KR20090032784A - Method for manufacturing of single-chain antibody against zearalenone using recombinant methylotrophic yeast - Google Patents
Method for manufacturing of single-chain antibody against zearalenone using recombinant methylotrophic yeast Download PDFInfo
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Abstract
The present invention relates to a method for producing a single-chain antibody against zearalenone, a type of fungal toxin, from recombinant methanoly yeast (recombinant methylotrophic yeast), the base sequence of cDNA or yeast preferred codon of the geralenone antibody Fv portion The optimized geralenone single-chain antibody gene was amplified by PCR, cloned into an expression vector containing an alcohol oxidase (AOX) promoter, transformed into yeast ( Pichia pastoris ), and induced expression with methanol. The present invention relates to a method for efficiently producing stable antibodies having excellent expression rate, solubility and antigen affinity by allowing chain antibodies to be secreted into the medium.
Description
The present invention relates to a method for producing a single-chain antibody against zearalenone from recombinant methanoly yeast (recombinant methylotrophic yeast), more specifically, the base sequence of cDNA or yeast preferred codon of the geralenone antibody Fv portion The optimized geralenone single-chain antibody gene was amplified by PCR, cloned into an expression vector containing an alcohol oxidase (AOX) promoter, transformed into yeast ( Pichia pastoris ), and induced expression with methanol. The present invention relates to a method for efficiently producing a stable antibody having excellent expression rate, solubility and antigen affinity by allowing chain antibodies to be secreted into a medium.
In general, fungal toxin (mycotoxin) produced by the fungus of the genus Fusarium (mycotoxin) occurs in a variety of cereals, especially barley, wheat, corn, causing plant diseases, including red fungal diseases. As such, when the grains containing fungal toxins are directly consumed by humans or used as livestock feed, it is known to cause various mycotoxicosis.
One of the fungal toxins produced by this genus Fusarium , zearalenone, was first detected in grain feed in Ontario, Canada, in 1970. Thereafter, the geralenone grows as a packaging fungus in the crop to produce toxins, and these toxins have been reported to cause infertility of livestock by invading the reproductive organs as a result of experiments with pigs and other animals, leading to a decrease in productivity.
Therefore, a test is performed to determine whether the fungal toxin containing geralenone is contained in the process of harvesting, storing, and distributing cereals, and various chromatographic methods and immunoassay methods are used as detection methods for this. Among them, immunoassays using antibodies are more frequently used due to their simplicity of use, short detection time, and high specificity and sensitivity (Pestka, Food Technol, 49, 120-128, 1995).
Conventional techniques for producing antibodies for use in such immunoassays have been used to produce polyclonal or monoclonal antibodies using animal or hybridoma cells, but these techniques are lengthy to perform, costly and experimental. There was a problem that special facilities were needed to perform them (Yuan et al., Appl. Environ. Microbiol., 63, 263-269, 1997).
On the other hand, recently widely used recombinant antibody production techniques that will evaluate whether high-affinity antibody using the phage display technique or to the V H and V L of the F ab portion, or single chain antibodies (Fv part of the former antibody linker , scFv) by amplifying the polymerase chain reaction (PCR) technique, cloning the vector and introduced into the host to enable the mass production of the antibody expressed (McCafferty et al., Nature, 348, 552-554, 1990; Marks et al., J. Mol. Biol., 222, 581-597, 1991). Single-chain antibodies prepared through such recombinant antibody production techniques have been used mainly for medical purposes because they have a lower molecular weight but superior activity compared to whole antibodies (Leonardo et al., Prot. Exp. Purif., 37, 18-26, 2004). .
Recombinant antibody production technology using the microorganisms or flora and fauna as described above has been increasing in necessity and demand due to the recent development of biotechnology and its advantages, especially microbial expression system using E. coli or yeast culture It is widely used due to its short period of time, low cost, and high mass expression of foreign proteins.
