Method for rapidly screening chimeric antigen receptor by using alpaca phage natural library and human T cell strain
阅读说明：本技术 一种应用羊驼噬菌体天然文库和人t细胞株快速筛选嵌合抗原受体的方法 (Method for rapidly screening chimeric antigen receptor by using alpaca phage natural library and human T cell strain ) 是由 叶健斌 蔡冠星 汤赞 曾桂芳 陈洁莹 蔡车国 胡隽源 于 2021-09-30 设计创作，主要内容包括：本发明公开了一种应用羊驼噬菌体天然文库和人T细胞株快速筛选嵌合抗原受体的方法,涉及分子生物学和细胞生物学技术领域。本发明方法利用噬菌体文库展示、同源重组和细胞培养等技术,通过羊驼抗体天然文库进行多次淘选、验证和鉴定获得针对肿瘤抗原的VHH序列,进行质粒重组构建表达于人T细胞株Jurkat细胞快速验证该CAR是否有效,最后使用T细胞株Hut78细胞再次确认效果。本发明方法具有筛选速度快、低成本的特点,可快速筛选并评估出抗原受体CAR是否可以用于后续实验。(The invention discloses a method for rapidly screening a chimeric antigen receptor by applying an alpaca phage natural library and a human T cell strain, and relates to the technical field of molecular biology and cell biology. The method utilizes technologies such as phage library display, homologous recombination and cell culture, and the like, obtains a VHH sequence aiming at a tumor antigen through multiple times of panning, verification and identification of an alpaca antibody natural library, constructs and expresses the VHH sequence in a human T cell strain Jurkat cell through plasmid recombination, quickly verifies whether the CAR is effective, and finally reconfirms the effect by using the T cell strain Hut78 cell. The method has the characteristics of high screening speed and low cost, and can quickly screen and evaluate whether the antigen receptor CAR can be used for subsequent experiments.)
1. A method for rapidly screening a chimeric antigen receptor by using an alpaca bacteriophage natural library and a human T cell strain is characterized by comprising the following steps:
(1) coating the antigen on an immune tube overnight, sealing, adding an alpaca phage natural library to bind the antigen, washing unbound phage by PBST, eluting the bound phage by pancreatin, adding AEBSF to terminate elution, and obtaining a phage elution library;
(2) infecting SS320 with the phage elution library in the step (1) to amplify and preserve a bacterial library, then infecting, packaging and amplifying phage by using auxiliary phage and purifying to obtain a phage library;
(3) repeating the processes of the steps (1) to (2) for 2 to 3 times by using the phage library obtained in the step (2) to obtain a phage elution library;
(4) infecting the phage elution library obtained in the step (3) with SS320, then coating a plate, selecting a preset number of monoclonals for culture, adding auxiliary phage for phage expression, carrying out ELISA detection, selecting monoclonals corresponding to positive holes for ELISA verification again, and retaining the final positive monoclonals;
(5) culturing, amplifying, preserving and sequencing the positive monoclonal obtained finally in the step (4);
(6) inserting the alpaca nano antibody sequence confirmed to be correct by sequencing in the step (5) into a virus core vector with a CAR structure to construct a recombinant plasmid, transforming competent cells to obtain monoclone, breed conservation and sequencing identification;
(7) after the CAR recombinant plasmid obtained in the step (6) is electrically transferred to Jurkat cells to express CAR, the Jurkat cells are respectively co-cultured with target cells and non-target cells for 18h, and the difference value between CD69 expression and MFI of the Jurkat cells is detected by using an anti-human CD69 flow antibody;
(8) performing virus packaging concentration on the effectively activated CAR recombinant plasmid in the step (7), co-culturing the Hut78 cell and the target cell for 48h after the infected Hut78 cell expresses CAR, and detecting the death rate of the target cell by using a 7-AAD flow antibody;
(9) if the killing capacity is higher than that of the control group and has significant difference, the chimeric antigen receptor CAR can be judged to be used for subsequent experiments.
2. The method for rapidly screening the chimeric antigen receptor by using the alpaca bacteriophage natural library and the human T cell strain as claimed in claim 1, wherein the specific operation of the step (1) comprises:
1) coating the antigen on an immune tube, wherein the coating solution is CBS, the pH value is 9.6, and incubating for 16-20h at 4 ℃; removing supernatant, and washing with 2ml PBS for 3 times at room temperature; adding 2ml of confining liquid, and performing rotary incubation for 2 hours at room temperature; removing supernatant, and washing with 2ml PBS for 3 times at room temperature;
2) centrifuging the product of 1) to remove supernatant, adding 2ml PBS and 0.05ml of alpaca phage natural library, and keeping the titer at 10 or more13pfu/ml, rotating for 1h at room temperature; removing supernatant, washing the immune tube with 2ml of PBST buffer solution at room temperature for 20 times; removing supernatant, adding 1ml of 0.25mg/ml Trypsin solution, and performing rotary elution at room temperature for 30 min; the elution was stopped by adding 10. mu.l of 10% AEBSF and the solution in the immune tube was transferred to a new EP tube to obtain a phage elution library.
