CN117143859A

CN117143859A - Method for cloning human IgG heavy chain full-length gene by single B lymphocyte

Info

Publication number: CN117143859A
Application number: CN202311128586.5A
Authority: CN
Inventors: 王为民
Original assignee: Yinsha Biotechnology Hangzhou Co ltd
Current assignee: Yinsha Biotechnology Hangzhou Co ltd
Priority date: 2023-09-04
Filing date: 2023-09-04
Publication date: 2023-12-01

Abstract

The invention relates to a method for cloning human IgG heavy chain full-length gene by single B lymphocyte, which utilizes the separated secretory antibody IgG single B lymphocyte to directly amplify the full-length gene sequence of the antibody IgG heavy chain by single cell PCR technology of end-point method by designing specific PCR primer, thus obtaining the human functional IgG heavy chain full-length gene. The defect that only part of the sequence of the variable region gene of the IgG antibody can be obtained by cloning by the current sequencing method and the heavy chain variable region can be expressed after being recombined with the constant region in vitro is completely abandoned.

Description

Method for cloning human IgG heavy chain full-length gene by single B lymphocyte

Technical Field

The invention relates to the technical fields of genetic engineering, antibody engineering, cell engineering and the like, in particular to a method for efficiently cloning human antibody IgG heavy chain full-length genes by single B lymphocyte.

Background

The monoclonal antibody medicine as one kind of biological targeting medicine with unique advantages has the features of high specificity, high targeting performance and low toxic side effect, and may be used widely in clinical treatment.

The basic structure of an antibody molecule is composed of four polypeptide chains. Namely, the antibody heavy chain gene (H) consists of four parts of gene fragments of 65-100 variable regions (V), 2 variable regions (D), 6 binding regions (J) and constant regions (C) and consists of 2 identical H chains and L chains; the light chain (L) consists of three gene segments, the variable region (V), the binding region (J) and the constant region (C). The antibody heavy chain constant region is divided into CH1, CH2, CH3 and CH4 (depending on the type of antibody).

Human antibodies are classified into 5 classes according to the heavy chain constant region differences: igG (γ), igM (μ), igA (α), igD (δ), igE (epsilon). Wherein, igG accounts for 75% -80% of total serum immunoglobulin, and has the immune defense functions of specifically binding antigen, neutralizing toxin, preventing pathogen invasion, eliminating pathogenic microorganisms and the like.

The function of each partition of the immunoglobulin, the VH and VL regions, are responsible for recognizing and binding antigens, determining the specificity of the antibody and thus exerting an immune effect. The CH2 of IgG and the CH3 of IgG are responsible for activating complement, and the CH2/CH3 of IgG is responsible for binding to Fc receptors (FcRs) on the surface of effector cells (most notably NK cells), mediating effector cell killing of target cells (tumor cells, infected cells, pathogenic immune cells, etc.). Since different IgG subtypes have different affinity activities with different FcRs, this results in significantly different IgG subtypes with different functional activities in vitro and in vivo. For example, igG1 can bind to all IgG fcrs except FcRL 4; igG2 can bind to IgG FcRs other than fcyri, fcyriib and FcRL 4; igG3 can bind to all IgG fcrs; igG4 can bind to all IgGFcRs except fcyriiib, and thus the antibody C region sequence also plays an important role in therapy.

The fully human antibody prepared by the antibody genes of B lymphocytes in human peripheral blood has the advantages of strong specificity, high affinity, good safety and the like due to the natural selection of a human immune tolerance mechanism. The method fully utilizes human dominant natural resources (some high-efficiency antibodies cannot be generated in non-human species and can only be derived from human) to prepare the antibodies, so that the problem of immunogenicity of antibody drug application can be solved to the greatest extent, the maximum safety guarantee is provided for patients, research and development and investment risks are greatly reduced, and the patent medicine rate is greatly improved. Therefore, the monoclonal antibody is an important direction for developing monoclonal antibody medicines.

The existing technical schemes for preparing B lymphocyte to prepare human monoclonal antibody are 2, firstly, screening is carried out through phage expression library, namely, after cDNA fragment of coding human antibody gene is fused with coding gene of phage surface protein, the fused protein is presented on the surface of phage, and the displayed polypeptide or protein can keep relative space structure and biological activity and is displayed on the surface of phage. A group of phages into which various exogenous genes have been introduced constitutes a phage display library displaying various exogenous peptides. When an antigen is used for screening a phage display library, the antigen can be selectively combined with a certain exogenous peptide interacted with the antigen, so that a specific phage in the phage display library can be separated, and the sequence and biological function of the exogenous gene contained in the phage are researched and analyzed, so that the gene fragment of the antibody light chain or heavy chain with antigen binding activity can be obtained. As a discovery technique of antibody genes, the antibody IgG genes obtained by this method are often incomplete, eventually leading to a decrease in their affinity to the antigen, and later, it is necessary to further screen high affinity antibodies using antibody affinity maturation techniques, i.e. by constructing a library of mutants.

