CN114381472B - Nanobody total synthesis library and construction method thereof - Google Patents
Nanobody total synthesis library and construction method thereof Download PDFInfo
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Abstract
The invention discloses a total synthesis library of nano antibodies and a construction method thereof. The construction method of the invention comprises the following steps: s1, selecting a fixed antibody constant region sequence as a framework, statistically analyzing the reported nanometer antibody sequence, and determining the amino acid length of a CDRs region; s2, determining the randomized numbers of the amino acids in the CDR1 region and the CDR2 region according to the use frequency of the amino acids in the CDRs region, designing the randomized numbers of the amino acids with 3 lengths in the CDR3 region, and carrying out NNK randomization on all 3 CDR regions; s3, obtaining a complete DNA fragment by utilizing overlap extension PCR, cloning the complete DNA fragment to a vector, and converting the complete DNA fragment to escherichia coli to construct a total synthesis library of the nano antibody. The construction method of the invention has strong controllability, flexible design, large library capacity of the constructed total synthesis library of the nano antibody, and rich diversity, can be used as a screening platform for different antigens, and enriches the resources of the total synthesis library of the nano antibody.
Description
Technical Field
The invention belongs to the technical field of antibody library construction. More particularly, relates to a nanobody total synthesis library and a construction method thereof.
Background
An antibody is a protein capable of specifically recognizing its antigen, and a conventional antibody is composed of two identical heavy chains and two identical light chain polypeptides, the light chain having 3 complementarity determining regions: CDRL1, CDRL2 and CDRL3, respectively, the 3 complementarity determining regions on the heavy chain are CDRH1, CDRH2 and CDRH3, respectively, and these 6 CDR regions are the main regions of the antibody that recognize and bind to its antigen. It was found that in camelid animals there is a natural heavy chain antibody with only heavy chains and no light chains, and that the variable region of the heavy chain antibody is cloned to give nanobodies, also called single domain antibodies (VHH). The VHH has the advantages of high affinity, strong stability, good solubility, easy coupling transformation and the like, so that the VHH has wide application prospect in the fields of biomedicine, drug development and food safety detection.
The preparation of specific nanobodies requires panning of specific gene fragments from a VHH gene library. Immune libraries are the type of genetic library commonly used for screening nanobodies. The construction of immune libraries requires animal immunization, with periodic continuous injection of immunogens into camelids, resulting in an immune system that produces a sufficient immune response to the antigen and a continuous in vivo selection and antibody maturation process. Therefore, nanobody immune libraries constructed by immunizing camelids generally have relatively high affinity and specificity of the nanobody obtained.
However, the biggest drawback of immune libraries is the small diversity of genes, which, although easier to screen for genes specifically recognizing immune antigens, make it difficult to screen further for antibody genes against other antigens. The nano antibody for identifying the new antigen by panning needs to be re-immunized with animals to construct a library, and the workload is large and the period is relatively long, so that the immune library cannot be used as a widely applicable antibody discovery platform. Furthermore, in particular situations, such as where the antigen is highly toxic, lethal, or where some small molecule compounds are poorly or even not immunogenic, specific antibodies cannot be obtained by immunizing camelids, these drawbacks greatly limit the use of immune libraries. In order to solve the problems, a fully synthetic nanobody library which has the advantages of large storage capacity, rich diversity, low production cost, capability of screening various antigens and the like is generated.
The fully synthetic antibody library is constructed by splicing optimized antibody sequences serving as a framework with natural or artificial Complementarity Determining Regions (CDRs), and has become a supplement to natural libraries and immune libraries. However, there are few fully synthetic antibody libraries available in the prior art for screening nanobodies, such as Qin Xiufeng, which are synthesized by large-scale sequencing comparison of natural nanobody libraries to determine the number of framework sequences and amino acids in the CDRs region, and random introduction of mutations in the CDRs3 region using NKK randomization (Qin Xiufeng. Design, construction and identification of fully synthetic nanobody libraries [ D ]. University of Tianjin, 2017.); in the high-flying research, the nanobody synthesis library is constructed by introducing diversity into complementarity determining region 3 (CDR 3) through randomization of synthetic oligonucleotides based on camel conserved single domain antibody fragment (VHH) framework (high-flying. Construction of nanobody synthesis library and preliminary application [ D ]. Henan university, 2018.); research also shows that 3 groups of primers containing mutant bases added with CDRH regions are introduced to construct libraries (yellow Haiming, zhuang Xianhan. A synthetic antibody library, a construction method and application thereof [ P ].2021-01-26 ]) by adopting 1ZVH wild-type nanometer monoclonal antibody as a template; in order to enrich the resources of nanobody libraries, increase the opportunity of screening more antigen-specific nanobodies, and also need to construct nanobody total synthesis libraries with more abundant diversity.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings, and provides a total synthesis library of nano antibodies and a construction method thereof, which enrich the resources of the nano antibody library.
