CN112501273B - Nested PCR primer and kit for amplifying horse antibody and application thereof - Google Patents
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C07K2317/00—Immunoglobulins specific features
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Abstract
The invention relates to the fields of genetic engineering and antibody preparation, in particular to a nested PCR primer and a kit for amplifying a horse antibody and application thereof. The positive rate of antibody heavy chain and light chain genes matched by the nested PCR primer is about 56%, so that the method for separating the antibody genes from single B lymphocyte can be further used for preparing high-throughput equine antibodies. The method retains the natural pairing of the light chain and the heavy chain variable region, has the advantages of good gene diversity, high titer, good antibody affinity, strong specificity, short experimental period and the like, provides a wide approach for developing preventive and therapeutic drugs or diagnostic antibodies of monoclonal antibodies of infectious diseases and autoimmune diseases, and simultaneously provides a more efficient, simple and rapid method for obtaining antibodies of exogenous proteins for basic research of life science.
Description
Technical Field
The invention relates to the fields of genetic engineering and antibody preparation, in particular to a nested PCR primer and a kit for amplifying a horse antibody and application thereof.
Background
Antibodies (and their membrane-form B cell receptors) are key molecules for humoral immunity to protect the body from damage by specific pathogens, with a very rich diversity. The sources of antibody diversity include three aspects of combinatorial diversity, linkage diversity and somatic hypermutation. Wherein, the combinatorial diversity refers to the random combination among the genes of the germ line of the variable region of the antibody and the combination among the light and heavy chains with abundant possibility; linkage diversity refers to the diversity caused by inaccurate linkage between three (heavy chain) or two (light chain) germline genes of the antibody variable region; somatic high-frequency mutation refers to high-frequency point mutation of rearranged antibody genes after antigen stimulation, and the high-frequency point mutation can greatly improve the diversity of antibodies. This rich diversity allows the potential of antibodies to recognize almost any pathogen that the body may encounter. Therefore, the analysis of antibodies and their secretory cells or corresponding memory B cells is one of the important contents in the research of the immune protection of mammals against foreign pathogens or the research of autologous tumors and immune diseases.
Monoclonal antibodies (MAbs), abbreviated Monoclonal antibodies, are antibodies produced by only one type of immune cell. Compared with polyclonal antibodies (antibodies produced by various types of B cells), the monoclonal antibody has the characteristics of clear antigen binding sites and easiness in further immune reaction mechanism research. Currently, obtaining endogenous monoclonal antibodies is mainly achieved by two ways: the first monoclonal antibody large-scale preparation platform based on hybridoma technology is that in 1975, German scientist Kohler and England scientist Mil-stein fuse B lymphocytes producing antibodies with myeloma cells, and a hybridoma technology monoclonal antibody preparation technology is successfully established, and the technology also obtains the Nuobell medical and physiological prize in 1984; the second is two ways of isolating antibody gene cloning by single B fine lymphocyte. The hybridoma technology transforms B cells to obtain the monoclonal antibody, and because a stable cell line is difficult to generate, the obtained monoclonal antibody has extremely low efficiency and very long experimental period, and the method is mainly applied to mice and rarely applied in horses; the single B fine lymphocyte separation and antibody gene cloning technology reserves the natural pairing of a light chain heavy chain variable region, and has the advantages of good gene diversity, high efficiency, endogenesis and the like, but the technology is only applied to two species of human and monkey at present, and no report about the single B lymphocyte separation and antibody gene cloning is reported. In addition, the single-cell transcriptome mRNA full-length sequencing technology can theoretically obtain the antibody gene sequence of a single B lymphocyte, but the technology has the defects of high cost, high experimental throughput and the like, and the application of the technology to horse species is limited.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention relates to a nested PCR primer for amplifying a horse antibody, which comprises an outer primer and an inner primer for amplifying heavy chains and light chains of the antibody:
a) the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 1-7, and the nucleotide sequence of a downstream primer is shown as at least one of SEQ ID NO. 8-12;
b) an outer primer for amplifying a kappa chain of an antibody light chain, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 13-23, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 24;
c) the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 25-33, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 34;
d) the nucleotide sequence of an upstream primer of the inner primer for amplifying the heavy chain of the antibody is shown as SEQ ID NO. 35, and the nucleotide sequence of a downstream primer is shown as at least one of SEQ ID NO. 36-40; and the selection of the downstream primers SEQ ID NO. 36-40 and the selection of SEQ ID NO. 8-12 are in one-to-one correspondence in sequence;
e) an inner primer for amplifying a kappa chain of an antibody light chain, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 35, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 41;
f) the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 35, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 42.
According to a further aspect of the invention, the invention also relates to a kit containing the nested PCR primers as described above.
