CN117384277A - Humanized IgA antibody heavy chain expression plasmid and application thereof - Google Patents
Humanized IgA antibody heavy chain expression plasmid and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- 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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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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Abstract
The invention discloses a human IgA antibody heavy chain expression plasmid, which contains a human IgA (IgA 1/IgA 2) heavy chain constant region sequence and a heavy chain variable region homologous arm sequence, and a human IgA antibody heavy chain variable region coding gene is integrated into the human IgA antibody heavy chain expression plasmid through a homologous sequence carried by the human IgA antibody heavy chain coding gene and a homologous recombination method. The heavy chain expression plasmid of the humanized IgA antibody provided by the invention can be used for preparing the humanized IgA antibody by cotransfection with the light chain expression plasmid of the antibody, and has the advantages of high efficiency, time-saving preparation and high accuracy.
Description
Technical Field
The invention discloses a plasmid, and belongs to the technical field of nucleic acid molecules.
Background
Antibodies (abs) are important effector molecules that mediate liquid immunity, and are immunoglobulins (igs) that are produced by the immune system under antigen stimulation by B cells or plasma cells that proliferate and differentiate from memory B cells and that bind specifically to the corresponding antigen, and are mainly distributed in serum, as well as in tissue fluids, exocrine fluids, and certain cell membrane surfaces. The antibody has a heavy chain molecular weight of about 50-75 kDa and is composed of 450-550 amino acid residues, which can be classified into 5 classes according to the difference of heavy chain constant regions: mu, gamma, alpha, delta and epsilon chains, the different heavy and light chains make up the complete antibody molecule, known as IgM, igG, igA, igD and IgE, respectively.
IgA is of both serotypes and secretes. The serotypes are monomers and mainly exist in serum and account for 10% -15% of the total amount of serum immunoglobulin. Secretory IgA (sIgA) is a dimer, linked by a J chain, containing secretory components (secretory component, SC) and secreted into the exocrine fluid via mucosal epithelial cells. sites of sIgA synthesis and secretion are found primarily in the intestinal tract, respiratory tract, breast, salivary and lacrimal glands, and are found primarily in gastrointestinal and bronchial secretions, colostrum, saliva and tears. sIgA is a main antibody class in exocrine fluid, participates in local immunity of mucous membrane, and prevents pathogens from adhering to the cell surface through combination with corresponding pathogenic microorganisms, thereby playing an important role in local anti-infection and being the 'frontier' of organism anti-infection. sIgA also has a toxin-neutralizing effect on mucosal surfaces. Neonatal susceptibility to respiratory tract and gastrointestinal tract infections may be associated with insufficient IgA synthesis. Infants can obtain sIgA from mother's colostrum, an important natural passive immunity.
In humans, igA is classified into two subtypes IgA1 and IgA2, with about 90% of serotypes IgA being the IgA1 subtype and 10% being the IgA2 subtype, with the distribution ratio of the secretory IgA subtype to the secretory IgA subtype depending on different mucosal sites, igA1 is commonly found in nasal, bronchial, gastric and intestinal mucosa, igA2 is mainly present in the large intestine and female genital tract. The hinge region of IgA1 is longer than that of IgA2, and in addition, there are differences in the glycosylation levels of IgA1 and IgA2, and preclinical studies are being conducted on IgA of both subtypes.
The 2022 global antibody drug market size reaches 2200 billions of dollars, and the indications cover the fields of autoimmune diseases, tumors, infectious diseases, etc. thanks to the sales contribution of at least 125 antibody drugs. Artificial preparation of antibodies is an effective way to obtain antibodies in large quantities, and with the development of molecular biology, one has been able to prepare genetically engineered antibodies by antibody engineering techniques. The antibody discovery strategy can obtain natural paired fully human antibody genes from hybridoma technology, phage library technology, transgenic mouse technology, single cell PCR technology and single cell sequencing technology. The existing antibody therapeutic drugs are mainly of IgG type, and in recent years, igA has been attracting more and more attention as a novel therapeutic antibody. Although there is no IgA drug on the market in batches at present, a plurality of preclinical researches show that IgA has great application potential in the fields of mucosal anti-infection, autoimmune diseases and the like, and is hopeful to become a new therapeutic means. The construction of the heavy chain expression plasmid of the humanized IgA antibody provides a rapid and convenient method for researching therapeutic IgA medicaments.
The invention aims to provide a plasmid capable of conveniently and rapidly cloning and expressing a heavy chain of a human IgA antibody, and further provides a method for expressing the heavy chain of the human IgA antibody by using the plasmid.
Disclosure of Invention
In view of the above, the present invention firstly provides a human IgA antibody heavy chain expression plasmid in whichThe expression plasmid sequentially contains an antibiotic resistance screening gene, a CMV promoter, an antibody leader sequence element carrying a Kozak sequence element and an antibody heavy chain signal peptide, a connecting element, a heavy chain constant region, a post-transcriptional regulatory sequence and a polyA tail, wherein the downstream of the antibody leader sequence element is provided withHpa I cleavage site, located upstream of the heavy chain constant regionEcoR V cleavage site, said connecting element being located atHpa I cleavage siteEcoRBetween V enzyme cutting sites, the human IgA antibody heavy chain expression plasmid passes throughHpa I andEcoRand after the V double enzyme digestion is carried out, the two linearization ends are used for carrying out homologous recombination on the homologous recombination arms at the 5 '-end of the coding gene of the heavy chain variable region of the human IgA antibody and the homologous recombination arms at the 3' -end of the coding gene of the heavy chain variable region of the human IgA antibody respectively, the sequence of the homologous recombination arms at the 5 '-end of the coding gene of the heavy chain variable region of the human IgA antibody is shown as SEQ ID NO.13, the homologous recombination arms at the 3' -end of the coding gene of the heavy chain variable region of the human IgA antibody is shown as SEQ ID NO.14, and the sequence of the leader sequence element of the antibody is shown as SEQ ID NO. 4.