And secondly, directly obtaining an antibody heavy chain variable region gene by utilizing a B lymphocyte separation screening combined gene sequencing technology, then splicing with a fixed known antibody C region sequence, pairing, and screening an active monoclonal antibody after cell transfection. Because of the limitations of single cell transcriptome sequencing read length (150 bp), it is currently not possible to obtain matched heavy chain full-length genes from single B lymphocytes simultaneously, and therefore later it is necessary to splice antibody variable region gene fragments obtained from multiple single B sequencing by bioinformatics technology, and further splice with the existing fixed C region fragments, and perform activity screening after in vitro recombination. The process breaks the natural structure of the hinge region between the variable region and the constant region of the antibody light and heavy chain, the antibody heavy chain gene is subsequently required to be paired for verifying the activity of the antibody, the affinity of the antibody to the antigen is reduced due to the loss of the C region structure of the natural IgG antibody gene, the exertion of the antibody therapeutic effect is not facilitated, the risk of antibody initiation of antibody production is increased, and the prepared antibody does not belong to a truly human full-structure antibody.

In summary, cloning the full length of the heavy chain gene of IgG antibodies in human single B lymphocytes has not been achieved in the prior art, so that human IgG monoclonal antibodies with all natural structures are recombined in vitro.

RACE, a technique for rapid amplification of cDNA ends (rapid amplification of cDNA ends), is a technique for rapid amplification of the 5 'and 3' ends of cDNA from a sample based on reverse transcription PCR, and was invented by Frohman et al in 1988. Using RACE, the 5 'and 3' ends of the complete cDNA can be obtained by known partial cDNA sequences. The feature of RACE is that when only a single-sided sequence is known for designing a specific primer, the amplification of the full-length sequence of the gene can still be completed by applying the RACE technology, so that the RACE technology is also called single-sided PCR. Specific primers are designed according to known sequences, 5' RACE is utilized to obtain 5' end sequences (gene specific primers→5' end), and finally complete cDNA sequences of genes can be obtained.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for efficiently cloning human IgG heavy chain full-length genes using single B lymphocytes. In order to solve the problems in the prior art, the invention aims to provide a preparation method for efficiently cloning the heavy chain full-length gene of a human IgG antibody by using a single cell 5' RACE technology.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the invention provides a method for cloning human IgG heavy chain full-length gene by single B lymphocyte, which comprises the following steps:

(1) Adding PBS buffer solution without calcium and magnesium into human peripheral blood lymphocytes (Human peripheral blood B lymphocytes) to prepare suspension containing B lymphocytes;

(2) Carrying out first separation on the suspension containing the B lymphocytes in the step (1) by using a magnetic bead separation technology to obtain positive B lymphocyte (IgG secretion) suspension; performing secondary sorting on the positive B lymphocyte suspension by using a microfluidic technology to obtain single B lymphocytes;

(3) Lysing the single B lymphocytes in the step (2) by using a single cell lysate, and reversely transcribing the obtained lysate into cDNA by reverse transcriptase SMARTScribe Reverse Transcriptase, a 5' terminal linker sequence, an oligo dT primer and a random primer; in the reverse transcription process, a linker sequence is added to the 5 'end of RNA by utilizing the template conversion activity of reverse transcriptase SMARTScribe Reverse Transcriptase, so that the 5' end of cDNA generated by reverse transcription of the heavy chain gene of the IgG antibody is connected with a linker sequence which is designed independently.

The 5' terminal linker sequence is as follows:

5′GGACCTGGAGCATCCTTTTrGrG+G 3

wherein rG is a mono-deoxyguanine and +G is a locked nucleotide modified guanine;

(4) Performing PCR amplification by using the cDNA in the step (3) as a template and using a forward primer and a reverse primer to obtain the single B lymphocyte clone human IgG heavy chain full-length gene;

the sequence of the forward primer is as follows:

FP 5′GGACCTGGAGCATCCTTTT 3′

the sequence of the reverse primer is one of the following:

1-AP 5′TCATTTACCCGGAGACAG 3′

2-AP 5′CTCATTTACCCGGAGACA 3′

3-AP 5′CACTCATTTACCCGGAGAC 3′。

the invention provides a simple, rapid and efficient method for cloning the full-length gene sequence of the heavy chain of the human IgG antibody from single B lymphocyte. The invention adopts the method of directly cloning the full-length gene sequence of the heavy chain of the human IgG antibody by adopting an end-point method.

