CN111139264B - Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules - Google Patents
Method for constructing single-domain antibody library in mammalian cell line based on linear double-stranded DNA molecules Download PDFInfo
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Abstract
The invention provides a method for constructing a single-domain antibody library in a mammalian cell line based on linear double-stranded DNA molecules, which comprises the following steps: the method comprises the following steps: preparing upstream and downstream linear double-stranded DNA containing nucleotide sequences encoding random amino acid sequences of Complementarity Determining Regions (CDRs) of a single domain antibody; step two: and (3) transfecting the upstream and downstream linear double-stranded DNA obtained in the step one into a mammalian cell line, and connecting the upstream and downstream linear double-stranded DNA to generate full-length linear double-stranded DNA capable of generating the single-domain antibody. The invention develops a new method for establishing a single-domain antibody library in mammalian cells, and the complexity of the generated single-domain antibody library comes from 1) introducing random amino acid coding sequences into upper and lower linear double-stranded DNA in a PCR process; 2) random ligation reaction between the upstream and downstream linear double-stranded DNA occurs.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to construction of an antibody library, and particularly relates to a construction method of a single-domain antibody library.
Background
Antibodies (antibodies), also known as immunoglobulins (igs), are large proteins secreted mainly by plasma cells and used by the immune system to identify and neutralize foreign substances such as pathogens like bacteria, viruses, etc., and are found only in body fluids such as blood of vertebrates, and on the cell membrane surface of B cells thereof. Due to their ability to specifically recognize and bind to antigens, antibodies have been developed and used in the fields of disease diagnosis and treatment, molecular imaging, molecular biology, and the like.
Single-domain antibodies (sdabs), also known as nanobodies (nbs), are natural antibodies that are abundant in the serum of camelids and sharks. Unlike conventional antibodies, single domain antibodies have no light chain and recognition and binding of antigen can be accomplished by the heavy chain (heavy chain) alone. Single domain antibodies comprise a heavy chain variable region (VHH) and two constant regions (CH2, CH 3). The heavy chain variable region (VHH) is a domain that binds to an antigen, and the structure analysis result shows that the VHH has a radius of only 2.4 nm and a height of 4 nm, and is also called nanobody (nanobody). VHH has a molecular weight of only 12-15kDa and is the smallest unit known to bind antigen. The heavy chain variable region (VHH) contains three Complementary Determining Regions (CDRs), which are regions that directly bind to an antigen and determine the recognition specificity and binding ability of an antibody. The CDR amino acid sequences of different single domain antibodies differ, which together constitute the diversity of single domain antibodies.
Compared with conventional antibodies, single domain antibodies have the following advantages: 1) the immunogenicity is weak; 2) the solubility is high, and the tolerance is strong; 3) the tissue permeability is strong; 4) high expression efficiency and simple purification; 5) the transformation is easy. Currently, single domain antibodies against specific antigens are mainly obtained by screening using high-throughput technologies such as phage display (phage display), yeast display (yeast display), and the like. These high throughput techniques are based on the physical binding properties between the displayed single domain antibodies and the antigen, and are selected from a large pool of candidate random single domain antibodies, which have high affinity but are not capable of post-translational modification (e.g., glycosylation modification) of the displayed antibodies. At the same time, these high throughput techniques have not been achieved in mammalian cell lines. This greatly limits the application range, especially for screening of single domain antibodies targeting transmembrane proteins such as G protein-coupled receptor (GPCR), ion channel (ion channel), tyrosine kinase Receptor (RTK). Thus, in order to facilitate the establishment of a screening system for single domain antibodies in mammalian cells, there is a need to establish a method for constructing a library of single domain antibodies in mammalian cell lines.
To achieve transmembrane protein display screening, the Lerner topic group of the Scripps research institute in 2014 developed an autocrine-based signaling system in mammalian cell lines. This technique displays candidate antibodies on the surface of mammalian cells via the platelet derived growth factor receptor transmembrane domain (PDGFR-TM), binds to a functional reporter system for transmembrane proteins of interest, and screens transmembrane protein-targeting antibody agonists using flow cytometry and high throughput sequencing.
The autocrine signaling system realizes the antibody display and function screening in the mammalian cells for the first time: on the one hand, the natural state is maintained to the maximum extent by overexpression of the target transmembrane protein, rather than immobilized recombinant protein, in mammalian cells; on the other hand, function-based screening is achieved with a reporting system. However, compared to phage display technology, this system has not been able to achieve screening of high abundance antibody libraries, mainly because: the abundance of the CDR region of the antibody library in the system is obtained from a PCR product containing NNK degenerate codons (degenerate codons), and the PCR product is constructed, transformed and extracted by plasmids; plasmid transfection and slow virus library packaging; after a series of operations such as infection of mammalian cells, the abundance of the antibody library finally displayed on the cell surface is greatly reduced (the abundance is lost in the above steps). The abundance of the antibody library is a key factor for screening effective candidate antibodies. Therefore, after Lerner establishes the antibody display screening technology of mammalian cell lines, whether to establish a complex antibody library in mammalian cells is the key of current antibody drug development.
