Carrier fragment for positioning target protein on surface of cell membrane and application of carrier fragment

Abstract

The invention discloses a carrier for localizing a target protein on the surface of a cell membrane, the carrier comprises a Claudin18.2 1-27aa sequence, a PDGFR membrane localization sequence, multiple cloning sites and a gene for coding the target protein, the multiple cloning sites are located between the Claudin18.2 1-27aa sequence and the PDGFR membrane localization sequence, the gene for coding the target protein is inserted into the multiple cloning sites, and the nucleotide sequence of the Claudin18.2 1-27aa sequence is shown as SEQ ID No.6. The invention further discloses a preparation method of the carrier for localizing the target protein on the surface of the cell membrane. The molecular design of the vector fragment can efficiently and quickly locate the protein / polypeptide on the cell membrane surface, does not need to synthesize the full length of the protein, reduces the gene synthesis cost, and can be applied to production of liquid cell quality control products.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a carrier fragment for positioning a target protein on a cell membrane surface and an application thereof. Background Art

[0002] Claudin is a class of four-transmembrane proteins encoded by a multigene family and is an important component in the formation of tight junctions between cells. Claudin18 is a member of this family, and its encoding gene can form two subtypes of proteins, Claudin18.1 and Claudin18.2, through alternative splicing. Claudin18.1 is specifically expressed in normal and cancerous lung tissues, while Claudin18.2 is not expressed in any healthy tissue except the gastric mucosa, but is widely expressed in gastric cancer, especially the diffuse type in Lauren classification. Both the N-terminus and C-terminus of the Claudin18.2 protein are located inside the cell, and the entire protein is expressed on the cell membrane, which is an important structural component of cell tight junctions.

[0003] Claudin18.2 has a variety of clinical uses: 1. It is the target of companion diagnostics for the specific monoclonal antibody drug Zolbetuximab.

[0004] 2. Combination with Cadherin-17 can be an effective means of detecting and locating gastric cancer cells.

[0005] 3. Related to the treatment of pancreatic cancer, esophageal cancer, and non-small cell lung cancer.

[0006] Liquid cells are used as quality control products in immunohistochemical staining to eliminate experimental errors during the experiment. However, there are no cells that highly express Claudin18.2 in nature, and there are no natural cells that can be used as quality control products. Therefore, it is necessary to use genetic modification technology to construct cells that highly express Claudin18.2 as a positive control for liquid cell quality control products. At the same time, Claudin18.2 is a popular target for gastric cancer detection, diagnosis, and treatment, and its overexpression cell lines can be used as materials in diagnosis and treatment research and development.

[0007] Since Claudin18.2 is a four-transmembrane protein, the expression of multiple-transmembrane proteins is limited by the cell surface area. At the same time, unlike the nuclear localization signal (NLS) that can locate any protein in the nucleus, there is no recognized and efficient membrane localization signal to locate proteins / peptides outside the cell membrane. In addition to the above-mentioned Claudin18.2, there are other membrane proteins that can be used as liquid quality control products. Therefore, it is very necessary to provide a specific molecular design to efficiently and quickly locate the target protein on the cell membrane surface for use in the production of liquid cell quality control products. Summary of the invention

[0008] In order to solve one of the above problems, the present invention provides a vector fragment for localizing the target protein on the cell membrane surface and its application, which can achieve efficient and rapid localization of the protein / polypeptide on the cell membrane surface without the need to synthesize the full-length protein, thereby reducing the cost of gene synthesis.

[0009] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a vector fragment for localizing a target protein on the cell membrane surface, comprising a Claudin18.2 1-27aa sequence, a PDGFR membrane localization sequence, a multiple cloning site and a gene encoding the target protein, wherein the multiple cloning site is located between the Claudin18.2 1-27aa sequence and the PDGFR membrane localization sequence, and the gene encoding the target protein is inserted into the multiple cloning site, and the nucleotide sequence of the Claudin18.2 1-27aa sequence is shown in SEQ ID No. 6.