In one embodiment of the invention, in step (1), the human peripheral blood lymphocytes are commercially available or self-prepared. In one embodiment of the invention, the human peripheral blood lymphocytes are purchased from Shanghai enzyme-linked biotechnology Co., ltd., cat: ml098362 (containing different B lymphocytes). In another embodiment of the present invention, in step (1), the human peripheral blood lymphocytes are obtained by subjecting a human peripheral whole blood sample to density gradient separation (monocytes including B lymphocytes) using human lymphocyte separation fluid (Cytiva Situo Co., ficoll-Paque PREMUM, cat# 17544203).

Preferably, in step (1), the cell density of the suspension comprising B lymphocytes is 1X 10 ⁷ And each mL.

In one embodiment of the present invention, in step (2), the first sorting is: coupling the biotinylated anti-human IgG antibody with streptomycin affinity magnetic beads to obtain labeled magnetic beads; incubating the labeled magnetic beads with the suspension containing B lymphocytes in the step (1) at 2-8 ℃, separating the supernatant by a magnetic rack, and suspending the supernatant by a PBS buffer without calcium and magnesium to obtain positive human B lymphocyte suspension (B lymphocyte secreting IgG antibody). Further, the mass ratio of the biotinylated anti-human rabbit IgG antibody to the streptomycin affinity magnetic beads is 20 μg:1 mg; the mass of the streptomycin-affinity beads was 4 μg/mL based on the volume of the suspension containing B lymphocytes described in step (1).

The invention avoids the complicated operation that a plurality of fluorescent markers aiming at the specific antibodies of the human B cell surface antigen are needed when the flow cytometry is used for sorting at present, and obviously improves the sorting efficiency of the human single B lymphocyte.

In one embodiment of the present invention, in step (2), the second sorting is: diluting positive B lymphocyte suspension with PBS buffer without calcium and magnesium to cell concentration of 1×10 ⁴ Per mL-1×10 ⁶ Single cell sorting was performed in single cell mode using a single cell instrument. Sorting by a single cell instrument under the sorting pressure of less than 0.138bar into a 96-well plate pre-filled with single cell lysate, immediately freezing with dry ice, and placing into a refrigerator at-80 ℃ for standby.

In one embodiment of the present invention, in step (3), the reverse transcribed 3' primer is an oligo dT primer with a sequence length of 20. The 5' terminal linker sequence was 5' GGACCTGGAGCATCCTTTTTrGrG+G3 '. In the reverse transcription system, the concentration of the 5' -terminal linker sequence was 1.2uM, the concentration of the oligo dT primer was 5uM, and the concentration of the random primer was 1uM.

Further, in the step (4), the reaction system of the PCR amplification is: the 50. Mu.L system contained 6. Mu.L cDNA solution, 25. Mu.L 2X PrimeSTAR Max DNA polymerase premix, 0.2. Mu.M forward primer and 0.2. Mu.M reverse primer.

Further, in the step (4), the reaction steps of the PCR amplification are as follows:

(1) pre-denaturation at 94 ℃ for 20s;

(2) 5 PCR cycles: first round denaturation at 98℃for 10sec, first round annealing at 70℃for 10sec, first round extension at 72℃for 15sec;

(3) 30 PCR cycles: the second round denaturation at 98℃for 10sec, the second round annealing at 62-68℃for 10sec, and the second round extension at 72℃for 15sec.

Preferably the temperature of the second round of annealing is 64 ℃.

Compared with the prior art, the invention has the beneficial effects that: the invention adopts the technical scheme that cDNA is generated by transcription of mRNA in single B lymphocyte, and simultaneously, the template conversion characteristic of reverse rate enzyme is fully utilized, namely, the template conversion is only carried out at the 5 'end capable of completely reverse transcribing cDNA, so that the added 5' end joint sequence is used as a template, and the cDNA sequence is continuously synthesized. Therefore, a section of consensus sequence is added at the 5 'end of all the antibody gene cDNAs which can be completely transcribed selectively, the problem that the PCR primer is difficult to design because of the abundant sequence polymorphism of the 5' end of the human antibody gene is solved, and the cloning of the antibody IgG gene full-length sequence by utilizing the PCR technology is realized.