The invention provides a plasmid-independent high-abundance single-domain antibody library construction strategy in mammalian cells.
Disclosure of Invention
The invention provides a method for constructing a single-domain antibody library based on linear double-stranded DNA molecules, which comprises the following steps:
the method comprises the following steps: preparing linear double-stranded DNA comprising a nucleotide sequence encoding a random amino acid sequence of a Complementarity Determining Region (CDR) of a single domain antibody;
step two: transfecting the linear double-stranded DNA obtained in the first step into a mammalian cell. Preferred are HEK293T cells, CHO cells, HeLa cells, HepG2 cells, A549 cells, NIH-3T3 cells and the like.
The method for constructing the single domain antibody library provided by the invention is beneficial to realizing the establishment of a screening system of the single domain antibody in the mammalian cell, is beneficial to the posttranslational modification (such as O-linked glycosylation, N-linked glycosylation, disulfide bond formation, tyrosine sulfation and the like) of the single domain antibody in the mammalian cell, and is beneficial to the screening of the single domain antibody aiming at transmembrane proteins such as a target G protein-coupled receptor (GPCR), an ion channel (ion channel), a tyrosine kinase Receptor (RTK) and the like.
Preferably, in the first step, the linear double-stranded DNA containing a nucleotide sequence encoding a random amino acid sequence of a single domain antibody Complementarity Determining Region (CDR) comprises at least one of a random sequence of a single domain antibody complementarity determining region 1(CDR1), a random sequence of a single domain antibody complementarity determining region 2(CDR2), or a random sequence of a single domain antibody complementarity determining region 3(CDR 3). Preferably, the linear double-stranded DNA comprising a nucleotide sequence encoding a random amino acid sequence of a Complementarity Determining Region (CDR) of a single domain antibody further comprises a promoter and a nucleotide sequence of a poly A signal, so as to ensure smooth translation of the CDR. Preferred promoter sequences are mammalian cell-preferred promoters including the CMV promoter, EF1A promoter, CAG promoter, CBh promoter, and the like. Preferably, the linear double-stranded DNA comprising a nucleotide sequence encoding a random amino acid sequence of a Complementarity Determining Region (CDR) of a single domain antibody further comprises a single domain antibody framework region to ensure display of the CDR.
Preferably, in any of the above, in the first step, the number of linear double-stranded DNAs containing a nucleotide sequence encoding a random amino acid sequence of a Complementarity Determining Region (CDR) of the single domain antibody is at least 2. In step two, the 2 linear double-stranded DNAs containing nucleotide sequences encoding random amino acid sequences of Complementarity Determining Regions (CDRs) of the single domain antibody obtained are simultaneously transfected into mammalian cells. 2 pieces of the linear double-stranded DNA comprising a nucleotide sequence encoding a random amino acid sequence of a Complementarity Determining Region (CDR) of a single domain antibody are ligated in a mammalian cell. It is preferred in any of the above that at least one of the random sequence of CDR1, the random sequence of CDR2 or the random sequence of CDR3 is introduced into the linear double-stranded DNA containing a nucleotide sequence encoding a random amino acid sequence of Complementarity Determining Regions (CDRs) of a single domain antibody by a method of PCR. In a preferred embodiment of the present invention, the linear double-stranded DNA containing the nucleotide sequence encoding the single domain antibody Complementarity Determining Region (CDR) random amino acid sequence is introduced by PCR using three segments of CDR1 random sequence, CDR2 random sequence and CDR3 random sequence, and the introduction of two segments of linear double-stranded DNA is more convenient and accurate, i.e., one segment of the obtained two segments of linear double-stranded DNA contains one CDR random sequence and the other segment contains two CDR random sequences, the two segments of sequences are transfected simultaneously, the two segments of sequences are ligated in mammalian cells, and in vitro ligation can generate a highly abundant antibody library, and the medium in this process is plasmid, and is suitable for various display technologies (display technologies) based on bacteria, yeast, test tubes (test tubes), etc. But cannot be used in mammalian cell systems or produce severely reduced abundance. Constructing, transforming and extracting plasmids; plasmid transfection and slow virus library packaging; after a series of operations such as infection of mammalian cells, the abundance of the antibody library finally displayed on the cell surface is greatly reduced (the abundance is lost in the above steps). The invention overcomes the defects of the prior art and provides a plasmid-independent high-abundance single-domain antibody library construction strategy in mammalian cells.