According to yet another aspect of the present invention, the present invention also relates to a method for preparing an equine antibody, comprising:
a) obtaining horse peripheral blood mononuclear lymphocytes, wherein the horse is previously contacted with an antigenic substance;
b) sorting B cells capable of secreting/expressing a substance specifically recognizing the antigen from the peripheral blood mononuclear lymphocytes;
c) sequencing said B cells using the nested PCR primers of claim 1, or the kit of any one of claims 2 to 4, to obtain antibody sequences;
d) constructing a vector comprising the antibody sequence;
e) expressing the vector in a host cell, and recovering the antibody thus produced from the culture medium or from the cultured host cell.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a nested PCR primer for amplifying horse antibodies, the positive rate of antibody heavy chain and light chain genes which can be matched by the primer is about 56%, and further a method for separating antibody genes from single B lymphocyte is realized, and a high-throughput horse source antibody is prepared. The method retains the natural pairing of the light chain and the heavy chain variable region, has the advantages of good gene diversity, high titer, good antibody affinity, strong specificity, short experimental period and the like, provides a wide approach for developing preventive and therapeutic drugs or diagnostic antibodies of monoclonal antibodies of infectious diseases and autoimmune diseases, and simultaneously provides a more efficient, simple and rapid method for obtaining antibodies of exogenous proteins for basic research of life science.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a map of a B cell sorting specifically expressing an HA antibody according to one embodiment of the present invention;
FIG. 2 shows the result of PCR electrophoresis of the heavy chain gene of an equine single B cell antibody in accordance with an embodiment of the present invention; pos positive control group; 2-11, experimental group; neg is negative control group; m is Marker;
FIG. 3 shows PCR electrophoresis results of the light chain lambda gene of the equine single B cell antibody in accordance with one embodiment of the present invention; pos positive control group; 2-11, experimental group; neg is negative control group; m is Marker;
FIG. 4 shows the result of PCR electrophoresis of the light chain kappa gene of the equine single B-cell antibody in accordance with an embodiment of the present invention; pos positive control group; 2-11, experimental group; neg is negative control group; m is Marker;
FIG. 5 is a schematic representation of a linear Ig expression vector in one embodiment of the present invention;
FIG. 6 shows PCR electrophoresis results of heavy chain genes of a single B cell antibody of a comparative horse; 1, positive control group; 2-11, experimental group; 12, negative control group; m is Marker;
FIG. 7 shows the result of PCR electrophoresis of the kappa gene of the light chain of the antibody of a single B-cell of a comparative example horse; 1, positive control group; 2-11, experimental group; 12, negative control group; m is Marker;
FIG. 8 shows PCR electrophoresis results of light chain lambda gene of a single B-cell antibody of a comparative example horse; 1, positive control group; 2-11, experimental group; 12, negative control group; and M is Marker.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.
The invention relates to a nested PCR primer for amplifying a horse antibody, which comprises an outer primer and an inner primer for amplifying heavy chains and light chains of the antibody:
a) the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 1-7, and the nucleotide sequence of a downstream primer is shown as at least one of SEQ ID NO. 8-12;
b) an outer primer for amplifying a kappa chain of an antibody light chain, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 13-23, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 24;
c) the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 25-33, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 34;
d) the nucleotide sequence of an upstream primer of the inner primer for amplifying the heavy chain of the antibody is shown as SEQ ID NO. 35, and the nucleotide sequence of a downstream primer is shown as at least one of SEQ ID NO. 36-40; and the selection of the downstream primers SEQ ID NO. 36-40 and the selection of SEQ ID NO. 8-12 are in one-to-one correspondence in sequence;
e) an inner primer for amplifying a kappa chain of an antibody light chain, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 35, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 41;
f) the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 35, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 42.
The term "antibody" includes polyclonal and monoclonal antibodies, as well as antibody fragments thereof, and the term "antibody fragments" includes antigenic compound-binding fragments of such antibodies, including Fab, F (ab')2Fd, Fv, scFv, diabodies and antibody minimal recognition units, as well as single chain derivatives of these antibodies and fragments, such as scFv-Fc and the like. The type of antibody can be selected from IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgA, IgM, IgE, IgD. Furthermore, the term "antibody" includes naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, chimeric (chimeric), bifunctional (bifunctional), and related synthetic isomeric forms (isoforms). The term "antibody" is used interchangeably with "immunoglobulin".
Light chain constant region (C)L) Specifically included are both kappa and lambda types, generally representing the C-terminal half of the natural kappa or lambda light chains of natural antibodies. CL) Typically comprising about 110 amino acids representing an immunoglobulin domain.
Heavy chain constant region (C)H) Including about three-quarters or more of the antibody heavy chain, and is located at its C-terminus. In general, CHIncluding three or four immunoglobulin domains. According to CHThe antigenicity of the heavy chains is different, mu,Delta, gamma, epsilon and alpha, and the corresponding immunoglobulins are IgM, IgD, IgG, IgE and IgA, respectively.
Variable region (V or VR) refers to the variable region or domain of an antibody, specifically the heavy chain variable region (V)H) Or light chain variable region (V)L). Preferably, VR comprises a single immunoglobulin domain. Preferred VRs are those of equine immunoglobulins.
Each VR comprises so-called Complementarity Determining Regions (CDRs) that determine the binding characteristics of the antibody and are embedded in a so-called framework. Preferably, the VR comprises three CDRs, preferably CDR1, CDR2 and CDR3, embedded in a framework (FR 1-4). Thus, in a preferred embodiment, VR includes the following elements in the following N-terminal to C-terminal order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Typically VR comprises or preferably consists of a polypeptide which is the product of a V-gene fragment family member in combination with another gene fragment, such as a D and J gene fragment or a J gene fragment.
