CN110655576A - IL-6 recombinant monoclonal antibody and preparation method and application thereof - Google Patents
IL-6 recombinant monoclonal antibody and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a preparation method of an IL-6 recombinant monoclonal antibody, which comprises the following steps: immunizing a mouse by using IL-6, and preparing a single cell suspension by taking the spleen of the mouse after the immunization is finished; screening out memory B cells and plasma cells by a flow cytometer; respectively culturing the obtained cells, adding the obtained culture supernatant into a culture device coated with IL-6 for continuous culture, detecting cell strains secreting specific antibodies, and screening antibody clones capable of specifically recognizing antigen proteins; extracting total RNA from the obtained cell strain, performing reverse transcription to obtain cDNA, amplifying variable regions of heavy chains and light chains of the antibody, and respectively connecting the variable regions to a vector; then, cells are transfected to produce the IL-6 recombinant monoclonal antibody. The invention can obtain a large amount of antibody sequences through one-time screening, can obtain the gene information of the antibody, and solves the problems of low cell fusion rate and antibody gene loss in the hybridoma technology.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an IL-6 recombinant monoclonal antibody, and a preparation method and application thereof.
Background
Interleukin-6 (Interleukin-6), abbreviated as Interleukin 6(IL-6), is a pleiotropic cytokine with wide functions. The serum IL-6 concentration determination is widely used in the diagnosis of surgery, stress reaction, infection and other diseases, at present, the serum IL-6 concentration determination is mainly detected by immunological methods such as chemiluminescence method, immunofluorescence chromatography and the like, and because the IL-6 content in normal human bodies is extremely low (< 7pg/ml), the serum IL-6 concentration determination has higher requirements on core raw materials in a detection kit, namely antibodies, and the development of antibodies with high affinity is required by the market.
The basic principle of the present diagnostic kit is that spleen cells and myeloma cells are fused to obtain a hybridoma cell line, single cells are obtained by a limited gradient dilution method, then culture supernatant is detected, required positive cell strains are screened out, ascites is generated by injecting the hybridoma cells into a mouse body or the supernatant of a secretory antibody is obtained by an in vitro culture mode, and then the antibody with higher purity is obtained by affinity purification. The method has the advantages of low fusion efficiency, high randomness, low positive rate in the experimental process and multiple cell fusion, thereby increasing the workload; in addition, the culture of the hybridoma cells in vivo needs to be completed by means of experimental animals, the culture cost of the experimental animals is high, and the difference of individual animals is large, so that the batch difference is easy to cause, and the industrial production is not facilitated.
Disclosure of Invention
In view of the disadvantages of the prior art, it is an object of the present invention to provide a method for producing a recombinant IL-6 monoclonal antibody, which can obtain a large number of antibody sequences at a time and is easy to handle.
The preparation method of the IL-6 recombinant monoclonal antibody provided by the invention comprises the following steps:
(1) immunizing a mouse by using IL-6, and preparing a single cell suspension by taking the spleen of the mouse after the immunization is finished;
(2) screening out memory B cells and plasma cells by a flow cytometer;
(3) respectively culturing the cells obtained in the step (2), adding the obtained culture supernatant into a culture device coated with IL-6 for continuous culture, detecting cell strains secreting specific antibodies, and screening antibody clones capable of specifically recognizing antigen proteins;
(4) extracting total RNA from the cell strain screened in the step (3), carrying out reverse transcription to obtain cDNA, amplifying variable regions of heavy chains and light chains of the antibody, and respectively connecting the variable regions to a carrier; then, cells are transfected to produce the IL-6 recombinant monoclonal antibody.
The traditional technology for obtaining hybridoma cells by fusing spleen cells and myeloma cells and producing antibodies has the defects of low fusion efficiency and high randomness, so that the positive rate in the experimental process is low, and multiple cell fusions are needed, thereby increasing the workload. In addition, the culture of the hybridoma cells in vivo needs to be completed by means of experimental animals, the culture cost of the experimental animals is high, and the difference of individual animals is large, so that the batch difference is easy to cause, and the industrial production is not facilitated.