It is preferred in any of the above that the linear double-stranded DNA of the nucleotide sequence encoding a single domain antibody Complementarity Determining Region (CDR) random amino acid sequence comprises two stretches of an upstream product and a downstream product, and at least one of the CDR1 random sequence, the CDR2 random sequence or the CDR3 random sequence is distributed on the upstream product and/or the downstream product. Preferably, the upstream product comprises a CDR1 random sequence and a CDR2 random sequence, and the downstream product comprises a CDR3 random sequence. Preferably, the upstream product (i.e., upstream linear double stranded DNA) further comprises a promoter sequence. Preferably, the downstream product (i.e., downstream linear double stranded DNA) further comprises a poly a signal.
Preferably in any of the above, the upstream and downstream products are transfected into the cell as in step two, linked as an intact product expressing a single domain antibody. The ligation of two sequences is done in mammalian cells and in vitro ligation can generate a highly abundant antibody library, and the medium of this process is plasmid, which is suitable for various display technologies based on bacteria, yeast, test tube (test tube), etc. But cannot be used in mammalian cell systems or produce severely reduced abundance. Constructing, transforming and extracting plasmids; plasmid transfection and slow virus library packaging; after a series of operations such as infection of mammalian cells, the abundance of the antibody library finally displayed on the cell surface is greatly reduced (the abundance is lost in the above steps). The invention overcomes the defects of the prior art and provides a plasmid-independent high-abundance single-domain antibody library construction strategy in mammalian cells.
It is preferred in any of the above that the random sequence of CDR1 and/or the random sequence of CDR2 introduced into the primer sequence of linear double-stranded DNA comprises the random sequence of CDR of (mnn)xWherein n represents T, C, A, G one of four bases in a ratio of 1: 1: 1: 1; m represents C, A one of two bases, the ratio is 1: 1; x represents the number of mnn codons in the primer, and x is more than or equal to 2. (in fact mnn is the reverse complement of nnk, i.e. nnk on the antisense strand.) x preferably takes on the value 6, 7, 8 or 9, 10. It is further preferred that the primer sequences for introducing the random sequence of CDR1 and/or the random sequence of CDR2 into linear double-stranded DNA further comprise a single domain antibody framework region (non-variable region) to ensure CDR display of subsequent products.
Any of the above is preferred, wherein the random sequence of CDR1 and the random sequence of CDR2 are introduced into the upstream product by PCR using the primer sequences shown in Seq ID NO: 2. The primer sequences shown in Seq ID No. 2 contain a single domain antibody framework region (non-variable region), a random sequence encoding CDR2, a random sequence encoding CDR1, and a sequence complementary to the PCR template.
Any of the preferred embodiments above for introducing the random sequence of CDR1 and/or the random sequence of CDR2 into a primer sequence of linear double stranded DNA further comprises introducing the random sequence of CDR1 and the random sequence of CDR2 into the upstream product by PCR with a primer sequence as set forth in Seq ID No. 13. The primer sequence shown in Seq ID No. 13 comprises a single domain antibody framework region (non-variable region), a random sequence encoding CDR2, a random sequence encoding CDR1 and a sequence complementary to the PCR template, wherein the single domain antibody framework region (non-variable region) is another commonly used single domain antibody framework region, and the number of mnn sequences of CDR1 and/or CDR2 is different from that of Seq ID No. 2.
It is preferred in any of the above that the PCR primer sequence for introducing the random sequence of CDR3 into linear double-stranded DNA comprises a random sequence of CDR (nnk)yWherein n represents T, C, A, G one of four bases in a ratio of 1: 1: 1: 1; k represents T, G one of two bases in a ratio of 1: 1; y represents the number of nnk codons in the primer, and y is more than or equal to 2. Preferred values for Y are 7, 8, 9, 12, 15, 18, 20. It is further preferred that the PCR primer sequences for introducing the random sequence of CDR3 into linear double stranded DNA also include a single domain antibody framework region (non-variable region) to ensure CDR display of subsequent products. Any of the above is preferred, wherein the random sequence of CDR3 is introduced into the downstream product by PCR using the primer sequence shown in Seq ID NO 3. The primer sequence shown in Seq ID No. 3 contains a single domain antibody framework region (non-variable region), a random sequence encoding CDR3 and a sequence complementary to the PCR template.