3. The method for rapidly screening the chimeric antigen receptor by using the alpaca bacteriophage natural library and the human T cell strain as claimed in claim 1, wherein the specific operation of the step (2) comprises:
1) taking 1.8ml of strain to recover SS320, adding 2xYT to culture until OD600 of the bacterial liquid is 0.250; adding 500 mu l to 2ml of bacterial liquid of the phage elution library obtained in the step (3), and carrying out reaction at 37 ℃ and 220rpm for 30 min; uniformly coating all bacteria liquid on 1T 1502 xYT plate, drying, and culturing at 37 ℃ for 16-20 h; taking out the plate, adding 6ml of 2XYT, scraping the bacterial colony, collecting the bacterial liquid into a 15ml centrifugal tube to obtain an amplified bacterial sublibrary, and detecting OD600 before freezing, wherein the freezing volume is X ml which is 10/OD 600;
2) adding X ml of the bacterial sublibrary bacterial liquid into 100ml of 2XYT liquid culture medium, culturing at 37 ℃ and 220rpm until OD600 is 0.250, wherein X is 10/OD 600; adding helper phage MOI 10: continuously culturing at 1, 37 ℃ and 220rpm for 30 min; adding Kana with a final concentration of 50 μ g/ml and IPTG with a final concentration of 0.2mM, and culturing overnight at 30 ℃ and 220 rpm;
3) collecting 50ml of the cultured bacterial liquid to a centrifugal tube, centrifuging for 2000g for 10min, and collecting the supernatant; adding 1/4 volume pre-cooled PEG/NaCl solution, mixing, and ice-cooling for 30 min; centrifuging at 4 deg.C for 2000g for 20min, discarding supernatant, and inverting on paper for 2 min; adding 1ml PBS to resuspend and precipitate to an EP tube, centrifuging at 4 ℃ for 13500g for 20min, and collecting supernatant; adding 1/4 volume pre-cooled PEG/NaCl solution, mixing, and ice-cooling for 10 min; centrifuging at 4 ℃ for 13500g for 10min, discarding the supernatant, and adding 1ml PBS for resuspension and precipitation; the phage library was obtained by centrifugation at 13500g for 2min at 4 ℃ and transfer of the supernatant to a new 1.5ml centrifuge tube.
4. The method for rapid screening of chimeric antigen receptor according to claim 1, wherein the phage library is used in an amount of 0.05ml when repeating the steps (1) - (2) in step (3).
5. The method for rapidly screening the chimeric antigen receptor by using the alpaca bacteriophage natural library and the human T cell strain according to claim 1, wherein the specific operation of the step (4) comprises:
1) recovering 1.8ml of SS320 strain, and culturing until OD600 of the bacterial liquid is 0.250;
2) taking 100 mu l of the phage elution library obtained in the step (3), and diluting the phage elution library in a 1.5ml centrifugal tube by 10 times; diluting 10 mu l of the phage elution library obtained in the step (3) to 1ml, and diluting the phage elution library to 10E-9 in a gradient manner; then, 20 mu l of the phage elution library obtained in the step (3) is taken to be diluted to 200 mu l, the phage elution library is diluted to 10E-14 in a gradient way, and the diluted phage elution library is blown, beaten and mixed evenly;
3) adding 180 μ l of SS320 bacterial liquid with OD600 value of 0.5-0.55 into each dilution centrifuge tube, blowing and beating uniformly, standing and culturing at 37 deg.C for 30 min; uniformly coating the cultured bacterial liquid on Amp containing 2xYT-solid-100 mu g/ml, and culturing at 37 ℃ for 16-20 h; randomly picking 200 monoclonals from the culture medium plate to two sterile 96-well cell culture plates, adding 200 mul of 2XYT culture medium into each well, and culturing at 37 ℃ for 16 h;
4) adding 10 μ l of monoclonal bacterial liquid corresponding to the positive well and 140 μ l of 2XYT liquid culture medium into a new 96-well plate, performing static culture at 37 ℃ until OD600> is 0.125, and preserving the rest bacterial liquid at 4 ℃; adding 0.5 mul of helper phage into each hole, and continuously culturing for 30min at 37 ℃; adding 50 μ l2XYT, mixing, standing at 30 deg.C overnight, and culturing; coating the antigen on an enzyme label plate, simultaneously coating BSA (bovine serum albumin) in parallel as a control, and coating overnight at 4 ℃;
5) centrifuging the 96-well culture plate after overnight culture at 4 ℃ for 10min at 2000g, and storing at 4 ℃ for later use; discarding the liquid in the overnight coated ELISA plate, adding 200 μ L PBST into each hole, washing at room temperature for 1 time, adding 100 μ L of sealing liquid into each hole, and sealing at room temperature for 1 h; then adding 100 mu l of phase supernatant into each hole, and incubating for 2h at room temperature; discarding the solution, washing with 200 μ l PBST per well for 3 times, and standing for 2min each time; adding 100 mu of l M13 Bacteriophage Antibody-HRP into each hole, and incubating for 1h at room temperature; discarding the solution, washing with 200 μ l PBST per well for 6 times, and standing for 2min each time; discarding the solution, developing with 100 μ l of TMB single-component developing solution in each well in dark place, adding 100 μ l of stop solution in each well, reading OD450 value with an enzyme-linked immunosorbent assay, and recording and storing.