The invention aims to provide a construction method of a total synthesis library of nano antibodies.
It is another object of the present invention to provide a total synthetic library of nanobodies.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a construction method of a total synthesis library of nano antibodies, which comprises the steps of selecting a fixed antibody constant region sequence as a total synthesis library framework, statistically analyzing the reported nano antibody sequence, determining the amino acid length of a CDRs region and the randomized number of amino acids, statistically analyzing the reported nano antibody sequence, determining the amino acid length of the CDRs region, then determining the randomized number of amino acids of a CDR1 region and a CDR2 region according to the use frequency of the amino acids of the CDRs region, designing the randomized number of amino acids with 3 lengths because the CDR3 region is a key region combined with antigen, and then carrying out NNK randomization on all 3 CDR regions to construct 3 total synthesis nano antibody libraries.
The construction method of the nanobody total synthesis library comprises the following steps:
s1, selecting a fixed antibody constant region sequence as a framework, statistically analyzing the reported nanometer antibody sequence, and determining the amino acid length of a CDRs region;
s2, determining the randomized numbers of the amino acids in the CDR1 region and the CDR2 region according to the use frequency of the amino acids in the CDRs region, designing the randomized numbers of the amino acids with 3 lengths in the CDR3 region, and carrying out NNK randomization on all 3 CDR regions;
s3, obtaining a complete DNA fragment by utilizing overlap extension PCR, cloning the complete DNA fragment into a vector, and converting the complete DNA fragment into escherichia coli to construct a fully-synthesized nano antibody library.
Since VHH comprises 4 FR regions and 3 CDR regions. Among them, CDRs are more variable, and particularly amino acid sequences of CDR3 exhibit a large difference between different nanobodies, and are considered as critical regions determining antibody specificity, so that the originally constructed synthetic library is constructed by replacing the natural CDR3 region with a fragment of artificially synthesized CDR3 region, thus greatly increasing the diversity and storage capacity of the library. The nano antibody is mainly combined with antigen molecules by virtue of 3 CDR regions, and the diversity of the antibody is closely related to the diversity of the 3 CDR regions, so that the invention randomizes all the 3 CDR regions to construct a fully-synthesized nano antibody library with more abundant diversity, and the opportunity of screening more antigen-specific nano antibodies can be increased.
Further, the method for the NNK randomization mutation in step S2 includes the steps of:
1) Designing a primer group containing mutant bases, enabling the primer group to be used as templates for replication to obtain DNA fragments with randomized CDR1, CDR2 and CDR3 amino acids, and then obtaining complete DNA fragments through repeated overlapping extension PCR;
2) Cutting and digesting the complete DNA fragment in the step 1), connecting the DNA fragment into corresponding vectors, and sequencing;
3) Transforming the product of step 2) into E.coli cells.
Preferably, the scaffold selected in step S1 is the nanobody conserved framework cAbBCII10. The framework cabBCII10 is more stable and has relatively high expression level, and has antibody function under the condition of reducing or deleting a conserved disulfide bond.
Preferably, the nanobody conserved framework cAbBCII10 comprises an FR1 region with an amino acid sequence shown as SEQ ID NO.1, an FR2 region with an amino acid sequence shown as SEQ ID NO.2, an FR3 region with an amino acid sequence shown as SEQ ID NO.3 and an FR4 region with an amino acid sequence shown as SEQ ID NO. 4.
Preferably, the vector used for prokaryotic expression in step S3 is pComb3xss.
Preferably, the reaction system for cleavage in step 2) is as follows: mu.L of 10 XBuffer, 2. Mu.L of SfiI enzyme, 1. Mu.g of VHH or pComb3xss plasmid, 21. Mu.L of RNase-free deionized water, and a total volume of 50. Mu.L; and reacted overnight in a 50 ℃ water bath.
Preferably, E.coli TG1 competent cells are used in step S3.