[0010] In some embodiments of the present invention, the genes encoding the target proteins include Claudin18.2 protein gene and MUC-4 protein gene.

[0011] In some embodiments of the present invention, restriction sites EcoRI and BamHI sequences are added to both ends of the vector fragment, respectively. Restriction sites EcoRI and BamHI sequences are added to both ends of the vector fragment sequence, respectively, to provide restriction sites for cloning into a lentiviral vector.

[0012] The second aspect of the present invention provides a gene encoding the vector fragment described in the first aspect, the nucleotide sequence of the gene encoding the vector fragment that localizes the Claudin18.2 protein on the cell membrane surface is shown as SEQ ID No. 4, and the nucleotide sequence of the gene encoding the vector fragment that localizes the MUC-4 protein on the cell membrane surface is shown as SEQ ID No. 5.

[0013] The third aspect of the present invention provides a lentiviral vector comprising the gene described in the second aspect. In some embodiments of the present invention, the gene of the synthesized vector fragment is cloned into the plv-CMV-MCS-EF1α-Puro plasmid according to the restriction sites EcoRI and BamHI.

[0014] In some embodiments, the step of transforming the plasmid into Escherichia coli DH5α for expansion culture is also included.

[0015] A fourth aspect of the present invention provides a cell pool stably expressing a target protein, wherein the cell pool is constructed by one of lentiviral method, liposome transfection method or cell electroporation method.

[0016] The fifth aspect of the present invention provides the use of the vector fragment described in the first aspect as a vector tool for localizing the overexpressed target protein on the cell membrane surface.

[0017] The sixth aspect of the present invention provides an application of the vector fragment described in the first aspect as a vector tool for localizing an overexpressed target protein on the cell membrane surface in tumor drug research.

[0018] The seventh aspect of the present invention provides an application of the vector fragment described in the first aspect as a carrier tool for localizing the overexpressed target protein on the cell membrane surface in the preparation of an engineered cell line for immunoassay quality control products. In some embodiments of the present invention, the immunoassay quality control product is a liquid quality control product. In some embodiments of the present invention, the liquid quality control product is used for various immunoassays of cells or tissues, including but not limited to immunohistochemistry, immunofluorescence, and protein immunoblotting.

[0019] The eighth aspect of the present invention provides a method for localizing a target protein on the cell membrane surface, using the vector fragment described in the first aspect as a cell membrane localization signal molecule to localize the overexpressed target protein on the cell membrane surface.

[0020] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: multiple transmembrane proteins are limited by the cell surface area and cannot achieve high expression, such as Claudin18.2; although individual membrane proteins are single transmembrane proteins, they have huge molecular weights and high cost of overexpression gene synthesis, such as MUC-4. The present invention provides a vector fragment that can locate the overexpressed target protein on the cell membrane, including Claudin18.2 1-27aa sequence, PDGFR membrane localization sequence, multiple cloning site and gene encoding the target protein, the multiple cloning site is located between the Claudin18.2 1-27aa sequence and the PDGFR membrane localization sequence, and the gene encoding the target protein is inserted into the multiple cloning site. The structural molecule can efficiently and quickly locate the target protein fragment on the cell membrane surface, and is located outside the cell membrane, achieving high expression of envelope localization protein, and at the same time, it is not necessary to synthesize the full length of the protein, reducing the cost of gene synthesis; verified by Claudin18.2 protein and MUC-4 protein, both can be used for the construction of cell pools, and can also be used for liquid cell quality control products, and its application includes but is not limited to cell immunohistochemistry and protein immunoblotting. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A schematic diagram of the structure of the Claudin18.2 transmembrane protein is shown; Figure 2 The schematic diagram of the structure of the molecular design C in this application is shown; Figure 3The immunohistochemical results of HEK-293T cell pools expressing Claudin18.2 fragments with different molecular designs are shown; Figure 4 The results of Western blot experiments of HEK-293T cell pools expressing Claudin18.2 fragments with different molecular designs are shown; Figure 5 Shown are the staining results of HEK-293T-Claudin18.2-Molecular Design D cell pool cells as quality control; Figure 6 The immunohistochemical results of MUC-4 fragment overexpression in Jurkat cells are shown; Figure 7 The results of protein immunoblotting of Jurkat-MUC-4 cell pools are shown; Figure 8 The staining results of Jurkat-MUC-4 cell pool cells as quality control are shown; Fig. 9 An example diagram of the Claudin18.2 1-27aa +MCS+PDGFR sequence design is shown. DETAILED DESCRIPTION