6. The method for rapid screening of chimeric antigen receptor according to claim 1, wherein the recombinant plasmid constructed in step (6) can be used for PCR to obtain the target sequence, and the target sequence is inserted into the plasmid by homologous recombination; the virus core vector in the step (6) is lentivirus or adenovirus.
7. The method for rapid screening of chimeric antigen receptor according to claim 1, wherein the conditions for co-culturing the Jurkat cells and the target cells in step (7) are 96-well plate culture, the culture volume is 100 μ l, the ratio of Jurkat cells to target cells is 30000: 3000.
8. the method for rapidly screening chimeric antigen receptors by using the natural alpaca phage library and the human T cell strain according to claim 1, wherein during the coculture of the Hut78 cells and the target cells in the step (8), the Hut78 cells or the target cells need to be labeled.
9. The method for rapid screening of chimeric antigen receptors using natural library of alpaca phages and human T Cell lines according to claim 8, characterized in that target cells are stained with a staining marker such as CFSE or Cell Trace face red before plating, or with a staining marker such as anti-human CD45 or anti-human CD2 before plating Hut78 cells.
Technical Field
The invention relates to the technical field of molecular biology and cell biology, in particular to a method for rapidly screening a chimeric antigen receptor by applying an alpaca phage natural library and a human T cell strain.
Background
CAR-T, collectively known as Chimeric Antigen Receptor T-Cell Immunotherapy, refers to Chimeric Antigen Receptor T-Cell Immunotherapy. On 3/8.2017, FDA approved CAR-T therapy kymeriah (CTL-019) by Novartis (Novartis) for treatment of relapsed or refractory B-cell acute lymphoblastic leukemia in children, adolescents. On 18/10/2017, CAR-T therapy yescatta (KTE-C10) approved by the FDA for the treatment of certain types of adult large B-cell lymphoma, relapsed or refractory. As with all technologies, CAR-T technology also undergoes a lengthy evolution, and it is in this series of evolutions that CAR-T technology is gradually maturing.
The nano antibody, or named as alpaca antibody heavy chain variable region VHH, has the features of small size, stable structure, affinity capacity similar to that of scFv but lower immunogenicity. Therefore, the application of nanobodies to CAR-T cells is a suitable option.
Phage display technology is an in vitro screening technique for identifying proteins and other macromolecular ligands. The principle is to insert the DNA sequence of the foreign polypeptide or protein into the proper position of the phage gene, and the foreign protein is displayed on the surface of the progeny phage as the phage is passed. The displayed protein or polypeptide can maintain relative spatial structure and biological activity, and can be screened by using a target protein.
Therefore, using phage library display technology, we can theoretically quickly screen out VHH sequences directed against tumor antigens, perform plasmid recombination to construct CAR-T cells, and quickly verify whether the CAR-T cells are effective.
CD69 is a classical surface marker that is rapidly expressed early after lymphocytes are activated by antigen. By utilizing the characteristic, whether the CAR-T cell is activated by the target cell can be confirmed within 24 hours, and the effect of rapidly screening the CAR is achieved.
However, there is no report on the related art.
Disclosure of Invention
The technical problem to be solved by the invention is the defects in the background technology, and provides a method for quickly screening a chimeric antigen receptor by applying an alpaca bacteriophage natural library and a human T cell strain. Specifically, by utilizing technologies such as phage library display, homologous recombination and cell culture, a VHH sequence aiming at a tumor antigen is obtained by multiple times of panning, verification and identification of an alpaca antibody natural library, plasmid recombination is carried out to construct and express the VHH sequence in a human T cell strain Jurkat cell and quickly verify whether the CAR is effective or not, and finally the T cell strain Hut78 cell is used for reconfirming the effect, so that whether the CAR can be used for subsequent experiments or not can be evaluated.
