CN113105544A

CN113105544A - Fully human novel crown IgG4 single-chain antibody and application thereof

Info

Publication number: CN113105544A
Application number: CN202110547245.6A
Authority: CN
Inventors: 方涛; 程艳兵
Original assignee: Wuhan Feisha Genomics Medicine Co ltd
Current assignee: Wuhan Feisha Genomics Medicine Co ltd
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2021-07-13
Anticipated expiration: 2041-05-19
Also published as: CN113105544B

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a fully human novel crown IgG4 single-chain antibody and application thereof. The novel crown-specific IgG4 provided by the invention has 1 sequence, all sequences comprise VDJ regions, the 5 'end comprises an initiation codon ATG, the 3' end comprises a termination codon TGA, and the antibody sequence is a fully human sequence and can be safely applied to subsequent vaccine production, antibody drug development and other applications.

Description

Fully human novel crown IgG4 single-chain antibody and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a fully human novel crown IgG4 single-chain antibody and application thereof.

Background

The novel coronavirus is an enveloped, nonsegmented, positive-strand single-stranded RNA virus, the particle is circular or elliptical, the diameter is about 80-120 nm, and the coronavirus belongs to Betacononavirus of coronaviridae of Neuroviridae. The virion is enveloped by a lipid bilayer provided by the host cell, which contains nucleic acids and nucleocapsid proteins, three major proteins: envelope protein (E protein), membrane protein (M protein), and spike protein (S protein). The genome of each group of the virus is about thirty thousand nucleotides in length, and the gene sequence shows that SARS-CoV-2 belongs to a virus with longer branches in the beta (Betacononavirus Lineare beta, Sarbecovirus) evolutionary tree of the genus coronavirus B, and is similar to the coronavirus found in the Chinese horsetail bats, such as MERS-CoV or SARS-CoV. The biological genetic analysis of the virus shows that compared with SARS virus isolate AY274119 of human coronavirus, MERS virus isolates KC164505, JX869059 and the like are closer to SARS-CoV-2 virus in genetic relationship.

By the end of 2019, the disease outbreaks and thereafter spreads globally. The viral latency is about 3-7 days on average, and no more than 14 days at maximum. Most patients show the symptoms of the lower respiratory tract, the common clinical manifestations comprise symptoms such as fever, limb weakness, dry cough and the like, and other manifestations comprise nasal obstruction, rhinorrhea, headache, pharyngalgia, hemoptysis, expectoration, diarrhea and the like. Some patients only show low fever, slight hypodynamia and the like, and have no pulmonary inflammation. Some patients do not have any clinical manifestations. After severe viral infection, a variety of complications including Acute Respiratory Distress Syndrome (ARDS), septic shock, systemic inflammatory response syndrome, refractory metabolic acidosis, acute myocardial injury, and hemorrhagic coagulation dysfunction may be induced.

Aiming at the group pandemic endangering the whole human, the immune process of the human body after the infection of the new corona is disclosed, and the acquisition of the B cell receptor of the new corona virus is important for the screening of new corona specific drugs and the production research and development of virus vaccines. The B Cell Receptor (BCR) is a B cell antigen recognition determinant surface molecule, which is a membrane immunoglobulin (mIg) in nature. BCR has antigen binding specificity, the diversity of BCR of each individual is as high as 5 multiplied by 10^13, a BCR library with huge capacity is formed, and the individuals are endowed with huge potentials of recognizing various antigens and generating specific antibodies.

The structure of BCR includes heavy and light chains. The heavy chain (H) of the BCR consists of four gene segments of 65-100 variable regions (VH), 2 variable regions (DH), 6 binding regions (JH) and a constant region (CH); the light chain (L) is composed of three gene segments, a variable region, a binding region and a constant region. B cells in the development process form BCR with diversity as high as 1-2 x 10^11 under the action of recombinase (RAG1, RAG 2). Meanwhile, Complementary Determining Regions (CDRs) are formed therefrom: diversity in the amino acid sequences of the CDR1, CDR2, and CDR3 regions, particularly the gene encoding CDR3, due to its location at the junction of the light chain V, J or heavy chain V, D, J segments, can further increase the diversity of BCRs by v (d) J rearrangement and/or the loss or insertion of several nucleotides between the junctions of the two gene segments, resulting in a functional BCR-encoding gene (B cell clone).