Of these, E. coli is most commonly used as a protein-expressing host, which has the advantage of high expression rate, short incubation period, and low cost, but it is necessary to homogenize cells during recovery of expressed protein. There was a problem in that the activity of the protein is lost by forming and refolding the protein in order to use it.
On the other hand, yeasts such as Pichia genus are eukaryotic cell expression systems that are easy to culture and undergo post-translational modifications to produce stable forms of protein, and these yeasts in the genus Saccharomyces Higher expression rate compared to 12 g / L for tetanus toxin fragment C (Clare et al., Bio / Technol., 9, 455-460, 1991), 4.88 g / L for single-chain antibodies against colon cancer antigens (Leonardo et al., Prot. Exp. Purif., 37, 18-26, 2004).
In addition, there have been several reports that the expression rate of the protein was increased when the nucleotide sequence was optimized with yeast preference codon (Clare et al., Bio / Technol., 9, 455-460, 1991; Woo et al., Prot.Exp. Purif., 25, 270-282, 2002).
In addition, there have been reports of the expression of recombinant single-chain antibodies against the above-mentioned fungal toxin geralenone in E. coli and plants (Yuan et al., Appl. Environ. Microbiol., 63, 263-269, 1997; Yuan et al., Appl. Environ). Microbiol., 66, 3499-3505, 2000).
As described above, various researches on recombinant antibody production technology have been conducted, but until now, there has been no report on the method for producing a geralenone single-chain antibody using a yeast expression system, and in particular, the use of the single-chain antibody is mainly used in medicine. To this end, the present inventors have completed the present invention to produce antibodies secreted into the medium by supplementing the disadvantages of the existing expression system and increasing the expression rate for use in the immunoassay of geralenone. Reached.
Therefore, in order to solve the above problems, the recombinant yeast expression system ( Pichia pastoris ) using a strong AOX promoter under the control of a strong expression rate and having a high affinity characteristics, antigen-antibody reactions including detection It is an object of the present invention to provide a method for producing a geralenone single chain antibody suitable for use in all immunoassays.
In order to achieve the above object, the present invention
Amplifying the single-chain antibody gene against geralenone using multistep PCR; Cloning a single chain antibody gene for the amplified geralenone into an expression vector and confirming the presence thereof; Transforming the cloned expression vector into methanol magnetized yeast and then selecting the cloned expression vector; Expressing a single chain antibody against geralenone in the selected recombinant yeast; is achieved by providing a method for producing a single chain antibody against geralenon using recombinant methanolized magnetized yeast, characterized in that consisting of.
In addition, the present invention provides a recombinant methanol magnetizing yeast characterized in that the nucleotide sequence is optimized by using the cDNA or yeast preferred codon of the geralenone antibody Fv in the production method of the above-mentioned geralenone. It provides a method for producing a single chain antibody against the geralenone used.
In addition, in the above-described manufacturing method, the expression vector is pPIC9K having an AOX promoter and ampicillin and kanamycin resistance genes and having a secretory signal peptide gene, wherein the single expression for geralenone using recombinant methanolylated yeast It provides a method for producing a chain antibody.
In addition, in the above-described manufacturing method, the cloning to the expression vector is transformed into E. coli with the expression vector by electroporation, and then plasmid isolated from the transformed E. coli selected through the LB-kanamycin plate A method for producing a single-chain antibody against geralenone using recombinant methanolized magnetized yeast, characterized by cutting with restriction enzymes (EcoRI and NotI) to confirm whether the single-chain antibody gene against geralenone was properly cloned in the expression vector. to provide.
In addition, in the above-described production method, the transformation of the methanol magnetized yeast is performed by cutting the cloned expression vector with restriction enzyme (Pme I) and extracting the extracted DNA on an agarose gel, followed by electrophoresis of the DNA of the extract. Provided is a method for preparing a single chain antibody against geralenone using recombinant methanolized magnetized yeast, characterized in that it is introduced into methanol magnetized yeast by a puncture method.
In another aspect, the present invention provides a method for producing a single-chain antibody against geralenone using recombinant methanol magnetized yeast, wherein the methanol magnetized yeast is Pichia pastoris GS115.