In order to solve the above problems, the present invention proposes the following technical solutions:
the invention provides a method for rapidly screening a chimeric antigen receptor by applying an alpaca bacteriophage natural library and a human T cell strain, which comprises the following steps:
(1) coating the antigen on an immune tube overnight, sealing, adding an alpaca phage natural library to bind the antigen, washing unbound phage by PBST, eluting the bound phage by pancreatin, adding AEBSF to terminate elution, and obtaining a phage elution library;
(2) infecting SS320 with the phage elution library in the step (1) to amplify and preserve a bacterial library, then infecting, packaging and amplifying phage by using auxiliary phage and purifying to obtain a phage library;
(3) repeating the processes of the steps (1) to (2) for 2 to 3 times by using the phage library obtained in the step (2) to obtain a phage elution library;
(4) infecting the phage elution library obtained in the step (3) with SS320, then coating a plate, selecting a preset number of monoclonals for culture, adding auxiliary phage for phage expression, carrying out ELISA detection, selecting monoclonals corresponding to positive holes for ELISA verification again, and retaining the final positive monoclonals;
(5) culturing, amplifying, preserving and sequencing the positive monoclonal obtained finally in the step (4);
(6) inserting the alpaca nano antibody sequence confirmed to be correct by sequencing in the step (5) into a virus core vector with a CAR structure to construct a recombinant plasmid, transforming competent cells to obtain monoclone, breed conservation and sequencing identification;
(7) after the CAR recombinant plasmid obtained in the step (6) is electrically transferred to Jurkat cells to express CAR, the Jurkat cells are respectively co-cultured with target cells and non-target cells for 18h, and the difference value between CD69 expression and MFI of the Jurkat cells is detected by using an anti-human CD69 flow antibody;
(8) performing virus packaging concentration on the CAR recombinant plasmid effectively activated in the step (7), performing co-culture of Hut78 cells and target cells for 48h after the CAR is expressed by infected Hut78 cells, and detecting the death rate of the target cells by using 7-AAD flow antibodies
(9) If the killing capacity of the CAR cell is higher than that of the control group and has significant difference, the chimeric antigen receptor CAR is judged to be used in subsequent experiments.
Compared with the prior art, the invention can achieve the following technical effects:
the invention can rapidly screen and evaluate whether the chimeric antigen receptor CAR can be used for subsequent experiments at low cost by using a phage library display screening technology and the early activation characteristic of Jurkat cells. Specifically, by utilizing technologies such as phage library display, homologous recombination and cell culture, a VHH sequence aiming at a tumor antigen is obtained by multiple times of panning, verification and identification of an alpaca antibody natural library, plasmid recombination is carried out to construct and express the VHH sequence in a human T cell strain Jurkat cell to quickly verify whether the CAR is effective, and finally, the T cell strain Hut78 cell is used for reconfirming the effect. Furthermore, in the phage library display screening link, an alpaca VHH sequence specific to the tumor antigen can be rapidly screened out (only 14 days are needed at the fastest speed); the constructed modular virus plasmid with the CAR structure is utilized in the construction link of the recombinant plasmid, the recombinant plasmid can be quickly obtained (only two days are needed at the fastest), and finally the recombinant plasmid is co-cultured with target cells after being transformed into Jurkat cells through plasmid electricity to express the CAR, the CD69 expression of the Jurkat is detected, and the conditions of cell membrane expression, self-activation, the effect function and the like of the CAR (only two days are needed at the fastest) can be quickly evaluated.
Drawings
FIG. 1 results of positive rates after electroporation of Jurkat cells in the examples of the present invention.
FIG. 2 is a difference result of CD69MFI after co-culturing Jurkat cells with target cells and non-target cells in the example of the present invention.
FIG. 3 shows the results of the positive rates of Hut78 cells after viral infection in the examples of the present invention.