BCR sequencing is a sequencing technology which detects BCR heavy chains and light chains subjected to targeted amplification through a high-throughput sequencing technology, comprehensively analyzes a BCR gene rearrangement base sequence and the abundance of each sequence. BCR sequencing is commonly used for evaluating BCR gene rearrangement base sequences in all B cells of a certain species or cell immune reactions mediated by specific B cell activation caused by various immune related diseases and genetic mutation and abundance of each sequence, and is used for researching transcription conditions and interrelations of different B cell clones, so that deeper B cell function specificity is disclosed, and then relevant life phenomena such as humoral immune response tolerance, high-frequency mutation, antigen abnormality recognition in B cell response and the like are explained. The traditional BCR sequencing is to use a sequencer of Illumina to carry out a double-end sequencing method of 2 × 300bp or 2 × 150bp to sequence the BCR, the sequencing accuracy of the method is high, but for a part of BCR sequences with the length exceeding 600bp, the method can only obtain sequences at two ends, and the problem of deletion of key variable region sequences at the middle part exists.

After a subsequent II generation sequencing system is formally promoted by PacBio, the accuracy of reads can be improved by rolling circle sequencing depending on a unique CCS sequencing mode of a PacBio SMRT sequencing technology, and simultaneously, the enzyme reading length is greatly improved by combining the optimization of a polymerase reagent, so that the reading length of an insert fragment of more than 10kb can be ensured while high-precision HiFi reads are obtained, and the problem that the whole fragment region cannot be completely covered under the original secondary sequencing platforms such as Illumina and the like is solved. By performing HiFi sequencing, long read sequences with an accuracy of over 99.5% can be obtained.

At present, aiming at BCR immune reaction caused by infection of a new corona virus on a human body, a conventional method is to adopt a second-generation sequencing platform to sequence a variable region of an antibody and then screen a new corona related antibody, but the method is limited by the reading length of sequencing, only partial sequences of the variable region can be obtained, accurate antibody type identification and coding translation identification cannot be carried out, and the BCR full-length sequence after virus infection can be easily read by the BCR full-length amplification sequencing of the new corona virus based on a third-generation sequencing platform PacBio, so that the limitation of short reading length of the second-generation sequencing is broken through, the resolution capability aiming at the BCR antibody with the specificity of the new corona virus is improved, and the resolution and the accuracy of identification of the BCR antibody with the specificity of the new corona virus are greatly improved. Particularly, after a sequenl II next-generation sequencing system is formally promoted by PacBio corporation, the accuracy of reads can be improved through rolling circle sequencing depending on a unique HiFi sequencing mode of a PacBio SMRT sequencing technology, the accuracy of BCR antibody full-length sequencing by adopting the HiFi technology can reach more than 99.5 percent at present, and a new crown BCR antibody full-length sequence with extremely high accuracy can be obtained through HiFi sequencing, and the method is used for screening new crown related BCR antibody sequences, expression translation based on the full-length antibody sequences, subsequent new crown neutralization reaction evaluation and the like.

BCRs generally include membrane-bound immunoglobulin molecules and Ig- α/Ig- β signal transduction modules, which are linked by disulfide bonds. BCR comprises the following two parts: 1. membrane-bound immunoglobulins (mIg) of a certain subtype (IgD, IgM, IgA, IgG or IgE). These membrane-bound immunoglobulins and secreted immunoglobulin monomers are identical except for the hydrophobic membrane-bound region and the intracellular region at the C-terminus, with two heavy chains (IgHs) and two light chains (IgLs); 2. a signal transduction component: the Ig-alpha/Ig-beta heterodimer (CD79) is linked by disulfide bonds. Both subunits are transmembrane proteins with an activating motif for the Immunoreceptor Tyrosine (ITAM) in the intracellular domain.

The immunoglobulin IgG is a kind of immunoglobulin with the highest content in human serum and extracellular fluid, accounts for about 75-80% of total serum immunoglobulin, is a kind of immunoglobulin with the smallest molecular mass, and has a typical immunoglobulin monomer structure. In humans, IgG begins to synthesize 3 months after birth, approaching adult levels by 3-5 years. IgG is synthesized by plasma cells, and bone marrow hematopoietic stem cells initially differentiate into pre-B cells, further develop into immature B cells, where IgM can be expressed on the cell surface, after which IgD is sequentially expressed and development continues. Only immature B cells which can express IgM and cannot recognize self-antigen can continue to develop and mature into B cells with immune function, and the mature B cells migrate out of the spleen and lymph nodes through peripheral blood. B cells differentiate to proliferate into plasmablasts when subjected to antigen stimulation, and can be divided into two subgroups, B1 and B2, depending on whether T cells are required for antibody production, wherein B2 is a T-cell dependent cell that generates an immune response when stimulated by thymus-dependent antigens. IgG, which is produced by plasma cells differentiated from B2, has 4 subclasses, named IgG1, IgG2, IgG3, and IgG4, according to their content in serum, and each has a different immune function.

