Preparation method of ITGB5 specific antibody

Abstract

The invention discloses a preparation method of an ITGB5 specific antibody, which comprises the following steps: enriching ITGB5 antigens, enriching nano antibodies for identifying the ITGB5 antigens in an original immune library, carrying out competitive sorting through a flow sorter and a control antibody, sequencing enriched clones and carrying out monoclonal flow identification to obtain the ITGB5 specific antibody. Results show that the obtained part of monoclone can compete with a control antibody to be combined with a target antigen, the screening process is accurately controlled by adopting a flow analysis technology, the antibody with higher affinity is screened, the affinity of the antibody to ITGB5 is about two times that of a commercially available antibody, and the antibody has lower cross reactivity to other antigens, so that the antibody can be used for preparing the target antigen. The invention discloses a preparation method of an ITGB5 specific antibody.

Description

Technical Field

[0001] The present invention relates to the field of biology and medicine technology, in particular to a method for preparing an ITGB5-specific antibody. Background Art

[0002] The ITGB5 gene encodes a β subunit of integrin, which can bind to different α chains to form a variety of integrin heterodimers. The expression of ITGB5 in tumor cells and tissues is significantly increased, and it is involved in tumor cell proliferation, migration, invasion, adhesion and other processes, and promotes tumor angiogenesis and epithelial-mesenchymal transition (EMT) induced by transforming growth factor-β (TGF-β), which is a key factor in the formation and development of cancer. There are few related studies. Therefore, the development of antibody drugs targeting ITGB5 is crucial for tumor treatment. However, research on diagnostic or therapeutic antibodies for detecting the expression of ITGB5 and for treating ITGB5-related cancers with ITGB5 as a target is still relatively scarce. There is an unmet need in the art for antibodies with high affinity for ITGB5. Summary of the invention

[0003] 1. Technical issues to be resolved

[0004] In view of the deficiencies of the prior art, the present invention provides an antibody with high affinity for ITGB5, whose affinity for ITGB5 is about twice that of commercially available antibodies and has low cross-reactivity to other antigens, and a method for preparing an ITGB5-specific antibody.

[0005] (II) Technical solution

[0006] To achieve the above purpose, the present invention first uses ITGB5 antigen for enrichment, enriches nanoantibodies that recognize ITGB5 antigen in the original immune library, competitively sorts them with a flow sorter and a control antibody, and sequences the enriched clones and performs monoclonal flow cytometry identification. The results show that some of the obtained monoclones can compete with the control antibody for binding to the target antigen. The present invention uses flow analysis technology to accurately control the screening process and screen antibodies with higher affinity.

[0007] A method for preparing an ITGB5-specific antibody, characterized in that the specific preparation steps are as follows:

[0008] Step 1, yeast amplification: 37° resuscitation of yeast, Trp-glu, 30° culture for 48h;

[0009] Step 2, inducing antibodies: take 1×1010 amplified yeast, centrifuge and discard the supernatant, wash twice with PBS, and induce culture with Trp-gala medium at 25° for 60h;

[0010] Step 3, antigen sample preparation: Take 293T cells in good condition, take 2×105 cells to a 6-well plate, and culture overnight; in the afternoon of the next day, three wells were used as the control group without intervention, and the other three wells were taken with ITGB5human 2ug for transient transfection;

[0011] Step 4, first enrichment: discard the 293T culture medium in the 6-well plate, wash twice with PBS, take 1×1010 successfully induced yeast and add them to the 293T cell control group, and shake horizontally for 30 minutes;

[0012] Step 5, second enrichment: the supernatant was transferred to the wells of 293T cells transfected with ITGB5 and shaken horizontally for 30 min;

[0013] Step 6, amplification: discard the supernatant, wash off the unbound yeast with PBS, add Trp-glu medium for overnight culture, transfer the supernatant to a 15 ml centrifuge tube, and culture at 30° for 12-16 h;

[0014] Step 7, induction: collect the amplified yeast, wash twice with PBS, and induce culture with Trp-gala medium for 48h;

[0015] Step 8, plasmid extraction: extract yeast plasmid using a plasmid extraction kit;

[0016] Step 9, PCR: amplify the antibody fragment using a PCR kit;

[0017] Step 10, vector digestion: digest the PB3.2 vector with EcoRI / BamHI;

[0018] Step 11, connecting the amplified product to the vector: connecting the PCR product and the restriction digested vector using a ligase;

[0019] Step 12, transformation: transform the above-constructed vector using DH5@;

[0020] Step 13, extracting plasmid: picking a single clone for shaking, and then extracting the plasmid using a plasmid extraction kit;

[0021] Step 14, transfection of 293T cells: transfect 293T cells at 2ug per well; collect the supernatant after 48 hours, perform flow cytometry staining on cells overexpressing ITGB5, and screen positive clones;

[0022] Step 15, sequence acquisition: sequence the positive clone plasmid number to obtain the antibody sequence with the best affinity.

