CN111748035B - Novel FN3 antibody mutant and application thereof - Google Patents
Novel FN3 antibody mutant and application thereof Download PDFInfo
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Abstract
The application discloses a group of novel FN3 antibody mutants and application thereof. Wherein the mutant corresponding to the original FN3 antibody comprises the following mutation sites: a at position 85 by E and Y at position 87 by M. The amino acid sequence of the original FN3 antibody is shown as SEQ ID No: 2, the process is carried out; the mutant corresponding to the original FN3 antibody comprises the following mutation sites: y at position 35 by S, P at position 56 by L, A at position 85 by E and Y at position 87 by M. The amino acid sequence of the original FN3 antibody is shown as SEQ ID No: 2, the process is carried out; the mutant corresponding to the original FN3 antibody comprises the following mutation sites: p at position 56 is substituted by L, A at position 85 by E and Y at position 87 by M. The amino acid sequence of the original FN3 antibody is shown as SEQ ID No: 2, the preparation method is as follows.
Description
Technical Field
The invention relates to one or more novel FN3 scaffold antibodies and application thereof in reagent detection and medicine. Relates to the field of biological proteins and biotechnology.
Background
The FN3 scaffold antibody is based on human tenth fibronectin type III domain, and has the advantages of only 10kDa, small molecular weight, good permeability and stable structure. Consists of a highly stable beta-sheet region and three circular variable regions. The antibody-like scaffold protein has wide application prospect, can play an important role in basic research, and can be applied to clinical diagnosis and treatment. Compared with the traditional antibody medicine, the antibody medicine has many obvious advantages, including low cost, high intracellular stability, weak immune effect, easy oral absorption and other important properties, and has become one of the hot fields of medicine research and development. The HA4 antibody is a FN3 scaffold antibody discovered by phage display technology of John Wojcik et al, and can be specifically combined with SH2 subunit of human ABL1 protein. (John Wojcik et al. A post and highly specific FN3 monobody inhibitor soft he Abl SH2 domain NATURE STRUCTURAL & MOLECULAR BIOLOGY.17, 519-
Ph (or Ph') chromosome, or Philadelphia translocation, is a specific chromosomal translocation associated with Chronic Myelogenous Leukemia (CML). Wherein the long arm of chromosome 9 and the long arm of chromosome 22 of the cell are translocated with each other, specifically defined as t (9; 22) (q 34; q 11). This chromosomal translocation phenomenon is highly sensitive to chronic myelogenous leukemia, and 95% of patients with chronic myelogenous leukemia are detected with this chromosomal translocation (the remaining population either has an invisible translocation during the preparation of G-banding chromosomes or has other variable chromosomal translocations).
Chromosomal translocations are the major cause of chromosomal deletions in the philadelphia chromosome. The long-chain ABL1 gene (position q34) in chromosome nine is translocated in parallel with the long-chain BCR gene (position q11) in chromosome twenty-two to generate a new fusion gene (fusion gene). This chromosomal translocation is referred to as "t (9; 22) (q 34; q 11)" according to the Nomenclature of the International Nomenclature of Human cytogenetics (English).
This translocation produces an oncogenic BCR-ABL1 fusion gene located on the long chain of chromosome 22, which is thus shortened. This gene produces a BCR-ABL1 fusion protein. Since the molecular weight of this fusion gene is 185 to 210kDa, it is also referred to as p185 or p 210. ABL1 is a tyrosine kinase; in normal cells, it plays an important role in cell differentiation and cell cycle regulation. The BCR-ABL1 fusion gene results in constitutive activation of tyrosine kinases, leading to uncontrolled cellular proliferation and thus carcinogenesis.
John Wojcik et al demonstrated: the HA4 antibody in the cell can inhibit the phosphorylation process of ABL1 by combining with SH2 subunit of ABL1, inhibit the activity of STAT5 and inhibit the constitutive activation of tyrosine kinase. This suggests that the HA4 antibody may be a candidate for anticancer drugs, or may be used for the mechanism study of cancer caused by BCR-ABL1, and the like. Based on the previous research, we have screened HA4 mutants by biotechnology methods, named as KJDL2, KJDL7 and KJDL9 (all mutants are referred to as KJDL in the following text), and the new mutants have better affinity with ABL1 protein, thus becoming better candidates for anticancer drugs or detection kits. Meanwhile, the research also finds that A85E and Y87M are two key amino acid mutations influencing the affinity, and when the A85E and Y87M mutations occur in the amino acid sequence of HA4, the affinity of the novel antibody generated by combining the mutations at other sites with the ABL1 protein is improved.
Disclosure of Invention
The object of the present invention is to provide one or more mutation sites and mutated amino acids. The antibody-like body containing the mutation has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like.
It is another object of the present invention to provide a mutant amino acid sequence having a mutation from A to E at amino acid sequence position 85 and a mutation from Y to M at amino acid sequence position 87, the amino acid sequence before mutation being as set forth in SEQ ID No: 2, the preparation method is as follows. The antibody including the mutation has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like. Preferably, the amino acid sequence of the antibody is shown in SEQ ID No: 4, respectively.
It is another object of the present invention to provide a mutant form P to L at amino acid sequence position 56, A to E at amino acid sequence position 85, and Y to M at amino acid sequence position 87, the amino acid sequence before mutation being as set forth in SEQ ID No: 2, the preparation method is as follows. The antibody including the mutation has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like. Preferably, the amino acid sequence of the antibody is shown in SEQ ID No: shown in fig. 8.
It is another object of the present invention to provide a polypeptide having a mutation at amino acid sequence position 35 from Y to S, a mutation at amino acid sequence position 56 from P to L, a mutation at amino acid sequence position 85 from A to E, and a mutation at amino acid sequence position 87 from Y to M, wherein the amino acid sequence before mutation is as shown in SEQ ID No: 2, the preparation method is as follows. The antibody including the mutation has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like. Preferably, the amino acid sequence of the antibody is shown in SEQ ID No: and 6.