To date, the full length of IgG4BCR antibodies with novel corona specificity has not been obtained by screening.

Disclosure of Invention

The invention aims to provide a fully human new crown IgG4 single-chain antibody, which can be used for new crown related application such as new crown resistant drug development, vaccine production, detection marker development and the like after being directly expressed or genetically engineered into other antibody forms.

The invention provides a fully human novel crown IgG4 single-chain antibody, which comprises the amino acid sequence shown in SEQ ID No: 2, or a pharmaceutically acceptable salt thereof.

The invention also provides a gene sequence for encoding the novel crown IgG4 single-chain antibody, preferably, the gene sequence comprises the nucleotide sequence shown in SEQ ID No: 1.

The invention also provides a library containing the gene sequence in the novel crown IgG4 single-chain antibody.

The invention also provides a preparation method of the library, which is used for analyzing the BCR sequences shared by different new crown convalescent persons but not existing in normal people and screening out the IgG4BCR antibody sequences with new crown specificity.

Further, the analysis screening process is as follows: BCR full-length sequencing is carried out on the Xinguan rehabilitative persons and normal people, HiFi consistency correction is carried out on sequenced original data, a BCR full-length consistency sequence with the quality value of more than Q20 is obtained, and different classes of BCR antibody sequences in sequencing data of each sample are obtained after comparison with antibody constant region sequences in a BCR database.

The invention also provides an expression vector containing the gene sequence in the novel crown IgG4 single-chain antibody.

The invention also provides a host cell containing the gene sequence in the novel crown IgG4 single-chain antibody.

The invention also provides application of the new crown IgG4 single-chain antibody in preparation of new crown virus treatment drugs, drug carriers and detection markers.

Compared with the prior art, the invention has the beneficial effects that:

1. the BCR full-length amplification library-building sequencing method is used for carrying out BCR full-length amplification library-building sequencing on new crown rehabilitators and healthy people, a BCR full-length amplicon sequence with the quality value of more than Q20 is obtained, the obtained BCR sequence comprises a complete region from a promoter to a stop codon, the obtained sequence is fully human-derived, and subsequent expression verification can be carried out without further integration.

2. The method obtains a high-quality BCR sequence through PacBio HiFi sequencing, and after the sequence is compared with a database, the transcription direction correction and classification are carried out on the obtained BCR full-length sequence according to the conservation of the human BCR sequence in a constant region, so that a BCR antibody full-length database of different classes of Xinguan rehabilitators and database building crowds is constructed; the traditional second-generation BCR sequencing technology only carries out sequencing on partial variable regions, and can not accurately carry out clustering screening on antibodies.

3. Aiming at the obtained BCR sequences of different types, the invention directly translates the obtained antibody into a protein amino acid sequence by positioning the stop codon position on the constant region based on the consistency of the BCR sequences of the constant regions of different types of antibodies, and compares the antibodies on the protein level; traditional next-generation BCR sequencing methods do not involve the constant region of the BCR sequence, do not allow accurate translation, and can only compare at the DNA sequence level.

4. The invention finds BCR antibodies which are shared by new crown convalescent persons and are not existed in normal people by comparing the amino acid sequences of different types of antibody proteins of the new crown convalescent persons and the normal people, and the antibodies are new crown specific antibodies; the DNA sequence of the antibody is the full-length sequence of the fully human new crown specific antibody, and can be used for new crown related application in anti-new crown drug development, vaccine production, detection marker development and the like after being directly expressed or being modified into other antibody forms through genetic engineering; the traditional new crown related application is based on the expression after genetic engineering of partial sequences of variable regions, and because the sequences are not completely humanized and must be modified, some unknown safety problems may be introduced.