[0022] The following examples are used to demonstrate preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention and therefore can be considered as preferred embodiments of the present invention. However, it will be appreciated by those skilled in the art based on this specification that many modifications may be made to the specific embodiments disclosed herein and still achieve the same or similar results without departing from the spirit or scope of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs, and the materials cited herein and those cited by them are incorporated by reference. Those skilled in the art will recognize or will learn through routine experimentation that there are many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0024] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0025] Example 1 Construction of expression vector The human Claudin18 gene sequence (Gene ID: 51208) was searched in NCBI (National Center for Biotechnology Information), and the full sequence of Claudin18 isoform 2 (Claudin18.2) was saved.

[0026] Molecular design A: The complete sequence of Claudin18.2, the nucleotide sequence is shown in SEQ ID No.1.

[0027] Molecular design B: Claudin18.2 1-122aa sequence, the nucleotide sequence is shown in SEQ ID No. 2; including the N-terminus, the first transmembrane region, the first extracellular loop, the second transmembrane region, and the intracellular region, and its structure is as shown Figure 1 The framed part is shown.

[0028] Molecular design C: The signal peptide, Claudin18.2 28-80 aa and PDGFR membrane localization sequence are linked in sequence, i.e. Sig-Claudin18.2 28-80aa-PDGFR, wherein 28-80aa is the first extracellular loop of Claudin18.2, which is the region where the corresponding antibody recognizes the Claudin18.2 site. The nucleotide sequence is shown in SEQ ID No. 3, and its structure is shown in Figure 2 shown.

[0029] Molecular design D: Claudin18.2 1-80 aa was linked to the PDGFR membrane localization sequence for overexpression, i.e., Claudin18.2 1-80aa-PDGFR, whose nucleotide sequence is shown in SEQ ID No.4, wherein Claudin18.2 1-80aa includes the N-terminus, the first transmembrane region, and the first extracellular loop.

[0030] Restriction sites EcoRI and BamHI were added to both ends of the above four nucleotide sequences (SEQ ID No. 1-4) to provide restriction sites for cloning into lentiviral vectors.

[0031] The final sequence was synthesized by a gene synthesis company and cloned into plv-CMV-MCS-EF1α-Puro according to the restriction sites EcoRI and BamHI, which are plasmids designed for four different sequences of AD, corresponding to four protein membrane localization strategies.

[0032] The obtained four AD lentiviral plasmid dry powders were transformed into E. coli DH5α for expansion culture: (1) Centrifuge the plasmid powder at 5000 rpm for 1 min and add 20 μL ddH 2O deionized water to dissolve the plasmid; (2) Take 100 μL of competent cells and thaw on ice for 10 min, add 2 μL of plasmid, gently shake to mix, place on ice for 30 min, heat shock at 42°C for 60 s without stirring, and place on ice for 5 min; (3) Add 400 μL of antibiotic-free LB liquid medium and culture at 37°C with shaking at 180 rpm for 45 min; (4) Centrifuge at 6000 rpm for 5 min, leaving only 150 μL of supernatant to resuspend the bacterial pellet, and spread 50 μL and 100 μL respectively on the LB plate with the target plasmid resistance; (5) Incubate the plate in the normal direction for 1 h, then invert and incubate at 37°C for 14 h. (6) Pick a single colony and place it in LB liquid culture medium. Add ampicillin and shake at 220 rpm for 14 h. Extract the plasmid according to the instructions of the plasmid extraction kit.