The light chain variable region is encoded by a rearranged nucleic acid molecule and is kappa (V)κ) Or λ (V)λ) Two types. Maltogenic VκComprising a polypeptide, wherein said polypeptide is equine derived Vκ-the products of members of families 1 to 7 of a gene fragment. Maltogenic VλComprising a polypeptide, wherein said polypeptide is equine derived VλThe products of 1 to 8 family members of a gene fragment.
Heavy chain variable region (V)H) A heavy chain variable region encoded by a rearranged nucleic acid molecule. Maltogenic VHComprising a polypeptide, wherein said polypeptide is the product of a 1 st to 5 th family member of a equine derived HV-gene fragment. Preferred VHIs of equine origin VHPreferably equine derived V encoded by DNA amplified from equine B cellsH。
"horse" means in the present application an equine (Equidae) of the mammalia, Equus, Equidae, Oncomelidae, preferably Equus, for example, Maloideae E. (Equus), Equus E. (Asinus), zebra E. (Dolichophius), zebra E. (Hippotogris), Equus Americana (Americana) and Equus woody. Most preferably horse (e.f. caballus).
According to a further aspect of the invention, the invention also relates to a kit containing the nested PCR primers as described above.
In some embodiments, the kit further comprises a reverse transcription primer for synthesizing cDNA for reverse transcription B cells, the nucleotide sequence of the reverse transcription primer is shown as at least one of SEQ ID NO. 8-12, and the selection of the reverse transcription primer and the selection of SEQ ID NO. 8-12 are in sequence in one-to-one correspondence.
In some embodiments, the kit further comprises one or more of a reverse transcriptase, a random primer, dntps, a DNA polymerase, an amplification buffer, and water.
The invention also relates to the application of the nested PCR primer or the kit in horse antibody sequencing.
According to yet another aspect of the present invention, the present invention also relates to a method for preparing an equine antibody, comprising:
a) obtaining horse peripheral blood mononuclear lymphocytes, wherein the horse is previously contacted with an antigenic substance;
b) sorting B cells capable of secreting/expressing a substance specifically recognizing the antigen from the peripheral blood mononuclear lymphocytes;
c) sequencing said B cells using the nested PCR primers of claim 1, or the kit of any one of claims 2 to 4, to obtain antibody sequences;
d) constructing a vector comprising the antibody sequence;
e) expressing the vector in a host cell, and recovering the antibody thus produced from the culture medium or from the cultured host cell.
In the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage, animal virus, etc. Animal viruses that may be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). In some embodiments, regulatory elements commonly used in genetic engineering, such as enhancers, promoters, Internal Ribosome Entry Sites (IRES), and other expression control elements (e.g., transcription termination signals, or polyadenylation signals and poly-U sequences, etc.) are included in the vectors of the present invention.
The separated heavy chain and light chain variable region genes of the antibody can be respectively connected into a linear vector expression vector by an overlapping PCR method.
When the separated heavy chain and light chain variable region genes of the antibody are used for constructing a vector and expressing the antibody, if the traditional plasmid vector is adopted, the time consumption is long, the efficiency is low, and the method is not suitable for high-flux application.
The linear expression system vector consists of two parts, the first part consists of a promoter and tag DNA consisting of 20 bases, and the second part consists of a heavy chain fragment or light chain fragment constant region and a poly-polyA signal (e.g., BGH polyA) fragment.
The linear vector, preferably a linear Ig expression vector, consists of a promoter (e.g., CMV promoter), tag DNA, a heavy or light chain fragment constant region, and a poly PolyA signal (e.g., BGH PolyA) fragment (B-H, B-K, B-L).
Alternatively, the heavy chain linear expression vector is composed of two parts, the first part consisting of a promoter (e.g., CMV promoter), tag DNA, and the second part consisting of a heavy chain constant region and a poly PolyA signal (e.g., BGH PolyA) segment. And (3) connecting the separated heavy chain guide region and variable region gene of the antibody between the first part and the second part by overlapping PCR, thereby forming an antibody heavy chain linear expression system consisting of a promoter, tag DNA, Ig guide region, heavy chain variable region, heavy chain constant region and poly polyA signal fragment.
Alternatively, the light chain λ linear expression vector consists of two parts, the first part consisting of the promoter (e.g., CMV promoter), tag DNA, and the second part consisting of the light chain λ constant region and a poly PolyA signal (e.g., BGH PolyA) fragment. And (3) connecting the separated antibody light chain lambda guide region and variable region genes into the middle of the first part and the second part by overlapping PCR, thereby forming an antibody light chain lambda linear expression system consisting of a promoter, tag DNA, Ig guide region, light chain lambda variable region, light chain lambda constant region and poly-polyA signal fragment.
Alternatively, the light chain kappa linear expression vector is composed of two parts, the first part consisting of a promoter (e.g., CMV promoter), tag DNA, and the second part consisting of a light chain kappa constant region and a poly PolyA signal (e.g., BGH PolyA) fragment. And (3) linking the separated antibody light chain kappa guide region and variable region gene into the middle of the first part and the second part by overlapping PCR, thereby forming an antibody light chain kappa linear expression system consisting of a promoter, tag DNA, an Ig guide region, a light chain kappa variable region, a light chain kappa constant region and a poly-polyA signal fragment.
Further, the tag DNA sequence is:
ATCGACGTTGGACTCCAGAG。
methods for introducing vectors into host cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, liposome encapsulation of polynucleotides, biolistic injection, and direct microinjection of DNA into cells.
