CN113105544B

CN113105544B - New fully human crown IgG4 single chain antibody and application thereof

Info

Publication number: CN113105544B
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: 2023-06-16
Anticipated expiration: 2041-05-19
Also published as: CN113105544A

Abstract

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

Description

New fully human crown IgG4 single chain antibody and application thereof

Technical Field

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

Background

The novel coronavirus is a enveloped, non-segmented positive strand single strand RNA virus, the particles are round or oval, the diameter is about 80-120 nm, and the coronavirus belongs to the order of the net nest virus, the family of coronaviridae, betacoronavirus. The virion is surrounded by a lipid bilayer provided by the host cell, which contains nucleic acids and nucleocapsid proteins, three major proteins: envelope proteins (E proteins), membrane proteins (M proteins) and spike proteins (S proteins). The genome length of each group of the virus is about thirty thousand nucleotides, and the gene sequence shows that SARS-CoV-2 belongs to a virus with longer branches in the evolution tree of the B coronavirus genus pedigree beta (Betacoronavirus Lineage beta, sarbecovirus), which is similar to coronaviruses found in Chinese chrysanthemum head bats, such as MERS-CoV or SARS-CoV. Biological genetic analysis of the virus showed that SARS virus isolate AY274119, which is a similar genus of human coronavirus, is relationally closer to SARS-CoV-2 virus than MERS virus isolates KC164505, JX869059, etc.

At the end of 2019, the outbreak, which is followed by global spread. The viral latency period averages about 3-7 days, up to 14 days. Most patients have respiratory symptoms mainly, and common clinical manifestations include fever, limb weakness, dry cough and other symptoms, and other manifestations include nasal obstruction, nasal discharge, headache, pharyngalgia, hemoptysis, expectoration, diarrhea and the like. Some patients only show low fever, slight hypodynamia and the like, and have no pneumonia. There are also some patients without any clinical manifestations. After severe viral infection, various complications may be caused including Acute Respiratory Distress Syndrome (ARDS), septic shock, systemic inflammatory response syndrome, uncorrectable metabolic acidosis, acute myocardial injury, and clotting dysfunction.

Aiming at the population pandemic endangering the whole human, the immune process of the human body after infection of a new crown is revealed, and the B cell receptor of the new crown virus is obtained to screen the new crown specific medicine, and the production, the research and the development of the virus vaccine and the importance thereof. B Cell Receptor (BCR) is a B cell antigen recognition determining surface molecule, which is essentially a membrane surface immunoglobulin (membrane immunoglobulin, mIg). BCR has antigen binding specificity, and each individual BCR has diversity up to 5 x 10-13, so that a BCR library with huge capacity is formed, and the BCR library has huge potential for individual recognition of various antigens and generation of specific antibodies.

The structure of BCR includes heavy and light chains. The heavy chain (H) of BCR consists of four gene segments of 65-100 variable regions (VH), 2 variable regions (DH), 6 binding regions (JH) and constant regions (CH); the light chain (L) consists of three gene segments, the variable region, the binding region and the constant region. B cells in the development process form BCR with diversity up to 1-2 x 10-11 under the action of recombinant enzymes (RAG 1, RAG 2). Meanwhile, complementary determining regions (complementarities determining region, CDRs) are formed therefrom: the diversity of amino acid sequences in CDR1, CDR2 and CDR3 regions, particularly the genes encoding CDR3, can be further increased by rearrangement of V (D) J and/or loss or insertion of several nucleotides between the junctions of the two gene fragments, as they are located at the junctions of the light chain V, J or heavy chain V, D, J fragments, to form a BCR-encoding gene with function (B cell clone).

