn is easy to be glycosylated, the recognition of the antigen is influenced after glycosylation, and the probability is not too high; pro is prone to hydroxylation and disrupts the three-dimensional structure of the protein. Trp is easy to oxidize, and an over-strong hydrophobic interaction is easy to form after the probability is high, so the probability is not high; gly, Ser and Tyr play an important role in the recognition of antigen and antibody, so the probability is higher; cys is not included in the amino acid mixture because it will form additional disulfide bonds and contribute little to the recognition of antigen antibodies.
In one embodiment, the construction method further comprises quality control after obtaining the synthetic antibody library. The purpose of quality control is to see whether the synthetic antibody library was successfully constructed. And the quality control standard is that if the amino acid distribution of the mutated antibody CDR region meets the quantity distribution of each amino acid in the polypeptide, the synthetic antibody library is successfully constructed, and if the amino acid distribution does not meet the quantity distribution, the construction fails.
In one embodiment, the amino acid profile of the CDR regions of the antibody after mutation is obtained by: and (3) amplifying molecules in the library by PCR, sequencing the amplified product, and analyzing to obtain the distribution of each amino acid in the CDR region.
The present invention also provides a synthetic antibody library obtained by the above method.
In one embodiment, the library of synthetic antibodies may have a real library size of up to 3X 109More than cfu. For example, up to 1010cfu、1011cfu or 1012More than cfu.
The CDR length of the antibody constructed from the synthetic antibody library of the present invention is not particularly limited.
In one example, the antibodies constructed from the synthetic antibody library have amino acid lengths of CDRH1, CDRH2, and CDRH3 of 7, 8, and 14 amino acids, respectively. In other embodiments of the invention, the length of the CDRs from which the library is constructed may be different from the lengths exemplified above.
The invention also provides polynucleotides or polynucleotide libraries encoding one or more antibodies in the synthetic antibody libraries.
The invention also provides polynucleotides or polynucleotide libraries encoding all antibodies in the synthetic antibody library.
The nucleotide sequence of the polynucleotide is not particularly limited as long as the polynucleotide encodes an antibody CDR in which the probability of amino acids at each position satisfies the above requirements.
The polynucleotide can be synthesized by a conventional chemical synthesis technique by a gene synthesis company.
The invention also provides the polypeptide obtained by encoding the polynucleotide.
The invention also provides the use of the polynucleotide or polynucleotide library, the polypeptide, the synthetic antibody library in antibody in vitro screening and antibody discovery.
By screening the synthetic antibody library of the present invention, monoclonal antibody molecules useful in therapy, detection, and the like can be obtained.
For example, the synthetic antibody library may be used to screen antibodies against Spike full length protein S, fragment S1, or RBD of COVID 19.
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Nanobody 1ZVH is a publicly published natural antibody against lysozyme, and has a nucleotide sequence shown in SEQ ID NO.1 and an amino acid sequence shown in SEQ ID NO.2, wherein CDRH1 has 7 amino acid residues (27-33), CDRH2 has 8 amino acid residues (51-58), and CDRH3 has 18 residues (93-102).
DVQLVESGGGSVQAGGSLRLSCAASGYIASINYLGWFRQAPGKEREGVAAVSPAGGTPYYADSVKGRFTVSLDNAENTVYLQMNSLKPEDTALYYCAAARQGWYIPLNSYGYNYWGQGTQVTVSS(SEQ ID NO.2)
Wherein the underlines are CDRH1, CDRH2 and CDRH3 respectively.
Example the procedure for constructing a synthetic nanobody library is illustrated by the use of a known anti-lysozyme nanobody (from camel, PDB:1ZVH) as an antibody template. The overall flow is as follows: 1ZVH is used as the basis for constructing and synthesizing the nano monoclonal antibody, gene mutation is carried out on 3 hypervariable regions (namely CDRH1, CDRH2 and CDRH3) by a genetic engineering method, the polynucleotides are inserted into 7 sites of CDRH1 and 8 sites of CDRH2 one by a Kunkel mutation method, 13 or 14 polynucleotides are inserted between 99-112 sites of CDRH3 (shown in figure 1), and the mutated molecule (3 x 10)9cfu) display on the surface of filamentous phage to form a nanobody library, each molecule in the library has different CDR regions from the original nanobody, so that we can select antibody molecules binding to different antigens from the library.
EXAMPLE 1 obtaining template Single-stranded DNA molecules
1. Obtaining template 1ZVH
The amino acid sequence of the wild type nano monoclonal antibody of the template 1ZVH is obtained by searching a Protein three-dimensional structure database (Protein Data Bank), the coding DNA sequence of the wild type nano monoclonal antibody is obtained by reverse translation, and the sequence is chemically synthesized and then cloned to two Sfi I sites of a phage display vector pComb3XSS to form fusion Protein with p3 Protein of M13 phage on the vector. DNA sequencing ensures that neither the sequence nor the reading frame is in error.