The term "host cell" refers to a cell which can be used for introducing a vector, and includes, but is not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf 9. The host cell is preferably a eukaryotic cell, more preferably a mammalian cell. Host cells for expression are well known in the art as mammalian cells, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These cell lines include primarily Chinese Hamster Ovary (CHO) cells, NSO, SP2 cells, HeLa cells, Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatoma cells, a549 cells, 3T3 cells, 293 cells, and many other cell lines. Particularly preferred cell lines are selected by determining which cell line has a high expression level.
When a recombinant expression vector encoding a heavy chain or an antigen-binding site thereof, a light chain and/or an antigen-binding site thereof is introduced into a mammalian host cell, the antibody can be produced by culturing the host cell for a period of time sufficient to allow the antibody to be expressed in the host cell. Preferably, the antibody is secreted into the medium in which the host cell is grown.
The separation and purification can be carried out according to the characteristics of the antibody, for example, the antibody can be separated from other proteins by salting out according to the difference of protein hydrophobicity, and the antibody can be separated by ion exchange, electrophoresis and other technologies according to the difference of the charge of the antibody. The separation and purification method comprises precipitation, salting out, membrane technology, electrophoresis, chromatography and the like. Preferably, the antibody is purified by protein A or protein G affinity chromatography.
In some embodiments, the peripheral blood mononuclear lymphocytes are collected and isolated from the jugular vein of an equine.
In some embodiments, the B cell is at least one of a memory B cell, a plasma cell, a plasmablast, a B-2 cell.
In some embodiments, the method of sorting is flow cytometry.
In some embodiments, the B cells are memory B cells and the combination of B cell surface markers from flow sorting is:
AqVD-/CD3-/CD14-/CD16-/CD21 +/specific antigen +.
Specific antigen is meant to include the antigen against which the desired antibody is directed.
Specific antigen + indicates that the sorted B cells are capable of binding the specific antigen.
In some embodiments, the combination of sorting fluorescent labels used for flow sorting is:
AqVD-AmCyan-/CD3-FITC-/CD14-FITC-/CD16-FITC-/CD21-PE-Cy7 +/specific antigen-BV 421 +/specific antigen-APC +.
The combination of specific antigen-BV 421 +/specific antigen-APC + can reduce the detection of B cells containing non-specific antibody.
In some embodiments, the method further comprises step f):
performing one or more of affinity verification, antibody and the binding epitope verification of the antigen substance and antibody purification on the antibody generated in the step e).
The separation and purification can be carried out according to the characteristics of the antibody, for example, the antibody can be separated from other proteins by salting out according to the difference of protein hydrophobicity, and the antibody can be separated by ion exchange, electrophoresis and other technologies according to the difference of the charge of the antibody. The separation and purification method comprises precipitation, salting out, membrane technology, electrophoresis, chromatography and the like. Preferably, the antibody is purified by protein A or protein G affinity chromatography.
In some embodiments, the antigenic substance is a microorganism; further, the antigenic material is selected from one or more of equine influenza virus, equine infectious anemia virus, african horse sickness virus, equine viral arteritis and equine infectious pleuropneumonia pathogens and equine herpes virus type 1 and 3, or subunits thereof.
In some embodiments, the antigenic substance is selected from the group consisting of: human, simian, sheep, camel, horse, chicken, pig, rice, corn, wheat, and Arabidopsis thaliana.
The antigenic material may also be pretreated by attenuation or inactivation.
Embodiments of the present invention will be described in detail with reference to examples.
The primers used in the present invention are as follows:
first round nested PCR heavy chain PCR primer
First round nested PCR light chain kappa PCR primer
First round nested PCR light chain lambda PCR primer
Second round nest type PCR heavy chain variable region PCR primer
Second round nested PCR light chain kappa variable region PCR primers
Second round nested PCR light chain lambda variable region PCR primers
RT-PCR primer
Example 1 sample Collection and separation of PBMCs
B cell isolation and cryopreservation: the corresponding vaccines are inoculated for different experimental purposes, and the equine influenza virus is taken as an example for introduction. Two immunizations were used for all experimental horses. The first immunization, injecting antigen in neck muscle, 2ml each time. Secondary immunizations were performed at 4 weeks intervals. Collecting anticoagulated blood from jugular vein every 3 days after immunization, standing at room temperature for 30min, taking supernatant plasma, centrifuging at 4 deg.C 2000r/min for 10min, discarding supernatant, resuspending cell precipitate with 1640 culture medium, centrifuging again, repeating twice, subpackaging cells, suspending cells with cell freezing solution (fresh serum: DMEM ═ 9:1), and freezing with liquid nitrogen.
Example 2 sorting of B cells
Since large numbers of antigen-specific B lymphocytes are present in the blood of horses after receiving immunization or after the infection with infectious disease has healed, a flow cytometer will be used to sort individual antigen-specific B lymphocytes.
The cell fluorescent labeling adopted for sorting the antigen specific memory B lymphocytes containing the equine monoclonal antibody gene is combined according to the following mode:
sorting fluorescent label combination: AqVD-AmCyan-/CD3-FITC-/CD14-FITC-/CD16-FITC-/CD21-PE-Cy7 +/specific antigen-BV 421 +/specific antigen-APC +. Antigen-specific B lymphocyte sorting is shown in figure 1.