Preferably, in any of the above steps, in the first step, an upstream PCR product (the upstream PCR product in the present invention) comprising the CMV promoter, the single domain antibody complementarity determining region 1(CDR1), and the single domain antibody complementarity determining region 2(CDR2) is obtained by PCR reaction; and a downstream PCR product containing a single domain antibody complementarity determining region 3(CDR3), poly A signal (the downstream PCR product in the present invention is the downstream product); the upstream PCR product and the downstream PCR product are the linear double-stranded DNA molecules; purifying the upstream PCR product and the downstream PCR product; and step two, simultaneously transfecting the upstream PCR product and the purified product of the downstream PCR product into a mammalian cell, collecting the transfected cell after culture, extracting RNA and performing reverse transcription to obtain cDNA, performing PCR amplification on the single-domain antibody complementarity determining regions 1-3(CDR1-3), and performing High-throughput sequencing (HTS). The introduction of the CMV promoter and the poly A signal ensures the smooth progress of the transcription and translation process of the intracellular single domain antibody. The CDR segments are introduced into the linear double-chain DNA, so that the experimental method is simpler and more convenient. The two fragments were introduced into mammalian cells, which spontaneously triggered ligation of the two fragments (Li S, Su W, Zhang C. Linear double-stranded DNAs as innovative biological reagents in mammalian cells FEBS J.2019.doi: 10.1111/fess.14816.). Preferably, the CDR1 random sequence and the CDR2 random sequence are introduced into the upstream PCR product in step one using upstream PCR product 3' primers; the CDR3 random sequence was introduced into the downstream PCR product using the downstream PCR product 5' primer. Further preferably, the 3' primer of the upstream PCR product is a primer sequence shown in Seq ID NO. 2, and the random sequence of CDR1 and the random sequence of CDR2 are introduced into the upstream PCR product by using the primer sequence shown in Seq ID NO. 2; the 5' primer of the downstream PCR product is a primer sequence shown by Seq ID NO. 3, and the random sequence of the CDR3 is introduced into the downstream PCR product by using the primer sequence shown by Seq ID NO. 3.
Preferably in any of the above, the template of the PCR reaction comprises the nucleotide fragment of a) or b) as follows:
a) comprises the nucleotide sequence shown as Seq ID NO 7 and/or Seq ID NO 8;
b) the amino acid sequence obtained by the substitution and/or deletion and/or addition of one or more bases on Seq ID NO 7 and/or the substitution and/or deletion and/or addition of one or more bases on Seq ID NO 8 after the translation of the whole sequence comprises the random amino acid sequence of CDR1, CDR2 and CDR3 and the amino acid sequence of the framework region (conserved region) of the single domain antibody; a nucleotide sequence derived from a). The number of the templates may be one or more.
Any one of the above preferred templates for the PCR reaction comprise the nucleotide sequence shown in Seq ID No. 7 and/or Seq ID No. 8, and the number of templates is at least 2.
Any of the above is preferred, wherein the number of templates is 2, and the upstream template and the downstream template are the same, wherein the nucleotide sequence shown by Seq ID No. 7 is contained in the upstream template and the nucleotide sequence shown by Seq ID No. 8 is contained in the downstream template.
In any of the above, preferred embodiments regarding the template further include a derivative sequence obtained by substitution and/or deletion and/or addition of one or more bases to Seq ID NO 7, for example, a nucleotide sequence shown in Seq ID NO 11; and/or a derivative sequence obtained by substituting and/or deleting and/or adding one or more bases to Seq ID NO 8, for example, a nucleotide sequence shown in Seq ID NO 12.
The beneficial effect of the plurality of templates is that the template DNA pollution caused by the transfection of the residues of the template DNA into mammalian cells in the purification process of PCR products in the subsequent library building process can be effectively avoided. The complexity of the single domain antibody libraries generated by the present invention comes from 1) the introduction of random amino acid coding sequences into the upstream and downstream linear double stranded DNA (i.e., the upstream and downstream products) during the PCR process; 2) random ligation reaction between the upstream and downstream linear double-stranded DNA (i.e.the upstream and downstream products).
Thus, the present invention provides a method for the production of a single domain antibody library based on linear double stranded DNA molecules for mammalian intracellular ligation. The object of the present invention is to first introduce random nucleic acid sequences corresponding to CDR 1(CDR1) and CDR 2(CDR2) into an upstream PCR product by PCR reaction; random nucleic acid sequences corresponding to the complementarity determining region 3(CDR3) were introduced into the downstream PCR products by PCR reactions. Then the upstream and downstream PCR products are transfected into the mammalian cells simultaneously, and the complete PCR product capable of expressing the single-domain antibody is generated through the connection reaction between the PCR products in the cells. The present invention adds greater complexity by randomly joining together the random nucleic acid sequences corresponding to CDRs 1, 2 and CDR 3.