It is another object of the present invention to provide a human FN 3-like antibody mutant consisting of a highly stable beta sheet region and three circular variable regions including BC Loop, DE Loop, and FG Loop. The amino acid sequence of the BC Loop is SEQ ID NO: 9, wherein X1 is selected from any one of Y and S; the amino acid sequence of the DE Loop is SEQ ID NO: 9 amino acids 57-60; the amino acid sequence of FG Loop is SEQ ID NO: 9 from amino acids 90 to 102. The mutant antibody has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like.
It is another object of the present invention to provide a human FN 3-like antibody mutant consisting of a highly stable beta sheet region and three circular variable regions including BC Loop, DE Loop, and FG Loop. The amino acid sequence of the BC Loop is SEQ ID NO:10, wherein X1 is selected from any one of Y and S; the amino acid sequence of the DE Loop is SEQ ID NO:10 amino acids 57-60; the amino acid sequence of FG Loop is SEQ ID NO:10 from amino acids 90 to 102. The mutant antibody has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like.
Another objective of the present invention is to provide an amino acid sequence of a human FN 3-like antibody mutant, the amino acid sequence being as shown in SEQ ID NO: 4, the method is described in the specification. The antibody has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like.
Another objective of the present invention is to provide an amino acid sequence of a human FN 3-like antibody mutant, the amino acid sequence being as shown in SEQ ID NO: 6, and (3). The antibody has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like.
Another objective of the present invention is to provide an amino acid sequence of a human FN 3-like antibody mutant, the amino acid sequence being as shown in SEQ ID NO: 8, the method is described in the specification. The antibody has stronger affinity with ABL1 protein, and can be used as a candidate drug of an anti-cancer drug or applied to other fields of medicines and the like.
Another objective of the invention is to provide a method for preparing a human FN3 antibody mutant, wherein the antibody can be artificially synthesized, or the encoding gene can be synthesized, and then the antibody is biologically expressed to obtain a cell containing the FN3 antibody mutant, and the cell is cultured to obtain the FN3 antibody mutant. The cell can be a microbial cell, such as Escherichia coli, and specifically can be Escherichia coli BL 21.
It is another object of the present invention to provide a kit for testing the SH2 subunit of ABL1 protein or ABL1 protein or SH2 subunit of ABL1 fusion protein, the composition of which comprises an antibody-like body containing any one of the above combinations of mutations.
It is another object of the present invention to provide a kit for testing the SH2 subunit of ABL1 protein or ABL1 protein or SH2 subunit of ABL1 fusion protein, the composition of which comprises the amino acid sequence selected from SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO:10, wherein: SEQ ID NO: 9 or X1 in SEQ ID NO. 10 is selected from any one of Y and S.
It is another object of the present invention to provide a method for detecting ABL1 protein or other types of ABL1 fusion proteins, comprising the steps of: (As for the specific procedure, refer to example 5)
1) Coating ABL1 protein or other ABL1 fusion protein on an enzyme label plate, and sealing with a sealing liquid;
2) adding any one of the KJDL antibodies;
3) adding enzyme-labeled secondary antibody;
4) adding a developing solution for developing for 10 minutes, adding an isometric stop solution, putting the ELISA plate into an ELISA reader, and reading a light absorption value at 450 nm.
Another objective of the invention is to provide applications of the above mutant sites, mutant amino acids, or their combinations in preparing anticancer drugs or other medical fields.
Another object of the present invention is to provide the use of the above mutant site, mutant amino acid, or combination thereof in the preparation of a kit.
It is another object of the present invention to provide the use of the above mentioned mutant sites, mutant amino acids, or combinations thereof for detecting ABL1 protein or other types of ABL1 fusion proteins.
The invention also aims to provide the application of the amino acid sequence of any one of the antibodies in the preparation of anti-cancer drugs or other medical fields.
Another objective of the invention is to provide application of the amino acid sequence of any one of the antibodies in preparation of a kit.
Another objective of the invention is to provide application of the amino acid sequence of any one of the antibodies in detection of ABL1 protein or other types of ABL1 fusion proteins.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a diagram of the expression vector of HA 4;
FIG. 2 is a diagram of a universal expression vector for a KJDL mutant;
FIG. 3 is a graph showing the results of electrophoresis; the size of the target protein is 14.6kDa, and the size of the band in an observed electrophoretogram is proper when compared with the molecular weight standard of the protein, and the purity is about 70 percent. Wherein 1 is 200mM imidazole elution peak of HA4 antibody; 3 is 200mM imidazole elution peak of KJDL7 antibody; 5 is 200mM imidazole elution peak of KJDL9 antibody; 8 is the 200mM imidazole elution peak of KJDL2 antibody;
FIG. 4 is a diagram showing the amino acid sequence alignment of the KJDL mutant and HA 4;
FIG. 5 is a biacore affinity curve of KJDL7 mutant;
FIG. 6 is a graph of HA4 affinity;
FIG. 7 is a KJDL2 affinity plot.
Detailed Description
The following examples are presented to facilitate a better understanding of the present invention and are not intended to limit the invention thereto.
The present invention will be described in further detail with reference to specific embodiments. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The engineered strain used in all examples was E.coli BL 21.
The LB medium components are tryptone 10g/L, yeast extract 5g/L, NaCl 10 g/L.
Example 1 affinity test of HA4 protein with ABL1 protein.
1.1 construction and expression of the HA4 vector.
After a primer (a primer sequence is shown in a table 1) is designed and an HA4 encoding gene (a gene sequence is shown in SEQ ID NO: 1) is amplified, enzyme digestion is carried out, a gene coding sequence is constructed on a protein expression vector pET28a through a method of enzyme digestion and T4 connection, as shown in a figure 1, a gene sequence of an HA4 antibody is connected to an expression vector pET28a, then protein expression is induced by shaking bacteria, and an engineering strain containing the expression vector is cultured to express recombinant protein.