BCR sequencing is a sequencing technology for detecting heavy chains and light chains of BCR subjected to targeted amplification by a high-throughput sequencing technology, and comprehensively analyzing rearranged base sequences of BCR genes and abundance of each sequence. BCR sequencing is commonly used to evaluate the rearranged base sequences of BCR genes in cellular immune responses mediated by activation of all B cells or specific B cells of a species caused by various immune related diseases and genetic mutations, and the abundance of each sequence, for studying the transcription and interrelation of different B cell clones, thereby revealing a deeper level of B cell functional specificity, and further explaining the humoral immune response tolerance and the related life phenomena of high frequency mutation in recognition of antigen abnormalities in B cell responses. The traditional BCR sequencing adopts a double-end sequencing method of 2×300bp or 2×150bp by using an Illumina sequencer to sequence the BCR, the sequencing accuracy of the method is high, but for a BCR sequence with a part length exceeding 600bp, the method can only obtain sequences at two ends, and the problem of sequence deletion of a key variable region in the middle part exists.

After PacBIO corporation formally pushes out a new generation sequencing system of sequence II, depending on a CCS sequencing mode unique to the PacBIO SMRT sequencing technology, the accuracy of reads can be improved through rolling circle sequencing, meanwhile, the optimization of polymerase reagent is combined, enzyme reading length is greatly improved, the reading length of an inserted fragment with the length of more than 10kb can be ensured while high-precision HiFi reads is obtained, and the problem that the whole fragment area cannot be completely covered under the original two-generation sequencing platform such as Illumina is solved. By performing HiFi sequencing, long read sequences with an accuracy of 99.5% or more can be obtained.

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

BCR generally includes an immunoglobulin molecule and an Ig-a/Ig- β signal transduction component bound to a membrane, which are linked by disulfide bonds. BCR includes 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 C-terminal hydrophobic membrane-binding and intracellular regions, having two heavy chains (igh) and two light chains (IgLs); 2. signal transduction component: heterodimers of Ig-alpha/Ig-beta (CD 79) are linked by disulfide bonds. Both subunits are transmembrane proteins and have an immune receptor tyrosine activating motif (ITAM) in the intracellular region.

Immunoglobulin IgG is the highest content immunoglobulin in human serum and extracellular fluid, accounting for about 75% -80% of total immunoglobulin in serum, is the least molecular weight immunoglobulin, and has typical immunoglobulin monomer structure. In humans, igG starts to synthesize 3 months after birth, and is near adult level 3-5 years old. IgG is synthesized by plasma cells, bone marrow hematopoietic stem cells initially differentiate into pre-B cells, further developing into immature B cells, where IgM can be expressed on the cell surface, after which IgD is expressed sequentially and development continues. Only immature B cells which can express IgM and can not recognize autoantigens can continue to develop and mature into B cells with immune function, and the mature B cells migrate out through peripheral blood and enter spleen and lymph nodes. B cells differentiate and proliferate into plasmablasts upon antigen stimulation, and can be divided into two sub-populations B1 and B2, wherein B2 is a T-cell dependent cell that responds to an immune response upon stimulation with a thymus-dependent antigen, depending on whether T cells are required for antibody production. IgG, i.e. produced by plasma cells differentiated from B2, has 4 subclasses, designated IgG1, igG2, igG3 and IgG4, depending on their content in serum, and each has different immune functions.

So far, no new crown-specific IgG4BCR antibody full length could be obtained by screening.

Disclosure of Invention

The invention aims to provide a fully human new crown IgG4 single-chain antibody, which can be used as an anti-new crown drug development, vaccine production, detection marker development and the like for new crown related applications after the single-chain antibody itself specifically recognizing the new crown or a variable region sequence thereof is directly expressed or is subjected to genetic engineering to be transformed into other antibody forms.

The invention provides a novel fully human crown IgG4 single-chain antibody, which comprises the amino acid sequence shown in SEQ ID No:2, and a polypeptide having the amino acid sequence shown in 2.

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

The invention also provides a library comprising the gene sequences in the novel crown IgG4 single chain antibody.

The invention also provides a preparation method of the library, which is used for analyzing BCR sequences shared by different new crown rehabilitation people and not in normal people and screening out new crown-specific IgG4BCR antibody sequences.

Further, the analytical screening process is: performing BCR full-length sequencing on new crown rehabilitators and normal people, correcting the consistency of the sequenced original data by HiFi, obtaining a BCR full-length consistency sequence with the quality value of more than Q20, and comparing the BCR full-length consistency sequence with an antibody constant region sequence in a BCR database to obtain different types of BCR antibody sequences in sequencing data of each sample.