FIG. 4 shows the tumor killing effect of CAR-Hut78 cells selected by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments. It is apparent that the embodiments to be described below are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a method for rapidly screening a chimeric antigen receptor by applying an alpaca phage natural library and a human T cell strain, which comprises the following steps:
(1) coating the antigen on an immune tube overnight, sealing, adding an alpaca phage natural library to bind the antigen, washing unbound phage by PBST, eluting the bound phage by pancreatin, adding AEBSF to terminate elution, and obtaining a phage elution library;
(2) infecting SS320 with the phage elution library in the step (1) to amplify and preserve a bacterial library, then infecting, packaging and amplifying phage by using auxiliary phage and purifying to obtain a phage library;
(3) repeating the processes of the steps (1) to (2) for 2 to 3 times by using the phage library obtained in the step (2) to obtain a phage elution library;
(4) infecting the phage elution library obtained in the step (3) with SS320, then coating a plate, selecting a preset number of monoclonals for culture, adding auxiliary phage for phage expression, carrying out ELISA detection, selecting monoclonals corresponding to positive holes for ELISA verification again, and retaining the final positive monoclonals;
(5) culturing, amplifying, preserving and sequencing the positive monoclonal obtained finally in the step (4);
(6) inserting the alpaca nano antibody sequence confirmed to be correct by sequencing in the step (5) into a virus core vector with a CAR structure to construct a recombinant plasmid, transforming competent cells to obtain monoclone, breed conservation and sequencing identification;
(7) after the CAR recombinant plasmid obtained in the step (6) is electrically transferred to Jurkat cells to express CAR, the Jurkat cells are respectively co-cultured with target cells and non-target cells for 18h, and the difference value between CD69 expression and MFI of the Jurkat cells is detected by using an anti-human CD69 flow antibody;
(8) performing virus packaging concentration on the CAR recombinant plasmid effectively activated in the step (7), performing co-culture of Hut78 cells and target cells for 48h after the CAR is expressed by infected Hut78 cells, and detecting the death rate of the target cells by using 7-AAD flow antibodies
(9) If the killing capacity is higher than that of the control group and has significant difference, the chimeric antigen receptor CAR can be judged to be used for subsequent experiments.
The steps are described in detail below:
using phage library display technology, the VHH sequences of tumor antigens were panned multiple times through a natural library of alpaca antibodies:
1. the first screening process includes the following steps:
1) the antigen was removed from-80 ℃ and thawed on ice; coating the antigen on an immune tube (50 μ g/tube, coating solution CBS, pH9.6, 2 ml/tube), incubating at 4 deg.C and 7rpm/min for 16-20h by slow rotation; after incubation, the supernatant was removed, washed 3 times with 2ml PBS at room temperature (5 discard solutions inverted); adding 2ml of blocking solution (1xPBS, 0.1% Tween and 3% BSA), and rotating at room temperature for 2 h; remove supernatant, wash 3 times with 2ml PBS (invert 5 times discard);
2) centrifuging the product of 1) to remove supernatant, adding 2ml PBS, and 0.05ml natural library of alpaca bacteriophage (titer is greater than or equal to 10)13pfu/ml), rotate for 1h at room temperature; remove supernatant, wash the immune tubes 20 times at room temperature with 2ml PBST (0.1% Tween20) buffer (invert 5 discard); removing supernatant, adding 1ml of 0.25mg/ml Trypsin solution, and performing rotary elution at room temperature for 30 min; adding 10 μ l of 10% AEBSF to terminate elution, transferring the solution in the immune tube to a new EP tube to obtain a phage elution library, which is named as primary phage elution solution in the example;
3) recovering 1.8ml SS320 strain, supplementing 2xYT to 10ml, Tet 20 mug/ml, culturing for 3h-3.5h until OD600 is 0.250 (detected by an enzyme-linked immunosorbent assay (ELISA) instrument 300 mug); adding 500 μ l of primary phage eluent into 2ml of SS320 bacterial liquid, at 37 deg.C and 220rpm for 30 min; uniformly coating all bacteria liquid (containing primary phage eluent and SS320 bacteria liquid) into 1T 1502 xYT plate (100 mu g/ml Amp and 2% glucose), drying, and incubating for 16-20h at 37 ℃;
4) taking out the plate, adding 6ml of 2XYT (20 mu g/ml Tet) to scrape the bacterial colony, collecting the bacterial liquid into a 15ml centrifuge tube to obtain an amplified bacterial sublibrary, and detecting that the OD600 is 8.66 (the final concentration of frozen glycerol is 20%) before freezing, wherein the frozen volume is 0.577 ml;
5) adding 0.577ml of the cryopreserved bacterial liquid into 100ml of 2XYT liquid medium (containing 20. mu.g/ml Tet and 100. mu.g/ml Amp), and culturing at 37 ℃ and 220rpm until the OD600 reaches 0.250; adding helper phage MOI 10: continuously culturing at 1, 37 ℃ and 220rpm for 30 min; adding Kana with a final concentration of 50 μ g/ml and IPTG with a final concentration of 0.2mM, culturing overnight at 30 ℃ and 220 rpm;
6) collecting the cultured bacterial liquid to a 50ml centrifuge tube, centrifuging for 2000g for 10min, and collecting the supernatant; adding 1/4 volume pre-cooled PEG/NaCl solution, mixing, and ice-cooling for 30 min; centrifuging at 4 deg.C for 20min at 2000g, discarding supernatant, and inverting on paper for 2 min; adding 1ml PBS to resuspend and precipitate to an EP tube, centrifuging 13500g, performing 20min at 4 ℃, and collecting supernatant; adding 1/4 volume pre-cooled PEG/NaCl solution, mixing, and ice-cooling for 10 min; centrifuging at 13500g for 10min at 4 ℃, discarding the supernatant, and adding 1ml PBS for heavy suspension precipitation; the supernatant was transferred to a new 1.5ml centrifuge tube by centrifugation at 13500g again at 4 ℃ for 2min to obtain a phage library, which was named primary phage library in this example.