In addition, the present invention, in the above-described manufacturing method, the selection of the transformed yeast is first screened according to histidine (histidine) requirements, the selected transformed yeast again with a minimal dextrose (MD) plate and minimal Single spot for geralenone using recombinant methanol magnetized yeast, characterized by spotting on methanol (MM) plate to make methanol availability secondary screening with fast transforming yeast (Mut + ) and slow transforming yeast (Mut s ) It provides a method for producing a chain antibody.
In addition, in the above-described production method, the expression of the single-chain antibody against geralenone in the transformed yeast is first used for 48 hours using a 30 ℃ shaking incubator in buffered glycerol-complex medium (BMGY) yeast After culturing, centrifugation, and recovering the yeast, and again dispersed in a buffered methanol-complex medium (BMMY), and the methanol is added every 24 hours to induce the expression of the recombinant methanol magnetization yeast using geranol It provides a method for preparing a single chain antibody.
As described above, the method for producing a geralenone single-chain antibody using the recombinant methanolized yeast of the present invention utilizes a recombinant yeast expression system ( Pichia pastoris ) to compensate for the disadvantages of conventional polyclonal or monoclonal antibody production technology or E. coli expression system. This allows the production of single-chain antibodies against geralenone with high expression and high affinity under the control of strong AOX promoters. These single-chain antibodies can be used in all immunoassays that use antigen-antibody responses, including the detection of geralenone. It has the effect of ease.
In addition, the present invention by using a protein secretion system of recombinant methanol magnetizing yeast, cell homogeneous process is omitted, it is possible to reduce the time and cost, and to produce an antibody having excellent solubility and stability, and mass production of high-quality products This has another effect of supplying at a low price.
Hereinafter, the present invention will be described in more detail.
Firstly, the present invention relates to a method for producing a single chain antibody against geralenone by an improved method than the conventional method, wherein pPIC9K is designed to carry a signal peptide gene and regulate expression by an AOX promoter (US Invitrogen). Was used as the expression vector.
In addition, PCR, DNA electrophoresis, protein electrophoresis (SDS-PAGE), western blot, surface plasmon resonance (SDS-PAGE) to identify and measure the activity of single chain antibodies against geralenone in recombinant yeast. The study was carried out using molecular biological and optical methods such as SPR) analysis.
In addition, the variable heavy chain (V H ) and the variable light chain (V L ) of the single-chain antibody gene for geralenone are linked by linker {(Gly) 4 Ser} 3 and cDNA or yeast preference codon of the F. Oligo was prepared using the optimized gene sequence and cloned by amplification of the gene by multistep PCR.
In addition, in order to produce a single chain antibody against geralenone using recombinant yeast, first, glycerol in the medium is supplied to proliferate the cell, and then methanol is supplied to activate the AOX promoter to supply a single chain antibody to geralenone. Expression was induced to be secreted into the medium.
The Pichia yeast expression system to be used in the present invention has a high expression, short incubation time, and unlike E. coli, there is a post-translational modification process, so that the protein is stabilized and an active protein having excellent solubility can be produced. In particular, since the protein expressed using the signal peptide is secreted in the medium, the process of homogenizing the cells to obtain the desired protein is unnecessary, which shortens the experimental procedure and brings time and cost effect.
By producing a single chain antibody against geralenone as described above using a recombinant yeast expression system, it is possible to solve the difficulty of securing an existing antibody or a problem of a microbial expression system, thereby enabling mass production and producing a recombinant antibody with excellent yield as well as activity. By preparing the, the single-chain antibody thus prepared can be easily used in all fields using the antigen-antibody reaction, including the detection of geralenone, thereby bringing the effect of high industrial value.
Hereinafter, the manufacturing method of the present invention will be described through the following examples, which are only presented to aid the understanding of the present invention, and the present invention is not limited thereto.