1.2 culturing and expressing the recombinant protein.
The specific steps are that the engineering strain obtained in the step 1.1 is subjected to shaking overnight culture in LB liquid culture medium at 37 ℃, then the engineering strain is inoculated into a shake flask containing 1L of LB culture medium in a ratio of 1:100, the culture is carried out for 2h at 37 ℃, isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1mmol/L is added for induction, and the temperature is reduced to 16 ℃ for overnight culture. Centrifuging at 8000r/min for 10min to collect thallus, resuspending with bacteria breaking solution (20mM PB, 150mM NaCl, pH 8.0), ultrasonic crushing, centrifuging at 12000r/min for 30min to collect supernatant, and purifying.
1.3 purification of the recombinant protein.
Since the rear-end sequence of the recombinant protein obtained in the step 1.2 contains His fusion protein, the recombinant protein can be purified by using a His affinity chromatography column. The buffer used for affinity chromatography was 20mM PB, 150mM NaCl, pH 7.5, and the buffer used for elution was 20mM PB, 150mM NaCl, 500mM imidazole, pH 7.5. Collecting the eluted fractions, and detecting the expression of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (the chromatogram can be shown in figure 3) the size of the target protein is 14.6kDa, the size of the band in the observed electrophoretogram is compared with the standard of the protein molecular weight, and the target protein with proper size is selected to go to the next step.
1.4 the purified protein obtained in step 1.3 was tested for affinity using a biacore instrument.
Test protocol: the apparatus BIACORE T200 used was manufactured by GE.
An experimental chip: CM5 is available from GE under the trade designation BR-1005-30.
The target protein capture kit comprises: GST Capture Kit, available from GE under the reference BR-1002-23.
Target protein: c-Abl/ABL1 Protein, Human, Recombinant (GST tag) available from Italy Hooka, Cat #: 11199-H09B, and the target protein SH2 is a subunit of ABL1 protein and is a subunit combined with HA 4.
The following test results were obtained according to the instrument instructions and the kit protocol:
the specific test steps are divided into the following steps:
a) starting up: setting the reaction temperature to be 25 ℃, waiting for 30min, and starting the test when the yellow indicator lamp on the panel stops flashing to indicate that the temperature reaches the preset temperature.
b) The control software was turned on to prepare the buffer, 50mL 10 x HBS-EP + buffer, 450mL deionized water (filtered through a 0.22 μm filter) were measured and mixed in a 500mL buffer vial.
c) Putting in a CM5 chip. The anti-GST antibody was immobilized on a chip according to the GST Capture Kit instructions and blocked, then the recombinant protein ABL1 (GSTtag) was captured using the anti-GST antibody, and the unused epitope of the anti-GST antibody was blocked with a reagent and the recombinant GST protein in the middle.
d) The HA4 protein to be tested is loaded and the affinity is determined.
e) Fitting the affinity curve by using software to obtain kinetic data Ka、KdAffinity data KD. Since the affinity of HA4 to ABL1 is the base reference value for comparison with the affinity of other samples to ABL1, the affinity values were measured twice, respectively, see a and b of table 2.
Example 2: affinity of KJDL7 samples with ABL1 protein.
2.1 construction and expression of KJDL7 vector.
After a primer (a primer sequence is shown in a table 1) is designed and a KJDL7 encoding gene (a gene sequence is shown in SEQ ID NO: 3) is amplified, enzyme digestion is carried out, a gene coding sequence is constructed on a protein expression vector pET28a by a method of enzyme digestion and T4 connection, as shown in a figure 2, a gene sequence of a KJDL7 antibody is connected to an expression vector pET28a, then protein expression is induced by shaking bacteria, and an engineering strain containing the expression vector is cultured to express recombinant protein.
2.2 culturing and expressing the recombinant protein.
The specific steps are that the engineering strain obtained in the step 2.1 is shaken in an LB liquid culture medium at 37 ℃ for overnight culture, then is inoculated into a shake flask containing 1L of LB culture medium in a ratio of 1:100, is cultured for 2h at 37 ℃, is added with isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1mmol/L for induction, and is cooled to 16 ℃ for overnight culture. Centrifuging at 8000r/min for 10min to collect thallus, resuspending with bacteria breaking solution (20mM PB, 150mM NaCl, pH 8.0), ultrasonic crushing, centrifuging at 12000r/min for 30min to collect supernatant, and purifying.
2.3 purification of the recombinant protein.
Since the rear-end sequence of the recombinant protein obtained in step 2.2 contains His fusion protein, the recombinant protein can be purified by using a His affinity chromatography column. The buffer used for affinity chromatography was 20mM PB, 150mM NaCl, pH 7.5, and the buffer used for elution was 20mM PB, 150mM NaCl, 500mM imidazole, pH 7.5. Collecting the eluted fractions, and detecting the expression of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (the chromatogram can be shown in figure 3) the size of the target protein is 14.6kDa, the size of the band in the observed electrophoretogram is compared with the standard of the protein molecular weight, and the target protein with proper size is selected to go to the next step.
2.4 affinity testing of the purified protein from step 2.3 was performed using a biacore instrument.
Test protocol: the apparatus BIACORE T200 was manufactured by GE.
An experimental chip: CM5 is available from GE under the trade designation BR-1005-30.
The target protein capture kit comprises: GST Capture Kit, available from GE under the reference BR-1002-23.
Target protein: c-Abl/ABL1 Protein, Human, Recombinant (GST tag) available from Italy Hooka, Cat #: 11199-H09B, and the target protein SH2 is a subunit of ABL1 protein and is a subunit combined with KJDL 7.