The experimental method comprises the following steps: the cells were thawed, 2ml PBS/1% BSA was added, mixed well, centrifuged at 450g for 5min at 22 ℃. Discarding the supernatant, repeating twice, transferring into a flow tube, and transferring into 200 μ l/tube; adding corresponding antibody (BD, Biolegend) in dark, and standing at 4 deg.C for 45 min; adding 2ml PBS/1% BSA, mixing, centrifuging at 4 deg.C and 450g for 5min, and washing repeatedly for 3 times; resuspend with 200-. Individual B cells were sorted into 96-well plates (Eppendorf). 96-well plates contained 20. mu.l buffer: mu.l of 5 XPCR Buffer, 1.25. mu.l of DTT, 0.5. mu.l of RNAase out, 13.25. mu.l of water (Invitrogen). After sorting, a 96-well plate was quickly sealed (Axygen) and stored in a-80 ℃ freezer.
Example 3 Single B lymphocyte Gene amplification
The isolated single peripheral nuclear cells were subjected to immunoglobulin heavy and light chain variable region gene cloning.
1. Reverse transcription to synthesize cDNA:
cDNA was synthesized in a 20. mu.l reaction in 96-well PCR plates, 50 ng/. mu.l of Random hexamer Primers, 1.5. mu.l of 25mM dNTPs, and 50U Superscript III reverse transcriptase (Invitrogen, Carlsbad, Calif.), and reacted at 65 ℃ for 1 hour on a PCR instrument;
2. nested PCR amplification of antibody variable region genes:
the nested PCR primer provided by the invention is used for carrying out antibody variable regionA gene. PCR reactions IgH, Ig λ and Ig κ variable region genes were synthesized separately in a 96-well plate in 10 μ l reaction system, first round PCR: a10. mu.l system contained 1. mu.l of RT reaction product, 1 unit of HotStarTaq Plus enzyme (QIAGEN), 0.2mM dNTPs, and 0.5. mu.M IgH (V)H1-VH7)、Igκ(Vκ1-Vκ10) or Ig lambda (VL1-VL9) variable region primers, IgM, IgG, IgD, IgA and IgE, or Ig kappa or Ig lambda antibody constant region primers, reaction conditions: pre-denaturation 95 ℃ for 5min, followed by 35 PCR cycles, each cycle: 95 ℃ X30 sec, 55 ℃ (V)HAnd Vκ) Or 50 deg.C (V)λX 60sec, 72 ℃ x 90sec, and finally extension for 7min at 72 ℃.
Second round PCR: a50. mu.l system contained 3. mu.l of the first round PCR reaction product as template, 5 units of HotStarTaq Plus enzyme (QIAGEN), 0.2mM dNTPs, and 0.5. mu.M IgH (V)H-int)、Igκ(Vκ-int)or Igλ(VL-int) variable region primers, IgM, IgG, IgD, IgA and IgE, or Ig κ or Ig λ antibody variable region primers, reaction conditions: pre-denaturation 95 ℃ for 5min, followed by 35 PCR cycles, each cycle: 95 ℃ X30 sec, 58 ℃ (V)H)，60℃(Vκ) Or 64 ℃ (V)λ) X 60sec, 72 ℃ x 90sec, and finally extension for 7min at 72 ℃. Amplified VH，VκAnd VλThe variable region PCR products of (1.2%) were identified by electrophoresis on a 1.2% agarose gel. All reactions used total RNA extracted from PBMC as a positive control for PCR reaction quality control, and a PCR reaction system without template as a negative control.
The variable region genes of the heavy and light chains of the isolated cloned immunoglobulins were electrophoretically detected and are shown in FIG. 3.
3. Immunoglobulin heavy and light chain variable region gene sequence analysis
1) And (3) carrying out original sequencing sequence cleaning on the amplified immunoglobulin heavy chain and light chain variable region gene products by using Sequcher5.0, splicing, and outputting a fasta format for storage. Trime ends are used for automatically cleaning parts with poor sequencing quality, so that multi-strip bimodal sequences can be eliminated. And (3) splicing a plurality of primer sequencing results, removing the primer sequencing results one by one, regarding a sequence with a correct reading frame as a positive sequence, and finally outputting and storing the positive sequence in a fasta format file.
2) Antibody sequence V region gene analysis. This was done with the IMGT database (http:// www.imgt.org /). This is done using the "analysis your immunoglobulin μ lin (IG) or antibody nucleotide sequences" function under IMGT/VQUEST. The antibody heavy and light chains can be analyzed for V region subtype and identity, CDR1/CDR2/CDR3 length, etc., and the functional reading frame and sequence translation can be confirmed. According to the results of database analysis, the functional (productive) sequence of H/K or H/L derived from the same cDNA is picked up and combined for pairing for subsequent overlapping PCR.
We selected 80 equine single B cells for antibody V region gene PCR, with the following results:
we performed antibody gene amplification from 80 equine single B lymphocytes and obtained 48 antibody heavy chain genes, 6 antibody light chain kappa genes, and 59 antibody light chain lambda genes. The PCR positive rate of the heavy chain gene was 60%, the PCR positive rate of the antibody light chain kappa gene was 7.5%, and the positive rate of the antibody light chain lambda gene was 73.8%. In addition, 45 heavy chain genes in 48 amplified heavy chain genes can be matched with light chain genes, and the matching rate is 95.4%; 4 of the 6 amplified light chain kappa genes can be matched with the heavy chain gene, and the matching rate is 66.7 percent; and 40 of the 59 amplified light chain lambda genes can be matched with the heavy chain gene, and the matching rate is 67.8%.