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 novel crown IgG4 single-chain antibody in preparing novel coronavirus therapeutic drugs, drug carriers and detection markers.

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

1. according to the invention, a BCR full-length amplicon sequencing method is used for carrying out BCR full-length amplification library establishment sequencing on new crown rehabilitators and healthy people, so that a BCR full-length amplicon sequence with a 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 of a full human source, and subsequent expression verification can be carried out without further integration.

2. The invention obtains high-quality BCR sequences through PacBio HiFi sequencing, compares the high-quality BCR sequences with a database, and corrects and classifies the transcription direction of the obtained BCR full-length sequences according to the conservation of human BCR sequences in a constant region to construct a BCR antibody full-length database of different types of new crown rehabilitators and database building people; the traditional second-generation BCR sequencing technology only carries out sequencing of partial variable regions, and can not accurately carry out clustering screening of antibodies.

3. Aiming at the obtained BCR sequences of different types, the obtained antibodies are directly translated into protein amino acid sequences by locating the stop codon positions on the constant regions based on the consistency of the BCR sequences of constant regions of different antibody types, and the antibodies are compared at the protein level; traditional second-generation BCR sequencing methods do not involve the constant region of the BCR sequence, cannot perform accurate translation, and can only perform comparison at the level of the DNA sequence.

4. The invention finds out BCR antibodies shared by the new crown rehabilitation people and not in the normal people by comparing the amino acid sequences of different antibody proteins of the new crown rehabilitation people and the normal people, wherein the antibodies are new crown specific antibodies; the DNA sequence of the antibody is the full-length sequence of a fully human new crown specific antibody, and can be used as an anti-new crown drug development, vaccine production, detection marker development and the like for new crown related application after being directly expressed or genetically engineered into other antibody forms; the traditional new crown related application is to carry out expression after genetic engineering modification based on partial sequences of variable regions, and certain unknown safety problems can be introduced because the sequences are not fully human and have to be modified.

5. The invention designs a specific primer for the obtained new crown specific antibody sequence, wherein the primer amplification region comprises all regions from a promoter to a stop codon, the primer is used for carrying out PCR amplification by taking cDNA of a new crown resumpter as a template, then the amplified product is transferred into an escherichia coli expression vector, after a monoclonal is constructed, the first generation sequencing is carried out, the obtained target sequence is screened based on the size of an amplified fragment, the sequencing is carried out, a single new crown specific antibody DNA sequence is found in sequencing data, the obtained DNA sequence is a single pure fully human specific antibody DNA sequence, and the obtained DNA sequence can be directly used as an original DNA reactant for developing an anti-new crown drug, producing a vaccine and developing a detection marker without artificial synthesis.

Drawings

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

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

FIG. 2 is an electrophoretic representation of enrichment of novel crown related BCR sequences by amplification of specific primers of the present invention.

FIG. 3 is a schematic representation of the monoclonal preparation culture of the invention.

FIG. 4 is a diagram of a screening electrophoresis of the monoclonal amplification of the present invention.

FIG. 5 is a diagram of a single generation sequencing peak of the present invention.

Detailed Description

The following examples are illustrative of the invention but do not limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

As shown in FIG. 1, the fully human new crown single chain antibody provided by the invention is obtained by the following steps:

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

after obtaining high-quality BCR antibody sequences, comparing the high-quality BCR antibody sequences with an antibody database, constructing a BCR antibody constant region sequence to determine antibody types, and translating the BCR antibody sequences into protein amino acid sequences based on the termination codon positions because the same type of BCR antibodies have very high similarity and the termination codon positions translated during translation are consistent;

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

4. designing a specific primer based on the found specific sequence of the new crown, and carrying out amplification specificity enrichment on the antibody sequence related to the new crown;

5. transferring the antibody sequence into competent cells, preparing monoclonal, selecting monoclonal for amplification, selecting an amplification product with a specific fragment size for first-generation sequencing, and screening the obtained fully human new crown-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, the amino acid sequence after translation is shown as SEQ ID No: 2. All sequences contain a VDJ region, an initiation codon ATG is included at the 5 'end, a termination codon TGA is included at the 3' end, and the antibody sequence is a fully human sequence, so that the antibody can be safely applied to subsequent vaccine production, antibody drug research and development and other applications.