The following test results were obtained according to the instrument instructions and the kit protocol:
the specific test steps are divided into the following steps:
a) starting up: setting the reaction temperature to be 25 ℃, waiting for 30min, and starting the test when the yellow indicator lamp on the panel stops flashing to indicate that the temperature reaches the preset temperature.
b) The control software was turned on to prepare the buffer, 50mL 10 x HBS-EP + buffer, 450mL deionized water (filtered through a 0.22 μm filter) were measured and mixed in a 500mL buffer vial.
c) Putting in a CM5 chip. The anti-GST antibody was immobilized on a chip according to the GST Capture Kit instructions and blocked, then the recombinant protein ABL1 (GSTtag) was captured using the anti-GST antibody, and the unused epitope of the anti-GST antibody was blocked with a reagent and the recombinant GST protein in the middle.
d) The KJDL7 protein to be tested is loaded and the affinity is determined.
e) Fitting the affinity curve by using software to obtain kinetic data Ka、KdAffinity data KD. The affinity values are shown in Table 2.
2.5 comparison of the affinity of KJDL7 for ABL1 protein with the affinity of the original HA4 for ABL1 protein.
As shown in Table 2-a, the affinity constant K of HA4 was measuredDAffinity constant K for KJDL7 mutant at 2.73e-08D1.43e-08, which is obviously lower than HA4, the lower the affinity constant, the higher the affinity, which indicates that the affinity is obviously improved after the amino acid mutation of the key site. Affinity curves fitted to KJDL7 and HA4 are shown in FIGS. 5 and 6, 0 seconds and 180 seconds, respectivelyThe curve shows a sharp increase and decrease, respectively, due to the inconsistency of the buffer of the system with the buffer of the antibody protein to be determined, which does not affect the determination of the affinity and can be ignored. The radian of KJDL7 is greater, HA4 is straighter, and the slope of KJDL7 is greater during the rise of both affinity curves, and the slope represents the binding constant Ka. While incorporating the constant K in Table 2-aaIs also KJDL7>HA4 shows that after the amino acid mutation of the key site, the binding speed is obviously improved. The slope of the descending phase sample KJDL7 is less than HA4, and the descending slope represents the dissociation constant Kd. While the dissociation constant Kd in Table 2-a is also KJDL7<HA4, indicating that the off-rate of KJDL7 becomes slower compared to HA4, indicating that this mutant is less likely to dissociate once bound to ABL1 protein. The data demonstrate that after mutation at key amino acid sites, KJDL7 binds faster and dissociates more slowly than HA4 with higher affinity than HA 4.
Example 3: testing of affinity of KJDL9 samples to ABL1 protein
3.1 construction and expression of KJDL9 vector.
After a primer (a primer sequence is shown in a table 1) is designed and a KJDL9 encoding gene (a gene sequence is shown in SEQ ID NO: 5) is amplified, enzyme digestion is carried out, a gene coding sequence is constructed on a protein expression vector pET28a by a method of enzyme digestion and T4 connection, as shown in a figure 2, a gene sequence of a KJDL9 antibody is connected to an expression vector pET28a, then protein expression is induced by shaking bacteria, and an engineering strain containing the expression vector is cultured to express recombinant protein.
3.2 culturing and expressing the recombinant protein.
The specific steps are that the engineering strain obtained in the step 3.1 is subjected to shaking overnight culture in LB liquid culture medium at 37 ℃, then the engineering strain is inoculated into a shake flask containing 1L of LB culture medium in the inoculation amount of 1:100, the culture is carried out for 2h at 37 ℃, isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1mmol/L is added for induction, and the temperature is reduced to 16 ℃ for overnight culture. Centrifuging at 8000r/min for 10min to collect thallus, resuspending with bacteria breaking solution (20mM PB, 150mM NaCl, pH 8.0), ultrasonic crushing, centrifuging at 12000r/min for 30min to collect supernatant, and purifying.
3.3 purification of the recombinant protein.
Since the rear-end sequence of the recombinant protein obtained in step 3.2 contains His fusion protein, the recombinant protein can be purified by using a His affinity chromatography column. The buffer used for affinity chromatography was 20mM PB, 150mM NaCl, pH 7.5, and the buffer used for elution was 20mM PB, 150mM NaCl, 500mM imidazole, pH 7.5. Collecting the eluted fractions, and detecting the expression of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (the chromatogram can be shown in figure 3) the size of the target protein is 14.6kDa, the size of the band in the observed electrophoretogram is compared with the standard of the protein molecular weight, and the target protein with proper size is selected to go to the next step.
3.4 affinity testing of the purified protein from step 3.3 was performed using a biacore instrument.
Test protocol: the apparatus BIACORE T200 was manufactured by GE.
An experimental chip: CM5 is available from GE under the trade designation BR-1005-30.
The target protein capture kit comprises: GST Capture Kit, available from GE under the reference BR-1002-23.
Target protein: c-Abl/ABL1 Protein, Human, Recombinant (GST tag) available from Italy Hooka, Cat #: 11199-H09B, and the target protein SH2 is a subunit of ABL1 protein and is a subunit combined with KJDL 9.
The following test results were obtained according to the instrument instructions and the kit protocol:
the specific test steps are divided into the following steps:
a) starting up: setting the reaction temperature to be 25 ℃, waiting for 30min, and starting the test when the yellow indicator lamp on the panel stops flashing to indicate that the temperature reaches the preset temperature.
b) The control software was turned on to prepare the buffer, 50mL 10 x HBS-EP + buffer, 450mL deionized water (filtered through a 0.22 μm filter) were measured and mixed in a 500mL buffer vial.
c) Putting in a CM5 chip. The anti-GST antibody was immobilized on a chip according to the GST Capture Kit instructions and blocked, then the recombinant protein ABL1 (GSTtag) was captured using the anti-GST antibody, and the unused epitope of the anti-GST antibody was blocked with a reagent and the recombinant GST protein in the middle.
d) The HA4 protein to be tested is loaded and the affinity is determined.
e) Fitting the affinity curve by using software to obtain kinetic data Ka、KdAffinity data KD. The affinity values are shown in Table 2.