In a preferred embodiment, the heavy chain constant region encoding gene is as shown in SEQ ID NO.1 or as shown in SEQ ID NO. 2. The SEQ ID NO.1 is an IgA1 heavy chain sequence, and a human IgA antibody heavy chain expression plasmid with the sequence is named as: pAb-IGHA1; the SEQ ID NO.2 is an IgA2 heavy chain sequence, and a human IgA antibody heavy chain expression plasmid with the sequence is named as: pAb-IGHA2.
In another preferred embodiment, a multiple cloning site is provided between the heavy chain constant region and the post-transcriptional regulatory sequence, the sequence of which is shown in SEQ ID NO. 7.
In yet another preferred embodiment, the post-transcriptional regulatory sequence is a woodchuck hepatitis virus post-transcriptional regulatory sequence (GenBank: MQ 208857.1).
In a preferred embodiment, the antibiotic resistance selection gene is an ampicillin resistance selection gene.
In another preferred embodiment, the sequence of the linker element is shown in SEQ ID NO.5, and the linking element is designed to link the antibody leader element to the heavy chain constant region to maintain plasmid circularization prior to cleavage of the vector in the present invention, and the linker element is released after cleavage and linearized plasmid recovery is recovered. Therefore, the specific sequence content of the linking element is irrelevant to the function of the human IgA antibody heavy chain expression plasmid of the present invention, and any DNA fragment capable of fulfilling the above function can be used as the linking element of the present invention.
Secondly, the invention provides a preparation method of the humanized IgA antibody heavy chain expression plasmid, which comprises the following steps:
(1) Constructing a eukaryotic cell expression vector containing an antibiotic resistance selection gene, a CMV promoter, a post-transcriptional regulatory sequence, a polyA tail and a multiple cloning site, said eukaryotic cell expression vector being designated pAb in the present invention; wherein, downstream of the CMV promoter is arrangedEcoR I cleavage site, upstream of the post-transcriptional regulatory sequenceNot I cleavage site, the eukaryotic cell expression vector is subjected to the following steps ofEcoR I and is provided withNot I, two linearization ends are used for homologous recombination after double enzyme digestion;
(2) Constructing a linear fragment containing a 5' -terminal homologous recombination arm, an antibody leader sequence element, a connecting element, a heavy chain constant region and a 3' -terminal homologous recombination arm, wherein the 5' -terminal homologous recombination arm containsEcoR I cleavage site, the 3' -homologous recombination arm containsNot I cleavage site, downstream of the antibody leader sequence element is provided withHpa I cleavage site, located upstream of the heavy chain constant regionEcoR VAn enzyme cutting site;
(3) UsingEcoR I and is provided withNot I, carrying out double enzyme digestion on the eukaryotic cell expression vector obtained in the step (1) to obtain a double enzyme digestion linearization vector, wherein the double enzyme tangent linearization vector is used for homologous recombination;
(4) And (3) carrying out homologous recombination on the linearization vector obtained by double digestion in the step (3) and the linear fragment constructed in the step (2) to obtain the humanized IgA antibody heavy chain expression plasmid.
In a preferred embodiment, the sequence of the 5 'homologous recombination arm in step (2) is shown in SEQ ID NO.3 and the sequence of the 3' homologous recombination arm is shown in SEQ ID NO. 6.
In a more preferred embodiment, the linear fragment in step (2) is designated IGHA1 in the present invention as shown in SEQ ID NO.8 or IGHA2 in the present invention as shown in SEQ ID NO. 9.
In a specific embodiment of the present invention, the eukaryotic cell expression plasmid pAb described in the step (1) is constructed by using the commercial vector pcDNA3.4TOPO as a starting vector. Wherein use is made ofXbaI andEcor V after double cleavage of pcDNA3.4TOPO, the synthesis containedEcoR I、NotThe homologous recombination sequence of the I and other enzyme cutting sites with the sequence shown as SEQ ID NO.16 is inserted into a linearized pcDNA3.4TOPO vector through homologous recombination to obtain eukaryotic cell expression plasmid pAb.
In a specific embodiment of the invention, the plasmid pAb comprises: (1) The human cytomegalovirus early promoter element (CMV), a stronger promoter for expression in numerous mammalian cells, has a wide host range of applications, including chinese hamster ovary cells (Chinese Hamster Ovary, CHO); (2) The woodchuck hepatitis virus posttranscriptional regulatory sequence (WPRE) is an element for enhancing expression, and can increase the expression efficiency of exogenous fragments; (3) Poly A tail gene (poly A), which is helpful for mRNA transport from nucleus to cytoplasm, and prevents mRNA from being degraded by ribozyme in cells, and enhances mRNA stability; (4) Ampicillin resistance gene on LB plates containing ampicillin antibiotics, positive bacterial clones were able to grow with successful transformation. Plasmid pAb removes the original pcDNA3.4TOPO vectorEcoR V enzyme cutting site, which is convenient for the subsequent carrier construction.