According to the invention, only 10mL of peripheral blood sample is needed, the heavy chain full-length gene sequence of up to hundreds of IgG antibodies can be obtained within 1 day, compared with the current method of cloning only part of the variable region sequence (150 bp) of IgG by a single cell sequencing method, then analyzing and splicing the single cell sequencing result by a homology comparison method, and then compared with the method of fixing IgG constant region recombination, on one hand, the time required for cloning the antibody genes is greatly shortened; on the other hand, the natural structure of the antibody variable region is reserved, so that the antibody variable region has high affinity, the processes of affinity maturation and the like are omitted, and the immunogenicity is greatly reduced; in addition, the specificity of constant regions of different IgG antibodies is reserved, which is favorable for the clinical effect of therapeutic antibodies, thereby providing powerful technical support for preparing real fully human monoclonal antibody discovery projects.

The cloning method of the heavy chain full-length gene of the humanized IgG antibody comprises the steps of sorting, reverse transcription, amplification and the like. The method clones the full-length gene of the heavy chain of the IgG antibody from 1B lymphocyte, and retains the natural structure of the heavy chain of the antibody gene, so that the method has the advantages of good antibody affinity, strong specificity and the like, provides a new technical scheme for the discovery of the human IgG monoclonal antibody, and is a remarkable improvement on the existing human monoclonal antibody gene cloning technology.

The invention provides a cloning method of a high-efficiency full-human IgG antibody heavy chain full-length gene, and a person skilled in the art can refer to the content of the invention and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention.

Drawings

FIG. 1 is a schematic diagram of functional partitions of the full-length gene of the light and heavy chains of an IgG antibody, and the sequence difference structure of the gene length of the heavy chain of the IgG antibody amplified by the prior art and the technology of the patent application, wherein a represents that the prior art gene cloning technology can only directly obtain the partial sequence length of the gene length of the heavy chain of the IgG of the human antibody. b represents the full-length gene sequence of the IgG heavy chain of the human antibody obtained by direct amplification of the technology of the patent application.

FIG. 2 is a schematic diagram of the RACE technology for amplifying the heavy chain full length gene of an IgG antibody.

FIG. 3 is a flow chart of cloning a full length gene of a human single B lymphocyte IgG antibody;

FIG. 4 is a gel electrophoresis diagram of amplification of the heavy chain full length gene of an IgG antibody using forward primer FP and reverse primer 1-AP;

lanes 1 and 2, 3 and 4 are electrophoresis bands of the full-length gene sequence of human antibody IgG amplified by single B lymphocyte in situ PCR. Wherein the PCR annealing temperatures of lanes 2-4 are 68 ℃, 66 ℃ and 64 ℃, and the amplified target bands are indicated by arrows.

FIG. 5 is a gel electrophoresis diagram of the full length gene of the heavy chain of human IgG antibody amplified by using FP/2-AP as a primer: lanes 1 are 2kb molecular weight markers (Takara Shuzo Co., ltd., DL2,000DNA Marker number: 3427Q), and lanes 2 and 3 are H chain product electrophoresis bands of PCR amplified IgG. Lanes 2 and 3 of the PCR annealing temperatures were 66℃and 68℃with the amplified target bands indicated by the arrows.

FIG. 6 is a gel electrophoresis diagram of the full length gene of the heavy chain of human IgG antibody amplified by using FP/3-AP as a primer: lanes 1 are 2kb molecular weight markers (Takara Shuzo Co., ltd., DL2,000DNAMarker cat# 3427Q), and lanes 2, 3, 4 and 5 are H chain product electrophoresis bands of PCR amplified IgG. Wherein the PCR annealing temperature of lanes 2-5 is 62 ℃, 64 ℃, 66 ℃ and 68 ℃, and the amplified target bands are indicated by arrows.

Detailed Description

The test materials adopted by the invention are all common commercial products and can be purchased in the market. The invention is further illustrated by the following examples:

example 1: preparation of B lymphocyte suspensions

10mL of a purchased suspension of fresh human B lymphocytes (Shanghai enzyme-linked biotechnology Co., ltd., cat# mL 098362) was transferred to a 15mL sterile centrifuge tube, centrifuged at 200 Xg for 5 minutes, the supernatant was discarded, and 5mL of calcium-magnesium free PBS buffer (Cytiva Co., cat# SH 30256.01) was added to resuspend the cells; at the same time, 100. Mu.L of the above cell suspension was aspirated and appropriately diluted, and counted by a screen cell counter. Centrifuging at 200 Xg for 5min, discarding supernatant, adding calcium-magnesium-free PBS buffer according to cell count, and adjusting cell density to about 1×10 ⁷ And each mL. The materials used in the subsequent examples were taken from this example.