[0023] Further, the DH5@ transformation step is: using DH5α to transform the above-mentioned ligated vector (take out the DH5α competent cells from the -80°C refrigerator and put them in an ice bath for 5 minutes to melt the competent cells; take 5 μL of the ligated product into a new 1.5mL EP tube, add 25 μL of the melted DH5α competent cells, flick 2 to 3 times to mix, and put them in an ice bath for 25 minutes; place them in a 42°C water bath for heat shock for 90 seconds, and immediately place them on ice for 5 minutes;

[0024] Add 500 μL of LB liquid culture medium without ampicillin to the EP tube after ice bath, incubate in an incubator (37° C., 220 rpm) for 1 h, spread on an LB solid plate containing ampicillin, and invert and culture in a 37° C. incubator for 16 h).

[0025] (III) Beneficial effects

[0026] Compared with the prior art, the present invention provides an ITGB5-specific antibody, which is enriched by using ITGB5 antigen, and nano antibodies that recognize ITGB5 antigen in the original immune library are enriched, and competitive sorting is performed by a flow sorter and a control antibody, and the enriched clones are sequenced and monoclonal flow identification is performed, and the results show that some of the obtained monoclones can compete with the control antibody for binding to the target antigen; by using flow analysis technology to accurately control the screening process, antibodies with higher affinity are screened. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the overall screening described in an embodiment of the present invention;

[0028] Figure 2 After the second enrichment in steps 4, 5, 6, and 7 described in the embodiment of the present invention, flow cytometry verification of yeast flow staining (staining with Anti-mouse IgG-PE flow cytometry antibody) after 48 hours of induction culture in Trp-gala medium;

[0029] Figure 3 In step 14 of the embodiment of the present invention, the plasmid constructed in steps 9-13 is transferred into 293T, and the supernatant is collected and then flow cytometry staining of ITGB5 positive cells is performed, and the staining is compared with the best commercially available flow cytometry antibody (Biolgend). The results show that the positive rate of the present invention is significantly higher than that of the commercially available antibody by 2 times;

[0030] Figure 4 This is a schematic diagram of the flow cytometry screening process of antibodies expressed by a single clone plasmid in step 14 of the embodiment of the present invention;

[0031] Figure 5 This is a schematic diagram of verifying the binding of the screened antibody to liver cancer cells in step 15 described in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0033] A method for preparing an ITGB5-specific antibody, characterized in that the specific preparation steps are as follows:

[0034] Step 1, yeast amplification: 37° resuscitation of yeast, Trp-glu, 30° culture for 48h;

[0035] Step 2, inducing antibodies: take 1×1010 amplified yeast, centrifuge and discard the supernatant, wash twice with PBS, and induce culture with Trp-gala medium at 25° for 60h;

[0036] Step 3, antigen sample preparation: Take 293T cells in good condition, take 2×105 cells to a 6-well plate, and culture overnight; in the afternoon of the next day, three wells were used as the control group without intervention, and the other three wells were taken with ITGB5human 2ug for transient transfection;

[0037] Step 4, first enrichment: discard the 293T culture medium in the 6-well plate, wash twice with PBS, take 1×1010 successfully induced yeast and add them to the 293T cell control group, and shake horizontally for 30 minutes;

[0038] Step 5, second enrichment: the supernatant was transferred to the wells of 293T cells transfected with ITGB5 and shaken horizontally for 30 min;

[0039] Step 6, amplification: discard the supernatant, wash off the unbound yeast with PBS, add Trp-glu medium for overnight culture, transfer the supernatant to a 15 ml centrifuge tube, and culture at 30° for 12-16 h;

[0040] Step 7, induction: collect the amplified yeast, wash twice with PBS, and induce culture with Trp-gala medium for 48h;

[0041] Step 8, plasmid extraction: extract yeast plasmid using a plasmid extraction kit;

[0042] Step 9, PCR: amplify the antibody fragment using a PCR kit;

[0043] Step 10, vector digestion: digest the PB3.2 vector with EcoRI / BamHI;

[0044] Step 11, connecting the amplified product to the vector: connecting the PCR product and the restriction digested vector using a ligase;

[0045] Step 12, transformation: transform the above-constructed vector using DH5@;

[0046] Step 13, extracting plasmid: picking a single clone for shaking, and then extracting the plasmid using a plasmid extraction kit;

[0047] Step 14, transfection of 293T cells: transfect 293T cells at 2ug per well; collect the supernatant after 48 hours, perform flow cytometry staining on cells overexpressing ITGB5, and screen positive clones;

[0048] Step 15, sequence acquisition: sequence the positive clone plasmid number to obtain the antibody sequence with the best affinity.