3.5 comparison of the affinity of KJDL9 to ABL1 protein with the affinity of HA4 to ABL1 protein.
As shown in Table 2-b, the affinity constant K of HA4 was measuredDThe affinity constant K of the KJDL9 mutant was 4.92e-07D4.69e-08, significantly lower than HA4, and an order of magnitude lower. The lower the affinity constant, the higher the affinity, indicating that the affinity is significantly improved after the amino acid mutation at the key site.
Example 4: affinity of KJDL2 samples with ABL1 protein.
4.1 construction and expression of KJDL2 vector.
After a primer (a primer sequence is shown in a table 1) is designed and a KJDL2 encoding gene (a gene sequence is shown in SEQ ID NO: 7) is amplified, enzyme digestion is carried out, a gene coding sequence is constructed on a protein expression vector pET28a by a method of enzyme digestion and T4 connection, as shown in a figure 2, a gene sequence of a KJDL2 antibody is connected to an expression vector pET28a, then protein expression is induced by shaking bacteria, and an engineering strain containing the expression vector is cultured to express recombinant protein.
4.2 culturing and expressing the recombinant protein.
The specific steps are that the engineering strain obtained in the step 4.1 is shaken in an LB liquid culture medium at 37 ℃ for overnight culture, then is inoculated into a shake flask containing 1L of LB culture medium in a ratio of 1:100, is cultured for 2h at 37 ℃, is added with isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 1mmol/L for induction, and is cooled to 16 ℃ for overnight culture. Centrifuging at 8000r/min for 10min to collect thallus, resuspending with bacteria breaking solution (20mM PB, 150mM NaCl, pH 8.0), ultrasonic crushing, centrifuging at 12000r/min for 30min to collect supernatant, and purifying.
4.3 purification of the recombinant protein.
Since the rear-end sequence of the recombinant protein obtained in the step 4.2 contains His fusion protein, the recombinant protein can be purified by using a His affinity chromatography column. The buffer used for affinity chromatography was 20mM PB, 150mM NaCl, pH 7.5, and the buffer used for elution was 20mM PB, 150mM NaCl, 500mM imidazole, pH 7.5. Collecting the eluted fractions, and detecting the expression of the target protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (the chromatogram can be shown in figure 3) the size of the target protein is 14.6kDa, the size of the band in the observed electrophoretogram is compared with the standard of the protein molecular weight, and the target protein with proper size is selected to go to the next step.
4.4 affinity testing of the purified protein from step 4.3 was performed using a biacore instrument.
Test protocol: the apparatus BIACORE T200 was manufactured by GE.
An experimental chip: CM5 is available from GE under the trade designation BR-1005-30.
The target protein capture kit comprises: GST Capture Kit, available from GE under the reference BR-1002-23.
Target protein: c-Abl/ABL1 Protein, Human, Recombinant (GST tag) available from Italy Hooka, Cat #: 11199-H09B, and the target protein SH2 is a subunit of ABL1 protein and is a subunit combined with KJDL 2.
The following test results were obtained according to the instrument instructions and the kit protocol:
the specific test steps are divided into the following steps:
a) starting up: setting the reaction temperature to be 25 ℃, waiting for 30min, and starting the test when the yellow indicator lamp on the panel stops flashing to indicate that the temperature reaches the preset temperature.
b) The control software was turned on to prepare the buffer, 50mL 10 x HBS-EP + buffer, 450mL deionized water (filtered through a 0.22 μm filter) were measured and mixed in a 500mL buffer vial.
c) Putting in a CM5 chip. The anti-GST antibody was immobilized on a chip according to the GST Capture Kit instructions and blocked, then the recombinant protein ABL1 (GSTtag) was captured using the anti-GST antibody, and the unused epitope of the anti-GST antibody was blocked with a reagent and the recombinant GST protein in the middle.
d) The HA4 protein to be tested is loaded and the affinity is determined.
e) Fitting the affinity curve by using software to obtain kinetic data Ka、KdAffinity data KD. The affinity values are shown in Table 2.
Comparison of the affinity of 4.5KJDL2 for ABL1 protein with the affinity of HA4 for ABL1 protein.
As shown in Table 2-a, the affinity constant K of HA4 was measuredDAffinity constant K for KJDL2 mutant at 2.73e-08DIs 7.00E-09 and is obviously lower than HA4, and the lower the affinity constant, the higher the affinity, which shows that the affinity is obviously improved after the amino acid mutation of the key site. KJDL2 and HA4 fit affinity curves as shown in FIGS. 7 and 6, respectively, with a sharp increase and decrease in the 0 second and 180 second curves, respectively, due to the inconsistency between the system buffer and the buffer of the antibody protein to be tested, which does not affect the affinity determination and can be ignored. The radian of KJDL2 is greater, HA4 is straighter, and the slope of KJDL2 is significantly greater during the rise of both affinity curves, the slope representing the binding constant Ka. While incorporating the constant K in Table 2-aaIs also KJDL2>HA2 shows that after the amino acid mutation of the key site, the binding speed is obviously improved. The slope of the descending phase sample KJDL2 is less than HA4, and the descending slope represents the dissociation constant Kd. While dissociation constant K in Table 2-adIs also KJDL2<HA4, indicating that the off-rate of KJDL2 becomes slower compared to HA4, indicating that this mutant is less likely to dissociate once bound to ABL1 protein. The data demonstrate that after mutation at key amino acid sites, KJDL2 binds faster and dissociates more slowly than HA4 with higher affinity than HA 4.