Example 2: preparation of human peripheral blood mononuclear cell suspensions

10mL of peripheral blood is taken by using an anticoagulation tube, and after being mixed uniformly for 4-5 times by upside down, the anticoagulation tube is added with an equal volume of PBS buffer solution containing calcium and magnesium for uniform mixing, and the anticoagulation is obtained. 1 of a 50mL density separation centrifuge tube was prepared, 20mL of human lymph separation liquid (Cytiva Situofan Co., ficoll-Paque PREMICUM, cat. No. 17544203) was added, and the above anticoagulants were added to the upper layer of the lymph separation liquid, and the mixture was centrifuged horizontally at 19℃to 20℃and 400 Xg for 30 minutes.

The intermediate leucocyte layer was carefully aspirated using a bus pipette, added to 3 volumes of PBS buffer, mixed well, 150 Xg, centrifuged for 10min, the supernatant was discarded, and the procedure was repeated 1 time. Then, 6-8mL PBS buffer solution is used for suspending cells, thus obtaining the human peripheral blood mononuclear cell suspension.

Example 3: antibody and magnetic bead coupling

The biotinylated anti-human IgG antibody is coupled with streptavidin magnetic beads, and the biotinylated IgG antibody is coupled with the magnetic beads with the diameter of 1 mu m according to the ratio of 20 mu g to 1mg, and the magnetic beads are fully oscillated to be resuspended. The centrifuge tube was placed on a rotary mixer, and the mixture was mixed for 60min at room temperature for labeling. Then placing the above mixture containing magnetic beads in a magnetic rack (DynaMag, sieimer China Co., ltd.) ^TM -2 a magnetic stand; placed on 12321D) for 3min, and the supernatant was aspirated and discarded.

Then taking out the centrifuge tube containing the magnetic beads, adding 10 times of calcium-magnesium-free PBS buffer (Cytiva company, product number: SH 30256.01) for suspension cleaning; after the centrifuge tube was allowed to stand on a magnetic rack for 3min, the supernatant was aspirated and discarded. Taking out the centrifuge tube containing the magnetic beads from the magnetic rack, adding a proper amount of PBS buffer solution without calcium and magnesium for suspension, and placing at 4 ℃ for standby.

Example 4: magnetic bead selective adsorption of B cells

Antigen-specific B lymphocytes secreting IgG antibodies are sorted using labeled magnetic beads, in a specific step involving taking accurate counts of lymphocytes (concentration of about 1X 10 ⁷ And (3) a per mL) suspension (prepared in example 1 or example 2), wherein the ratio of the cell suspension to the magnetic beads is 1mL to 4ug, the magnetic beads are selected by adding the antibody conjugated cells of the anti-human IgG antibody, the antigen-specific B cells secreting the specific IgG antibody are selected by rotating and incubating at 2-8 ℃ for 20min, then the magnetic bead conjugated cells are adsorbed by a magnetic rack, and the supernatant is discarded by using a pipette. And then taking out the centrifuge tube containing the magnetic bead adsorption cells from the magnetic frame, suspending positive B lymphocytes by using a proper amount of PBS buffer solution without calcium and magnesium, counting, and storing at 4 ℃ for further sorting into single cells.

Example 5: sorting single B cells

The obtained B lymphocyte suspension is separated into single cells by using a microfluidic single cell separation technology, and the method specifically comprises the following steps: according to the cell count results of example 4, use is made ofThe B lymphocyte suspension was diluted appropriately with calcium-magnesium-free PBS buffer to maintain a cell concentration of 1X 10 ⁴ personal/mL-1X 10 ⁶ In the range of individual/mL. Single B lymphocytes in the above cell suspension were sorted in a single cell mode using a single cell meter into 96-well PCR plates containing 10 μl of single cell lysate (Shanghai Yubo biotechnology limited, cat#, YB-339H) and immediately placed in dry ice and liquid nitrogen for quick freezing and then stored at-80 ℃ for later use.