The invention solves the problem of low fusion efficiency of hybridoma technology, can obtain a large amount of antibody sequences through one-time screening, and lays a foundation for screening high-affinity and high-specificity antibodies in the later period. Meanwhile, the invention can obtain the gene information of the antibody, and solves the problems of unstable hybridoma technology and the risk of losing antibody genes. In addition, after the antibody gene obtained by the invention is recombined and expressed, a uniform monoclonal antibody can be obtained, and the control of the batch-to-batch difference of the antibody is facilitated.
Compared with the technology of screening antibodies by phage display technology, the single cell sorting technology of the invention is an antibody gene directly obtained from B lymphocytes, and the heavy chain and the light chain of the antibody are original combinations, so that the antibody with better affinity can be obtained.
Compared with ribosome display technology, the invention does not relate to an unstable complex system such as 'protein-ribosome-mRNA', and the operation mode is simpler. Therefore, the monoclonal antibody technology can better meet the requirements of the diagnostic reagent on the affinity, the specificity, the uniformity and the like of the antibody, and has high operability and popularization value.
As an alternative embodiment of the present invention, in step (1), the mouse is a Balb/c mouse.
As an optional technical scheme of the invention, in the step (1), the IL-6 is obtained by expressing IL-6 protein in an Escherichia coli system and purifying.
In step (2), memory B cells and plasma cells are screened by flow cytometry using cell surface antibody labeling with antibodies against mouse CD45R/B220, CD19, CD27, CD38, or CD 138. Preferably, in step (2), the plasma cells are screened by flow cytometry using an antibody against mouse CD138, which is a surface marker specific for plasma cell differentiation, for cell surface antibody labeling.
As an alternative embodiment of the invention, in step (3), the cell culture is carried out by using 384 micro-well plates, and/or the culture device coated with IL-6 is 384 micro-well plates.
As an alternative embodiment of the present invention, in step (4), the amplification is performed by nested PCR amplification.
As an alternative embodiment of the present invention, in step (4), the transfection of the cells is carried out by transfection of CHO cells.
As a preferred embodiment of the present invention, in step (4), after cell transfection, selection of stable cell strains is also performed to obtain cell strains capable of producing recombinant monoclonal antibodies in large quantities, and then the culture medium and culture conditions are optimized to increase the antibody production of the cells.
Another objective of the invention is to provide the IL-6 recombinant monoclonal antibody prepared by the above method, wherein the amino acid sequences of the heavy chain and the light chain of the IL-6 recombinant monoclonal antibody are selected from one of the following combinations:
the heavy chain has the sequence shown in SEQ ID No: 1, and the light chain has the sequence shown in SEQ ID No: 2 is shown in the specification;
the heavy chain has the sequence shown in SEQ ID No: 3, the sequence of the light chain is shown as SEQ ID No: 4 is shown in the specification;
the heavy chain has the sequence shown in SEQ ID No: 5, the sequence of the light chain is shown as SEQ ID No: 6 is shown in the specification;
the heavy chain has the sequence shown in SEQ ID No: 7, the sequence of the light chain is shown as SEQ ID No: shown in fig. 8.
The invention also aims to provide the application of the IL-6 recombinant monoclonal antibody in preparing immunodiagnostic preparations.
The invention has the beneficial effects that:
the invention can obtain a large amount of antibody sequences through one-time screening, can obtain the gene information of the antibody, and solves the problems of low cell fusion rate and antibody gene loss in the hybridoma technology. The recombinant antibody obtained by the invention has the advantages of convenient amplification, controllable batch difference and lower cost, and can better meet the requirements of diagnostic reagents on the stability and the repeatability of the antibody. The invention creatively applies the recombinant monoclonal antibody to the immunodiagnosis reagent, breaks through the current situation that the recombinant monoclonal antibody is only applied to the preparation of monoclonal antibody medicines.
Drawings
FIGS. 1 to 2 are graphs showing the results of the test of the antibody obtained in example 1 of the present invention.
Detailed Description
The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
The term "vector" as used herein when referring to a polynucleotide refers to any molecule (e.g., nucleic acid, plasmid, or virus, etc.) that is used to transfer nucleotide-encoding information into a host cell. The term "expression vector" or "expression cassette" refers to a vector suitable for expressing a gene of interest (a nucleotide sequence to be expressed) in a host cell, and generally includes portions of the gene of interest, a promoter, a terminator, a marker gene, and the like.