The invention provides a total synthesis library of nano-antibodies, which is obtained by the construction method of the total synthesis library of the nano-antibodies.
Preferably, the library capacity of the nanobody total synthesis library is not less than 1.0X10 8 。
Preferably, the nanobody total synthesis library is CDRs-12AA, CDRs-15AA and CDRs-19AA.
The invention has the following beneficial effects:
the invention provides a total synthesis library of nano-antibodies and a construction method thereof, wherein a fixed antibody constant region sequence is selected as a framework, 3 length amino acid randomization numbers are designed for a CDR3 region, and then NNK randomization is carried out on all 3 CDR regions to construct the total synthesis library of the nano-antibodies. Unlike conventional construction method, the method of the present invention randomizes only one CDR3 region, and the method of the present invention randomizes all three CDR regions during construction, and needs multiple overlapping extension PCR, and the randomized three regions are complicated and difficult during multiple overlapping extension PCR.
The construction method of the invention and the nanobody total synthesis library constructed by the method have the following advantages: the nano antibody total synthesis library constructed by the NNK mutation method can be used as a widely applicable antibody discovery platform to screen specific nano antibodies, so that the nano antibody total synthesis library with more abundant diversity is constructed, the opportunity of screening more antigen specific nano antibodies can be increased, and the problem that the corresponding immune library cannot be constructed to obtain the required nano antibodies due to the fact that immunity cannot be realized or immunity fails due to antigen factors is solved.
Drawings
FIG. 1 is a schematic diagram of nanobody gene splicing;
FIG. 2 is a graph of statistical analysis of the number of amino acid randomizations for CDR1, CDR2 and CDR 3;
FIG. 3 is a DNA electrophoresis diagram of the overlapping extension PCR stage;
FIG. 4 shows the gene insertion rate detection of the fully synthetic nanobody library;
FIG. 5 shows the detection of the insertion accuracy and diversity of the synthetic nanobody library genes.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 selection of Total synthetic antibody library framework and determination of the number of amino acid randomizations of the CDRs region
The fully synthetic antibody library framework employed in the present invention selects the nanobody conserved framework cAbBCII10 as the framework of the synthetic library from the Conrath team's study (Dirk Saerens, mirille Pellis, remy Loris, els Pardon, mireille Dumoulin, andre Matagne, lode Wyns, serge Mu yldermans, katja Conrath. Identity of a Universal VHH Framework to Gr aft Non-canonical Antigen-binding Loops of Camel Single-domain Antibodies [ J ]. Journal of Molecular Biology,2005,352 (3):).
Reported nanobody sequences were analyzed using clustalw (https:// www.ebi.ac.uk) and multiple sequence alignment software Jalview to determine the amino acid length of the CDRs regions and the number of amino acid randomizations, as shown in figure 1.
The sequence alignment shows that: the most common antibody sequences with the length of 8 amino acids are in the CDR1 and CDR2 regions, and the 27 th site of the CDR1 is found to be up to 82% in proportion by statistics, so that the 27 th amino acid is determined as G, and the final randomized number of the amino acids in the CDR1 region is determined as 7; the 56 th and 65 th positions at the two ends of the CDR2 are respectively determined as I and T, and the randomized number of the amino acids in the CDR2 region is determined as 6; positions 105, 106 and 117 at the two ends of CDR3 are determined as A, A and Y respectively, three different numbers of amino acids are designed for randomization (12, 15 and 19 amino acids) because the CDR3 region is a key part for binding antigen, and NNK (N stands for 4 bases A, T, C and G, K stands for 2 bases T and G) is introduced at each position for randomization, so that library diversity is increased, and the quality of the total synthesized nanobody library is improved.
EXAMPLE 2 overlap extension PCR amplification of nanobody full Length fragments
Determining the amino acid randomization number of the CDRs according to bioinformatics and sequence comparison results, carrying out NNK randomization on all 3 CDR regions, and constructing 3 total synthetic phage display nanobody libraries by using a statistical analysis chart of the amino acid randomization numbers of CDR1, CDR2 and CDR3 as shown in figure 2.
And amplifying and splicing by using overlap extension PCR to obtain the full-length gene fragment of the nano antibody, and carrying out 3 rounds of PCR. The 1 st round of PCR amplification yielded 4 short DNA fragments, FR1-CDR1, FR2-CDR2, FR3 and CDR3-FR4 regions, respectively. 2 nd round of PCR amplification to obtain 2 DNA fragments of FR1-CDR2 and FR3-FR4 region, 3 rd round of PCR amplification to obtain complete nano antibody gene fragments, and further recovering the complete nano antibody gene fragments by using a gel cutting recovery kit to obtain 3 nano antibody gene fragments of about 400 bp.