2. Second round of screening
1) The antigen was removed from-80 ℃ and thawed on ice; coating the antigen on an immune tube (50 μ g/tube, coating solution CBS, pH9.6, 2 ml/tube), incubating at 4 deg.C and 7rpm/min for 16-20h by slow rotation; after incubation, the supernatant was removed, washed 3 times with 2ml PBS at room temperature (5 discard solutions inverted); adding 2ml of blocking solution (1xPBS, 0.1% Tween and 3% BSA), and rotating at room temperature for 2 h; remove supernatant, wash 3 times with 2ml PBS (invert 5 times discard);
2) centrifuging the product of 1) to remove supernatant, adding 2ml PBS, adding 0.05ml (titer is greater than or equal to 10) of primary phage library13pfu/ml), rotate for 1h at room temperature; remove supernatant, wash the immune tube 20 times (reverse 5 discard) with 2ml of PBST (0.1% Tween20) buffer at room temperature; removing supernatant, adding 1ml of 0.25mg/ml Trypsin solution, and performing rotary elution at room temperature for 30 min; adding 10 μ l of 10% AEBSF to terminate elution, transferring the solution in the immune tube to a new EP tube to obtain a phage elution library, which is named as secondary phage elution solution in the example;
3) recovering 1.8ml SS320 strain, adding 2XYT to 10ml and Tet 20 μ g/ml to 1.8ml strain, culturing for 3h-3.5h until OD600 is 0.250 (detected by microplate reader 300 μ l); adding 500 μ l of secondary phage eluate into 2ml of SS320 bacterial liquid, at 37 deg.C and 220rpm for 30 min; uniformly coating all bacteria liquid on 1T 1502 xYT plate (100 mu g/ml Amp and 2% glucose), drying, and drying at 37 ℃ for 16-20 h;
4) taking out the plate, adding 6ml of 2XYT (20 mu g/ml Tet) to scrape the bacterial colony, collecting the bacterial liquid into a 15ml centrifuge tube to obtain an amplified bacterial sublibrary, and detecting that the OD600 before freezing is 5.78 (the final concentration of frozen glycerol is 20%) and the frozen volume is 0.865 ml;
5) adding 0.865ml of the cryopreserved bacterial liquid into 100ml of 2XYT liquid culture medium (containing 20 mu g/ml Tet and 100 mu g/ml Amp), and culturing at 37 ℃ and 220rpm until OD600 reaches 0.250; adding helper phage MOI 10: continuously culturing at 1, 37 ℃ and 220rpm for 30 min; adding Kana with a final concentration of 50 μ g/ml and IPTG with a final concentration of 0.2mM, culturing overnight at 30 ℃ and 220 rpm;
6) collecting the cultured bacterial liquid to a 50ml centrifuge tube, centrifuging for 2000g for 10min, and collecting the supernatant; adding 1/4 volume pre-cooled PEG/NaCl solution, mixing, and ice-cooling for 30 min; centrifuging at 4 deg.C for 20min at 2000g, discarding supernatant, and inverting on paper for 2 min; adding 1ml PBS to resuspend and precipitate to an EP tube, centrifuging 13500g, performing 20min at 4 ℃, and collecting supernatant; adding 1/4 volume pre-cooled PEG/NaCl solution, mixing, and ice-cooling for 10 min; centrifuging at 13500g for 10min at 4 ℃, discarding the supernatant, and adding 1ml PBS for heavy suspension precipitation; the supernatant was transferred to a new 1.5ml centrifuge tube by centrifugation at 13500g again at 4 ℃ for 2min to obtain a phage library, which was named as a secondary phage library in this example.