2.1 demonstration experiment of 2.1ZVH
The phage display vector (1ZVH/pComb3XSS) is transformed into Escherichia coli XL1-Blue, and XL1-Blue is infected with helper phage M13KO7(NEB) to secrete phage particles displaying 1ZVH wild type nano monoclonal antibody. The binding of 1ZVH to lysozyme protein was detected by ELISA to ensure the display of 1 ZVH.
3.1 preparation of 3.1ZVH Single-stranded DNA
Plasmid DNA of 1ZVH/pComb3XSS was used to transform E.coli CJ236 cells to generate uracil-inserted single-stranded DNA as a template for a gene mutation library, the purified single-stranded DNA was purified by a standard method, and the concentration and purity of the purified single-stranded DNA were examined by UV spectrophotometer and electrophoresis.
Example 2 construction of Nanobody library
1. Preparation of double-chain hybrid library molecule-Kunkel mutation method
The single-stranded 1ZVH/pComb3XSS DNA prepared above is taken as a template, 3 groups of primers containing mutant bases are added, a mutant primer of CDRH1 is shown as SEQ ID NO.3, a mutant primer of CDRH2 is shown as SEQ ID NO.4, a mutant primer of CDRH3 is shown as SEQ ID NO.5, the primers are respectively paired with CDRH1, CDRH2 and CDRH3 on 1ZVH (shown in figure 1), and under the action of DNA polymerase and ligase, complementary, closed-loop and mutated heteroduplex DNA molecules (namely library molecules) are synthesized.
2. Obtaining libraries
The heteroduplex DNA molecules synthesized above are introduced into Escherichia coli TG1 cells (pre-infected with helper phage M13K07) by electroporation, the efficiency of electrotransformation is determined by sequence dilution method, and the transformant obtained by electrotransformation, i.e. the library capacity of the constructed library, is calculated. Calculated, the storage capacity is 3 multiplied by 109cfu。
3. QC of libraries
PCR amplification library molecules, sequencing the amplification result by using a second generation DNA sequencing (NGS) method to obtain 20112 results, analyzing all CDR regions, and checking whether the distribution of each amino acid at each site meets the design. The results are shown in tables 1, 2 and 3
TABLE 1 comparison of CDR H1 site amino acid residue ratios and Design (Design)
Figure BDA0002730660590000081
Figure BDA0002730660590000091
TABLE 2 CDR H2 amino acid residue ratios at each position and comparison with Design (Design)
Figure BDA0002730660590000092
TABLE 3 CDR H3 amino acid residue ratios at each position and comparison with Design (Design)
Figure BDA0002730660590000093
Figure BDA0002730660590000101
TABLE 3 comparison of the amino acid residue ratios at the positions of CDR H3 and Design (Design)
Figure BDA0002730660590000102
Example 3 Effect of the antibody library constructed in the discovery of antibodies
The libraries herein may be a source of antibodies directed against different target molecules, which antibodies are the lead molecules for therapeutic or detectable antibodies. The role of the constructed antibody library in antibody discovery is demonstrated below by taking the Spike protein of the novel crown COVID19 as an example.
1. Obtaining of antigens
The Spike full length protein S and fragments S1 and RBD of COVID19 were purchased from commercial companies.
2. Screening of libraries and preliminary validation of Positive clones
The S, S1 and RBD proteins are taken as antigens respectively, and the constructed nano antibody library is screened by a standard ELISA method. After 4 rounds of screening, 3 proteins are enriched with phage antibodies. The monoclonal antibody was isolated and ELISA was performed to confirm the binding of the antigen to the phage antibody and the positive molecules were sent to third party companies for DNA sequencing. Sequencing results show that 5 monoclonal antibodies are screened against the S protein, 1 monoclonal antibody is screened against the S1, and 3 monoclonal antibodies are screened against the RBD.
3. Expression and purification of Positive clones
The coding DNA of the positive cloning molecule is respectively subcloned into a protein expression vector pET22b and transformed into Escherichia coli BL 21. Expression and purification of proteins by standard methods
4. Identification of Positive clones
After the concentration of the positive antibody protein is tested by an ultraviolet spectrophotometer, the purity and the molecular weight of the positive antibody protein are detected by SDS-PAGE, the concentration data are shown in Table 4, and the result of the SDS-PAGE is shown in FIG. 2.