According to the specific nucleotide or amino acid sequence in the novel crown-specific IgG4 of the invention, the nucleotide sequence of the same antibody light-heavy chain gene or the nucleotide sequence encoding the same amino acid can be artificially synthesized in vitro, thereby obtaining the same antibody gene or being used for modifying the related gene to obtain the IgG4 antibody or the related protein.

In the present invention, a library comprising the gene sequences described in the novel crown IgG4 single chain antibody described above is proposed, which library is: the BCR full-length amplicon library establishment sequencing of the new crown rehabilitation people and the healthy people is carried out, so that the BCR full-length sequence with the quality value of more than Q20 is obtained, and the BCR antibody libraries of the new crown rehabilitation people and the healthy people are respectively constructed; and (3) comparing the sequence with a database, correcting and classifying the transcription direction of the obtained BCR full-length sequence according to the conservation of the human BCR sequence in a constant region, and constructing a BCR antibody full-length database of different types of new crown rehabilitation people and database building people.

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 common knowledge in the art.

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

Embodiment one: BCR full length amplification sequencing for new crown rehabilitation and normal population

BCR full-length amplification sequencing of new crown rehabilitation and normal population is performed by referring to the patent method (publication number CN111662970 a) of earlier application, and the main flow is as follows:

1. total RNA extraction from Whole blood sample

1mL of fresh whole blood sample is taken, triZol LS is used for Total RNA extraction of the whole blood sample, nanidoo 2000C is used for measuring the concentration and purity of the RNA sample after extraction, agilent 2100 is used for measuring the integrity of the sample, and a sample reaching a qualified standard (Total amount >1 mug, integrity RIN value > 7) is used for subsequent experiments.

2. Synthesis of cDNA first Strand in Total RNA

The experimental operation flow is as follows:

1) Oligo dT reverse transcription primer was bound to poly (A) as shown in Table 1.

Table 1:

mixing, instantaneous centrifuging, incubating at 70deg.C for 5min, and immediately placing on ice.

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

Table 2:

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

Full-length amplification of BCR cDNA

The full-length amplification of the BCR cDNA comprises two rounds of semi-nested amplification reaction, the first round of amplification is used for carrying out preliminary enrichment of the BCR sequence, and the second round of amplification adopts an internal nested primer, so that the specificity of the amplification is further improved, and the amplified band is single.

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

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

Table 3:

mixing thoroughly, centrifuging instantaneously, and placing on a PCR instrument for PCR reaction: 98 ℃ for 2min;98℃for 20s, 65℃for 15s, 72℃for 45s,18cycles; and at 72℃for 5min.

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

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

Table 4:

the 10 groups of mixed primers are all required to be amplified independently, so that the stability of the amplification reaction can be improved.

Mixing thoroughly, centrifuging instantaneously, and placing on a PCR instrument for PCR reaction: 98 ℃ for 2min;98 ℃ for 20s, 65 ℃ for 15s, 72 ℃ for 30s,20cycles; and at 72℃for 5min.

After the reaction, the amplified product was subjected to bead purification according to the instructions of the AMPure magnetic beads, and finally eluted with 10. Mu.L of elution buffer, 1. Mu.L of the purified product was taken and diluted 5-fold with nuclease-free water, followed by quantitative Qubit.

Bcr full-length amplicon fragment cocktail

And (3) carrying out equivalent sample mixing on different amplification products of the same sample according to the quantitative result of the Qubit, wherein the total amount of the mixed samples is required to be more than 1 mug, and the mixed samples are used for subsequent library establishment.

5. Library construction

1) End repair

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

Table 5:

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

After the completion of the reaction, 1X magnetic bead purification was performed according to the instructions of the AMPure magnetic beads, and the enzyme and Buffer added during the reaction were removed, and finally, the fragment ends were eluted with 14. Mu.L of elution Buffer to obtain the cohesive ends of the fragment ends plus A.