The invention also provides a method for preparing the heavy chain of the human IgA antibody by using the expression plasmid of the heavy chain of the human IgA antibody, which comprises the following steps:
(1) UsingHpa I andEcoRv, carrying out double enzyme digestion on the heavy chain expression plasmid of the humanized IgA antibody to obtain a linearization plasmid;
(2) Construction of a humanized IgA antibody to be expressedA linear fragment of a chain variable region, wherein the 5' -end of the linear fragment of a heavy chain variable region is provided with a linearization plasmid obtained in the step (1)Hpa I, a 5 '-end homologous recombination arm of homologous recombination occurs at the enzyme cutting end, and a linearization plasmid obtained in the step (1) is arranged at the 3' -end of the heavy chain variable region linear fragmentEcoRA 3' -end homologous recombination arm in which homologous recombination occurs at the V enzyme cleavage end;
(3) Carrying out homologous recombination on the linearization plasmid obtained in the step (1) and the heavy chain variable region linear fragment obtained in the step (2) to obtain a human IgA antibody heavy chain expression plasmid containing a heavy chain variable region of a human IgA antibody to be expressed;
(4) Expressing the heavy chain expression plasmid of the human IgA antibody obtained in the step (3), and recovering an expression product.
In a preferred embodiment of the present invention, the heavy chain variable region linear fragment of the human IgA antibody in the construction step (2) is amplified by PCR, wherein the upstream primer of the PCR amplification is formed by connecting a 5 '-end homologous recombination arm and two sections of a heavy chain variable region specific binding region in series, the downstream primer of the PCR amplification is formed by connecting a 3' -end homologous recombination arm and two sections of a heavy chain variable region specific binding region in series, the sequence of the 5 '-end homologous recombination arm of the upstream primer of the PCR amplification is shown as SEQ ID NO.13, and the sequence of the 5' -end homologous recombination arm of the downstream primer of the PCR amplification is shown as SEQ ID NO. 14.
The 6-base (CAGTGT) of the 3 '-end of the 5' -end homologous recombination arm sequence is added into the primer for filling inHpa I cleavage of the human IgA antibody heavy chain expression plasmid results in deletion of the 6 th base of the antibody leader sequence; the two bases (GC) at the 3 '-end of the 3' -end homologous recombination arm sequence are added to the primer to complement the two bases at the beginning of the deleted antibody constant region due to EcoR V cleavage of the human IgA antibody heavy chain expression plasmid.
In a specific method of the invention for preparing an anti-novel coronavirus S protein antibody ZW2G10 (CN 114031685A), the sequence of the upstream primer for amplifying the heavy chain variable region of the antibody ZW2G10 is shown as SEQ ID NO.10, and the sequence of the downstream primer for amplifying the heavy chain variable region of the antibody ZW2G10 is shown as SEQ ID NO. 11.
Finally, the invention provides a kit for preparing a heavy chain of a humanized IgA antibody, comprising:
(1) The above-mentioned humanized IgA antibody heavy chain expression plasmid;
(2) The upstream primer and the downstream primer for PCR amplification of the heavy chain variable region of the human IgA antibody are formed by connecting two sections of a 5 '-end homologous recombination arm and a heavy chain variable region specific binding region in series, the downstream primer for PCR amplification is formed by connecting two sections of a 3' -end homologous recombination arm and a heavy chain variable region specific binding region in series, the sequence of the 5 '-end homologous recombination arm of the upstream primer for PCR amplification is shown as SEQ ID NO.13, and the sequence of the 5' -end homologous recombination arm of the downstream primer for PCR amplification is shown as SEQ ID NO.14
(3) The agent for homologous recombination between the expression plasmid for the heavy chain of the human IgA antibody described in (1) and the heavy chain variable region of the human IgA antibody described in (2) is a conventional homologous recombination agent in the art, and the homologous recombination can be carried out by the homologous recombination agent of the present invention.
In a specific kit for preparing the anti-novel coronavirus S protein antibody ZW2G10 (CN 114031685A), the sequence of an upstream primer for amplifying the heavy chain variable region of the antibody ZW2G10 is shown as SEQ ID NO.10, and the sequence of a downstream primer for amplifying the heavy chain variable region of the antibody ZW2G10 is shown as SEQ ID NO. 11.
The invention constructs a heavy chain expression plasmid of a human IgA antibody, and introduces synonymous mutation into the heavy chain expression plasmid of the human IgA antibodyHpaI andEcoRV restriction enzyme site, can insert the antibody variable region into IGHA carrier on the basis of not changing the heavy chain variable region sequence of antibody, is suitable for carrier construction of all heavy chain variable regions of human antibody, and the homology arm in primer for amplifying antibody variable region is also the core invention point of the invention.
Compared with the traditional method, the carrier and the method for preparing the human IgA antibody heavy chain provided by the invention have the advantages that the following scheme is adopted:
1. time saving: the signal peptide and the constant region are directly constructed on the carrier, and the process of overlapping extension PCR to obtain the full-length antibody gene is not needed, so that the problem of sequence mutation caused by the PCR process is effectively avoided.
2. High flux: the antibody variable region sequences obtained by single cell PCR technology or single cell sequencing technology can be directly recovered by a 96-well plate and connected to a double-digested vector by homologous recombination.