5. The invention designs specific primers for the obtained new crown specific antibody sequence, the amplification region of the primers comprises all regions from a promoter to a stop codon, the primers are used for PCR amplification by taking cDNA of a new crown rehabilitator as a template, an amplification product is transferred to an escherichia coli expression vector, one generation sequencing is carried out after monoclonal construction, a target sequence obtained by screening based on the size of an amplification fragment is sequenced, a single new crown specific antibody DNA sequence is found in sequencing data, the obtained DNA sequence is a single pure human specific antibody DNA sequence, and the primer can be directly used as an original DNA reactant for anti-new crown drug development, vaccine production and marker detection development without artificial synthesis.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of the screening and obtaining principle of the novel crown-related specific antibody of the present invention.

FIG. 2 is a schematic diagram of electrophoresis of the specific primer amplification enriched new corona-associated BCR sequence according to the present invention.

FIG. 3 is a schematic diagram of the monoclonal production culture of the present invention.

FIG. 4 is an electrophoretogram of the monoclonal amplification screen of the present invention.

FIG. 5 is a graph of a single-generation sequencing peak of the invention.

Detailed Description

The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

As shown in FIG. 1, the process for obtaining fully human novel crown single-chain antibody provided by the invention is as follows:

1. the BCR full-length amplicon library building sequencing of the new coronary rehabilitative persons and the healthy people is carried out, so that a BCR full-length sequence with the quality value of more than Q20 is obtained, and BCR antibody libraries of the new coronary rehabilitative persons and the healthy people are respectively built;

after obtaining a high-quality BCR antibody sequence, comparing the obtained high-quality BCR antibody sequence with an antibody database, constructing a BCR antibody constant region sequence, determining the antibody class, and translating the BCR antibody sequence into a protein amino acid sequence based on the stop codon position because the similarity of the BCR antibodies of the same type is very high and the positions of translation stop codons are consistent during translation;

3. through comparing BCR protein amino acid sequences shared by new crown convalescent people but not shared by healthy people, different types of BCR sequences related to new crown specificity are found;

4. designing a specific primer based on the found new crown specific sequence, and amplifying the specific enrichment new crown related antibody sequence;

5. transferring the antibody sequence to competent cells, preparing monoclone, selecting the monoclone for amplification, selecting an amplification product with a specific fragment size for first-generation sequencing, and screening the obtained fully-human-derived new corona related BCR sequence based on the result of the first-generation sequencing.

In the invention, 1 new crown-specific IgG4 sequence is obtained, and the obtained new crown-specific IgG4 sequence is shown as SEQ ID No: 1, and the translated amino acid sequence is shown as SEQ ID No: 2, respectively. All sequences comprise a VDJ region, the 5 'end comprises an initiation codon ATG, the 3' end comprises a termination codon TGA, and the antibody sequence is a fully human sequence and can be safely applied to subsequent vaccine production, antibody drug development and other applications.

According to the specific nucleotide or amino acid sequence in the novel crown-specific IgG4, the same nucleotide sequence of the light and heavy chain genes of the antibody or the nucleotide sequence encoding the same amino acid can be artificially synthesized in vitro, so that the same antibody gene can be obtained or the IgG4 antibody or related protein can be obtained by modifying the related gene.

In the present invention, a library comprising the gene sequence of the novel single-chain antibody of crown IgG4 is provided, wherein the library is: establishing a library for sequencing BCR full-length amplicons of new coronary rehabilitators and healthy people to obtain a BCR full-length sequence with a quality value of more than Q20, and respectively establishing BCR antibody libraries of the new coronary rehabilitators and the healthy people; after comparison with a database, the obtained BCR full-length sequence is corrected and classified in the transcription direction according to the conservation of the human BCR sequence in a constant region, and different types of BCR antibody full-length databases of Xinguan rehabilitators and database building crowds are constructed.

In the present invention, an expression vector comprising the gene sequence is proposed, and the vector may be a prokaryotic cell expression vector, a eukaryotic cell expression vector, or an insect cell expression vector according to the general knowledge in the art.

In the present invention, a host cell comprising said expression vector is proposed, which may be a prokaryotic expression cell, a eukaryotic expression cell or an insect cell, said prokaryotic expression cell being preferably E.coli, according to common knowledge in the art.