Example 6: reverse transcription synthesis of cDNA

Single B lymphocytes synthesize cDNA by reverse transcription. After the aforementioned single B lymphocytes containing the lysate were subjected to lysis at room temperature for 5 minutes, a reverse transcription premix, in which 1.2. Mu.M of 5' -terminal linker sequence, 5. Mu.M oligo dT primer, 1. Mu.M random primer, dNTPs (1 mM each) and 1. Mu.L reverse transcriptase (Bao Ri doctor technologies (Beijing Co., ltd.; cat.) were added, and cDNA was synthesized according to a 20. Mu.l reaction system, was run on a PCR machine at 42℃for 40 minutes; preserving at 70deg.C for 15min at 4deg.C; or placing the prepared cDNA at-80 ℃ for preservation.

Example 7: PCR amplification of IgG antibody heavy chain

The reverse primer 1-AP and the forward primer FP which are synthesized by taking cDNA generated by single B lymphocyte reverse transcription as a template and designing the full-length gene of the IgG heavy chain form a primer pair, the high-fidelity PCR enzyme (Bao Ri doctor materials technology (Beijing)) is available from the company,max DNA Polymerase, cargo number: R045Q), the heavy chain full-length gene fragment of the IgG antibody heavy chain in a single B cell was cloned using end-point PCR.

PCR reaction system: the 50. Mu.L system contained 6. Mu.L cDNA as template, 25. Mu.L 2X PrimeSTAR Max DNA polymerase premix, and 0.2. Mu.M IgG antibody forward and reverse primers.

Reaction conditions: pre-denatured at 94 deg.c, 20S,

5 PCR cycles, 98 ℃ x 10sec,70 ℃ x 10sec,72 ℃ x 15sec;

30 PCR cycles, 98 ℃ x 10sec, x ℃ x 10sec,72 ℃ x 15sec; primer pair: primer pair:

x is 64 ℃, 66 ℃ and 68℃ respectively

Forward primer FP 5'GGACCTGGAGCATCCTTTT 3'

Reverse sequence 1-AP 5'TCATTTACCCGGAGACAG 3'

The amplified IgG heavy chain full-length gene fragment was detected by agarose gel electrophoresis at 1.2%, and the results are shown in FIG. 4.

Example 8: PCR amplification of IgG antibody heavy chain

PCR reaction condition system: the 50. Mu.L system contained 6. Mu.L cDNA as template, 25. Mu.L 2X PrimeSTAR Max DNA polymerase premix, and 0.2. Mu.M IgG antibody forward and reverse primers.

Reaction conditions: pre-denatured at 94 deg.c, 20S,

5 PCR cycles, 98 ℃ x 10sec,70 ℃ x 10sec,72 ℃ x 15sec;

30 PCR cycles, 98 ℃ x 10sec, x ℃ x 10sec,72 ℃ x 15sec; primer pair:

x is 66℃or 68℃respectively.

Forward primer FP 5'GGACCTGGAGCATCCTTTT 3'

Reverse sequence 2-AP 5'CTCATTTACCCGGAGACA 3'

The amplified IgG heavy chain full-length gene fragment was detected by agarose gel electrophoresis at 1.2%, and the result is shown in FIG. 5.

Example 9: PCR amplification of IgG antibody heavy chain

In single B showerThe cNDA generated by the back transcription of the barcell is used as a template, and a primer pair consisting of a reverse primer 1-AP and a forward primer FP which are designed and synthesized by the full-length gene of the IgG heavy chain is prepared by the method (Bao Ri doctor materials technology (Beijing) Co., ltd.),max DNA Polymerase, cargo number: R045Q), the heavy chain full-length gene fragment of the IgG antibody heavy chain in a single B cell was cloned using end-point PCR.

Reaction conditions: pre-denatured at 94 deg.c, 20S,

5 PCR cycles, 98 ℃ x 10sec,70 ℃ x 10sec,72 ℃ x 15sec;

30 PCR cycles, 98 ℃ x 10sec, x ℃ x 10sec,72 ℃ x 15sec;

x is 62 ℃, 64 ℃, 66 ℃ or 68 ℃, respectively.

Primer pair:

forward primer FP 5'GGACCTGGAGCATCCTTTT 3'

Reverse sequence 3-AP 5'CACTCATTTACCCGGAGAC 3'

The amplified fragment of the IgG heavy chain full-length gene was detected by agarose gel electrophoresis at 1.2%, and the results are shown in FIG. 6.