3. Simple and convenient: the colony after homologous recombination is directly selected for sequence determination confirmation without cloning PCR identification, and the accuracy is more than 90%.
4. The vector provided by the invention can be used for constructing IgA type antibody expression vectors of all heavy chain variable regions of human antibodies, and is a universal type human IgA antibody heavy chain expression vector.
Drawings
FIG. 1 shows a schematic diagram of the construction flow of pAb-IGHA expression vectors;
FIG. 2 shows the electrophoretogram of pAb-IGHA1 and pAb-IGHA2 plasmids;
FIG. 3 is a schematic diagram of the construction flow of an antibody expression vector of the example;
FIG. 4 shows an example antibody heavy chain variable region nucleic acid electrophoresis pattern;
FIG. 5 is an SDS-PAGE electrophoresis of purified example antibodies;
FIG. 6 shows an ELISA analysis of example antibodies.
Detailed Description
The invention is further described in the following specific examples of construction procedures for a recombinant chain expression plasmid of antibody ZW2G10-IgA against novel coronavirus S protein, and the advantages and features of the invention will be more apparent from the description. These examples are only exemplary and do not limit the scope of the invention in any way, which is defined by the claims.
The reagents involved in the examples
Hpa I、EcoR V、EcoR I and is provided withNotI restriction endonuclease and homologous recombinase NEBuilder are available from NEB Co. The gel recovery kit was purchased from OMEGA company. HiTrap LambdaFabSelect from Cytiva. HRP-labeled rabbit anti-human Lambda chain secondary antibodies were purchased from Abcam corporation. The 2X Phanta Max Master Mix polymerase system was purchased from Nanjinozan corporation. Expi293F cell culture medium and transfection reagents were purchased from Thermo Fisher. Example antibody ZW2G10 (see CN114031685 a) primers and sequencing work for the heavy chain variable region gene were all synthesized and done by the biological company limited. Top10 competent cells were purchased from Bomaide.
Construction examples: design and construction of heavy chain expression plasmid of universal humanized IgA antibody
1. Construction of eukaryotic expression plasmid pAb
The adoption of a suitable expression vector is the key for successful expression of exogenous genes, and the expression vector must be provided with: strong promoters, highly efficient transcription termination sequences, expression enhancing introns or elements.
The design of the invention is based on eukaryotic cell expression plasmid pAb constructed in the early stage of the laboratory, the plasmid is transformed from a commercial vector pcDNA3.4TOPO (Thermofisher, A14697), and the transformation process is as follows:
the synthesis comprisesEcoR I、NotI, etc. (SEQ ID NO. 16), usingXbaI andEcor V after double cleavage of pcDNA3.4 TOPO, a linearized pcDNA3.4 TOPO vector was inserted by homologous recombination to give eukaryotic cell expression plasmid pAb. In SEQ ID NO.16, CCGATCCAGCCTCCGGACTCTAGAGAATTCAATTGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCGCGCCCAAGCTTGGGAGGGTTCGATCCCTACCGGTTAGTAATGAGTTTAATCTCGACAATCAACCTCTGG the darkened sequence is pcDNA3.4 TOPO vector homology arm and the underlined sequence isEcoR I and is provided withNotI cleavage site, italic pcDNA3.4TOPO original sequence, other cleavage site also includes:Xba i cleavage site (19 th to 24 th positions),Bamh I cleavage site (60 th to 65 th positions),Hind III cleavage site (72-77).
The plasmid pAb obtained by homologous recombination comprises: (1) Human cytomegalovirus early promoter element (CMV), which is a relatively strong promoter for expression in a wide range of mammalian cells, and which has a wide range of host applications, including chinese hamster ovary cells (Chinese Hamster Ovary, CHO); (2) The woodchuck hepatitis virus posttranscriptional regulatory sequence (WPRE, genBank: MQ 208857.1) is an element for enhancing expression, and can increase the expression efficiency of exogenous fragments; (3) Poly A tail gene (poly A), which is helpful for mRNA transport from nucleus to cytoplasm, and prevents mRNA from being degraded by ribozyme in cells, and enhances mRNA stability; (4) Ampicillin resistance gene on LB plates containing ampicillin antibiotics, positive bacterial clones were able to grow with successful transformation. Plasmid pAb removes the original pcDNA3.4TOPO vectorEcoR V enzyme cutting site, which is convenient for the subsequent carrier construction.
2. Construction of the sequence of interest for homologous recombination with pAb
(1) IgA1 heavy chain constant region sequences (http:// www.imgt.org/ligmdb/view. Actionid=AK 027379, see SEQ ID NO. 1) and IgA2 heavy chain constant region sequences (http:// www.imgt.org/ligmdb/view. Actionid=BC 066594, see SEQ ID NO. 2) are found in the IMGT database, respectively.