[0033] The extracted plasmid was sequenced and verified by a gene sequencing company to ensure the correctness of the sequence.

[0034] Example 2 Lentivirus packaging (1) One day before the four AD lentivirus packaging, HEK-293T (human embryonic kidney) cells were passaged and 4×10 6 The cells were cultured in T75 culture flasks with DMEM + 10% FBS overnight. (2) On the day of lentiviral packaging, replace the HEK-293T culture medium with serum-free DMEM; (3) The total amount of DNA was 20 μg, with a mass ratio of pxPAX2 (10703 bp): pMD2G (5824 bp): plv-CMV-target gene-EF1α-Puro = 2:1:2, mixed with 40 μL of liposomes in 1 mL of culture medium and incubated at room temperature for 15 min; (4) Add the DNA-liposome mixture directly to the culture medium containing HEK-293T cells, shake to mix, and culture overnight; (5) On the next day, the culture medium of HEK-293T cells was changed to DMEM + 10% FBS and cultured overnight; (6) After 48 hours of infection, collect the cell culture supernatant into a 15 mL centrifuge tube and centrifuge at 500 g for 5 min; (7) Filter the supernatant using a 0.45 μM filter and a 10 mL syringe that has been sterilized at high temperature into a new 15 mL centrifuge tube, i.e., the virus solution, and store it at -80°C. Four venom solutions were obtained for the four AD molecular designs.

[0035] Example 3 Construction of HEK-293T-Claudin18.2 cell pool by lentiviral infection (1) One day before infection, HEK-293T cells were passaged in DMEM + 10% FBS culture medium; (2) On the day of infection, the culture medium was replaced with serum-free DMEM; (3) Count 3 × 10 6 cells in one well of a six-well plate, prepare four wells, add 1.5 mL of different virus venom solutions respectively, and culture overnight; (4) Change the culture medium to DMEM + 10% FBS and continue culturing for 3-4 days; (5) On the 5th / 6th day of lentiviral infection, add 0.5 μg / mL puromycin, a selective antibiotic, to the culture medium, and then perform subculture and observe cell viability every 3-4 days. The cell viability will first decrease (a large number of floating aggregated cells), and then slowly recover. When the cell viability recovers to 95% and is stably subcultured twice, it is the HEK-293T cell pool that stably expresses the target sequence.

[0036] Example 4 Preparation of cell wax blocks of HEK-293T-Claudin18.2 cell pool (1) Collect four HEK-293T cell pools A and B into appropriate centrifuge tubes and fix them with 10 mL of 10% formalin fixative for 2 h to 24 h; (2) After the cells are fixed, centrifuge at about 2500 rpm for 3 to 10 minutes, pour off the fixative, add about 1 mL of 75% ethanol, blow off the cells, then add about 9 mL of 75% ethanol to wash the cells, centrifuge at 3000 rpm for 5 minutes, and remove the supernatant; (3) Slowly and gently add about 10 mL of 95% ethanol to the centrifuge tube and let it stand for dehydration for 2 to 6 hours; (4) Pour off the supernatant, loosen the fixed cell clumps, and then use a pipette to remove the cell clumps from the centrifuge tube; (5) Place the cell mass in an embedding box, add it to anhydrous ethanol, incubate for 0.5 to 2 hours, remove the embedding box, drain it, and then place it in anhydrous ethanol and incubate it for 0.5 to 24 hours. (6) Take out the embedding box from the anhydrous ethanol, drain it, and then put it into xylene for transparency. Incubate it for 0.5 to 24 hours. Take out the embedding box, drain it, and then put it into xylene for incubation for 0.5 to 24 hours. (7) Place the embedding box in the wax pool of the embedding machine for wax immersion. After wax immersion overnight, embedding is performed. After embedding is completed, the prepared wax block is obtained.