The first embodiment is as follows: BCR full-length amplification sequencing for new coronary rehabilitators and normal population

BCR full-length amplification sequencing of the Xinguan convalescent people and the normal people is carried out by referring to a patent method (publication number CN111662970A) applied in the previous period, and the main process is as follows:

1. extraction of Total RNA from Whole blood sample

Taking 1mL of fresh whole blood sample, using Trizol LS to extract Total RNA of the whole blood sample, using Nanidrop 2000C to determine the concentration and purity of the RNA sample after extraction, using Agilent 2100 to determine the integrity of the sample, and using the sample which reaches the qualified standard (the Total amount is more than 1 mug, the integrity RIN value is more than 7) to perform subsequent experiments.

2. Synthesis of first Strand of cDNA in Total RNA

The experimental operation flow is as follows:

1) oligo dT reverse transcription primer binds to poly (A) as shown in Table 1.

Table 1:

flick and mix evenly, centrifuge instantaneously, incubate 5min at 70 ℃ and immediately put on ice.

2) The first strand of cDNA was synthesized by reverse transcription and the reactions in Table 2 were prepared.

Table 2:

flicking, mixing, centrifuging, and incubating at 42 deg.C for 75 min. Immediately after completion of the reaction, the reaction mixture was placed on ice, 1uL of BCR Template Switching Oligo was added, gently mixed, centrifuged instantaneously, and incubated at 42 ℃ for 15 min.

Full Length amplification of BCR cDNA

The full-length amplification of BCR cDNA comprises two rounds of semi-nested amplification reactions, wherein the first round of amplification is used for carrying out primary enrichment on BCR sequences, and an internal nested primer is adopted during the second round of amplification, so that the specificity of amplification is further improved, and an amplification band is single.

1) First round PCR amplification of the full Length of the BCR cDNA

A new 0.2mL PCR tube was taken and the reagents in Table 3 were added.

Table 3:

fully and uniformly mixing, performing instantaneous centrifugation, and placing on a PCR instrument for PCR reaction: 2min at 98 ℃; 18cycles at 98 ℃ for 20s, 65 ℃ for 15s, 72 ℃ for 45 s; 5min at 72 ℃.

2) Second round PCR amplification of the full Length of the BCR cDNA

A new 0.2mL PCR tube was taken and the reagents in Table 4 were added.

Table 4:

10 sets of mixed primers need to be amplified independently, so that the stability of the amplification reaction can be improved.

Fully and uniformly mixing, performing instantaneous centrifugation, and placing on a PCR instrument for PCR reaction: 2min at 98 ℃; 20cycles at 98 ℃ for 20s, 65 ℃ for 15s, 72 ℃ for 30 s; 5min at 72 ℃.

And after the reaction is finished, carrying out magnetic bead purification on the amplified product according to the AMPure magnetic bead instruction, finally eluting by using 10 mu L of elution buffer solution, taking 1 mu L of purified product, diluting by 5 times by using nuclease-free water, and then carrying out Qubit quantification.

BCR full-Length amplicon fragment cocktail

According to the quantitive result of the Qubit, carrying out equal-quantity sample mixing on different amplification products of the same sample, wherein the total amount of the mixed samples is required to be more than 1 mu g, and the mixed samples are used for building a library later.

5. Library construction

1) Tip repair

Mu.g of the whole genome amplicon sample was taken and subjected to the preparation of the end-repair reaction system to prepare the reactions in Table 5.

Table 5:

mixing, centrifuging, and incubating at 20 deg.C for 30 min.

After the reaction is finished, 1X magnetic bead purification is carried out according to the AMPure magnetic bead instruction, the enzyme and Buffer added during the reaction are removed, and finally 14 mu L of elution Buffer solution is used for elution to obtain the sticky end with the fragment end added with A.

2) Sequencing connector with barcode

After the end repair and the addition of A, a sequencing linker with barcode matched with the end of A is added, and the connection of the linker can be realized under the action of ligase. The reaction system is shown in Table 6.

Table 6:

mixing, centrifuging instantly, incubating at 20 deg.C for 60min, incubating at 65 deg.C for 10min after reaction, and placing on ice. Exonuclease digestion was performed and the reaction system is shown in table 7.

Table 7:

mixing, centrifuging instantly, incubating at 37 deg.C for 60min, and placing on ice. Bead purification was performed according to AMPure bead instructions and finally eluted with 20 μ L elution buffer to obtain a dumbbell-shaped circular library suitable for use in a PacBio sequencing platform.

3) Library quality inspection and on-machine sequencing

Taking 1 mu L of library to carry out Qubit quantification to obtain the concentration of the library; 1 μ L of the library was analyzed for fragment size of Agilent 2100, and the full-length amplified library was subjected to mixed sequencing on a PacBio sequence II sequencing platform, resulting in about 60G of sequencing data for each sample.