Drawings
FIG. 1 is a schematic diagram of the intracellular linkage of linear double-stranded DNA in the preferred embodiment 1 of the present invention
FIG. 2 is a schematic diagram of the 3' primer of the upstream PCR product in the preferred embodiment 1 of the present invention
FIG. 3 is an electrophoretogram of an upstream PCR product and a downstream PCR product in preferred embodiment 1 of the present invention
FIG. 4 is a schematic diagram of a high throughput sequencing process in preferred embodiment 1 of the present invention
FIG. 5 is a diagram showing the nucleotide sequence base distribution analysis of the single domain antibody library obtained in preferred embodiment 1 of the present invention
Detailed Description
The present invention is further described with reference to specific examples, which enable one skilled in the art to practice the invention with reference to the description. However, the present invention is not limited to the following examples. In the following examples, unless otherwise specified, all methods are conventional.
The present invention utilizes linear double-stranded DNA as a molecular element, introduces a random nucleic acid sequence into a PCR product through a PCR reaction to form a random nucleic acid sequence of Complementary Determining Regions (CDRs), then randomly combines different complementary determining regions through a ligation reaction in a cell by transfecting upstream and downstream PCR products into a mammalian cell, as shown in FIG. 1, and simultaneously forms a complete single domain antibody expression PCR product. Thereby creating a single domain antibody library within the mammalian cell.
KOD DNA polymerase was purchased from Toyobo Co; RNAioso Plus total RNA extraction reagent, PrimeScriptTMRT reagent Kit with gDNA Eraser reverse transcription Kit purchased from Takara company; agarose gel (Agarose) was purchased from bio-engineering (shanghai) gmbh; the DNA purification and recovery kit is purchased from Tianzhu Biochemical technology (Beijing) Co., Ltd; lipo2000 transfection reagent was purchased from Thermo Fisher;
the DNA PCR-Free Sample Preparation Kit is purchased from Illumina, HEK293T cells are purchased from Shanghai cell bank of Chinese academy, and plasmids are routinely used in pUC57 molecular biology, and commercial products can be directly purchased. The reagents used in the present invention are commercially available products unless otherwise specified. Unless otherwise specified, the methods are described in the literature and in the reference books on molecular cloning.
Example 1
1. Preparation of plasmid template
A928 bp double-stranded DNA sequence as shown in Seq ID NO:7 was synthesized and constructed (by conventional AT cloning method) into a plasmid pUC 57. The "pUC 57-upstream template" plasmid was obtained. The plasmid sequence of "pUC 57-upstream template" is shown in Seq ID NO: 9.
A637 bp double-stranded DNA sequence shown in Seq ID NO:8 was synthesized and constructed (by the conventional AT cloning method) into pUC57 plasmid. A "pUC 57-downstream template" plasmid was obtained. The sequence of the "pUC 57-downstream template" plasmid is shown in Seq ID NO: 10.
2. PCR amplification yielded an upstream PCR product containing the CMV promoter, complementarity determining region 1(CDR1), and complementarity determining region 2(CDR2) sequences.
The "pUC 57-upstream template" plasmid was used as a template, and the PCR template sequence was located at 417-1344 sites of the plasmid. An upstream PCR product comprising the CMV promoter, complementarity determining region 1(CDR1), complementarity determining region 2(CDR2) sequences was obtained by PCR amplification using KOD DNA polymerase.
The PCR reaction conditions are as follows:
preheating: 94 ℃ for 2 min;
and (3) cyclic reaction: 94 ℃, 30 sec; 55 ℃ for 30 sec; at 68 ℃ for 2 min; the above process is carried out for 35 cycles;
extension: 68 ℃ for 10 min.
The primer sequences are nucleotide sequences shown as Seq ID NO 1 and Seq ID NO 2. The template sequence is shown in Seq ID NO: 7. FIG. 2 is a schematic diagram showing the sequences of the primers shown in Seq ID No. 2, in which region 1 is a single domain antibody framework region (non-variable region), region 2 is a sequence encoding CDR2 (single domain antibody CDR2 random sequence), region 3 is a single domain antibody framework region (non-variable region), region 4 is a sequence encoding CDR1 (single domain antibody CDR1 random sequence), and region 5 is a sequence complementary to the template. Region 5 is responsible for binding to the template and regions 1 to 4 serve as templates for generating random CDR1 and CDR2 sequences. The primer sequences shown in Seq ID NO:2 were designed and synthesized by GeneWiz corporation.
3. The downstream PCR product containing the complementarity determining region 3(CDR3) and poly A signal was obtained by PCR amplification.
Using the "pUC 57-downstream template" plasmid as a template, the PCR template sequence was located at the 417-1053 site of the plasmid. A downstream PCR product containing the complementarity determining region 3(CDR3), poly A signal sequence was obtained by PCR amplification using KOD DNA polymerase.