[0037] Example 5 Immunohistochemical Identification of HEK-293T-Claudin18.2 Cell Pool (1) Baking: Cut the embedded wax blocks of the four HEK-293T cell pools into 1-2 μm slices and bake them in an oven at 60°C for about 1 h. (2) Antigen retrieval: After dewaxing and hydration, the sections were retrieval at high temperature and normal pressure (pH 9.0) and boiled at 100°C for 15 min. (3) Sealing: After cooling to room temperature, rinse with tap water three times and then seal with 3% H 2 O 2 Block endogenous peroxidase and allow to act at room temperature for about 10 minutes; (4) Draw circles: After rinsing with tap water three times, use an immunohistochemistry pen to draw circles around the cell slices and soak them in PBST for 3 to 5 minutes; (5) Primary antibody incubation: shake off the excess liquid on the slices, add about 100 μL of 1:200 diluted rabbit anti-human Claudin18.2 monoclonal antibody (Hangzhou Bailing Biotechnology, cat#BX50272, clone#BP6249) on the slices, and incubate at room temperature for 30 min; (6) Secondary antibody incubation: Wash the sections with PBST three times, shake off the excess liquid on the sections, add about 100 μL of HRP Polymer Conjugated Goat Anti Mouse + Rabbit IgG (Hangzhou Bailing Biotechnology, cat#BX10001) on the sections, and incubate at room temperature for 30 min. (7) Color development: Wash the sections with PBST three times, and prepare DAB color development solution using DAB chromogen (Hangzhou Bailing Biotechnology, cat#BX10002) and DAB Buffer (Hangzhou Bailing Biotechnology, cat#BX10003). Add about 100 μL of the color development solution on the sections and color for 2 min; (8) Restaining: After rinsing the sections with tap water for 3 times, use hematoxylin to restain for 3 to 6 minutes. After rinsing with tap water, immerse the sections in PBST to blue for 1 minute. (9) Dehydration, transparency and sealing of sections for routine immunohistochemistry; (10) Place the stained slide under a microscope for observation and photography.

[0038] The experimental results are shown in Figure 3 As shown, Figure A is the full-length expression fragment of Claudin18.2. It can be seen that the cell membrane shows obvious positive staining, but the intensity is weak, indicating that Claudin18.2, as a four-transmembrane protein, can be overexpressed on the cell membrane, but it may be limited by the cell membrane surface area and the expression intensity is not high.

[0039] Figure B shows the expression of Claudin18.2 1-122aa, which is the 1-122 amino acids at the N-terminus of Claudin18.2, including two transmembrane regions and the first extracellular loop in the middle. However, no positive staining was observed by immunohistochemistry. The reason may be that a certain sequence at the C-terminus of Claudin18.2 plays a certain role in its envelope localization, which will not be explored in depth here.

[0040] Figure C shows a cell pool overexpressing the signal peptide, Claudin18.2 28-80 aa (first extracellular loop) and PDGFR membrane localization sequence. It can be seen that some cells are positive in immunohistochemistry experiments, and the intensity is weak, indicating that this display system is not suitable for overexpression of the first extracellular loop of Claudin18.2. The reason may be that one end of it is not fixed, and a ring structure with fixed ends cannot be formed, and it cannot present a spatial conformation similar to that of the natural protein, so the antibody cannot recognize it.

[0041] Figure D shows that Claudin18.2 1-80 aa (N-terminus, first transmembrane region and first extracellular loop) linked to the PDGFR membrane localization sequence was overexpressed in cells, and immunohistochemical staining showed strong positive results on the cell membrane.

[0042] Figure E shows HEK-293T cells, which showed completely negative immunohistochemical staining, that is, the cells did not express Claudin18.2 and could be used as host cells for overexpression.