Wherein, the 2 nd round and the 3 rd round of segment splicing are both required to be performed with two times of PCR to obtain DNA segments, taking the splicing of FR1-CDR1 and FR2-CDR2 as an example, the 1 st round of PCR is only required to be performed for 10 cycles, the 2 nd round of PCR takes the 1 st PCR product as a template, and the two times of PCR systems and reaction conditions are shown in the following tables 1-4.
TABLE 1 st PCR reaction System
TABLE 21 st PCR reaction conditions
TABLE 3 PCR reaction System at 2 nd time
TABLE 4 first PCR reaction conditions
In the construction method, three regions are randomized, repeated overlap extension PCR is needed, two short segments containing NNK randomization are needed to obtain a complete VHH segment through overlap extension PCR, and meanwhile, the three randomized regions are complicated and more difficult in repeated overlap extension PCR. The results are shown in fig. 3, from left to right: the left panel shows the 4 short DNA fragments obtained by round 1 PCR amplification; the middle is the DNA fragment generated by the 2 nd round of PCR amplification; the right panel shows the full length fragment of VHH obtained from round 3 of two PCR.
EXAMPLE 3 construction and evaluation of fully synthetic nanobody libraries
Double digestion reaction of VHH gene fragment and pComb3xss vector, digestion of pComb3xss phagemid vector and VHH gene fragment with restriction enzyme sfienzyme, the digestion reaction system is as follows: mu.L of 10 XBuffer, 2. Mu.L of SfiI enzyme, 1. Mu.g of VHH or pComb3xss plasmid, 21. Mu.L of RNase-free deionized water, and a total volume of 50. Mu.L were reacted overnight in a water bath at 50 ℃. And (3) identifying whether enzyme digestion is complete or not by agarose gel electrophoresis, respectively recovering the VHH gene and the pComb3xss fragment by using a DNA purification kit and a gel digestion recovery kit, quantifying the Nanodrop 2000C and storing the fragments at the temperature of minus 20 ℃ for later use.
Connection of VHH gene fragment and pComb3xss vector, PCR tube, gun head, tweezers and other reagent consumable materials needed for high temperature sterilization in advance, and the following system is prepared according to the T4 DNA ligase reaction kit: 2. Mu.L of 10 XT 4 library Buffer, VHH/pComb3xss (m/m: 30/100), 2. Mu.L of T4 DNA library, and RNase-free deionized water were added to a total volume of 20. Mu.L. The reaction was carried out at 16℃overnight, 10 reactions were collected the next day, and the products were quantified by Nanodrop 2000C using a clean recovery kit for DNA purification.
Electrotransformation of ligation products: 10 50. Mu.L of E.coli TG1 competent cells were thawed on ice for 10min, and 1. Mu.L of ligation product was added separately and gently mixed. The competent cell mixture was slowly transferred to a cooled 0.1cm electric cuvette, transformed at 1.8kV, and immediately resuspended in 1mL of 37℃incubation SOC medium, and shaken at 37℃for 45min at 200 rpm. mu.L of the culture was diluted 10-fold in ase:Sub>A gradient, and the dilutions of each gradient were plated on LB-A plates, incubated overnight at 37℃and the colony count of the plates and the size of the gene library were calculated.
Identification of nanobody gene library: 10 individual clones were randomly picked from each of the 3 libraries, and the positive insertion rate of the constructed library was 100% by colony PCR detection, and the colony PCR results of each library are shown in FIG. 4. Meanwhile, 30 monoclonal sequencing is randomly selected, and correctness and diversity of CDRs are detected, wherein the result is shown in figure 5, and the sequence analysis of the monoclonal is randomly selected, wherein the sequence of an FR1 region is shown as SEQ ID NO.1, the sequence of an FR2 region is shown as SEQ ID NO.2, the sequence of an FR3 region is shown as SEQ ID NO.3, the sequence of an FR4 region is shown as SEQ ID NO.4, and the specific sequences are shown in the following table 5; the number of the amino acids of the CDRs is consistent with the design, 1 termination codon except TAG is contained, and the other 29 monoclone has different DNA sequences of the CDRs and codes different amino acid sequences, which shows that the diversity is good.