Example 5: detection of ABL1 protein content by using KJDL7 mutant
The instrument used in this experiment: incubator, wash trigger and ELIASA.
Reagents and sources used in this experiment were: ABL1 protein was purchased from KJDL7 monoclonal antibody, HRP-labeled anti-His-tag murine monoclonal antibody and TMB monocomponent color developing solution in Chinesia.
Reagents used in this experiment:
phosphate Buffered Saline (PBS): weighing 8.0g NaCl,0.2g KH2PO4,2.96g Na2HPO 4.12H 2O, adding 1000ml distilled water into a measuring cylinder, and adjusting the pH value to 7.5;
sample dilution (PBST): 100ml PBS with 0.1ml Tween-20.
Wash solution (PBST): 1000ml PBS with 1ml Tween-20.
Stopping liquid: 1mol/L H2SO 2 4. The sulfuric acid was added to the water with constant stirring. The reaction is largely exothermic.
Coating buffer solution: 1.5g of Na2CO3,2.93g of NaHCO3,0.2g of NaN3 (optional) were weighed out and 1000ml of distilled water were added to the flask, pH 9.6.
Sealing liquid: 3g of bovine serum albumin was dissolved in 100ml of coating buffer.
The KJDL7 mutant used in the experiment is used for detecting the content of ABL1 protein by an ELISA method, and the specific experimental steps are as follows:
5.1 coating: ABL1 protein 0.2mg/mL was diluted with coating buffer to 0 well, 1: 200. 1:1000, 1:5000 per well 100ul, 3 replicate wells per concentration 4 ℃, wet box inner wrap overnight.
5.2 washing the plate: the sample was spun off, then 300ul of wash solution was added with a calandria gun and poured off, the plate was washed 4 times and two drops were thrown in the newspaper.
5.3 sealing: 120ul of confining liquid is added into each hole, the temperature in the box is 37 ℃ and the time is 2h, and then the confining liquid is thrown off.
5.4 His-tagged KJDL7 antibody was added at a 1:200 dilution of 100ul per well and incubated in the wet box at 37 ℃ for 1 h.
5.5 washing the plate: the sample was spun off, then 300ul of wash solution was added with a calandria gun and poured off, the plate was washed 4 times and two drops were thrown in the newspaper.
5.6 adding HRP-labeled anti-His tag antibody 1:500 dilution, each well 100ul, placed at 37 degrees C, wet box temperature 1h incubation.
5.7 washing the plate: the sample was spun off, then 300ul of wash solution was added with a calandria gun and poured off, the plate was washed 4 times and two drops were thrown in the newspaper.
5.8 adding TMB single-component color developing solution, 100ul per hole, developing for 20min in a dark place, and adding equal volume of stop solution.
5.9 color comparison: the detection type was set to absorb light on the microplate reader, the wavelength was set to 450nm, and the plate was then read selectively. The OD of the sample at 450nm for light absorption was determined.
Table 1: primer sequence Listing
Table 2: biacore measurement a of the sample in the example first measurement
b second measurement result
Table 3: antibody name and corresponding mutation site and amino acid changes
| Name of antibody | Mutation site of amino acid and change of amino acid |
| KJDL2 | P56L，A85E，Y87M |
| KJDL7 | A85E，Y87M |
| KJDL9 | Y35S，P56L，A85E，Y87M |
Table 4: OD value of ABL1 protein concentration
According to ELISA detection results, when the concentration of the ABL1 protein is 200ng/mL, the measured OD value is 0.255, while the OD value measured by the ABL1 protein without coating is only 0.129, and the two are very different remarkably through differential analysis, so that the lowest detection concentration can be judged to be 200 ng/mL. The KJDL7 antibody can be used for detecting ABL1 protein. The detection method is only illustrative and is not limited to this one detection method.
Example 6: amino acid sequence alignment of HA4 and KJDL antibodies
As shown in the attached FIG. 4, the sequence alignment of the three mutants shows that the mutation sites of the three mutants are A85E and Y87M, and the research proves that: the 87 th amino acid is a key amino acid position, and the mutant HA4-Y87A completely loses the affinity as long as the 87 th amino acid is mutated into alanine. However, the 87 th amino acid was mutated to methionine for the first time. In addition, the 85-position amino acid is changed into glutamic acid from alanine, KJDL7 has the two mutation sites, the affinity is obviously improved, KJDL2 increases the 56-position amino acid from proline to leucine on the basis of the two mutation sites of KJDL7, and the affinity is further improved. And KJDL9 has the amino acid 35 changed from tyrosine to serine at three mutation sites of KJDL 2. The affinity of this mutant was also higher than that of HA 4. A summary of the mutation points of the KJDL antibodies is shown in Table 3.
Although the present application has been described in detail with respect to the general description and the specific examples, it will be apparent to those skilled in the art that certain changes and modifications may be made based on the present application. Accordingly, such modifications and improvements are intended to be within the scope of this invention as claimed.