[0043] It can be seen that the molecular design of Claudin18.2 1-27 aa (N-terminus, first transmembrane region) and PDGFR membrane localization sequence can highly express the first extracellular loop of Claudin18.2 (28-80aa) on the cell membrane.

[0044] Example 6: Protein immunoblotting detection of HEK-293T-Claudin18.2 cell pool (1) SDS-PAGE Prepare 4%~20% gradient gel, pour it into the gel casting machine, and insert a 15-hole comb. After the gel solidifies, put the gel into the electrophoresis solution, add protein markers and HEK-293T cell lysates with different molecular designs to the 15-hole comb. Perform electrophoresis at 80V for 40min; 120V for 80min.

[0045] (2) Transfer After the electrophoresis is finished, pry open the glass plate to take out the gel, and place it in the electrotransfer buffer to equilibrate at room temperature for more than 30 minutes. First, put the transfer clamp in place, with the anode side facing down, and spread a sponge. Put two filter papers on the sponge, use a glass rod to push out the bubbles, and spread the membrane, landing on one side first, then slowly lowering it, and then use a glass rod to push out the bubbles (do not touch the membrane with your hands during the process). The same is true for laying the gel. Cover with filter paper, add a small amount of electrotransfer solution on it, and use a glass rod to push out the bubbles. Finally, add another sponge, close the transfer clamp, and insert it into the electrotransfer tank in the correct electrode direction. Transfer the membrane with electrotransfer solution at room temperature for 15 minutes. Take out the PVDF membrane after transfer, add methanol (the membrane is completely immersed in methanol) and treat it for 5 minutes.

[0046] (3) Closed The PVDF membrane was cut into two parts after transfer and blocked with 5% skim milk solution at 100 rpm and room temperature for 1 h.

[0047] (4) Primary antibody incubation After washing the membrane with TBST, add 1:1000 diluted Claudin18.2 rabbit monoclonal antibody (Hangzhou Bailing Biotechnology, Cat#BX50272, clone#BP6249) to one of the PVDF membranes; add 1:1000 diluted actin internal reference antibody (Hangzhou Bailing Biotechnology, Cat#BX00063, clone#RR667) to the other, and incubate at room temperature for 1 h at 100 rpm.

[0048] (5) Secondary antibody incubation After washing the membrane with TBST, 1:4000 goat anti-rabbit IgG-HRP (Thermo, Cat#31460) was added and incubated at 100 rpm for 1 h at room temperature.

[0049] (6) Add ECL colorimetric solution After washing the membrane with TBST, ECL substrate development solution (manufacturer: Millipore, Cat# WBKLS0500) was added.

[0050] (7) Exposure The developed PVDF membrane was placed in a gel imager for exposure.

[0051] WB test results Figure 4As shown, the left side is the staining result of Claudin18.2, and the right side is the staining result of the internal reference Actin. Among them, lane 1 is the wild-type HEK-293T cell lysate, and Claudin18.2 is not stained. Lanes 2-5 correspond to the cell pool lysates of different molecular designs AD. The theoretical molecular weight of molecular design A is 29kDa, and the theoretical molecular weights of molecular designs B, C, and D are all around 14kDa. The WB result position is consistent with the theory. Actin, as an internal reference protein, has consistent expression intensity in the five lysates. The protein bands in lanes 3 and 4 are weak, 2 is slightly stronger, and 5 is the strongest, indicating that the protein expression level of molecular design D is the highest, which is consistent with the immunohistochemistry results.

[0052] Example 7 Application of HEK-293T-Claudin18.2-Molecular Design D Cell Pool as Quality Control Use a pipette to drop 1~2μL of HEK-293T-Claudin18.2-Molecular Design D cell pool cell suspension on the edge of the slide of gastric cancer tissue section, and drop HEK-293T wild-type cell suspension as a control. After drying the slice, detect it according to the conventional LYNX480 immunohistochemical staining process. For the preparation process of cell suspension, please refer to the patent "CN112611614B-A method for preparing a semi-solid cell suspension and its application".