(2) Under the condition of not changing the amino acid sequence of the heavy chain constant region of the IgA antibody, the invention introduces an enzyme cutting site through synonymous mutation to modify the vector. The general biotechnology company of the delegated Anhui synthesizes a target sequence for inserting pAb plasmid, and the design of the target sequence is as follows: from the 5 'end, there is first the pAb vector 5' homology arm, followed by the gene encoding the antibody heavy chain leader sequence downstream ofHpaI (GTT AAC) andEcor V (GAT A C) cleavage site, wherein an irrelevant sequence is arranged between the two cleavage sites, which is convenient for recovery after the vector is cleaved in cloning application, and the vector is used for cloningEcoR V (GAT A C) downstream of the cleavage site is the gene encoding the IgA1 or IgA2 heavy chain constant region, and finally the 3' homology arm of the pAb vector. IGHA1 has a sequence shown in SEQ ID NO.8, IGHA2 has a sequence shown in SEQ ID NO.9, and the synthetic sequence has the functions shown in Table 1:
TABLE 1 IGHA 1/A2 sequence annotation
3. Homologous recombination of target sequence and pAb vector
The synthesized target sequence is prepared through the designed homology arm and channel in the sequenceEcoR I and is provided withNotAnd (3) carrying out homologous recombination on the pAb vector subjected to enzyme digestion to obtain universal plasmids pAb-IGHA1 and pAb-IGHA2 containing the IgA heavy chain constant region, wherein the vector maps are shown in figure 1. The method comprises the following specific steps:
(1) pAb plasmid double enzyme digestion
The cleavage system is described in Table 2:
TABLE 2 double cleavage reaction System
The enzyme was digested for 15 min at 37℃and gel was recovered and purified using OMEGA Gel Extraction Kit. The process is as follows: performing agarose gel electrophoresis on the enzyme-digested product, cutting off gel containing a target gene, cutting off redundant parts as far as possible, retaining a target strip, placing the cut gel block into a 1.5 mL centrifuge tube, weighing the gel block, adding Binding Buffer (the mass of the gel block is 1 mg, the volume of the gel block can be regarded as 1 mu L, and adding Binding Buffer solution of 1 mu L) into the gel block, and placing the gel block into a 37 ℃ metal bath until the gel block is completely dissolved; adding the gel block fully dissolved in the Binding Buffer into an adsorption column, centrifuging at 12000 rpm for 1 min, discarding the waste liquid in the collecting pipe, placing the adsorption column into the collecting pipe again, adding 300 mu L of Binding Buffer into the adsorption column, centrifuging at 12000 rpm for 1 min, and discarding the waste liquid in the collecting pipe; 700. Mu.L of Wash Buffer was added to the above-mentioned column to which DNA fragments had been bound, and the column was centrifuged at 12000 rpm for 1 min, the waste liquid in the collection tube was discarded, and the column was replaced in the collection tube, and this step was repeated twice. Placing the adsorption column in a collecting tube again, centrifuging at 12000 rpm for 2 min, and drying at room temperature for 5-10 min to evaporate ethanol in Wash Buffer; placing the above adsorption column into a new 1.5 mL centrifuge tube, and dripping 40 μl ddH at 60deg.C into the center of the adsorption film 2 O was incubated for 5 min to allow the DNA to dissolve well in ddH 2 After O, DNA was collected by centrifugation at 12000 rpm for 2 minAnd (3) liquid.
(2) Homologous recombination of the sequence of interest with the pAb plasmid
NEBuilder assembly kit was used and the procedure was followed to ligate the fragments of interest. The homologous recombination reaction system is described in Table 3.
TABLE 3 homologous recombination reaction System
The mixture was gently mixed on ice, connected at 50℃for 15 min, and the system was then placed on ice. 5. Mu.L of the ligation product was added to Top10 competent cells, and after ice-bath for 30 min and heat shock for 60 s at 42℃and ice-bath for 2 min, the mixture was shake cultured for 20 min and centrifuged at 4000 rpm for 3 min, resuspended in 100. Mu.L of LB medium, spread on ampicillin-resistant LB plates, and incubated overnight at 37 ℃.6 monoclonal colonies with good growth state are picked up by a sterile gun head and streaked on an ampicillin resistance LB plate, and placed in a 37 ℃ incubator for culture of 6 h, and sequencing verification is carried out on the monoclonal colonies. The plasmid with correct sequence was extracted.
4. Agarose nucleic acid electrophoresis confirmed the above plasmid: 1 g agarose is dissolved in 100mL 1 xTAE, heated to boiling, and then added with 5. Mu.L GV-II nucleic acid dye to shake uniformly, the electrophoresis apparatus is assembled, and the electrophoresis gel is poured into and waits for solidification for about 30 min. The comb was pulled out, 5. Mu.L of 500 ng plasmid was mixed with 1. Mu.L of 6 Xloading buffer and added into the well, DL-10000 DNA marker was added with 7. Mu.L, 180V to carry out electrophoresis, after 20 min, the gel was run out, the gel was put on a plastic film, and the nucleic acid imager was imaged. The full length of pAb-IGHA1 is 7738 bp, the full length of pAb-IGHA2 is 7699bp, and the band size of the nucleic acid electrophoretogram is as expected, as shown in FIG. 2.
5. Obtaining the heavy chain expression plasmid of the universal humanized IgA antibody and preserving the heavy chain expression plasmid for later use.
Application examples: preparation of antibody ZW2G10 of novel coronavirus S protein
The construction of the IgA1/IgA2 heavy chain expression plasmid of antibody ZW2G10 (CN 114031685A) against the novel coronavirus S protein is exemplified as shown in FIG. 3. ZW2G10 is an antibody with a Lambda type light chain, and the ZW2G10 IgA1/IgA2 heavy chain expression plasmid and the light chain expression plasmid are co-transfected and purified to obtain antibodies ZW2G10-IgA1 and ZW2G10-IgA2.