The PCR reaction conditions are as follows:
preheating: 94 ℃ for 2 min;
and (3) cyclic reaction: 94 ℃, 30 sec; 55 ℃ for 30 sec; at 68 ℃ for 2 min; the above process is carried out for 35 cycles;
extension: 68 ℃ for 10 min.
The primer sequences are nucleotide sequences shown as Seq ID NO 3 and Seq ID NO 4. The template sequence is shown in Seq ID NO: 8. The sequence shown in Seq ID No. 3 is the downstream product 5' primer, which includes a binding region complementary to the template sequence, a sequence encoding CDR3 (random sequence of single domain antibody CDR3), and a single domain antibody framework region (non-variable region). The sequence shown in Seq ID NO:3 was designed and synthesized by GeneWiz corporation.
4. And (5) electrophoresis and recovery of PCR products.
The PCR product was electrophoresed on 1% agarose gel, and as shown in FIG. 3, the excised target gel was subjected to electrophoresis, and the PCR product was recovered using a DNA purification recovery kit. The method comprises the following specific steps: adding equal volume of sol solution PC into the rubber block, and placing in water bath at 50 ℃ for 10min to dissolve the rubber block; centrifuging at 12000rpm for 1min to allow the above solution to pass through adsorption column CB2, and discarding the liquid in the collection tube; adding 600 mul of rinsing liquid PW into an adsorption column CB2, centrifuging at 12000rpm for 1min, discarding the liquid in a collecting pipe, and repeatedly rinsing once; centrifuging at 12000rpm for 2min to remove residual rinsing solution on the adsorption column; placing adsorption column CB2 into a new 1.5ml EP tube, adding 50 μ l sterile water to dissolve the PCR product, centrifuging at 12000rpm for 2min, and collecting the PCR product; the concentration of the PCR product was determined and stored at-20 ℃ until use.
5. Transfection of HEK293T cells.
Spreading HEK293T cells in good growth state in a 6-well plate, and preferably ensuring that the cell density of the subcultured cells reaches 60-80% the next day; and co-transferring the amplified upstream and downstream linear double-stranded DNA into cells. The transfection procedure was as follows: transfection system for 6-well plate cells (500. mu.L/well): 2 sterile 1.5mL centrifuge tubes were filled with 250. mu.L of serum-free medium. Adding a transfection reagent Lipo 20004 mu L into one tube; the other tube was simultaneously filled with 500ng of upstream linear double-stranded DNA and 500ng of downstream linear double-stranded DNA, and each tube was mixed well. Finally, combining the two tubes into one tube, fully blowing, uniformly mixing, and standing for 20min at room temperature. Then, the liquid in the centrifuge tube was added dropwise to the cells and mixed well. The cells were incubated at 37 ℃ with 5% CO2The cultivation was continued in the incubator for 48 h.
6. Total RNA extraction
After pouring out the cell culture medium, the cells were washed twice with PBS, and after the remaining PBS was aspirated, 1ml of RNAioso Plus was added to each well. 1) Homogenizing and phase-splitting: 1ml of the cell lysed RNAasso Plus was transferred to a RNase free 1.5ml centrifuge tube and allowed to stand at room temperature for 5 min. Adding 200 μ l of chloroform, shaking vigorously, standing at room temperature for 3min, and centrifuging at 4 deg.C at 12000rpm × 15 min. 2) RNA precipitation: the centrifuged liquid was divided into three layers, the uppermost layer of liquid was aspirated (the tip of the pipette did not touch the other layers), transferred to a new EP tube, added with equal amount of isopropanol, left at room temperature for 10min, and centrifuged at 12000rpm × 10min at 4 ℃. 3) Washing and precipitating: the supernatant was discarded (care was taken not to discard the white precipitate), 1ml of 75% ethanol was added and inverted twice, and centrifuged at 7500rpm X5 min at 4 ℃. The supernatant was discarded, dried in air, dissolved in a suitable amount of RNase-free water, and stored at-70 ℃ for half a year (stable).
7. cDNA Synthesis
cDNA Synthesis from PrimeScriptTMRT reagent Kit with gDNA Eraser reverse transcription Kit. The method comprises the following steps: 1) removing the genomic DNA reaction. The system is as follows (10. mu.l system): 5 XgDNA Eraser Buffer 2.0. mu.l; gDNA Eraser 1.0. mu.l; total RNA 1. mu.g; RNase Free dH2O to 10. mu.l. 42 ℃ for 2 minutes. 2) And (5) reverse transcription reaction. The new 10. mu.l system was formulated as follows: PrimeScript RT Enzyme Mix I1.0. mu.l; RT Primer Mix 1.0. mu.l; 5 XPrimeScript Buffer 24.0. mu.l; RNase Free dH2O4.0. mu.l. Adding the newly prepared 10 mul system into the 10 mul system in the 1) and mixing evenly. The cDNA was obtained at 37 ℃ for 15 minutes and 85 ℃ for 5 seconds. The cDNA was stored at-20 ℃.