2) Sequencing adapter with barcode

After the end repair and the addition of A, adding a sequencing joint with barcode matched with the end A, and realizing the joint connection under the action of ligase. The reaction system is shown in Table 6.

Table 6:

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

Table 7:

mixing, instantaneous centrifuging, incubating at 37deg.C for 60min, and placing on ice. The purification of the beads was performed according to the instructions of AMPure magnetic beads, and finally, 20. Mu.L of elution buffer was used to obtain a dumbbell-shaped circular library suitable for use in a PacBio sequencing platform.

3) Library quality inspection and on-machine sequencing

1 mu L of library is taken for Qubit quantification to obtain library concentration; 1 μl of library was taken for fragment size analysis of Agilent 2100, and the full-length amplified library was mixed sequenced on a PacBIO sequence II sequencing platform, with about 60G of sequencing data obtained for each sample.

Embodiment two: the immune repertoire analysis of the BCR of the new crown rehabilitation person and the normal population, and the new crown specific IgG4 single chain antibody is obtained

After carrying out HiFi consistency correction on the sequenced original data, obtaining a BCR full-length consistency sequence with the quality value being more than Q20, and comparing the sequence with an antibody constant region sequence in a BCR database, obtaining BCR antibody sequences of different categories in each sample sequenced data, and simultaneously translating the DNA sequence into a protein polypeptide sequence based on the stop codon position of the constant region. By comparing the BCR polypeptide sequences shared by the new crown rehabilitation and not in the normal population, the full length of the antibody relevant to the new crown specificity is obtained. The obtained novel crown specific IgG4 sequence 1 comprises a VDJ region, an initiation codon ATG is included at the 5 'end, a termination codon TGA is included at the 3' end, and the antibody sequence is a fully human sequence, so that the novel crown specific IgG4 sequence can be safely applied to subsequent vaccine production, antibody drug research and development and other applications. The obtained novel crown specific IgG4 sequence is shown as SEQ ID No:1, the amino acid sequence after translation is shown as SEQ ID No: 2.

Embodiment III: construction of monoclonal BCR antibody library based on design of specific primers with New crown rehabilitation sample

IgG4 antibody sequences screened based on the antibody library are specifically enriched by PCR amplification on primers with the specificity of Ji Te outside the initiation codon and the termination codon. Because of the characteristic of multiple recombination of the antibody, the obtained amplified product is a set of antibody sequences matched with the primer, the first generation sequencing and subsequent application cannot be directly performed, the antibody sequences are required to be subjected to cloning experiments, monoclonal antibodies of single antibody sequences are obtained, and the monoclonal antibodies are selected for sequencing, so that the obtained monoclonal sequences are verified to be target antibodies. The specific flow is as follows:

1. design of target antibody IgG4 antibody sequence primer

The primers were designed based on sequencing the screened target sequence antibody, and the target fragment amplified by the primers comprises an initiation codon region and a termination codon region, and covers the whole length of the whole antibody expression region, and the specific sequence is shown in Table 8.

Table 8: igG4 antibody sequence primers

Wherein primer_ID_01-02 is an R-terminal universal Primer, and all IgG4 antibody sequences in the constant region can be matched with the Primer, and the Primer are mixed together to be used as the R-terminal universal Primer when in use; primer_ID_03-04 is an F-terminal Primer, the Primer sequence is designed based on the screened novel crown related specific antibody, and the Primer sequence and the R-terminal Primer are combined together to form a pair of primers for amplifying the target antibody sequence.

2. Amplification enrichment to obtain new crown related specific antibody

The amplified template used in this step was a single-stranded cDNA sample of a new crown rehabilitation sample. A new 0.2mL PCR tube was taken and the reagents shown in Table 9 were added.

Table 9:

mixing thoroughly, centrifuging instantaneously, and placing on a PCR instrument for PCR reaction: 98 ℃ for 2min;98℃for 20s, 65℃for 15s, 72℃for 120s,35cycles; and at 72℃for 5min.