[0053] The staining results are imaged by a scanner, such as Figure 5 As shown, the middle tissue is Claudin18.2-positive gastric cancer tissue, the two cell spots on the left side of the tissue are HEK-293T wild-type cells, and the two cell spots on the right side of the tissue are HEK-293T-Claudin18.2-Molecular Design D Cell Pool cells. The positive tissue part stained brown in the upper left corner corresponds to HEK-293T-Claudin18.2-Molecular Design D cells stained brown; the other positive tissue parts stained blue correspond to HEK-293T wild-type cells stained blue. It can be seen that adding HEK-293T-Claudin18.2-Molecular Design D Cell Pool cells as cell quality control products at the edge of the tissue can achieve quality control of tissue staining.

[0054] Example 8 Molecular Design D and Expression Verification of MUC-4 Protein Mucin 4 (MUC-4) is a large membrane-anchored glycoprotein of the mucin family. It is expressed in epithelial cells of various normal tissues such as the lung, bronchus, stomach, colon and cervix, and is abnormally expressed in many tumors, including ovarian cancer, lung cancer, pancreatic cancer and breast cancer. MUC-4 is cleaved into two subunits at the gly-asp-pro-his (gdph) site in an autocatalytic manner: an extracellular α subunit (500-850kda) with a large molecular weight and high glycosylation, and a β subunit (80-90kda) anchored to the cell membrane and extending into the cytoplasm. The clinical use of MUC-4 can be used as a diagnostic basis for lung cancer, esophageal squamous cell carcinoma, etc., and it is also a potential tumor antigen for immunotherapy of pancreatic cancer. Because the molecular weight of the MUC-4 protein is too large, the cost of gene synthesis is high when it is over-expressed, so the immunogenic region can be over-expressed on the cell membrane and used as a liquid cell quality control for positive control.

[0055] MUC-4 was selected as the validation protein to verify whether the membrane localization scheme of molecular design D was applicable to the expression of its protein fragment.

[0056] Claudin18.2 1-27 aa (N-terminus, first transmembrane region) was linked to the MUC-4 immunogen sequence region (1550-1650aa), and then linked to the PDGFR membrane localization sequence to obtain Claudin18.2 1-27aa-MUC-4-PDGFR, whose nucleotide sequence is shown in SEQ ID No.5.

[0057] EcoRI and BamHI sequences were added to both ends to provide restriction sites for cloning into lentiviral vectors, and lentiviral plasmids were constructed with reference to Example 1. According to Examples 2-5, lentiviral packaging, infection, cell pool wax block preparation, and immunohistochemical identification were performed. Lentivirus infection was performed in Jurkat, and rabbit anti-human MUC-4 monoclonal antibody (Abcam, ab214220) was used for immunohistochemical primary antibody incubation at a dilution of 1:200, and the other conditions were the same.

[0058] The experimental results are as follows Figure 6 As shown, Figure A is the immunohistochemical staining of wild-type Jurkat cells, which are MUC-4 negative cells with no brown positive staining. Figure B is the Jurkat cells overexpressing the MUC-4 fragment, which can be seen with strong positive staining on the cell membrane and partial positive staining in the cytoplasm, which is the staining of the MUC-4 protein fragment. The results prove that the molecular design can locate the MUC-4 fragment on the cell membrane surface.

[0059] Example 9. Protein immunoblotting detection of Jurkat-MUC-4 cell pool Referring to Example 6, the Jurkat-MUC-4 cell lysate was subjected to protein immunoblotting detection, and the primary antibody used was rabbit anti-human MUC-4 monoclonal antibody (Abcam, ab214220) at a dilution of 1:1000, and the other conditions were the same.