<Example 1>
Amplification of single chain antibody genes against geralenone and cloning into expression vectors
Before amplifying single chain antibody genes for geralenone, first obtain the variable heavy chain (V H ) gene sequence (GenBank Accession No. U74671) and the variable light chain (V L ) gene sequence (GenBank Accession No. U74672). A linker sequence of {(Gly) 4 Ser} 3 was inserted between V H and V L , a 6x-His sequence was inserted at the C-terminal, and restriction enzyme cleavage sites of EcoRI and NotI were inserted at both ends of the vector. Multiple cloning sites facilitate gene cloning. As shown in Table 1 and Table 2, scFv-Zen const1 having the cDNA gene sequence of the geralenone antibody Fv portion, and scFv-Zen const2 optimized for the nucleotide sequence with yeast preferred codons without any amino acid change, respectively. 12 oligonucleotides were synthesized, respectively.
To perform multi-step PCR using Vent DNA polymerase (US New England Biolab, product number 254L) with the oligos of Tables 1 and 2, 769 bp geralenone single-chain antibody finally under the conditions as shown in Table 3 below. The gene was amplified. At this time, the primers used for the final PCR are shown in Tables 1 and 2.
In addition, ScFv-Zen const3 and 4 were cloned into the expression vector of scFv-Zen const1 and 2, and the nucleotide sequence was identified. As a template, the 6x-His base sequence was prepared using primers prepared as shown in Tables 4 and 5. PCR was cloned again without. The expression vector was a pPIC9K (Invitrogen, Inc., USA) as shown in Figure 1 attached to the AOX promoter and ampicillin and kanamycin resistance genes with a secretion signal peptide gene.
PCR products of the single-chain antibody gene for the amplified 769 bp geralenone was confirmed on the agarose gel by DNA electrophoresis as shown in Figure 2, the four PCR products were purified by gel extraction, respectively, EcoRI and NotI The reaction was cleaved at 37 ° C. and then ligation with an expression vector using T4 DNA ligase (Promega, USA).
2 shows a result of amplifying a single-chain antibody gene against geralenone through a multi-step polymerase chain reaction (PCR). Each lane is as follows.
Lane 2: single
Lane 3: single-
Lane 5: single
Lane 6: single chain antibody gene 4 (scFv-Zen const4) against geralenone
<Example 2>
Confirmation of cloning by transformation of E. coli
E. coli was transformed with the expression vector prepared in Example 1 by electroporation (electroporaion, 2.5 kV, 200 μs, 2.5 msec), and then transformed E. coli was selected from the LB-kanamycin plate. The plasmid isolated from the transformed Escherichia coli was digested with restriction enzymes (EcoRI and NotI) to confirm whether the single-chain antibody gene for geralenone was properly cloned in the expression vector on the gel as shown in FIG. After sequencing (Automatic Sequencer 3730xl) all the gene sequences were correctly identified. ScFv-Zen cont3 and 4 were cloned into expression vectors in the same manner as in Example 1, by amplifying a single-chain antibody gene for geralenone by using a plasmid whose base sequence was identified as a template to prepare a primer so that his-tag was absent. .
3 is a result of performing DNA electrophoresis by cutting a plasmid isolated from transgenic Escherichia coli with restriction enzymes to confirm that the single-chain antibody gene for geralenone was properly cloned into the expression vector. Each lane is as follows. .
Lanes 2-5: Transgenic E. coli introduced with single chain antibody gene 1 (scFv-Zen const1) against geralenone
Lane 6-10: transgenic E. coli introduced with the single-chain antibody gene 2 (scFv-Zen const2) against geralenone;
Lane 12-16: Transgenic E. coli introduced with single chain antibody gene 3 (scFv-Zen const3) against geralenone
Lanes 17-20: Transgenic E. coli with single chain antibody gene 4 (scFv-Zen const4) against geralenone
<Example 3>
Transformation of Yeast and Selection of Recombinant Yeast
The expression vectors cloned with the four types of ScFv-Zen genes cloned through Example 1 and 2 were digested with restriction enzymes (Pme I) and extracted on an agarose gel, followed by electroporation (1.5). kV, 200 μs, 2.5 msec) was introduced into Pichia pastoris GS115, a methanol magnetizing yeast, and transformed and plated onto minimal dextrose (MD) plates to be screened first according to histidine requirements. The transformed yeast selected as described above is spotted on a minimal dextrose (MD) plate and a minimal methanol (MM) plate, so that the methanol availability is high into the transformed yeast (Mut + ) and the slow transformed yeast (Mut s ). Secondary screening.
