CN109207488B

CN109207488B - Construction method and application of PE38KDEL gene expression plasmid mediated by tumor specific promoter

Info

Publication number: CN109207488B
Application number: CN201811138316.1A
Authority: CN
Inventors: 施维; 郭琼; 吴江
Original assignee: Jilin University
Current assignee: Jilin University
Priority date: 2018-09-28
Filing date: 2018-09-28
Publication date: 2020-12-01
Anticipated expiration: 2038-09-28
Also published as: CN109207488A

Abstract

The invention provides a construction method and application of a PE38KDEL gene expression plasmid mediated by a tumor specific promoter, belonging to the technical field of gene targeting treatment of tumors, wherein the PE38KDEL gene has a nucleotide sequence shown as SEQ ID No. 1. The invention provides a recombinant plasmid containing the PE38KDEL gene, wherein pGL3-Basic is used as an original plasmid of the recombinant plasmid, a tumor specific promoter is inserted into a multiple cloning site of pGL3-Basic, and the PE38KDEL gene is used for replacing an original luciferase gene; the tumor specific promoter is a SERPINB3 promoter or a CEACAM6 promoter. The recombinant plasmid can inhibit and kill tumor cells in a targeted manner, so that the recombinant plasmid can be applied to the preparation of tumor targeted gene therapy medicines, and can provide a new means for tumor targeted gene therapy.

Description

Construction method and application of PE38KDEL gene expression plasmid mediated by tumor specific promoter

Technical Field

The invention relates to the technical field of gene-targeted tumor treatment, in particular to a method for regulating and controlling PE38KDEL toxin targeting inhibition and killing tumor cells by utilizing a tumor cell specific expression gene promoter.

Background

According to The famous medical journal "Lancet" (The Lancet), new cancer cases reach 200 million people by 2030, and The expenditure of anticancer drugs increases 15% each year. With the increasing morbidity and mortality, cancer has become the leading cause of death worldwide. Cancer treatment methods are gradually transitioning from traditional surgery, radiation therapy, chemotherapy, immunotherapy, to gene therapy. The principle of gene therapy is that according to the molecular biological characteristics of different cancers, a specific exogenous target gene segment is introduced into a tumor cell and expressed by utilizing a gene transfer technology so as to correct or compensate a defective gene, thereby inhibiting and killing the tumor cell and achieving the purpose of treating the cancers. The target gene therapy uses the over-expressed tumor specific gene as a target spot to inhibit the proliferation and the metastasis of tumor cells, so the target gene therapy has good specificity and small damage to normal cells. Two key factors of tumor gene therapy are the specific targeting property and safety of the gene, and the development characteristics of precise medical treatment are met.

Pseudomonas aeruginosa exotoxin (PEA) is secreted by Pseudomonas aeruginosa and exerts a cytotoxic effect through ADP ribosylation EF-2 His 699, with the enzyme active region at the C-terminal and the binding region at the N-terminal. PEA is a single-chain protein comprising 613 amino acid residues, and has 3 functional regions. Ia (1-252) is a binding region, II (253-364) is a transmembrane region, III (400-613) is an ADP ribosylase region, and the function of Ib region is not very clear. His 440 and Glu 553 of PEA are involved in the process of ribosylation. His 440 binds to NAD via AMP, while the C-terminus of Glu 553 may utilize hydrogen bonding on Tyr 481 and Glu 546 to allow Tyr 481 to bind to NAD. The process of killing target cell with the toxin includes the steps of cutting C end of Lys 613 with carboxypeptidase in culture liquid or periplasm; the Ia region binds to the alpha-2 macroglobulin receptor on the cell and is endocytosed into the cell and then transported from the inclusion body to the golgi apparatus; ③ the peptide bond between 279 and 280 amino acid residues of region II is cleaved by furin; the disulfide bond between 265 and 287 residues is reduced; 609-612 sequence REDL is combined with intracellular receptor vesicle, C-terminal 37000 fragment of relative molecular mass is transported from Golgi apparatus to endoplasmic reticulum; sixthly, mediating intracellular transport of the toxin by 280-313 amino acid residues; seventhly, the amino acid sequence of 400-602 enables EF-2 to generate ADP ribosylation, so that the synthesis of cell protein is inhibited, and cell death is caused.

At present, PEA toxin is mostly applied to immunotoxin therapy in the aspect of tumor therapy. The method can kill tumor cells in a targeted manner, but still has the defects of poor solubility, high preparation cost and non-durable drug effect of immunotoxin, and the gene therapy of the tumor by using the tumor specific expression gene to regulate and control the PE38KDEL toxin can effectively avoid the defects, enhance the drug action time and reduce the treatment cost.

Disclosure of Invention

The invention aims to provide a construction method and application of a PE38KDEL gene expression plasmid mediated by a tumor specific promoter, so as to inhibit the synthesis of tumor cell proteins and realize the inhibition and killing of tumor cells.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a PE38KDEL gene with the effects of inhibiting and killing tumor cells, wherein the PE38KDEL gene has a nucleotide sequence shown as SEQ ID No. 1.

Preferably, the upstream primer for amplifying the PE38KDEL gene PCR has a nucleotide sequence shown as SEQ ID No.2 in a sequence table; the downstream primer for amplifying the PE38KDEL gene PCR has a nucleotide sequence shown as SEQ ID No.3 in the sequence table.

The invention provides a recombinant plasmid containing the PE38KDEL gene in the scheme, wherein pGL3-Basic is taken as an original plasmid of the recombinant plasmid, a tumor-specific promoter is inserted into a multiple cloning site of pGL3-Basic, and the PE38KDEL gene is used for replacing an original luciferase gene; the tumor specific promoter is a SERPINB3 promoter or a CEACAM6 promoter.