----sequence info-------
>SEQ1-HA4-DNA
>SEQ2-HA4
GSSVSSVPTKLEVVAATPTSLLISWDAPMSSSSVYYYRITYGETGGNSPVQEFTVPYSSSTATISGLSPGVDYTITVYAW GEDSAGYMFMYSPISINYRTC
>SEQ3-KJDL7
>SEQ4-KJDL7(A85E+Y87M)
GSSVSSVPTKLEVVAATPTSLLISWDAPMSSSSVYYYRITYGETGGNSPVQEFTVPYSSSTATISGLSPGVDYTITVYAW GEDSEGMMFMYSPISINYRTC
>SEQ5-KJDL9
>SEQ6-KJDL9(Y35S+P56L+A85E+Y87M)
GSSVSSVPTKLEVVAATPTSLLISWDAPMSSSSVSYYRITYGETGGNSPVQEFTVLYSSSTATISGLSPGVDYTITVYAW GEDSEGMMFMYSPISINYRTC
>SEQ7-KJDL2
>SEQ8-KJDL2(P56L+A85E+Y87M)
GSSVSSVPTKLEVVAATPTSLLISWDAPMSSSSVYYYRITYGETGGNSPVQEFTVLYSSSTATISGLSPGVDYTITVYAW GEDSEGMMFMYSPISINYRTC
>seq9-general-Seq id no:9(x2-P)
GSSVSSVPTKLEVVAATPTSLLISWDAPMSSSSVX1YYRITYGETGGNSPVQEFTVPYSSSTATISGLSPGVDYTITVYAWGE DSEGMMFMYSPISINYRTC
>seq10-general Seq id no:10(x2-L)
GSSVSSVPTKLEVVAATPTSLLISWDAPMSSSSVX1YYRITYGETGGNSPVQEFTVLYSSSTATISGLSPGVDYTITVYA WGEDSEGMMFMYSPISINYRTC。
Sequence listing
<110> Kochia-Davida Biotechnology Ltd
<120> a novel group of FN3 antibody mutants and uses thereof
<141> 2020-05-15
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 306
<212> DNA
<213> Artificial Sequence
<400> 1
ggcagctctg tgagtagcgt tccgaccaaa ctggaagtgg ttgcagcaac cccgacgagc 60
ctgctgattt cttgggatgc cccgatgtct agtagctctg tgtattacta tcgtatcacc 120
tacggtgaaa cgggcggtaa cagcccggtg caggaattta cggttccgta tagtagctct 180
accgcgacga ttagtggcct gagcccgggt gtggattaca ccatcacggt ttatgcatgg 240
ggcgaagata gcgcgggtta tatgttcatg tattctccga ttagtatcaa ttaccgcacc 300
tgctaa 306
<210> 2
<211> 101
<212> PRT
<213> Artificial Sequence
<400> 2
Gly Ser Ser Val Ser Ser Val Pro Thr Lys Leu Glu Val Val Ala Ala
1 5 10 15
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Met Ser Ser Ser
20 25 30
Ser Val Tyr Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
35 40 45
Pro Val Gln Glu Phe Thr Val Pro Tyr Ser Ser Ser Thr Ala Thr Ile
50 55 60
Ser Gly Leu Ser Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Trp
65 70 75 80
Gly Glu Asp Ser Ala Gly Tyr Met Phe Met Tyr Ser Pro Ile Ser Ile
85 90 95
Asn Tyr Arg Thr Cys
100
<210> 3
<211> 306
<212> DNA
<213> Artificial Sequence
<400> 3
ggcagctctg tgagtagcgt tccgaccaaa ctggaagtgg ttgcagcaac cccgacgagc 60
ctgctgattt cttgggatgc cccgatgtct agtagctctg tgtattacta tcgtatcacc 120
tacggtgaaa cgggcggtaa cagcccggtg caggaattta cggttccgta tagtagctct 180
accgcgacga ttagtggcct gagcccgggt gtggattaca ccatcacggt ttatgcatgg 240
ggcgaagata gcgagggtat gatgttcatg tattctccga ttagtatcaa ttaccgcacc 300
tgctaa 306
<210> 4
<211> 101
<212> PRT
<213> Artificial Sequence
<400> 4
Gly Ser Ser Val Ser Ser Val Pro Thr Lys Leu Glu Val Val Ala Ala
1 5 10 15
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Met Ser Ser Ser
20 25 30
Ser Val Tyr Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
35 40 45
Pro Val Gln Glu Phe Thr Val Pro Tyr Ser Ser Ser Thr Ala Thr Ile
50 55 60
Ser Gly Leu Ser Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Trp
65 70 75 80
Gly Glu Asp Ser Glu Gly Met Met Phe Met Tyr Ser Pro Ile Ser Ile
85 90 95
Asn Tyr Arg Thr Cys
100
<210> 5
<211> 306
<212> DNA
<213> Artificial Sequence
<400> 5
ggcagctctg tgagtagcgt tccgaccaaa ctggaagtgg ttgcagcaac cccgacgagc 60
ctgctgattt cttgggatgc cccgatgtct agtagctctg tgtcttacta tcgtatcacc 120
tacggtgaaa cgggcggtaa cagcccggtg caggaattta cggttctgta tagtagctct 180
accgcgacga ttagtggcct gagcccgggt gtggattaca ccatcacggt ttatgcatgg 240
ggcgaagata gcgagggtat gatgttcatg tattctccga ttagtatcaa ttaccgcacc 300
tgctaa 306
<210> 6
<211> 101
<212> PRT
<213> Artificial Sequence
<400> 6
Gly Ser Ser Val Ser Ser Val Pro Thr Lys Leu Glu Val Val Ala Ala
1 5 10 15
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Met Ser Ser Ser
20 25 30
Ser Val Ser Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
35 40 45
Pro Val Gln Glu Phe Thr Val Leu Tyr Ser Ser Ser Thr Ala Thr Ile
50 55 60
Ser Gly Leu Ser Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Trp
65 70 75 80
Gly Glu Asp Ser Glu Gly Met Met Phe Met Tyr Ser Pro Ile Ser Ile
85 90 95
Asn Tyr Arg Thr Cys
100
<210> 7
<211> 306
<212> DNA
<213> Artificial Sequence
<400> 7
ggcagctctg tgagtagcgt tccgaccaaa ctggaagtgg ttgcagcaac cccgacgagc 60
ctgctgattt cttgggatgc cccgatgtct agtagctctg tgtattacta tcgtatcacc 120
tacggtgaaa cgggcggtaa cagcccggtg caggaattta cggttctgta tagtagctct 180
accgcgacga ttagtggcct gagcccgggt gtggattaca ccatcacggt ttatgcatgg 240
ggcgaagata gcgagggtat gatgttcatg tattctccga ttagtatcaa ttaccgcacc 300
tgctaa 306
<210> 8
<211> 101
<212> PRT
<213> Artificial Sequence
<400> 8
Gly Ser Ser Val Ser Ser Val Pro Thr Lys Leu Glu Val Val Ala Ala
1 5 10 15
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Met Ser Ser Ser
20 25 30
Ser Val Tyr Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
35 40 45
Pro Val Gln Glu Phe Thr Val Leu Tyr Ser Ser Ser Thr Ala Thr Ile
50 55 60
Ser Gly Leu Ser Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Trp
65 70 75 80
Gly Glu Asp Ser Glu Gly Met Met Phe Met Tyr Ser Pro Ile Ser Ile