1. Restriction linearization and recovery of plasmids pAb-IGHA1 and pAb-IGHA2
Plasmids pAb-IGHA1 and pAb-IGHA2 were double digested with the corresponding restriction enzymes and linearized. The double cleavage reaction system is shown in Table 4.
TABLE 4 double cleavage reaction System
The enzyme was digested for 15 min at 37℃and gel was recovered and purified using OMEGA Gel Extraction Kit. The process is as follows: performing agarose gel electrophoresis on the enzyme-digested product, cutting off gel containing a target gene, cutting off redundant parts as far as possible, retaining a target strip, placing the cut gel block into a 1.5 mL centrifuge tube, weighing the gel block, adding Binding Buffer (the mass of the gel block is 1 mg, the volume of the gel block can be regarded as 1 mu L, and adding Binding Buffer solution of 1 mu L) into the gel block, and placing the gel block into a 37 ℃ metal bath until the gel block is completely dissolved; adding the gel block fully dissolved in the Binding Buffer into an adsorption column, centrifuging at 12000 rpm for 1 min, discarding the waste liquid in the collecting pipe, placing the adsorption column into the collecting pipe again, adding 300 mu L of Binding Buffer into the adsorption column, centrifuging at 12000 rpm for 1 min, and discarding the waste liquid in the collecting pipe; 700. Mu.L of Wash Buffer was added to the above-mentioned column to which DNA fragments had been bound, and the column was centrifuged at 12000 rpm for 1 min, the waste liquid in the collection tube was discarded, and the column was replaced in the collection tube, and this step was repeated twice. Placing the adsorption column in a collecting tube again, centrifuging at 12000 rpm for 2 min, and drying at room temperature for 5-10 min to evaporate ethanol in Wash Buffer; placing the above adsorption column into a new 1.5 mL centrifuge tube, and dripping 40 μl ddH at 60deg.C into the center of the adsorption film 2 O was incubated for 5 min to allow the DNA to dissolve well in ddH 2 After O, the DNA solution was collected by centrifugation at 12000 rpm for 2 min.
2. Amplification of antibody heavy chain variable region genes
By single-thinAfter obtaining the variable region gene of the antibody by cell PCR or single cell sequencing, PCR amplification was performed on the antibody ZW2G10 heavy chain variable region gene (see SEQ ID NO. 12) by using a 2X Phanta Max Master Mix system through heavy chain variable region primers. The heavy chain variable region primer comprises 5' homology arms (SEQ ID NO.13, CTAATTTTAAAAGGTGTT) of linearization vector pAb-IGHA1/IGHA2, respectivelyCAGTGT) And 3' homology arm (SEQ ID NO.14, GAAGACCTTGGGGCTGGTCGGGGAT)GC) Wherein, the 5' end primer sequence is shown in SEQ ID NO.10, and the sequence is as follows:CTAATTTT AAAAGGTGTTCAGTGTGAAGTGCAGCTGGTGGAGTCT underlined is pAb-IGHA1/IGHA2HpaI upstream homology arm, bolded 6 bases (CAGTGT) for alignment of antibody leader sequence; the 3' -end primer sequence is shown in SEQ ID NO.11, and the sequence is as follows:GAAGACCTTGGGGCTGGTCGGGGATGCTGAGGAGACGGTGACCGT underlined is pAb-IGHA1/IGHA2EcoThe homology arm at the downstream of RV is thickened by 2 basesGC) For complementing the start of the IgA constant region.
The PCR reaction system is as follows: 2X Phanta Max Master Mix. Mu.L, 5 'and 3' primers were 2.5. Mu.L (10. Mu.M), ZW2G10-IgG plasmid template 10 ng, respectively, and water was made up to 50. Mu.L. The reaction conditions are as follows: pre-denaturation at 95℃for 3 min, followed by 15 s at 95℃for 15 s, 20 s at 60℃for 72℃for 30 cycles and finally extension at 72℃for 5 min. Gel electrophoresis results showed that the antibody heavy chain variable region gene (about 400 bp) was well amplified and the size was as expected, as shown in fig. 4.
3. Recombinant transformation and positive clone screening
NEBuilder assembly kit was used and the procedure was followed to ligate the fragments of interest. The homologous recombination reaction system is shown in Table 5.
TABLE 5 homologous recombination reaction System
The mixture was gently mixed on ice, connected at 50℃for 15 min, and the system was then placed on ice. 5. Mu.L of the ligation product was added to Top10 competent cells, and after ice-bath for 30 min and heat shock for 60 s at 42℃and ice-bath for 2 min, the mixture was shake cultured for 20 min and centrifuged at 4000 rpm for 3 min, resuspended in 100. Mu.L of LB medium, spread on ampicillin-resistant LB plates, and incubated overnight at 37 ℃.6 monoclonal colonies with good growth state are picked up by a sterile gun head and streaked on an ampicillin resistance LB plate, and then placed in a 37 ℃ incubator for culture of 6 h, and sequencing verification is carried out on the monoclonal colonies (the sequence of a sequencing primer is shown as SEQ ID NO. 15). By comparison, the ZW2G10-IgA1 and ZW2G10-IgA2 heavy chain sequences are 6 according to expectations, and the positive rate is 100%.