The term "transfection" as used herein refers to the uptake of foreign or exogenous DNA by a cell, and the technique can be used to introduce one or more exogenous DNA moieties into a suitable host cell. Cells can be induced by physicochemical means (e.g., by calcium chloride treatment) to be in a physiological state that is optimal for uptake and containment of foreign DNA, i.e., "competent".
Example 1
The method comprises the following main experimental steps:
1. expression of purified IL-6 protein: the Escherichia coli system is adopted to express and purify IL-6 protein, simultaneously the purity, concentration and activity of the antigen are characterized, and high-purity and high-activity antigen is selected as immunogen.
2. Preparation of splenocytes: the Balb/c mouse is immunized by the antigen, and the spleen of the mouse is taken after the immunization is finished to prepare single cell suspension.
3. Obtaining single cells: cell surface antibody labeling was performed using antibodies against mouse CD45R/B220, CD19, CD27, and CD38, and memory B cells and plasma cells were screened by flow cytometry.
4. And (3) detecting the antibody titer: spreading the screened cells in 384 micro porous plates for culture, adding the culture supernatant into micro porous plates coated with antigens in advance, detecting cell strains secreting specific antibodies, screening antibody clones capable of specifically recognizing antigen proteins,
5. preparing a recombinant clone: taking cell strain producing high affinity antibody, extracting total RNA of B lymphocyte, reverse transcribing into cDNA, amplifying variable regions of heavy chain and light chain of antibody by nested PCR, then connecting to vector, transfecting HEK293T cell to express, and obtaining cell strain capable of producing recombinant monoclonal antibody in large scale.
6. Screening recombinant clones: selecting pairing according to the affinity and the recognition sites of the antibody, then measuring a calibration curve and high and low value samples, screening out a proper antibody, stably transfecting to CHO cells, and screening out a stable cell line; the production process is determined by optimizing the culture medium, increasing the antibody yield of the cells under culture conditions, and then repeatedly producing 3 batches of antibodies.
On the basis of the main experimental steps, the related specific method comprises the following steps:
inserting the gene shown as SEQ ID No.9 into a vector of pGEX-6P-1, transferring the gene into E.coli BL21 by a CaCl2 chemical conversion method, sequencing, inoculating the recombinant Escherichia coli with correct sequencing into an LB liquid culture medium according to 1% of the inoculation amount, performing shake culture at 37 ℃ until the OD is about 0.6, adding IPTG (isopropyl-beta-thiogalactoside) to the final concentration of 0.5mM, continuing culturing for 4 hours, performing 6000g centrifugation to collect thalli, crushing the Escherichia coli by an ultrasonic crusher, performing 20000g, performing centrifugation at 4 ℃ to collect supernatant, purifying the supernatant by a Ni-NTA chromatographic column to obtain recombinant IL-6 protein, and detecting the activity of the recombinant IL-6 protein by a double antibody sandwich ELISA method (Table 1).
The recombinant protein and Freund's complete adjuvant are mixed uniformly to immunize mice, each mouse is immunized with 50ug of protein each time, each mouse is immunized once every two weeks, after five times of immunization, the spleen of the mouse is taken under aseptic condition, and then single spleen cell suspension is prepared. And (3) taking a proper amount of anti-CD 16 and anti-CD 32 monoclonal antibodies and sample cells, fully and uniformly mixing, and standing for 15min at 4 ℃. The fcrs on the B cell surface were all bound by anti-CD 16 or anti-CD 32 monoclonal antibodies, thereby preventing non-specific binding of subsequent fluorescein-conjugated antibodies to fcrs. After the blocking, 10. mu.L of the corresponding fluorescein conjugated antibody CD138-FITC labeled was taken and mixed in a 1.5ml EP tube containing 990. mu.L of sample cells, 100. mu.l of the mixture was added to 9 sample wells, mixed, and stained at room temperature in the dark for 20 min. Adding 1mL FACSBuffer to wash away unbound antibody, centrifuging at 4 deg.C for 5min, discarding supernatant, resuspending the precipitate with 200 μ L FACSBuffer or 2% paraformaldehyde, performing on-machine analysis as soon as possible, and differentiating lymphocyte subpopulations by adjusting voltage of FSC and SSC to obtain plasma cells. And spreading the screened cells in 384 micro-porous plates for culture, adding a culture medium into the micro-porous plates coated with the antigens in advance, culturing for 3 days, taking culture supernatant to detect titer, and screening out antibody clones capable of specifically recognizing the IL-6 protein.