Our experimental results show that most of the horse antibody light chain genes are lambda genes, and only a few are kappa genes. The results are consistent with the ratio of kappa gene to lambda gene in the horse light chain gene being about 1:20 as reported in the literature (Ford JE, Home WA, Gibson DM. light chain isotope alignment in the horse light chain. characterization of Ig kappa genes. the Journal of immunology.1994; 153: 1099-111).
From the results, the designed PCR primer for the equine single B cell antibody gene can effectively amplify the equine antibody gene to obtain a monoclonal antibody sequence, and the positive rate of obtaining the matchable heavy chain and light chain genes of the antibody is about 56%.
Example 4 construction of antibody heavy and light chain Gene Linear expression System
The variable region gene positive to sequence analysis is put on a linear expression vector for expression.
First, linear Ig expression vector construction (see fig. 4):
1. heavy chain linear Ig expression vector composition: a CMV promoter, an Ig guide region, a heavy chain variable region, a heavy chain constant region and a BGH poly (A) signal peptide fragment.
2. Light chain kappa (κ) linear Ig expression vector composition: a CMV promoter, an Ig leader, a light chain kappa variable region, a light chain kappa constant region, and a BGH poly (a) signal peptide fragment.
3. Light chain lambda (λ) linear Ig expression vector construction: a CMV promoter, an Ig guide region, a light chain lambda variable region, a light chain lambda constant region and a BGH poly (A) signal peptide fragment.
4. The above fragments were assembled together by OverlappingPCR, respectively, using Pyrobest DNA polymerase (Takara), 50. mu.l system containing 1. mu.l of PCR product, 1. mu.l each of forward and reverse primers, C-H heavy chain, C-K light chain kappa, C-L light chain lambda and B-H, B-K, B-L1. mu.l each, 10 XBuffer 5. mu.l, 1. mu.l of dNTPs, and the remainder using ddH2And (4) complementing O. Reaction conditions are as follows: pre-denaturation at 94 ℃ for 3min, followed by 30 PCR cycles, each cycle: 94 ℃ X30 sec, 63 ℃ (V)H) Or 55 deg.C (V)κAnd Vλ) X 30sec, 72 ℃ x 120sec, and finally an extension of 10min at 72 ℃. The PCR product was then purified using a PCR purification kit (Qiagen) and then transfected.
5. CO-transfecting the paired heavy and light chain gene expression vectors with human embryonic kidney HEK 293i or 293F cells with FuGENE (promega E2692) PolyFect (Qiagen, Valencia, Calif.) transfection reagent, replacing fresh medium 6-8 hours after transfection, and replacing fresh medium at 37 ℃ with 5% CO2Culturing in an incubator for 72-96 hours and collecting cell supernatant.
Second, ELISA detection
1. Coating: ELISA plates (costar) were prepared and the antigen was diluted with coating buffer, 100. mu.l of antigen dilution was added to each well. 4 ℃ overnight.
2. And (3) sealing: the cells were washed 5 times using a plate washer (PBST), 250. mu.l of blocking solution was added to each well, and incubated at 37 ℃ for 1 hour.
3. Adding a primary antibody: the cells were washed 5 times using a plate washer (PBST), and cell culture supernatant was added thereto at 100. mu.l per well and incubated at 37 ℃ for 1 hour.
4. Adding a secondary antibody: the plate was washed 5 times with a plate washer (PBST), and a secondary antibody (HRP-labeled goat anti-human IgG) was added to each well in an amount of 100. mu.l, followed by incubation at 37 ℃ for 1 hour.
5. Color development: washing with a plate washing machine (PBST) for 5 times, adding 100 μ l of color developing solution (color developing solution A and B1:1, mixing, and preparing).
6. And (4) terminating: after 5 minutes at room temperature, stop solution was added thereto in an amount of 50. mu.l per well.
7. Reading by a microplate reader, wherein the dual wavelength is 450 and 630 nm.
Enzyme digestion of the Overlapping PCR product with ELISA detection positive is connected to the special expression vector pcDNA of the antibodyTM3.3-
TA 
Kit(InvitrogenTMThe goods number is: k830001).
Example 5 antibody production and purification
Transfection paired heavy and light chain Gene expression vectors HEK 293 or 293T cells were CO-transfected with either calcium phosphate precipitation or PolyFect (Qiagen, Valencia, Calif.) transfection reagent, fresh medium was changed 8-12 hours after transfection, Medium DMEM (Gibco) was added with heat-inactivated 10% fetal bovine serum (Gibco) and penicillin 100U/ml, and 5% CO at 37 deg.C2Culturing in an incubator for 48-72 hours.