4. Eukaryotic expression and purification of antibodies
Firstly, extracting plasmids with correct sequencing; co-transfection of paired light and heavy chain plasmids into Expi293F cells, 5% CO 2 Continuously culturing for 120 hours at 37 ℃ and 120 rpm; collecting the cultured cell supernatant, centrifuging at 4deg.C and 600 g for 5 min, centrifuging at 8000 g for 10 min, filtering with 0.22 μm filter membrane, and standing at 4deg.C. The antibody was then purified using HiTrap LambdaFabSelect affinity column: the affinity column is connected into an AKTA pure of a protein purifier at a low flow rate, then the affinity column is washed clean by washing pure water with 5 column volumes, 20% ethanol in the affinity column is washed clean, and then the balancing solution PBS is taken away until the UV value is stable, and then the UV setting is zeroed. The filtered cell supernatants were loaded at the recommended flow rates and after the column was collected, equilibration with PBS was continued until the UV values were stable. The antibody in the affinity column was eluted with 0.1M glycine at pH2.7, the elution peak was collected and the eluate was neutralized to pH 6.0 with Tris-HCl solution at pH 9.0. The successfully purified antibodies were concentrated and pipetted into PBS through a 50 kDa ultrafiltration tube at 4℃4000 g.
SDS-PAGE analysis of the purified antibodies was consistent with the IgG type antibody of ZW2G10, and the antibodies of the examples were well expressed and purified. The reducing Buffer contained a reducing agent DTT which opened disulfide bonds in the antibody, allowing separation of the antibody heavy and light chains, igA1 and IgA2 antibodies heavy chains of approximately 60-65 kDa and light chains (the sequence of the light chains and the vector is described in patent ZL 202210023619.9) of approximately 25 kDa, with the band sizes being as expected (FIG. 5).
5. ELISA determination of binding Activity of purified antibodies ZW2G10-IgA1 and ZW2G10-IgA2
In 96-well microplatesmu.L of antigen (New coronavirus B.1.1.529S) containing 2. Mu.g/mL was added to each well ECD Protein), incubating overnight at 4 ℃; after washing the microplate 3 times with PBST using a plate washer, 100 μl of blocking solution was added to each well and incubated at 37 ℃ for 1 h; after washing the microplate 3 times with PBST using a plate washer, diluting the antibody to a concentration of 4 mug/mL as a primary well, continuously carrying out 3-time gradient dilution, setting three compound wells per well of 100 mu L, and incubating at 37 ℃ for 1 h; after washing the microwell plates 3 times with PBST using a plate washer, 100 μl of HRP-labeled anti-human antibody lambda chain murine mab (1:5000 dilution) was added to each well and incubated at 37 ℃ for 1 h; after washing the microplate 3 times with PBST using a plate washer, 100. Mu.L of TMB single-component substrate solution was added to each well, incubated at 37℃in the absence of light for 3 min, and then 50. Mu.L of stop solution was added to each well to terminate the chromogenic reaction; using a microplate reader, detecting the absorbance at 450 nm wavelengths with 630 nm as reference wavelength, absorbance = OD of the chromogenic sample 450-630 The method comprises the steps of carrying out a first treatment on the surface of the The resulting data were processed, analyzed using GraphPad Prism8 software and plotted (fig. 6). Measurement of EC of ZW2G10-IgG, ZW2G10-IgA1 and ZW2G10-IgA2 50 57.53 ng/mL, 43.55 ng/mL and 164.7. 164.7 ng/mL, respectively, which indicated that ZW2G10-IgG, ZW2G10-IgA1 and ZW2G10-IgA2 were all against S of the novel coronavirus B.1.1.529 ECD The protein has good binding activity, and the binding function of the IgA antibody and the antigen is not affected through the change of a heavy chain constant region.
Claims (12)
1. A human IgA antibody heavy chain expression plasmid is characterized in that the human IgA antibody heavy chain expression plasmid sequentially comprises an antibiotic resistance screening gene, a CMV promoter, an antibody leader sequence element carrying a Kozak sequence element and an antibody heavy chain signal peptide, a connecting element, a heavy chain constant region, a post-transcriptional regulatory sequence and a polyA tail, wherein the downstream of the antibody leader sequence element is provided withHpa I cleavage site, located upstream of the heavy chain constant regionEcoRV cleavage site, the connecting element is positioned atHpa I cleavage siteEcoBetween RV cleavage sites, the human IgA antibody heavy chain expression plasmid passes throughHpa I andEcoafter RV double cleavage the linker element is removed, two linearization ends are used for eachHomologous recombination is carried out on the homologous recombination arm at the 5 '-end of the coding gene of the heavy chain variable region of the human IgA antibody and the homologous recombination arm at the 3' -end of the coding gene of the heavy chain variable region of the human IgA antibody, the sequence of the homologous recombination arm at the 5 '-end of the coding gene of the heavy chain variable region of the human IgA antibody is shown as SEQ ID NO.13, the homologous recombination arm at the 3' -end of the coding gene of the heavy chain variable region of the human IgA antibody is shown as SEQ ID NO.14, and the sequence of the leader sequence element of the antibody is shown as SEQ ID NO. 4.
2. The humanized IgA antibody heavy chain expression plasmid of claim 1 wherein the heavy chain constant region encoding gene is set forth in SEQ ID No.1 or set forth in SEQ ID No. 2.
3. The humanized IgA antibody expression plasmid system of claim 1 wherein a multiple cloning site is provided between the heavy chain constant region and the post-transcriptional regulatory sequence, the multiple cloning site having the sequence shown in SEQ ID No. 7.