Thus, 7 clones of each of four types of scFv-Zen-derived transformed yeast were cultured, and genomic DNA was isolated using a genomic DNA extraction kit (RBC, Taiwan). The genomic DNA was isolated from the 5'AOX primer. PCR using (5'-gactggttccaattgacaagc-3 ') and 3'AOX primer (5'-gcaaatggcattctgacatcc-3'), and then in the yeast genome of the single-chain antibody gene for geralenone on agarose gel as shown in FIG. The integration was confirmed. As shown in the accompanying FIG. 4, two bands of 1.3 kb and 2.2 kb were Mut + , and one band of 1.3 kb was Mut s , and the result was selected from the transformed yeast selected on the MD / MM plate. Matched.
Figure 4 is a result of performing PCR by separating the genomic DNA of the transformed yeast to confirm that the recombinant expression vector is correctly introduced into the yeast, each lane is as follows.
Lanes 4-10: Transformed yeast introduced with single chain antibody gene 1 (scFv-Zen const1) against geralenone
Lane 11: expression vector
Lane 12: Expression vector cloned of single-chain antibody gene 1 (scFv-Zen const1) against geralenone
Lanes 14-20: transgenic yeast with single chain antibody gene 2 (scFv-Zen const2) directed against geralenone
Lanes 22-28: transgenic yeasts having introduced single-chain antibody gene 3 (scFv-Zen const3) against geralenone;
Lanes 30-36: transgenic yeasts having introduced single-chain antibody gene 4 (scFv-Zen const4) against geralenone
<Example 4>
Expression of Single Chain Antibodies to Geralenon Using Recombinant Yeast
In order to express a single chain antibody against geralenone in the transformed yeast as in Example 3, the transformed yeast was first incubated in a buffered glycerol-complex medium (BMGY) using a 30 ° C. shaking incubator for 48 hours. After centrifugation, yeast was recovered and dispersed again in buffered methanol-complex medium (BMMY), methanol was added every 24 hours to induce expression, and the medium was taken every 24 hours to precipitate the secreted antibody protein. It was confirmed by electrophoresis (SDS-PAGE) and Western blot.
As a result, as shown in FIGS. 5 and 6, the transgenic yeasts into which the ScFv-Zen const1, 2, 3, and 4 genes were introduced were all expressed and secreted with excellent expression of scFv-Zen protein having a desired size of about 27 kD. And, it was confirmed that the antibody through the Western blot using the protein-L-HRP conjugate. In addition, there was no difference in expression rate between the two transgenic yeasts of scFv-Zen cont1 and 2 into which the nucleotide-optimized genes of cDNA or yeast preferred codons of his-tag-geralenone antibody Fv were introduced. In the transgenic yeast with scFv-Zen cont3 and 4 without -tag, the expression rate of the transformed yeast with scFv-Zen cont4 introduced with yeast preference codon was excellent.