The expression supernatant was collected by purification, centrifuged to remove cell debris, and 1mL of pH 8.0, 0.1moL/L phosphate buffer was added and the pH was adjusted to 9.0 with pH 9.0, 1moL/L TRIS-HCL. Adding the cell supernatant to Protein G or Protein A S which has been equilibrated with 0.1moL/L phosphate buffer solution pH 8.0The column was washed with the above buffer in a sepharose CL 4B protein column (GE Healthcare) until no contaminating proteins were detected in the effluent. Eluting with citric acid buffer solution of pH 3.0, collecting eluate, immediately neutralizing with 1moL/L TRIS-HCL buffer solution of pH 8.5, and dialyzing with PBS of pH 7.2 and 0.01M for 72 h. Sampling and measuring OD on an ultraviolet spectrophotometer260、OD280Calculating the protein content, storing at 4 deg.C,
antibody expression detection:
1. sample preparation: mu.l of the antibody solution was added to 5. mu.l of 5 Xsample buffer and the mixture was boiled in a boiling water bath for 5min to denature the protein.
2. Adding TGS buffer solution into the electrophoresis tank, mounting the prepared protein glue (4% -15%) on a rack, placing the rack into the electrophoresis tank, adding fresh TGS into the inner tank until the position of the sample adding hole is submerged, and pulling out the comb.
3. Sequentially spotted, MARKER was stained with the pre-stained protein MARKER (BIO-RAD). Then run at 200V for 30min to allow the protein bands to separate sufficiently.
4. After glue running, cutting off the glue of SDS, placing the glue on the upper right corner of the glue into a staining solution, staining for 2 hours on a shaking table at room temperature, then destaining the glue overnight in the solution or until the glue bottom color is removed until a strip can be seen clearly, and taking a picture.
5. The glue for doing Western cuts off the upper right corner, and the NC membrane, the glue, the conducting strip and the filter paper which are sequentially placed on the membrane rotating instrument, and the membrane corner corresponding to the upper right corner of the glue is cut off by paying attention to the membrane corner, so that no bubble exists between the sandwich layers. And (5) transferring the membrane for 30 min. Then the membrane is taken down and put into a sealing solution and put on a shaking bed to be sealed for 2h at room temperature or overnight at 4 ℃. At the end of blocking, the membrane was washed twice with PBST for 5 minutes each. Add primary antibody (AP-labeled goat anti-human IgG H + K) and incubate for 2H on a shaker at room temperature. Washed twice with PBST for 5 minutes each. Adding a chromogenic substrate, placing the mixture in a dark place for developing for 20 minutes, and taking a picture.
A comparative graph of vector antibody expression levels of the linear expression vector and the plasmid gene was calculated by the excel standard curve method (see FIG. 5).
Example 6 preparation of neutralizing antibodies against equine influenza Virus of Whole equine origin
Equine influenza virus (EIA) is an important infectious disease pathogen that infects equine animals and causes equine influenza. By utilizing the method provided by the invention, the monoclonal neutralizing antibody of the equine anti-EIA, which is combined with the EIA surface membrane protein hemagglutinin HA protein, is prepared, and the antibody can be used for preventing and treating hepatitis C and identifying HCV.
The preparation method comprises the following steps:
(1) detecting and finding horses infected by EIA or horses inoculated with EIA vaccine;
(2) separating mononuclear cells in venous blood of an EIA infected horse or an EIA vaccine inoculated horse, then carrying out memory B cell sorting by using flow cytometry, sorting specific B cells containing HA antibodies by using a fluorescent marker combination, and putting the specific B cells into a 96-well PCR plate to enable each well to contain one B cell;
(3) amplifying the nucleotide fragments of the variable regions of the light chain and the heavy chain of the antibody in the single B cell obtained in the step (2) by using single-cell RT-PCR;
(4) fusing the nucleotide fragments of the variable regions of the light chain and the heavy chain of the antibody obtained in the step (3) into an expression vector containing a human antibody constant region to form a recombinant expression vector, and then introducing the recombinant expression vector into a host cell for expression;
(5) the monoclonal neutralizing antibody of the invention against EIA with binding activity and neutralizing activity is obtained by screening through an antibody screening platform.
The test result of the prepared antibody binding activity shows that the antibody can still be combined with the antigen after the antibody which is expressed and purified is diluted by more than 2000 times, and has extremely strong binding activity. The result of the affinity activity test shows that the EC of the antibody50＝0.003191μg/μL。
Comparative example
In designing primers, the inventors designed multiple sets of primers and repeatedly verified, but the positive rates were all low, and except the primer combination claimed in the present invention, a set of primers with good effect is as follows:
first round nested PCR heavy chain PCR primer
First round nested PCR light chain kappa PCR primer
First round nested PCR light chain lambda PCR primer
Second round nested PCR heavy chain constant region PCR primer
Second round nested PCR light chain kappa constant region PCR primers, second round nested PCR light chain lambda constant region PCR primers, RT-PCR primers the same as in the example.