8. PCR amplification of complementarity determining regions 1-3(CDR1-3)
The above-mentioned cDNA was used as a template, and the complementarity determining regions 1 to 3 on the cDNA were amplified by PCR using KOD DNA polymerase.
The PCR reaction conditions are as follows:
preheating: 94 ℃ for 2 min;
and (3) cyclic reaction: 94 ℃, 30 sec; 55 ℃ for 30 sec; at 68 ℃ for 1 min; the above process is carried out for 35 cycles;
extension: 68 ℃ for 10 min.
The PCR product was electrophoresed and recovered in the same manner as in step 3. The sequencing primer sequences are nucleotide sequences shown as Seq ID NO 5 and Seq ID NO 6.
9. High throughput sequencing of PCR products
Application of
The DNA PCR-Free Sample Preparation Kit was used to pool the PCR products obtained in the previous step and perform PE250 sequencing on a NovaSeq6000 sequencer.
10. High throughput sequencing data processing
Splitting each sample data from sequencing data according to a Barcode sequence and a PCR amplification primer sequence, cutting off the Barcode and the primer sequence, and splicing reads of each sample by using FLASH to obtain a spliced sequence which is original Tags data (Raw Tags); the Raw Tags obtained by splicing need to be filtered to obtain high-quality tag data (Clean Tags). Single domain antibody complementarity determining regions 1-3(CDR1-3) nucleic acid sequences were extracted using the Perl program, as shown in FIG. 4.
11. Results
The results of amino acid sequence analysis show that the method of the present invention can generate random amino acid sequences in the complementarity determining regions 1-3 of a single domain antibody. Thus, the present invention successfully establishes a highly complex single domain antibody library in mammalian cells.
By analyzing the nucleic acid sequences, a total of 22172 different single domain antibody sequences were obtained in this experiment. The nucleic acid sequence corresponding to CDR1 in these sequences was analyzed in combination and found to contain 8 consecutive codons and to substantially conform to the NNK degenerate base distribution law (as shown in FIG. 5, A). Theoretically, the NNK degenerate base distribution rule is that N represents 25% of the probability of A, T, C, G occurring at four bases, and K represents 50% of the probability of T, G occurring at two bases. Similarly, the nucleic acid sequences corresponding to CDR2 and CDR3 of these sequences contain 9 and 7 consecutive codons, respectively, and both conform substantially to the NNK degenerate base distribution law (fig. 5B, C). This indicates that the high throughput sequencing results remain consistent with the experimental design, namely: CDR1, CDR2, CDR3 encode 8, 9, and 7 random NNK amino acid sequences, respectively.
The nucleotide sequences of the CDR1-3 regions of the 22172 kinds of single-domain antibodies were translated into amino acid sequences, and the proportions of the various amino acids were analyzed to find that the theoretical proportions of the amino acids were almost similar to those of NNK-encoded amino acids (Table 1). These results indicate that this protocol can successfully establish a high abundance single domain antibody library in mammalian cells.
The scheme can be flexibly implemented. The number of random amino acids encoded by the CDRs 1, 2 and/or 3 can be varied by designing and synthesizing new primers, i.e., the length of the CDRs can be varied. For example, a longer CDR sequence can be generated by synthesizing primers containing more degenerate bases of NNK, thereby generating a single domain antibody library containing longer CDR regions.
TABLE 1 percentage amino acids encoded by CDR regions of the Single Domain antibody library
With the increase of the experimental scale, more HEK293T cells are transfected, more PCR products are prepared for high-throughput sequencing, and the high-throughput sequencing depth is increased, so that more single-domain antibody sequences are obtained, namely, a more abundant single-domain antibody library is obtained.
Remarking:
in the primers of the invention (and in the random sequences of CDRs 1, 2, 3):
n represents T, C, A, G one of four bases in the ratio of 1: 1: 1: 1; ② k represents T, G one of two bases, the proportion is 1: 1; ③ m represents C, A one of the two bases, the ratio is 1: 1.