5 is a result of confirming the
Figure 5-
Lane 1: size label (medium protein)
Lane 2: voice control
Lanes 3-9: single-chain antibody against geralenone expressed in
Lane 10-17: Western Blot In Lanes 2-9
Figure 5-b
Lane 1: size label (medium protein)
Lanes 2-8: single-
Lane 9: Figure 5-Western Blot of
Lanes 10-16: Western Blots in Lanes 2-8
FIG. 6 shows the results of confirming
Figure 6-A
Lane 1: size label (medium protein)
Lane 2: voice control
Lane 3-9:
Lane 10-17: Western Blot In Lanes 2-9
Fig 6-I
Lane 1: size label (medium protein)
Lanes 2-8: single-chain antibody against geralenone expressed in
Lane 9: Figure 6-Western Blot of
Lanes 10-16: Western Blots in Lanes 2-8
Example 5
Determination of activity of single chain antibodies against geralenone
The activity of single-chain antibodies (scFv-Zen) against the four types of geralenone expressed through Example 4 was compared by measuring the reactivity to the antigen using SPR (Surface Plasmon Resonance) analysis. The gold substrate to be used for the SPR was produced by the following method.
50 nm thick gold using ion sputter (Polaron Co., E5000, UK) on cover glass (BK7, n = 1.522, 18 ㅧ 18mm 2 , d = 0.13mm, Matsunami, Japan) washed with ethanol and acetone. The gold-covered cover glass was immersed in a solution in which 1.0 mM of 16-mercaptohexadecanoic acid (MHDA, Sigma-Aldrich, 90%) was dissolved in ethanol for about 12 hours to prepare a gold chip having MHDA. 0.4 M EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide, Sigma-Aldrich, 98%) and 0.1 M NHS (N-hydroxysuccinimide ester, Sigma-Aldrich, 97%) aqueous solution After immersing in each 20 minutes, the cells were washed / dried and scFv-Zen was immobilized in 1 ml PBS (pH 7.4) buffer.
The SPR was measured by the formation of single-chain antibodies against four types of geralenone on the gold chip, respectively, and the reaction with 10 μM geralenone as a change in the resonance angle with time, and a monoclonal antibody against geralenone. The measurement was carried out in the same manner using (mAb), and the MHDA chip that did not form scFv-Zen was used as a negative control and also measured in the same manner, and the results are shown in FIG. 7.
7 is a graph showing the change of the resonance angle with time using the surface plasmon resonance (SPR) analysis of the activity of the single-chain antibody (scFv-Zen) to the final prepared geralenon, as a result scFv-1, 2 The resonance angles of,, 3 and 4 shifted by 0.048, 0.017, 0.0021 and 0.048 degrees after washing, respectively. ScFv-2 and 3 showed similar reactivity with monoclonal antibodies to geralenone, and scFv-1 and 4 had better reactivity with monoclonal antibodies. That is, single chain antibodies against geralenone produced from recombinant methanolized yeasts showed an affinity for antigens similar to or better than monoclonal antibodies.
1 is a view showing the structure of the yeast expression vector introduced with a single chain gene (scFv-Zen) for the geralenon of the present invention
2 is a diagram showing the result of amplifying a single-chain antibody gene against geralenone of the present invention through a multi-step polymerase chain reaction (PCR).
3 is a diagram showing the results of DNA electrophoresis by cutting a plasmid isolated from transgenic Escherichia coli with restriction enzymes to confirm whether the single-chain antibody gene for geralenone was properly cloned into an expression vector.
Figure 4 is a diagram showing the results of performing PCR by separating the genomic DNA of the transformed yeast to confirm that the recombinant expression vector is properly introduced into the yeast of the present invention
5 is a diagram showing the results of confirming the
6 is a diagram showing the results confirmed by protein electrophoresis and Western blot for
7 is a graph showing the change in the resonance angle with time using the surface plasmon resonance (SPR) analysis of the activity of the single-chain antibody (scFv-Zen) for the final prepared geralenone
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| CN117106092A (en) * | 2023-10-25 | 2023-11-24 | 华南农业大学 | Nanometer antibody for resisting zearalenone and zearalanol and application thereof |
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| CN117106092A (en) * | 2023-10-25 | 2023-11-24 | 华南农业大学 | Nanometer antibody for resisting zearalenone and zearalanol and application thereof |
| CN117106092B (en) * | 2023-10-25 | 2024-01-05 | 华南农业大学 | Nanometer antibody for resisting zearalenone and zearalanol and application thereof |
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