ELISA detection
1) Recovering 1.8ml SS320 strain, supplementing 2xYT to 10ml and Tet 20 mug/ml, and culturing for 3h-3.5h until OD600 is 0.250;
2) taking 100 mu l of phage eluate obtained in the second round of screening, and diluting in a 1.5ml centrifuge tube by 10 times; then continuously taking 10 mu l of phage eluate obtained in the second round of screening to dilute to 1ml, and diluting to 10E-9 in a gradient manner; finally, continuously taking 20 mu l of phage eluate obtained in the second round of screening to dilute to 200 mu l, diluting to 10E-14 in a gradient manner, and uniformly pumping;
3) adding 180 μ l of SS320 bacterial liquid with OD600 value of 0.5-0.55 into each dilution centrifuge tube, uniformly blowing, dividing into two parts, continuously culturing at 37 ℃ and 220rpm for 30 min; one part is kept standing at 37 ℃ for further culture for 30min
4) Respectively and uniformly coating the bacterial liquid obtained by culture on Amp containing 2xYT-solid-100 mu g/ml, and culturing for 16-20h at 37 ℃;
5) randomly picking 200 single clones from the culture medium plate respectively into two sterile 96-well cell culture plates, adding 200. mu.l of 2XYT culture medium (100. mu.g/ml Amp and 20. mu.g/ml Tet) into each well, and culturing at 37 ℃ for 16 h;
6) adding 10 μ l of monoclonal bacterial liquid corresponding to the positive well and 140 μ l of 2XYT liquid culture medium into a new 96-well plate, performing static culture at 37 ℃ until OD600> is 0.125, and preserving the rest bacterial liquid at 4 ℃; adding 0.5 μ l of helper phage (0.5 × 200+19.9ml, 50 μ l per well) per well, and culturing at 37 deg.C for 30 min; adding 50 μ l2XYT (kana 500 μ g/ml, Amp 500 μ g/ml, IPTG 2mM), mixing, standing at 30 deg.C for overnight culture; coating an enzyme label plate (1 ng/mu l, coating liquid is CBS, pH9.6, 100 mu l/hole) with AG antigen, simultaneously coating BSA in parallel as a control, and coating overnight at 4 ℃;
7) centrifuging the 96-well culture plate after overnight culture at 4 ℃ for 10min at 2000g, and storing at 4 ℃ for later use; discarding the liquid in the overnight-coated ELISA plate, adding 200 μ L PBST into each hole, washing at room temperature for 1 time, adding 100 μ L blocking liquid (1xPBS, 0.1% Tween, 3% BSA) into each hole, and blocking at room temperature for 1 h; then adding 100 mu l of phase supernatant into each hole, and incubating for 2h at room temperature; discarding the solution, washing with 200 μ l PBST per well for 3 times, and standing for 2min each time; 100 mu l M13 Bacteriophage Antibody-HRP (diluted 1:8000 in the blocking solution) was added to each well and incubated for 1h at room temperature; discarding the solution, washing with 200 μ l PBST per well for 6 times, and standing for 2min each time; discarding liquid, developing with 100 μ l TMB single-component developing solution in each well in dark place, and adding 100 μ l stop solution (2M H)2SO4) And reading OD450 values by using a microplate reader, and recording and storing. The results of ELISA assays in this example are shown in tables 1-2.
Table 1: antigen group OD450 detection result
Antigens
1
2
3
4
5
6
7
8
9
10
11
12
A
0.0852
0.2867
0.3471
0.515
0.0781
0.1328
0.3134
0.8732
0.3813
0.0442
0.0775
0.0635
B
0.0879
0.1205
0.9271
0.9078
0.4129
0.1082
0.8979
0.3713
0.1174
0.8802
0.163
0.3489
C
0.0506
1.1979
0.0572
0.3152
1.2159
0.4036
0.8466
1.0246
0.1896
0.479
1.0949
0.1982
D
0.5582
1.3025
0.0722
0.9486
0.0524
0.2023
0.4969
0.4147
0.9803
1.0919
0.4297
0.1449
E
0.3353
0.9108
0.6719
0.6481
1.106
0.8093
0.653
0.7264
0.3855
0.173
1.0467
0.242
F
0.5716
0.9287
1.5326
0.7827
0.9918
0.1207
0.7994
0.7911
0.3276
0.1358
1.1765
0.1419
G
0.4675
0.2171
1.1663
1.1448
0.6157
1.1782
0.0628
1.5184
1.1311
0.1932
0.0472
0.1617
H
0.2085
0.7848
0.9402
1.4156
0.0857
0.2128
0.3418
0.0542
0.5118
0.1354
0.0454
0.7976
Table 2: OD450 detection result of BSA control group
BSA
1
2
3
4
5
6
7
8
9
10
11
12
A
0.09
0.272
0.08
0.2394
0.0936
0.1002
0.1723
0.6839
0.1597
0.0819
0.0958
0.0882
B
0.1043
0.1128
0.0765
0.5519
0.277
0.1123
0.5298
0.1935
0.0892
0.3847
0.1178
0.0841
C
0.096
1.0868
0.099
0.1524
1.0999
0.2009
0.9481
0.6693
0.1392
0.11
0.9264
0.0997
D
0.2227
0.9323
0.087
0.6821
0.2455
0.1025
0.3676
0.1385
0.4381
0.7286
0.305
0.1043
E
0.1839
0.6309
0.1511
0.5236
1.0318
0.0985
0.1572
0.547
0.2532
0.1154
0.21
0.1389
F
0.4954
0.106
1.5057
0.3859
0.1152
0.1107
0.5856
0.2088
0.185
0.0911
0.0874
0.1156
G
0.256
0.142
0.7205
0.8834
0.3699
0.618
0.0901
1.1743
0.1208
0.1479
0.0888
0.1315
H
0.162
0.6706
0.497
1.3204
0.0997
0.1273
0.1973
0.1036
0.1508
0.1205
0.1575
0.2016
4. Construction of recombinant plasmids
PCR primers were designed according to the requirements of the recombination Kit (One Step Cloning Kit, Vazyme, C112), and the inserts were obtained by performing PCR reaction using the PCR Kit (PrimeSTAR Mix, TAKARA, cat. R045) and the library of the screened bacteria as a template (in this example, a secondary phage library detected by ELISA) according to the parameters in tables 3 to 4 below.