Example 10: sequencing and functional analysis of cloned human IgG heavy chain Gene sequences

The full-length gene fragment of the IgG heavy chain obtained by PCR amplification is detected by agarose gel electrophoresis of 1.2%, the largest DNA fragment is cut under an ultraviolet lamp, and the DNA fragment is purified and recovered by a DNA adsorption column. The recovered purified PCR product is sampled to a sequencing company for sequencing the PCR product, and the sequenced original sequence is converted into a fasta format for storage. The H chain of the cloned IgG antibodies was sequenced using the "Analyse your Immunoglobulin (IG) or antibody nucleotide sequences" (https:// www.imgt.org/IMGT_vquest/input) tool under the IMGT/VQUEST option in the IMGT database (http:// www.imgt.org /).

Examples 6-8 each successfully amplified the full length human IgG heavy chain gene sequence, and the sequencing results of the amplification were analyzed for antibody sequence structure, as exemplified in example 1.

20230801 the nucleotide sequence of which is shown in SEQ ID NO. 1

20230801 detailed results of sequence analysis Using IMGT/V-QUEST

Hyphae (-) indicate nucleotide identity and dots (.) indicate gaps

The reference sequence is human IG heavy chain sequence in a database in IMGT

The following is an IMGT auto-annotation

V-D-J-REGION

137..494

/CDR_length＝"[8.6.14]"

/FR_length＝"[25.17.38.11]"

/nucleotide sequence

ctggaactcctggtcgagtctgggggaggcgtagtccagcctgggaggtccctcagactctcctgtacagcctctgggttcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtggaagccgttgatcatgcaggtcgtagcacatactacgcagactccgagaagggccggttcaccatctccagagacaattccaagaacacagtgtatttggaaacagacaccctggaaactgaagacacggcggtatattcctgtgggaagggctgccctctggctttggagatggttttctcgatggggcctgggaaccctcgtcaccgtctcctcag

/translation

LELLVESGGGVVQPGRSLRLSCTASGFTFSSYAMSWVRQAPGKGLEWKPLIMQVVAHTTQTPRRAGSPSPETIPRTQCIWKQTPWKLKTRRYIPVGRAALWLWRWFSRWGLGTLVTVSS

V-REGION

137..424

/allele＝"Homsap IGHV3-33*02 F or Homsap IGHV3-7*01 F or HomsapIGHV3-7*03 F"

/gene＝"Homsap IGHV3-33 or Homsap IGHV3-7"

/nucleotide sequence

ctggaactcctggtcgagtctgggggaggcgtagtccagcctgggaggtccctcagactctcctgtacagcctctgggttcacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtggaagccgttgatcatgcaggtcgtagcacatactacgcagactccgagaagggccggttcaccatctccagagacaattccaagaacacagtgtatttggaaacagacaccctggaaactgaagacacggcggtatattcctgtggga

/translation

LELLVESGGGVVQPGRSLRLSCTASGFTFSSYAMSWVRQAPGKGLEWKPLIMQVVAHTTQTPRRAGSPSPETIPRTQCIWKQTPWKLKTRRYIPVG

FR1-IMGT

137..211

/AA_IMGT＝"AA 1 to 26,AA 10 is missing"

/nucleotide sequence

ctggaactcctggtcgagtctgggggaggcgtagtccagcctgggaggtccctcagactctcctgtacagcctct

/translation

LELLVESGGGVVQPGRSLRLSCTAS

1st-CYS

200..202

/nucleotide sequence

tgt

/translation

C

CDR1-IMGT

212..235

/AA_IMGT＝"AA 27 to 38,AA 31,32,33 and 34 are missing"

/nucleotide sequence

gggttcacctttagcagctatgcc

/translation

GFTFSSYA

FR2-IMGT

236..286

/AA_IMGT＝"AA 39 to 55"

/nucleotide sequence

atgagctgggtccgccaggctccagggaaggggctggagtggaagccgttg

/translation

MSWVRQAPGKGLEWKPL

CONSERVED-TRP

242..244

/nucleotide sequence

tgg

/translation

W

CDR2-IMGT

287..304

/AA_IMGT＝"AA 56 to 65,AA 59,60,61 and 62 are missing"

/nucleotide sequence

atcatgcaggtcgtagca

/translation

IMQVVA

FR3-IMGT

305..418

/AA_IMGT＝"AA 66 to 104,AA 73 is missing"

/nucleotide sequence

catactacgcagactccgagaagggccggttcaccatctccagagacaattccaagaacacagtgtatttggaaacagacaccctggaaactgaagacacggcggtatattcct

/translation

HTTQTPRRAGSPSPETIPRTQCIWKQTPWKLKTRRYIP

CDR3-IMGT

419..460

/AA_IMGT＝"AA 105 to 117,AA 112.1 is added"