Extracting total RNA of B lymphocyte by TRIzol method, reverse transcription into cDNA by RT-PCR method, amplifying variable regions of heavy chain and light chain of antibody by nested PCR and sequencing, then respectively connecting to pCDNA3.4 vector, transient transfection to HEK293T cell, purifying cell culture supernatant by Protein A column to obtain antibody for identifying IL-6.
TABLE 1
TABLE 2
Example 2
Adding the purified antibody into a 96-well plate coated with protein G in advance, adding IL-6 protein labeled with biotin and HRP labeled with streptavidin for measuring antibody affinity (table 2), selecting pairing according to the antibody affinity and the recognition sites, detecting an IL-6 calibration curve, and screening out appropriate paired antibodies, namely cell numbers No. 2, No. 3 and No. 5. The corresponding plasmid was stably transfected into CHO cells, and then 3 batches of antibody were repeatedly produced, confirming the production process.
Example 3
The detection results of 30 human serum samples, which were coated on 96-well plates with antibodies No. 2 and No. 3 as capture antibodies, and HRP labeled with antibody No. 5 as labeled antibodies, on a chemiluminescence platform, and compared with hospital measurement values are shown in fig. 1 and fig. 2.
Sequence listing
<110> Wuhan Huamei bioengineering Co., Ltd
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Claims (10)
1. A preparation method of an IL-6 recombinant monoclonal antibody is characterized by comprising the following steps:
(1) immunizing a mouse by using IL-6, and preparing a single cell suspension by taking the spleen of the mouse after the immunization is finished;
(2) screening out memory B cells and plasma cells by a flow cytometer;
(3) respectively culturing the cells obtained in the step (2), adding the obtained culture supernatant into a culture device coated with IL-6 for continuous culture, detecting cell strains secreting specific antibodies, and screening antibody clones capable of specifically recognizing antigen proteins;
(4) extracting total RNA from the cell strain screened in the step (3), carrying out reverse transcription to obtain cDNA, amplifying variable regions of heavy chains and light chains of the antibody, and respectively connecting the variable regions to a carrier; then, cells are transfected to produce the IL-6 recombinant monoclonal antibody.
2. The method according to claim 1, wherein in the step (1), the mouse is a Balb/c mouse.
3. The method according to claim 1, wherein in step (1), the IL-6 is obtained by expressing IL-6 protein in E.coli system and purifying.
4. The method according to claim 1, wherein in the step (2), the memory B cells and plasma cells are screened by flow cytometry using an antibody against mouse CD45R/B220, CD19, CD27, CD38 or CD138 for cell surface antibody labeling.
5. The method according to claim 1, wherein in the step (3), the culturing of the cells is carried out in 384-well plates, and/or the IL-6-coated culturing apparatus is 384-well plates.
6. The method according to claim 1, wherein in the step (4), nested PCR amplification is used for the amplification.
7. The method according to claim 1, wherein the transfection is carried out by transfecting CHO cells in the step (4).
8. The method according to claim 1, wherein in step (4), after the cell transfection, the cell line is selected by selecting a stable cell line to obtain a cell line capable of producing recombinant monoclonal antibodies in large quantities, and then the culture medium and culture conditions are optimized to increase the antibody production of the cells.
An IL-6 recombinant monoclonal antibody, wherein the IL-6 recombinant monoclonal antibody is produced by the production method according to any one of claims 1 to 8, wherein the amino acid sequences of the heavy chain and the light chain of the IL-6 recombinant monoclonal antibody are selected from one of the following combinations:
the heavy chain has the sequence shown in SEQ ID No: 1, and the light chain has the sequence shown in SEQ ID No: 2 is shown in the specification;
the heavy chain has the sequence shown in SEQ ID No: 3, the sequence of the light chain is shown as SEQ ID No: 4 is shown in the specification;
the heavy chain has the sequence shown in SEQ ID No: 5, the sequence of the light chain is shown as SEQ ID No: 6 is shown in the specification;
the heavy chain has the sequence shown in SEQ ID No: 7, the sequence of the light chain is shown as SEQ ID No: shown in fig. 8.
10. Use of the recombinant monoclonal antibody against IL-6 produced by the production method according to any one of claims 1 to 8 or the recombinant monoclonal antibody against IL-6 according to claim 9 for the production of an immunodiagnostic agent.
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