And after the reaction is finished, performing electrophoresis detection, wherein a schematic diagram of an electrophoresis result is shown in fig. 2. And cutting gel to obtain target specific fragments with the fragment size of 1.5-2 k.

3. Monoclonal preparation of target fragment

This step uses the reagents in table 10.

Table 10:

preparing an LB solid medium: 8 g of the product is taken and dissolved in 250mL of distilled water, and the mixture is autoclaved for 15 minutes at 121 ℃, 250uL of ampicillin is added until hands are not scalded, and the mixture is poured into flat plates (about 15mL of each flat plate) for standby.

Preparing an LB liquid culture medium: 1 g of the product is taken and dissolved in 40mL of distilled water, the mixture is sterilized at 121 ℃ for 15 minutes under high pressure, and the mixture is packaged in a 2mL sterile centrifuge tube for standby.

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

Table 11:

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

Taking Fast-T1 competent cells out of the temperature of-70 ℃, rapidly placing the competent cells on ice for melting, adding 20 mu L of competent cells into a target carrier to connect a reaction product, uniformly mixing the walls of the flick tube (avoiding sucking with a gun), and standing on the ice for 30min.

After heat shock in a 42 ℃ water bath for 30s, the mixture is quickly placed on ice for standing for 2min, and the centrifuge tube is not shaken.

200. Mu.L of LB liquid medium (without antibiotics) was added to the centrifuge tube, and after mixing, the mixture was placed in a shaking table at 37℃and at 200rpm for 5min of resuscitation.

After resuscitating, 200 μl of the mixture is directly taken out after reversing and mixing, the mixture is coated on an LB solid medium plate containing ampicillin, the plate is positively placed in an incubator at 37 ℃ for 10min, after bacterial liquid is completely absorbed, the plate is inverted, the plate is cultured overnight, and the monoclonal preparation culture result is schematically shown in FIG. 3.

4. Monoclonal screening and sequencing identification

Selecting a monoclonal colony on a flat-plate culture medium, and uniformly mixing the monoclonal colony with 10 mu L of ddH2O to serve as a template; amplification was performed using 2X Rapid Taq Master Mix (Vazyme #P222), and the reaction system is shown in Table 12.

Table 12:

the amplification reaction program shown in Table 13 was carried out on a PCR instrument.

Table 13:

and (3) detecting the amplified product obtained by amplification by gel electrophoresis to obtain an electrophoresis chart shown in fig. 4, selecting the amplified product with high amplified band brightness, single amplified band and fragment size of 1.5-5 k for double-end monoclonal first-generation sequencing identification, comparing the sequence of the first-generation sequencing with the target antibody sequence according to the sequence of the first-generation sequencing, and selecting a monoclonal with completely consistent sequence for further amplification, so that a high-purity single novel crown related antibody specific sequence is obtained, and can be directly transferred into a pseudovirus system for subsequent antibody titer verification or directly transferred into an antibody expression system for expression.

Sequence listing

<110> Wuhan Feisha genome medicine Co., ltd

<120> New crown IgG4 single chain antibody of fully human origin and use thereof

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ggacaagggc ttgagtggat gggactcatc aaccctagtg gtggtgacac gagctacaca 240

cagaagttcc agggcagaat caccatgacc agggacacgt ccacgagcac attctacatg 300

gagctgagaa gcctgggaac tgaagacacg gccgtctatt actgtgcgag agcgggggtt 360

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tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacg 660

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gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 900

gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 960

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accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1200

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260

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Met Asp Trp Thr Trp Arg Val Phe Cys Leu Leu Ala Val Val Pro Gly

1 5 10 15

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 fully human crown IgG4 single chain antibody, which has an amino acid sequence as set forth in SEQ ID NO: 2.

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

3. The novel crown IgG4 single chain antibody gene of claim 2, having a nucleotide sequence as set forth in SEQ ID NO: 1.

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

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

6. The use of the novel coronal IgG4 single-chain antibody of claim 1 for preparing a novel coronavirus therapeutic drug, a drug carrier and a detection marker.