Example two: BCR immune repertoire analysis of new crown convalescent and normal population to obtain new crown specific IgG4 single-chain antibody

And performing HiFi consistency correction on the sequenced original data to obtain a BCR full-length consistency sequence with the quality value of more than Q20, comparing the sequence with an antibody constant region sequence in a BCR database to obtain different classes of BCR antibody sequences in the sequenced data of each sample, and translating the DNA sequence into a protein polypeptide sequence based on the stop codon position of the constant region. By comparing BCR polypeptide sequences shared by neocoronal convalescent persons but not present in normal persons, the full length of antibodies specifically related to neocoronal is obtained. The sequence of the new crown-specific IgG4 obtained at this time is 1, all sequences comprise a VDJ region, the 5 'end comprises an initiation codon ATG, the 3' end comprises a termination codon TGA, and the antibody sequence is a fully human sequence and can be safely applied to subsequent vaccine production, antibody drug development and other applications. The sequence of the obtained novel crown-specific IgG4 is shown in SEQ ID No: 1, and the translated amino acid sequence is shown as SEQ ID No: 2, respectively.

Example three: construction of monoclonal BCR antibody library based on specific primer design of Xinguan rehabilitative patient sample

Based on IgG4 antibody sequences screened by an antibody library, specific primers are designed outside the start codon and the stop codon, and target antibody sequences are specifically enriched by PCR amplification. Because the antibody has the characteristic of multiple recombination, the obtained amplification product is a collection of antibody sequences matched with the primers, and cannot be directly subjected to first-generation sequencing and subsequent application, and the antibody sequences are subjected to cloning experiments to obtain a single antibody sequence monoclonal, and the single antibody sequence is selected for sequencing to verify that the obtained monoclonal sequence is the target antibody. The specific process is as follows:

1. primer design for target antibody IgG4 antibody sequence

Primers are designed based on target sequence antibodies screened by sequencing, target fragments amplified by the primers comprise a start codon region and a stop codon region, the target fragments cover the whole length of an expression region of the whole antibody, and specific sequences are shown in table 8.

Table 8: IgG4 antibody sequence primer

The Primer _ ID _ 01-02 is an R-terminal universal Primer, the Primer is in a constant region, all IgG4 antibody sequences can be matched with the Primer, and the Primer are mixed together to be used as the R-terminal universal Primer when in use; the Primer _ ID _ 03-04 is an F-terminal Primer, the Primer sequence is designed based on the screened new crown related specific antibody, and the R-terminal Primer and the Primer are combined together to form a pair of primers for amplification of a target antibody sequence.

2. Amplification and enrichment to obtain new crown related specific antibody

The amplification template used in the step is a single-stranded cDNA sample of a Xinguan rehabilitative patient sample. A new 0.2mL PCR tube was taken and the reagents shown in Table 9 were added.

Table 9:

fully and uniformly mixing, performing instantaneous centrifugation, and placing on a PCR instrument for PCR reaction: 2min at 98 ℃; 20s at 98 ℃, 15s at 65 ℃, 120s at 72 ℃ and 35 cycles; 5min at 72 ℃.

Electrophoresis detection is carried out after the reaction is finished, and a schematic diagram of the electrophoresis result is shown in FIG. 2. And cutting the gel to obtain a target specific fragment with the fragment size of 1.5-2 k.

3. Monoclonal preparation of target fragments

The reagents in Table 10 were used in this step.

Table 10:

preparing an LB solid culture medium: 8 g of the product is taken and dissolved in 250mL of distilled water, sterilized for 15 minutes at 121 ℃ under high pressure, added with 250uL of ampicillin when hands are not scalded, mixed evenly and poured into plates for standby (each plate is about 15 mL).

Preparing an LB liquid culture medium: 1 g of the product is taken and dissolved in 40mL of distilled water, sterilized under high pressure at 121 ℃ for 15 minutes and subpackaged into 2mL of sterile centrifuge tubes for later use.

After the medium was prepared, a carrier ligation reaction was performed to prepare a reaction system as shown in Table 11.

Table 11:

and (3) flicking the tube bottom, mixing uniformly, performing low-speed instantaneous centrifugation, collecting all liquid at the bottom of the centrifugal tube, and reacting for 5min at the room temperature of 25 ℃. After the reaction was completed, the centrifuge tube was placed on ice.