[0049] In the specific implementation process, the DH5@ transformation steps are as follows: using DH5@ to transform the above-constructed vector, take out the DH5α competent cells from the -80°C refrigerator, and ice-bathe for 5 minutes to melt the competent cells; take 5μL of the ligation product into a new 1.5mL EP tube, add 25μL of the melted DH5α competent cells, flick 2-3 times to mix, and ice-bathe for 25 minutes; place in a 42°C water bath for heat shock for 90s, and immediately place on ice for 5 minutes;

[0050] Add 500 μL of LB liquid culture medium without ampicillin to the EP tube after ice bath, incubate in an incubator (37° C., 220 rpm) for 1 h, spread on an LB solid plate containing ampicillin, and culture upside down in a 37° C. incubator for 16 h.

[0051] The gene sequence numbers described in step 15 during the specific implementation process are as follows:

[0052] GDPSWLATMETDTLLLWVLLLWVPGSTGDEFQVQLQESGGGLVQAGGSLRLSCAASGSISPYAVMG

[0053] WYRQAPGKEREFVASIAYGSSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVEQPK

[0054] QIPYPIPSVRWHLYWGQGTQVTVSSGSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMIS

[0055] LSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFK

[0056] CKVNNKDLPAPIERTISKPKGSVRAPQVYVLPHQKKND

[0057] The specific applications of ITGB5-specific antibodies are as follows:

[0058] In view of the current situation that there are few antibodies targeting ITGB5 on the market, there is a lack of nano antibodies with potential therapeutic potential, and the affinity and sensitivity are low. Based on the previous study that identified ITGB5 as a potential target for liver cancer immunotherapy, this study screened nano antibodies targeting ITGB5 with high affinity and sensitivity through yeast antibody display technology. Its future specific applications are as follows: 1. Flow cytometry detection of antibodies for ITGB5 antigen; 2. Nano antibodies have the advantages of small molecular weight, high stability, and strong tissue penetration. Therefore, the high-affinity antibodies screened and prepared in this study may be used as antibody drugs for liver cancer immunotherapy; 3. The antibody sequences obtained in this study can be constructed onto CAR vectors, laying the foundation for subsequent CAR-T and CAR-NK cell treatment of liver cancer.

[0059] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing an ITGB5-specific antibody, characterized in that: The specific preparation steps are as follows: Step 1, yeast amplification: 37° resuscitation of yeast, Trp-glu, 30° culture for 48h; Step 2, inducing antibodies: take 1×1010 amplified yeast, centrifuge and discard the supernatant, wash twice with PBS, and induce culture with Trp-gala medium at 25° for 60h; Step 3, antigen sample preparation: Take 293T cells in good condition, take 2×105 cells to a 6-well plate, and culture overnight; in the afternoon of the next day, three wells were used as the control group without intervention, and the other three wells were taken with ITGB5human 2ug for transient transfection; Step 4, first enrichment: discard the 293T culture medium in the 6-well plate, wash twice with PBS, take 1×1010 successfully induced yeast and add them to the 293T cell control group, and shake horizontally for 30 minutes; Step 5, second enrichment: the supernatant was transferred to the wells of 293T cells transfected with ITGB5 and shaken horizontally for 30 min; Step 6, amplification: discard the supernatant, wash off the unbound yeast with PBS, add Trp-glu medium for overnight culture, transfer the supernatant to a 15 ml centrifuge tube, and culture at 30° for 12-16 h; Step 7, induction: collect the amplified yeast, wash twice with PBS, and induce culture with Trp-gala medium for 48h; Step 8, plasmid extraction: extract yeast plasmid using a plasmid extraction kit; Step 9, PCR: amplify the antibody fragment using a PCR kit; Step 10, vector digestion: digest the PB3.2 vector with EcoRI / BamHI; Step 11, connecting the amplified product to the vector: connecting the PCR product and the restriction digested vector using a ligase; Step 12, transformation: Use DH5@ to transform the above-constructed vector. Step 13, extracting plasmid: picking a single clone for shaking, and then extracting the plasmid using a plasmid extraction kit; Step 14, transfection of 293T cells: transfect 293T cells at 2ug per well; collect the supernatant after 48 hours, perform flow cytometry staining on cells overexpressing ITGB5, and screen positive clones; Step 15, sequence acquisition: sequence the positive clone plasmid number to obtain the antibody sequence with the best affinity.

2. The method for preparing an ITGB5-specific antibody according to claim 1, characterized in that: The transformation described in step 12 is to transform the above-mentioned ligated vector using DH5α, specifically, take out DH5α competent cells from a -80°C refrigerator, and melt the competent cells in an ice bath for 5 minutes; take 5 μL of the ligated product into a new 1.5mL EP tube, add 25 μL of the melted DH5α competent cells, flick 2 to 3 times to mix, and ice bath for 25 minutes; place in a 42°C water bath for heat shock for 90 seconds, and immediately place on ice for 5 minutes; Add 500 μL of LB liquid culture medium without ampicillin to the EP tube after ice bath, incubate in an incubator at 37°C, 220 rpm for 1 h, spread on an LB solid plate containing ampicillin, and invert and culture in a 37°C incubator for 16 h.

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