TABLE 3PCR primers
Primer-F
5’-gctcggccctctagacatatgATGGCGGTGCAGCTGGTG-3’
Primer-R
5’-ggtagtggtacgcgtgctagcGCGTGCGCCTGAGGAGAC-3’
TABLE 4 PCR reaction System
Volume of
Mix
25μl
Bacterial liquid
10μl
Primer-F
2μl
Primer-R
2μl
ddH2O
up to 50μl
TABLE 5 PCR reaction conditions
And carrying out double enzyme digestion to obtain the linearized vector according to the requirement of the enzyme digestion kit. The reaction system was prepared according to the parameters of Table 6 below, and incubated at 37 ℃ for 30min for recombination.
TABLE 6 restriction enzyme recombination reaction System
Volume of
Linearized vector (175.8 ng/. mu.L)
1μl
Insert 3CL-G1
1μl
5xCE II buffer
4μl
Exnase II
2μl
ddH2O
12μl
5. Plasmid transformed competent cells
Thawing competent cells on ice, adding 20 μ l of recombinant product to 100 μ l of competent cells, mixing, standing on ice for 30min, heat-shocking at 42 deg.C for 45s, and standing on ice for 2 min. Adding 150. mu.l of 2XYT, culturing at 37 ℃ and 220rpm for 1h, taking 30. mu.l of coated plate, picking single clone the next day, amplifying, preserving, extracting plasmid and sequencing for identification.
Jurkat cell-target cell coculture
Jurkat cells were subcultured using RMPI1640 plus 15% FBS, cells were harvested before electroporation, washed 3 times with physiological saline, and finally resuspended in Opti-MEM and plasmid DNA was added for electroporation. After 4h, the Jurkat cells are respectively cultured with the target cells and the non-target cells for 18h, the MFI of the Jurkat cells CD69 is detected in a flow mode, and whether the Jurkat cells are activated or not is judged according to the difference of the CD69MFI of the target cell group and the non-target cell group. As shown in FIGS. 1-2, in this example, FIG. 1 shows that the percentage of cells expressing EGFP, i.e., the positive rate of CAR, was successfully expressed after the plasmid was electroporated into Jurkat cells; FIG. 2 shows the difference between the mean fluorescence intensities of flow-assayed CD69 after coculture of Jurkat cells with the target and non-target cell groups, respectively, and all cells greater than the control group (Mock) were considered successful in activation and were selected for further validation.
Co-culture of Hut78 cells with target cells
The actively activated CAR recombinant plasmid was picked out to package the virus. Hut78 cells were cultured for passaging using IMDM plus 20% FBS. Viral infection expresses CAR while Cell Trace face red (Thermo, C34564, C34572) is used for staining before target Cell plating. The mortality of tumor cells was examined by 7-AAD staining after 48h of co-culture of Hut78 cells with target cells.
Whether the selected chimeric antigen receptor CAR can be used for subsequent experiments is judged according to the death rate of tumor cells.
The results after culture are shown in fig. 3-4, and in this example, fig. 3 shows that the percentage of cells expressing EGFP, i.e., the positive rate of CAR, was successfully expressed after viral infection of Hut78 cells; FIG. 4 shows the percentage of dead tumor cells, i.e., the mortality, of all tumor cells after 48h of co-culture of Hut78 cells with target cells. The chimeric antigen receptor CAR in this example was 3CL-C4, which was considered to be useful in subsequent experiments with a significantly higher mortality than the control group (Mock).
In the above embodiments, the descriptions of the respective embodiments have respective emphasis, and for parts that are not described in detail in a certain embodiment, reference may be made to related descriptions of other embodiments.
While the invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.