Claims (5)
1. a method for constructing a single-domain antibody library based on linear double-stranded DNA molecules comprises the following steps:
the method comprises the following steps: preparing linear double-stranded DNA comprising a nucleotide sequence encoding a random amino acid sequence of a Complementarity Determining Region (CDR) of a single domain antibody, the number of the linear double-stranded DNA encoding a nucleotide sequence of a random amino acid sequence of a Complementarity Determining Region (CDR) of a single domain antibody being at least 2; the linear double-stranded DNA containing a nucleotide sequence encoding a single domain antibody Complementarity Determining Region (CDR) random amino acid sequence comprises at least one of a single domain antibody complementarity determining region 1(CDR1) random sequence, a single domain antibody complementarity determining region 2(CDR2) random sequence, or a single domain antibody complementarity determining region 3(CDR3) random sequence; the random sequence of CDR1 and/or the random sequence of CDRs comprised by the random sequence of CDR2 is (mnn)xWherein n represents T, C, A, G one of four bases in a ratio of 1: 1: 1: 1; m represents C, A one of two bases, the ratio is 1: 1; x represents the number of mnn codons in the primer, and x is more than or equal to 2; CDR3 random sequence the CDR random sequence contained is (nnk)yWherein n represents T,C. A, G at a ratio of 1: 1: 1: 1; k represents T, G one of two bases in a ratio of 1: 1; y represents the number of nnk codons in the primer, and y is more than or equal to 2;
step two: transfecting the linear double-stranded DNA obtained in the first step into a mammalian cell, wherein the at least 2 linear double-stranded DNAs encoding nucleotide sequences of the random amino acid sequences of the Complementarity Determining Regions (CDRs) of the single domain antibody are linked in the mammalian cell to form a complete product expressing the single domain antibody;
introducing by means of PCR at least one of the CDR1 random sequence, the CDR2 random sequence or the CDR3 random sequence into the linear double-stranded DNA comprising a nucleotide sequence encoding a single domain antibody Complementarity Determining Region (CDR) random amino acid sequence; the linear double-stranded DNA of the nucleotide sequence coding for the single domain antibody Complementarity Determining Region (CDR) random amino acid sequence comprises two sequences of an upstream product and a downstream product, wherein the CDR1 random sequence and the CDR2 random sequence are distributed in the upstream product; the CDR3 random sequence is distributed on the downstream product;
the primers for the PCR reaction include random peptides and single domain antibody backbone portions: in the primer sequence corresponding to the upstream product, the random peptide sequence is (mnn)x(ii) said random peptides are contained in said random sequence of CDR1 and said random sequence of CDR2, respectively, said single domain antibody backbone moiety comprises bases 1 to 9 and bases 37 to 87 of the nucleotide sequence set forth in Seq ID No. 2; in the primer sequence corresponding to the downstream product, the random peptide sequence is (nnk)yThe single domain antibody scaffold moiety comprises bases 1 to 102 of the nucleotide sequence set forth in Seq ID No. 3;
the site of linkage between the upstream product and the downstream product in mammalian cells is the 1 st base in the nucleotide sequence shown by Seq ID NO. 2 in the upstream product and the 1 st base in the nucleotide sequence shown by Seq ID NO. 3 in the downstream product.
2. The method of constructing a single domain antibody library of claim 1 wherein the CDR1 random sequence is addedThe primer sequence for introducing the random sequence of CDR2 into the linear double-stranded DNA contains the random sequence of CDR of (mnn)xWherein n represents T, C, A, G one of four bases in a ratio of 1: 1: 1: 1; m represents C, A one of two bases, the ratio is 1: 1; x represents the number of mnn codons in the primer, and x is more than or equal to 2.
3. The method of claim 2, wherein the PCR primer sequence for introducing the random CDR3 sequence into the linear double-stranded DNA comprises a random CDR sequence of (nnk)yWherein n represents T, C, A, G one of four bases in a ratio of 1: 1: 1: 1; k represents T, G one of two bases in a ratio of 1: 1; y represents the number of nnk codons in the primer, and y is more than or equal to 2.
4. The method of constructing a single domain antibody library of any one of claims 1 to 3,
respectively amplifying by PCR reaction to obtain upstream PCR products containing a CMV promoter, a single-domain antibody complementarity determining region 1(CDR1) and a single-domain antibody complementarity determining region 2(CDR 2); and a downstream PCR product containing single domain antibody complementarity determining region 3(CDR3), poly a signal; the upstream PCR product and the downstream PCR product are the linear double-stranded DNA molecules; purifying the upstream PCR product and the downstream PCR product;
and step two, simultaneously transfecting the upstream PCR product and the purified product of the downstream PCR product into a mammalian cell, culturing the transfected cell, collecting the transfected cell for extracting RNA and performing reverse transcription to obtain cDNA, and finally performing PCR amplification on the single-domain antibody complementarity determining regions 1-3(CDR1-3) for deep sequencing.
5. The method for constructing a single domain antibody library according to claim 4, wherein the template for the PCR reaction comprises the nucleotide sequence set forth in Seq ID No. 7 and/or Seq ID No. 8.
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