Table 4 uv spectrophotometer test concentration results
Name of Nano antibody Concentration (mg/ml) Volume of
RBD-B7 0.137 1ml
S1-A11 0.351 1ml
S-D3 0.336 1ml
S-D8 0.165 1ml
S-D9 0.327 1ml
S-F12 0.939 1ml
S-G6 0.372 1ml
Positive antibody Protein was checked for aggregation by Size Exclusion Chromatography (SEC) using the Acquiy Acr system from Waters, and Column Xbridge Protein BEH SEC Column (
Figure BDA0002730660590000111
3.5 μm,7.8mm X300 mm), 1XPBS buffer, flow rate 0.5 ml/min. The results are shown in FIG. 3, where none of the positive antibody proteins aggregated.
Positive antibody proteins were tested for their affinity to the respective antigen using a biofilm interference assay (BLI). Biofilm interferometry was performed using an Octet RED96 instrument. Biotinylated-labeled antigens were diluted to a final concentration of 2. mu.g/mL with PBS-T (1xPBS, 0.005% (v/v) Tween20), the diluted proteins were immobilized to streptavidin labels at 30 degrees, equilibrated (baseline) in PBS-T for 120s, then bound (association) to different concentrations of positive antibody protein for 300s, and the sensors were then soaked in PBS-T solution for dissociation. Data Analysis 10.0 software was used for Data processing, fitting according to 1:1 stoichiometry. The positive antibody protein affinities and some biophysical properties are shown in FIG. 4 and Table 5.
TABLE 5 summary of positive antibody protein affinities and partial biophysical properties
Figure BDA0002730660590000121
The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the invention set forth herein, as well as variations of the methods of the invention, will be apparent to persons skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Sequence listing
<110> Shanghai Asian-Linked antibody medicine Co., Ltd
<120> a synthetic antibody library, and its construction method and use
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 375
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gatgttcagc tggttgaaag cggtggcggt agtgtgcaag ccggtggtag tctgcgtctg 60
agctgtgcag caagtggata tatcgcgagc atcaattatc taggctggtt tcgtcaagcc 120
ccgggcaaag aacgcgaagg agttgctgca gttagtccag ccggtggtac cccatactac 180
gccgatagcg tgaaaggccg cttcaccgtt agtctggata acgcggagaa tacggtgtat 240
ctgcagatga acagtctgaa accggaagac accgcgctgt actattgtgc ggccgcgcgc 300
cagggctggt atattccgct gaacagctat ggctataact actggggcca aggcacgcaa 360
gttaccgtta gcagc 375
<210> 2
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Ala Ser Ile Asn
20 25 30
Tyr Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Ala Val Ser Pro Ala Gly Gly Thr Pro Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Leu Asp Asn Ala Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ala Ala Arg Gln Gly Trp Tyr Ile Pro Leu Asn Ser Tyr Gly Tyr
100 105 110
Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125

Claims (10)

1. A method of constructing a library of synthetic antibodies, said method comprising: the nucleotide sequences of the CDRs of the template antibody were mutated such that the probability of each amino acid at each position in all the mutated antibody CDRs is as follows:
Figure FDA0002730660580000011
2. the method of construction according to claim 1, wherein the mutation method comprises the steps of:
1) designing a primer group containing mutant bases, and copying by using uracil-containing single-chain DNA of a template antibody as a template to generate heteroduplex DNA molecules;
2) transforming the heteroduplex DNA molecule of step 1) into E.coli cells.
3. The method according to claim 2, wherein the probabilities of the respective amino acids in the mutant base-containing primer set are as follows: 1% of Met; asn 1%; pro 1%; 1% of Trp; gly 10 percent; 10% of Ser; tyr 16 percent; ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Gln, Arg, Thr, Val are each 5%; cys is 0.
4. The method of claim 1, wherein the template antibody is in a form selected from the group consisting of IgG, scFv, Fab, F (ab')2Or one or more of the nano antibodies.
5. The method for constructing the antibody library of claim 1, wherein the method further comprises the step of performing quality control on the obtained synthetic antibody library, wherein the quality control criteria are as follows: the amino acid distribution of the mutated antibody CDR is successful if it matches the probability distribution of each amino acid of claim 1, and failed if it does not match.
6. A synthetic antibody library obtained by the method of any one of claims 1 to 5.
7. The synthetic antibody library of claim 6, wherein the library capacity of the synthetic antibody library is up to 3x 109More than cfu.
8. A polynucleotide or polynucleotide library encoding one or more antibodies in the synthetic antibody library.
9. An isolated polypeptide encoded by the polynucleotide of claim 8.
10. Use of the synthetic antibody library of claim 6 or 7, the polynucleotide or polynucleotide library of claim 8, the polypeptide of claim 9 for in vitro screening of antibodies or for antibody discovery.
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CN107629125A (en) * 2017-09-07 2018-01-26 北京大学 A kind of anti-CD147 nano antibodies, its production method and application
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