TABLE 5 FR1-4 region sequence listing
The quality evaluation table of the total synthesis library of the nanobody is shown in the following table 6, 3 CDR regions are completely randomized by selecting a nanobody conservative framework cabBCII10, 3 length CDR3 amino acid randomization numbers are designed, 3 total synthesis nanobody libraries are constructed, and the library capacity of the three total synthesis nanobody libraries can reach 1.6x10 8 The VHH gene insertion rate is 100%, and the fully synthetic nanobody library constructed by the method is more beneficial to screening various analytes.
TABLE 6 evaluation of quality of nanobody total synthetic library
The invention designs 3 length amino acid randomization numbers for CDR3 regions, then carries out NNK randomization for all 3 CDR regions, builds a fully-synthesized nano antibody library with more abundant diversity, and increases the opportunity of screening more antigen-specific nano antibodies. By the method, only one region of CDR3 is randomized, and the sequence diversity of the constructed nano-synthetic library is lower than that of a nano-antibody total-synthetic library which is constructed by all randomization of 3 CDR regions.
The invention randomizes three CDR regions when constructing a nanometer synthetic library, and needs to carry out repeated overlap extension PCR, and two short segments containing NNK randomization need to be subjected to overlap extension PCR to obtain a complete VHH segment, and the randomization of the three regions is complicated and more difficult when carrying out repeated overlap extension PCR. Therefore, the total synthesis library of the nano-antibodies can be used as an antibody discovery platform widely applied to screen specific nano-antibodies, and the problem that the corresponding immune library cannot be constructed to obtain the required nano-antibodies due to the fact that the immune is unavailable or the immune fails due to antigen factors is solved.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (6)
1. The construction method of the total synthesis library of the nano antibody is characterized by comprising the following steps of:
s1, selecting a nanobody conserved framework cAbBCII10 as a framework, wherein the nanobody conserved framework cAbBCII10 comprises an FR1 region with an amino acid sequence shown as SEQ ID NO.1, an FR2 region with an amino acid sequence shown as SEQ ID NO.2, an FR3 region with an amino acid sequence shown as SEQ ID NO.3 and an FR4 region with an amino acid sequence shown as SEQ ID NO.4, and determining the amino acid length of CDRs region by statistically analyzing the reported nanobody sequences;
s2, determining the randomized numbers of the amino acids in the CDR1 region and the CDR2 region according to the use frequency of the amino acids in the CDRs, designing the randomized numbers of the amino acids with 3 lengths in the CDR3 region, and carrying out NNK randomization on all 3 CDR regions, wherein the randomized numbers of the amino acids in the CDR1 region are determined to be 7, the randomized numbers of the amino acids in the CDR2 region are determined to be 6, and the randomized numbers of the amino acids in the CDR3 region are respectively determined to be 12, 15 and 19;
s3, obtaining a complete DNA fragment by utilizing overlap extension PCR, cloning the complete DNA fragment to a vector, and converting the complete DNA fragment to escherichia coli to construct a total synthesis library of the nano antibody.
2. The method of construction according to claim 1, wherein the method of NNK randomization mutation in step S2 comprises the steps of:
1) Designing a primer group containing mutant bases, enabling the primer group to be used as templates for replication to obtain DNA fragments with randomized CDR1, CDR2 and CDR3 amino acids, and then obtaining complete DNA fragments through repeated overlapping extension PCR;
2) Cutting and digesting the complete DNA fragment in the step 1), connecting the DNA fragment into corresponding vectors, and sequencing;
3) Transforming the product of step 2) into E.coli cells.
3. The method of claim 1, wherein the vector used in step S3 is pComb3xss.
4. The construction method according to claim 2, wherein the cleavage reaction system in step 2) is as follows: mu.L of 10 XBuffer, 2. Mu.L of SfiI enzyme, 1. Mu.g of VHH or pComb3xss plasmid, 21. Mu.L of RNase-free deionized water, and a total volume of 50. Mu.L; and reacted overnight in a 50 ℃ water bath.
5. The method of claim 1, wherein E.coli TG1 competent cells are used in step S3.
6. A nanobody total synthesis library, which is obtained by the construction method according to any one of claims 1 to 5, wherein the library capacity of the nanobody total synthesis library is not less than 1.0X10 8 The total synthesis library of the nano antibody is CDRs-12AA, CDRs-15AA and CDRs-19AA.
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