[0060] WB test results Figure 7 As shown, lane 1 is Jurkat cell lysate, and lane 2 is Jurkat-MUC-4 cell lysate. Actin is located at about 43kDa, and the membrane-localized fragment plus the MUC-4 fragment is about 18kDa. Jurkat cells did not express MUC-4, and the Jurkat-MUC-4 cell pool showed strong expression of the MUC-4 fragment. The WB results were consistent with the theory and the immunohistochemical results.

[0061] Example 10 Application of Jurkat-MUC-4 cell pool as quality control product Use a pipette to drop 1-2 μL of Jurkat-MUC-4 cell pool cell suspension on the edge of the slide of gastric cancer tissue section, and add Jurkat wild-type cell suspension as a control. After drying the slide, perform detection according to the conventional LYNX480 immunohistochemical staining process.

[0062] The staining results are imaged by a scanner, such as Figure 8 As shown, the middle is the MUC-4 positive tissue esophagus, the two cell spots on the left side of the tissue are Jurkat wild-type cells, and the two cell spots on the right side of the tissue are Jurkat-MUC-4 cell pool cells. The positive tissue part stained brown corresponds to the brown staining of Jurkat-MUC-4 cells; the positive tissue part stained blue corresponds to the blue staining of Jurkat wild-type cells. It can be seen that adding Jurkat-MUC-4 cell pool cells as cell quality control products at the edge of the tissue can achieve quality control of tissue staining.

[0063] In summary, Claudin18.2 1-27aa is sequentially linked to the target protein: Claudin18.2 28-80aa or MUC-4 1550-1650aa, and the PDGFR membrane localization sequence, e.g. Fig. 9 As shown, the target protein: Claudin18.2 28-80aa or MUC-4 1550-1650aa can be located on the cell membrane for the construction of liquid cell quality control products.

[0064] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined in the present application.

Claims

1. A vector fragment for localizing a target protein on the cell membrane surface, characterized in that: It includes a Claudin18.2 1-27aa sequence, a PDGFR membrane localization sequence, a multiple cloning site and a gene encoding a target protein. The multiple cloning site is located between the Claudin18.2 1-27aa sequence and the PDGFR membrane localization sequence. The gene encoding the target protein is inserted into the multiple cloning site. The nucleotide sequence of the Claudin18.2 1-27aa sequence is shown in SEQ ID No.

6.

2. The vector fragment for localizing a target protein on the cell membrane surface according to claim 1, characterized in that: The genes encoding the target protein include Claudin18.2 protein gene and MUC-4 protein gene.

3. The vector fragment for localizing a target protein on the cell membrane surface according to claim 2, characterized in that: The restriction sites EcoRI and BamHI sequences were added to both ends of the vector fragment.

4. The gene encoding the vector fragment according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the vector fragment that localizes the Claudin18.2 protein on the cell membrane surface is shown in SEQ ID No. 4, and the nucleotide sequence of the gene encoding the vector fragment that localizes the MUC-4 protein on the cell membrane surface is shown in SEQ ID No.

5.

5. A cell pool stably expressing a target protein, characterized in that: Containing the vector fragment according to claim 1.

6. The cell pool stably expressing a target protein according to claim 5, characterized in that: The cell pool is constructed by one of lentiviral method, liposome transfection method or cell electroporation method.

7. Use of the vector fragment according to claim 1 as a vector tool for localizing an overexpressed target protein on the cell membrane surface.

8. Use of the vector fragment according to claim 1 as a vector tool for localizing an overexpressed target protein on the cell membrane surface in tumor drug research.

9. Use of the vector fragment according to claim 1 as a carrier tool for localizing an overexpressed target protein on the cell membrane surface in the preparation of an engineered cell line for immunoassay quality control products.

10. A method for localizing a target protein on a cell membrane surface, characterized in that: The vector fragment described in claim 1 is used as a cell membrane localization signal molecule to localize the overexpressed target protein on the cell membrane surface.

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