Taking out Fast-T1 competent cells from-70 deg.C, rapidly thawing on ice, adding 20 μ L of competent cells into target vector to connect reaction product, flicking tube wall, mixing (avoiding sucking with gun), and standing on ice for 30 min.

After heat shock in 42 ℃ water bath for 30s, the tube was quickly placed on ice and left for 2min without shaking the tube.

Adding 200 μ L LB liquid culture medium (without antibiotic) into the centrifuge tube, mixing, and recovering in a shaker at 37 deg.C and 200rpm for 5 min.

After recovery, 200 μ L of the suspension was directly applied to LB solid medium plate containing ampicillin, the plate was placed in an incubator at 37 ℃ for 10min, after the bacterial solution was completely absorbed, the plate was inverted and cultured overnight, and the results of the monoclonal preparative culture are shown in FIG. 3.

4. Monoclonal screening and sequencing identification

Selecting a monoclonal colony on a plate culture medium to be evenly mixed in 10 mu L ddH2O to be used as a template; amplification was performed using 2 × Rapid Taq Master Mix (Vazyme # P222), and the reaction system is shown in Table 12.

Table 12:

the PCR product was placed on a PCR machine to perform the amplification reaction procedure shown in Table 13.

Table 13:

carrying out gel electrophoresis detection on the amplification product obtained by amplification to obtain an electrophoresis chart shown in figure 4, selecting the amplification product with high amplification band brightness, single amplification band and fragment size of 1.5-5 k to carry out double-end monoclonal first-generation sequencing identification, wherein a sequencing peak chart is shown in figure 5, carrying out consistency comparison on a first-generation sequencing sequence and a target antibody sequence, and selecting a monoclonal with completely consistent sequence to further carry out amplification, so that a high-purity single new crown related antibody specific sequence is obtained, can be directly transferred into a pseudovirus system to carry out subsequent antibody titer verification, and can also be directly transferred into an antibody expression system to carry out expression.

Sequence listing

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<120> fully human novel crown IgG4 single-chain antibody and application thereof

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Ala His Ser Gln Glu Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Thr Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser His Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Leu Ile Asn Pro Ser Gly Gly Asp Thr Ser Tyr Thr

65 70 75 80

Gln Lys Phe Gln Gly Arg Ile Thr Met Thr Arg Asp Thr Ser Thr Ser

85 90 95

Thr Phe Tyr Met Glu Leu Arg Ser Leu Gly Thr Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ala Gly Val Asp Trp Asp Asp Arg Gly Asp Ala

115 120 125

Phe Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

145 150 155 160

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr

210 215 220

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val

225 230 235 240

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

340 345 350

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Leu Gly Lys

465

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tacgtgccaa gcatcctcg 19

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcactcattt acccagagac 20

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

agcaccatgg actggacct 19

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atggactgga cctggagggt cttc 24

Claims

1. A novel single-chain antibody of crown IgG4 of fully human origin, comprising the amino acid sequence as shown in SEQ ID No: 2, or a pharmaceutically acceptable salt thereof.

2. The gene sequence encoding the novel crown IgG4 single chain antibody of claim 1.

3. The novel crown IgG4 single chain antibody according to claim 2, comprising the amino acid sequence as set forth in SEQ ID No: 1.

4. A library comprising the gene sequence of the novel crown IgG4 single chain antibody of claim 2.

5. The method of library generation according to claim 4, wherein BCR sequences common to different neocoronal convalescent individuals but not present in the normal population are analyzed and screened for neocoronal-specific IgG4BCR antibody sequences.

6. The method of claim 5, wherein the analytical screening process is: BCR full-length sequencing is carried out on the Xinguan rehabilitative persons and normal people, HiFi consistency correction is carried out on sequenced original data, a BCR full-length consistency sequence with the quality value of more than Q20 is obtained, and different classes of BCR antibody sequences in sequencing data of each sample are obtained after comparison with antibody constant region sequences in a BCR database.

7. An expression vector comprising the gene sequence of the novel crown IgG4 single chain antibody of claim 3.

8. A host cell comprising the gene sequence of the novel crown IgG4 single chain antibody of claim 3.

9. The use of the novel single chain antibody of crown IgG4 of claim 1 in the preparation of novel therapeutic coronaviruses, pharmaceutical vectors and detection markers.