The result of the antibody V region gene PCR performed by the primer is as follows:
it can be seen that for VH36 bands are amplified, the positive rate is 45%, and the sequencing result shows that the heavy chain gene of the horse antibody is shown (figure 6); for VκAmplifying 0 positive bands, and not using (figure 7); for Vλ27 bands are amplified, the positive rate is 33.75%, and the sequencing result is the horse antibody light chain gene (figure 8). The technical effect is far inferior to that of the examples.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> river university; harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center for animal health and epidemiology)
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Claims (10)
1. Nested PCR primers for amplification of a horse antibody, comprising outer and inner primers for amplification of the heavy and light chains of the antibody:
a) the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 1-7, and the nucleotide sequence of a downstream primer is shown as at least one of SEQ ID NO. 8-12;
b) an outer primer for amplifying a kappa chain of an antibody light chain, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 13-23, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 24;
c) the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 25-33, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 34;
d) the nucleotide sequence of an upstream primer of the inner primer for amplifying the heavy chain of the antibody is shown as SEQ ID NO. 35, and the nucleotide sequence of a downstream primer is shown as at least one of SEQ ID NO. 36-40; and the selection of the downstream primers SEQ ID NO. 36-40 and the selection of SEQ ID NO. 8-12 are in one-to-one correspondence in sequence;
e) an inner primer for amplifying a kappa chain of an antibody light chain, wherein the nucleotide sequence of an upstream primer is shown as SEQ ID NO. 35, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 41;
f) the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 35, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 42.
2. A kit comprising the nested PCR primer of claim 1.
3. The kit of claim 2, further comprising one or more of a reverse transcription primer for synthesizing cDNA from the reverse transcribed B cells, a reverse transcriptase, a random primer, dntps, a DNA polymerase, an amplification buffer, and water;
the nucleotide sequence of the reverse transcription primer comprises at least one of SEQ ID NO 48-49 and SEQ ID NO 43-47, and the selection of the SEQ ID NO 43-47 in the reverse transcription primer is in one-to-one correspondence with the selection of the SEQ ID NO 8-12 in sequence.
4. Use of the nested PCR primers of claim 1, or the kit of claim 2 or 3, for sequencing horse antibodies.
5. A method for producing an equine antibody, comprising:
a) obtaining horse peripheral blood mononuclear lymphocytes, wherein the horse is previously contacted with an antigenic substance;
b) sorting B cells capable of secreting/expressing a substance specifically recognizing the antigen from the peripheral blood mononuclear lymphocytes;
c) sequencing said B cells using the nested PCR primers of claim 1, or the kit of any one of claims 2 to 4, to obtain antibody sequences;
d) constructing a vector comprising the antibody sequence;
e) expressing the vector in a host cell, and recovering the antibody thus produced from the culture medium or from the cultured host cell.
6. The method of claim 5, wherein the peripheral blood mononuclear lymphocytes are collected and isolated from the jugular vein of horse.
7. The method of claim 5, wherein the method of sorting is flow cytometry.
8. The method of claim 7, wherein the B cells are memory B cells and the combination of flow sorted B cell surface markers is:
AqVD-/CD3-/CD14-/CD16-/CD21 +/specific antigen +.
9. A method according to any one of claims 5 to 8, characterized in that the method further comprises step f):
performing one or more of affinity verification, antibody and the binding epitope verification of the antigen substance and antibody purification on the antibody generated in the step e).
10. The method according to any one of claims 5 to 8, wherein the antigenic material is selected from one or more of equine influenza virus, equine infectious anemia virus, African horse sickness virus, equine viral arteritis and equine infectious pleuropneumonia pathogens and equine herpes virus types 1 and 3, or subunits thereof.
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| CN2020107317840 | 2020-07-27 |
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| CN102277451A (en) * | 2011-08-17 | 2011-12-14 | 中国农业科学院哈尔滨兽医研究所 | Universal nest-polymerase chain reaction (PCR) primer group for use in amplification of gp90 gene in main classic strain of equine infectious anemia virus (EIAV) |
| CN106086009A (en) * | 2016-06-17 | 2016-11-09 | 中国农业科学院哈尔滨兽医研究所 | A kind of method utilizing single B cell round pcr to produce full pig resource monoclonal antibody |
| CN107760690A (en) * | 2017-10-25 | 2018-03-06 | 珠海泰诺麦博生物技术有限公司 | A kind of preparation method and application of high flux human antibody |
| WO2019215112A1 (en) * | 2018-05-09 | 2019-11-14 | Lifetaq-Analytics Gmbh | In situ cell analysis in cell culture system |
| CN111378650A (en) * | 2018-12-28 | 2020-07-07 | 先声生物医药科技有限公司 | Combined primer for nested amplification and application thereof |
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2020
- 2020-07-27 CN CN202010731784.0A patent/CN111876476A/en not_active Withdrawn
- 2020-12-16 CN CN202011488629.7A patent/CN112501273B/en active Active
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| CN102277451A (en) * | 2011-08-17 | 2011-12-14 | 中国农业科学院哈尔滨兽医研究所 | Universal nest-polymerase chain reaction (PCR) primer group for use in amplification of gp90 gene in main classic strain of equine infectious anemia virus (EIAV) |
| CN106086009A (en) * | 2016-06-17 | 2016-11-09 | 中国农业科学院哈尔滨兽医研究所 | A kind of method utilizing single B cell round pcr to produce full pig resource monoclonal antibody |
| CN107760690A (en) * | 2017-10-25 | 2018-03-06 | 珠海泰诺麦博生物技术有限公司 | A kind of preparation method and application of high flux human antibody |
| WO2019215112A1 (en) * | 2018-05-09 | 2019-11-14 | Lifetaq-Analytics Gmbh | In situ cell analysis in cell culture system |
| CN111378650A (en) * | 2018-12-28 | 2020-07-07 | 先声生物医药科技有限公司 | Combined primer for nested amplification and application thereof |
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