4. The humanized IgA antibody heavy chain expression plasmid of claim 1 wherein the post-transcriptional regulatory sequence is a woodchuck hepatitis virus post-transcriptional regulatory sequence.
5. The humanized IgA antibody heavy chain expression plasmid of claim 1 wherein the antibiotic resistance selection gene is an ampicillin resistance selection gene.
6. The heavy chain expression plasmid of a human IgA antibody of claim 1 wherein the sequence of the linking element is set forth in SEQ ID No. 5.
7. A method of preparing a heavy chain expression plasmid for a human IgA antibody according to any one of claims 1 to 6 comprising the steps of:
(1) Construction of eukaryotic cells containing an antibiotic resistance selection Gene, a CMV promoter, a post-transcriptional regulatory sequence, and a polyA tailAn expression vector, wherein, downstream of the CMV promoterEcoR I cleavage site, upstream of the post-transcriptional regulatory sequenceNot I cleavage site, the eukaryotic cell expression vector is subjected to the following steps ofEcoR I and is provided withNot I, two linearization ends are used for homologous recombination after double enzyme digestion;
(2) Constructing a linear fragment containing a 5' -terminal homologous recombination arm, an antibody leader sequence element, a connecting element, a heavy chain constant region, a multiple cloning site and a 3' -terminal homologous recombination arm, wherein the 5' -terminal homologous recombination arm containsEcoR I cleavage site, the 3' -homologous recombination arm containsNot I cleavage site, downstream of the antibody leader sequence element is provided withHpa I cleavage site, located upstream of the heavy chain constant regionEcoR V cleavage site, said linear fragment being for homologous recombination;
(3) UsingEcoR I and is provided withNot I, carrying out double enzyme digestion on the eukaryotic cell expression vector obtained in the step (1) to obtain a double enzyme digestion linearization vector, wherein the double enzyme tangent linearization vector is used for homologous recombination;
(4) And (3) carrying out homologous recombination on the linear fragment obtained in the step (2) and the double-enzyme tangential vector obtained in the step (3) to obtain the humanized IgA antibody heavy chain expression plasmid.
8. The method according to claim 7, wherein the sequence of the 5 '-end homologous recombination arm in step (2) is shown in SEQ ID NO.3, and the sequence of the 3' -end homologous recombination arm is shown in SEQ ID NO. 6.
9. The method of claim 8, wherein the linear fragment in step (2) has a sequence shown in SEQ ID NO.8 or SEQ ID NO. 9.
10. A method of producing a human IgA antibody heavy chain using the human IgA antibody heavy chain expression plasmid of any one of claims 1 to 6, comprising the steps of:
(1) UsingHpa I andEcoRV will said personDouble enzyme digestion of the heavy chain expression plasmid of the source IgA antibody to obtain a linearization plasmid;
(2) Constructing a heavy chain variable region linear fragment of a human IgA antibody to be expressed, wherein a linearization plasmid obtained in the step (1) is arranged at the 5' -end of the heavy chain variable region linear fragmentHpa I, a 5 '-end homologous recombination arm of homologous recombination occurs at the enzyme cutting end, and a linearization plasmid obtained in the step (1) is arranged at the 3' -end of the heavy chain variable region linear fragmentEcoA 3' -end homologous recombination arm for carrying out homologous recombination on RV enzyme cutting ends;
(3) Carrying out homologous recombination on the linearization plasmid obtained in the step (1) and the heavy chain variable region linear fragment obtained in the step (2) to obtain a human IgA antibody heavy chain expression plasmid containing a heavy chain variable region of a human IgA antibody to be expressed;
(4) Expressing the heavy chain expression plasmid of the human IgA antibody obtained in the step (3), and recovering an expression product.
11. The method according to claim 10, wherein the heavy chain variable region linear fragment of the human IgA antibody of step (2) is constructed by PCR amplification, wherein the upstream primer of the PCR amplification is formed by concatenating two segments of a 5 'end homologous recombination arm and a heavy chain variable region specific binding region, the downstream primer of the PCR amplification is formed by concatenating two segments of a 3' end homologous recombination arm and a heavy chain variable region specific binding region, the sequence of the 5 'end homologous recombination arm of the upstream primer of the PCR amplification is shown in SEQ ID No.13, and the sequence of the 5' end homologous recombination arm of the downstream primer of the PCR amplification is shown in SEQ ID No. 14.
12. A kit for preparing a heavy chain of a human IgA antibody, the kit comprising:
(1) The human IgA antibody heavy chain expression plasmid of any one of claims 1-6;
(2) The PCR amplification method comprises the steps of amplifying an upstream primer and a downstream primer of a heavy chain variable region of a human IgA antibody by PCR, wherein the upstream primer of the PCR amplification is formed by connecting a 5 '-end homologous recombination arm with two sections of a heavy chain variable region specific binding region in series, the downstream primer of the PCR amplification is formed by connecting a 3' -end homologous recombination arm with two sections of a heavy chain variable region specific binding region in series, the sequence of the homologous recombination arm of the 5 '-end of the upstream primer of the PCR amplification is shown as SEQ ID NO.13, and the sequence of the homologous recombination arm of the 5' -end of the downstream primer of the PCR amplification is shown as SEQ ID NO. 14;
(3) A reagent for causing homologous recombination between the human IgA antibody heavy chain expression plasmid of (1) and the human IgA antibody heavy chain variable region of (2).
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