85 90 95
Asn Tyr Arg Thr Cys
100
<210> 9
<211> 101
<212> PRT
<213> Artificial Sequence
<400> 9
Gly Ser Ser Val Ser Ser Val Pro Thr Lys Leu Glu Val Val Ala Ala
1 5 10 15
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Met Ser Ser Ser
20 25 30
Ser Val Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
35 40 45
Pro Val Gln Glu Phe Thr Val Pro Tyr Ser Ser Ser Thr Ala Thr Ile
50 55 60
Ser Gly Leu Ser Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Trp
65 70 75 80
Gly Glu Asp Ser Glu Gly Met Met Phe Met Tyr Ser Pro Ile Ser Ile
85 90 95
Asn Tyr Arg Thr Cys
100
<210> 10
<211> 101
<212> PRT
<213> Artificial Sequence
<400> 10
Gly Ser Ser Val Ser Ser Val Pro Thr Lys Leu Glu Val Val Ala Ala
1 5 10 15
Thr Pro Thr Ser Leu Leu Ile Ser Trp Asp Ala Pro Met Ser Ser Ser
20 25 30
Ser Val Xaa Tyr Tyr Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser
35 40 45
Pro Val Gln Glu Phe Thr Val Leu Tyr Ser Ser Ser Thr Ala Thr Ile
50 55 60
Ser Gly Leu Ser Pro Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Trp
65 70 75 80
Gly Glu Asp Ser Glu Gly Met Met Phe Met Tyr Ser Pro Ile Ser Ile
85 90 95
Asn Tyr Arg Thr Cys
100
Claims (10)
1. A human-derived FN 3-like antibody mutant, comprising the following mutation sites corresponding to the original FN 3-like antibody: a substitution at position 85 with E and a substitution at position 87 with M; the amino acid sequence of the original FN3 antibody is shown as SEQ ID No: 2, the preparation method is as follows.
2. A human-derived FN 3-like antibody mutant, comprising the following mutation sites corresponding to the original FN 3-like antibody: y at position 35 substituted by S, P at position 56 substituted by L, A at position 85 substituted by E and Y at position 87 substituted by M; the amino acid sequence of the original FN3 antibody is shown as SEQ ID No: 2, the preparation method is as follows.
3. A human-derived FN 3-like antibody mutant, comprising the following mutation sites corresponding to the original FN 3-like antibody: p at position 56 substituted by L, a at position 85 substituted by E and Y at position 87 substituted by M; the amino acid sequence of the original FN3 antibody is shown as SEQ ID No: 2, the preparation method is as follows.
4. The FN 3-like antibody mutant of any of claims 1-3, which is producible from biological material.
5. The FN 3-like antibody mutant of claim 4, wherein the biological material is a microorganism.
6. The FN 3-like antibody mutant of claim 5, wherein the biological material is e.
7. A test method for detecting ABL1 protein for non-disease diagnostic purposes comprising the steps of:
1) coating the ABL1 protein on an enzyme label plate, and sealing with a sealing solution;
2) adding the FN 3-like antibody mutant of any of claims 1-3.
8. The method of claim 7, further comprising the steps of:
1) adding enzyme-labeled secondary antibody;
2) adding a developing solution for developing for 10 minutes, adding an isometric stop solution, putting the ELISA plate into an ELISA reader, and reading a light absorption value at 450 nm.
9. A kit for testing the SH2 subunit of ABL1 protein or ABL1 protein or SH2 subunit of ABL1 fusion protein, which comprises the FN3 type antibody mutant as claimed in any one of claims 1 to 3.
10. Use of the FN 3-like antibody mutant of any of claims 1-6 in the preparation of a kit.
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| US7598352B2 (en) * | 2000-11-17 | 2009-10-06 | University Of Rochester | Method of identifying polypeptide monobodies which bind to target proteins and use thereof |
| CN102076713A (en) * | 2008-05-02 | 2011-05-25 | 诺瓦提斯公司 | Improved fibronectin-based binding molecules and uses thereof |
| CN108179152A (en) * | 2017-12-29 | 2018-06-19 | 大连大学 | A kind of method that antigen substitute is prepared based on the dual anti-former epitope of III type structure domain views of people's fibronectin |
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2020
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| US7598352B2 (en) * | 2000-11-17 | 2009-10-06 | University Of Rochester | Method of identifying polypeptide monobodies which bind to target proteins and use thereof |
| CN102076713A (en) * | 2008-05-02 | 2011-05-25 | 诺瓦提斯公司 | Improved fibronectin-based binding molecules and uses thereof |
| CN108179152A (en) * | 2017-12-29 | 2018-06-19 | 大连大学 | A kind of method that antigen substitute is prepared based on the dual anti-former epitope of III type structure domain views of people's fibronectin |
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| Allosteric Inhibition of Bcr-Abl Kinase by High Affinity Monobody Inhibitors Directed to the Src Homology 2 (SH2)-Kinase Interface;Wojcik J等;《Journal of Molecular Biology》;20150224;第291卷(第16期);8836-8847 * |
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