/nucleotide sequence

gtgggaagggctgccctctggctttggagatggttttctcga

/translation

VGRAALWLWRWFSR

JUNCTION

416..463

/in_frame

/nucleotide sequence

cctgtgggaagggctgccctctggctttggagatggttttctcgatgg

/translation

PVGRAALWLWRWFSRW

(N-D)-REGION

425..443

/nucleotide sequence

agggctgccctctggcttt

/translation

RAALWL

J-REGION

444..494

/allele＝"Homsap IGHJ5*02 F"

/gene＝"Homsap IGHJ5"

/codon_start＝3

/nucleotide sequence

ggagatggttttctcgatggggcctgggaaccctcgtcaccgtctcctcag

/translation

RWFSRWGLGTLVTVSS

J-TRP

461..463

/nucleotide sequence

tgg

/translation

W

FR4-IMGT

461..494

/AA_IMGT＝"AA 118 to 128"

/nucleotide sequence

tggggcctgggaaccctcgtcaccgtctcctcag

/translation

WGLGTLVTVSS

Claims

1. A method for cloning a full length human IgG heavy chain gene from a single B lymphocyte, said method comprising:

(2) Performing first separation on the suspension containing the B lymphocytes in the step (1) by using a magnetic bead separation technology to obtain positive B lymphocyte suspension; performing secondary sorting on the positive B lymphocyte suspension by using a microfluidic technology to obtain single B lymphocytes;

(3) Lysing the single B lymphocytes in the step (2) by using a single cell lysate, and reversely transcribing the obtained lysate into cDNA by reverse transcriptase SMARTScribe Reverse Transcriptase, a 5' terminal linker sequence, an oligo dT primer and a random primer;

the 5' terminal linker sequence is as follows:

5′GGACCTGGAGCATCCTTTTrGrG+G 3

the sequence of the forward primer is as follows:

FP 5′GGACCTGGAGCATCCTTTT 3′

the sequence of the reverse primer is one of the following:

1-AP 5′TCATTTACCCGGAGACAG 3′

2-AP 5′CTCATTTACCCGGAGACA 3′

3-AP 5′CACTCATTTACCCGGAGAC 3′。

2. the method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 1, wherein: the suspension containing B lymphocytes in step (1) has a cell density of 1X 10 ⁷ And each mL.

3. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 1, wherein: in the step (2), the first sorting is as follows: coupling the biotinylated anti-human IgG antibody with streptomycin affinity magnetic beads to obtain labeled magnetic beads; incubating the marked magnetic beads with the suspension containing the B lymphocytes in the step (1) at 2-8 ℃, separating the supernatant by a magnetic frame, and suspending the supernatant by a PBS buffer solution without calcium and magnesium to obtain positive human B lymphocyte suspension.

4. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 3, wherein: the mass ratio of the biotinylated anti-human IgG antibody to the streptomycin affinity magnetic beads is 20 mug:1 mg.

5. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 3, wherein: the mass of the streptomycin affinity beads was 4 μg/mL based on the volume of the suspension containing B lymphocytes.

6. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 1, wherein: in the step (2), the second sorting is as follows: diluting positive B lymphocyte suspension with PBS buffer without calcium and magnesium to cell concentration of 1×10 ⁴ Per mL-1×10 ⁶ Single cell sorting was performed in single cell mode using a single cell instrument.

7. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 1, wherein: in the reverse transcription system, the concentration of the 5' -terminal linker sequence is 1.2uM, the concentration of the oligo dT primer is 5uM, and the concentration of the random primer is 1uM.

8. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 1, wherein: in the step (4), the reaction system of the PCR amplification is as follows: the 50. Mu.L system contained 6. Mu.L cDNA solution, 25. Mu.L 2X PrimeSTAR Max DNA polymerase premix, 0.2. Mu.M forward primer and 0.2. Mu.M reverse primer.

9. The method of cloning human IgG heavy chain full length genes by single B lymphocytes of claim 1, wherein: in the step (4), the reaction steps of the PCR amplification are as follows:

(1) pre-denaturation at 94 ℃ for 20s;

(2) 5 PCR cycles: first round denaturation at 98℃for 10sec, first round annealing at 70℃for 5sec, first round extension at 72℃for 15sec;

(3) 30 PCR cycles: the second round denaturation at 98℃for 10sec, the second round annealing at 62-68℃for 5sec, and the second round extension at 72℃for 15sec.

10. The method of cloning a full length human IgG heavy chain gene from a single B lymphocyte of claim 9, wherein: the temperature of the second round of annealing was 64 ℃.