Preferably, the SERPINB3 promoter has a nucleotide sequence shown as SEQ ID No. 4.

Preferably, the CEACAM6 promoter has a nucleotide sequence shown as SEQ ID No. 5.

Preferably, the tumor-specific promoter is inserted between the NheI and NcoI cleavage sites on the original plasmid pGL 3-Basic.

Preferably, the PE38KDEL gene is inserted between the NcoI and XbaI cleavage sites on the original plasmid pGL 3-Basic.

The invention provides a construction method of the recombinant plasmid in the scheme, which comprises the following steps:

1) after being respectively subjected to double enzyme digestion and recovery, pGL3-Basic and a SERPINB3 promoter or a CEACAM6 promoter are connected to obtain a pSERPINB3-Basic plasmid or a pCEACAM6-Basic plasmid;

2) carrying out double digestion recovery on the pSERPINB3-Basic plasmid or the pCEACAM6-Basic plasmid obtained in the step 1) and the PE38KDEL gene respectively, and then connecting to obtain a recombinant plasmid pSERPINB3-PE38KDEL plasmid or pCEACAM6-PE38KDEL plasmid.

The invention provides application of the PE38KDEL gene or the recombinant plasmid in the scheme in preparation of a medicament for targeted inhibition and killing of tumor cells.

The invention provides a recombinant strain of the recombinant plasmid in the scheme.

The invention has the beneficial technical effects that: the gene with the functions of inhibiting and killing tumor cells selected by the invention is a PE38KDEL gene, and the gene removes the Ia region structural domain (delta 1-250) and Ib region structural domain partial sequence (delta 365-380) of the original PEA toxin, so that the binding capacity of the gene and the cells is lost, and the immunogenicity of the toxin is reduced. Meanwhile, the carboxyl terminal REDLK sequence of PEA toxin molecule is replaced by KDEL sequence, thus improving the killing power of PEA toxin molecule on tumor cells. The invention constructs the recombinant plasmid of the tumor specific gene promoter regulating PE38KDEL gene by means of molecular cloning, and the recombinant plasmid is a eukaryotic expression vector. The PE38KDEL gene is highly expressed in tumor cells, but is not expressed or is lowly expressed in normal cells, so that the synthesis of specific inhibition tumor cell proteins is realized, the cells are induced to generate cycle block and apoptosis, and the targeting of gene therapy is realized.

Description of the drawings:

FIG. 1 is a schematic diagram of PEA and PE38KDEL structures;

FIG. 2-a is a flow chart of construction of recombinant plasmid pSERPINB3-PE38 KDEL;

FIG. 2-b is a flow chart of construction of recombinant plasmid pCEACAM6-PE38 KDEL;

FIG. 3 shows the alignment of the SERPINB3 promoter sequence;

FIG. 4 is a protein quantitation plot;

FIG. 5-a shows luciferase assay of SERPINB3 promoter expression activity in different cells;

FIG. 5-b shows luciferase assay of CEACAM6 promoter expression activity in different cells;

FIG. 6-a is luciferase assay of different doses of pSERPINB3-PE38KDEL for inhibition of protein synthesis in different cells;

FIG. 6-b is a luciferase assay for inhibition of protein synthesis in different cells using different doses of pCEACAM6-PE38 KDEL;

FIG. 7-a is a flow cytometry detection graph of the cycle distribution of human tongue squamous cell carcinoma cells TCA8113 by pSERPINB3-PE38KDEL, wherein Control is a Control group, PEI is a PEI group only added with a transfection reagent, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 7-b is a histogram of the periodic distribution of pSERPINB3-PE38KDEL to human tongue squamous cell carcinoma cells TCA8113, wherein Control is a Control group, PEI is a PEI group added with a transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 7-c is a flow cytometry examination of pSERPINB3-PE38KDEL on human hepatocyte LO2 cycle distribution, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 7-d is a histogram of the periodic distribution of pSERPINB3-PE38KDEL on human hepatocytes LO2, in which Control is a Control group, PEI is a PEI group added with a transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 8-a shows the effect of pSERPINB3-PE38KDEL on human tongue squamous cell carcinoma cell TCA8113 apoptosis, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 8-b shows the effect of pSERPINB3-PE38KDEL on apoptosis of human hepatocytes LO2, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 8-c shows the effect of pCEACAM6-PE38KDEL on human tongue squamous cell carcinoma cell TCA8113 apoptosis, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group;

FIG. 8-d shows the effect of pCEACAM6-PE38KDEL on apoptosis of human normal cells LO2, in which Control is a Control group, PEI is a group to which only a transfection reagent PEI was added, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group.

Detailed Description

The invention provides a PE38KDEL gene with the effects of inhibiting and killing tumor cells, wherein the PE38KDEL gene has a nucleotide sequence shown as SEQ ID No. 1; the upstream primer of the PE38KDEL gene PCR amplification has a nucleotide sequence shown as SEQ ID No.2 in a sequence table; the downstream primer of the PE38KDEL gene PCR amplification has a nucleotide sequence shown as SEQ ID No.3 in a sequence table.

In the invention, the PE38KDEL gene with the functions of inhibiting and killing tumor cells removes the Ia region structural domain (delta 1-250) and the Ib region structural domain partial sequence (delta 365-380) of the original PEA toxin, so that the binding capacity of the Ia region structural domain and the Ib region structural domain partial sequence with the cells is lost, and the immunogenicity of the toxin is reduced. Meanwhile, the carboxyl terminal REDLK sequence of PEA toxin molecule is replaced by KDEL sequence, the lethality of PEA and PE38KDEL on tumor cells is improved, and the structural schematic diagram of PEA and PE38KDEL is shown in figure 1.

In the invention, the design templates of the upstream primer and the downstream primer are plasmids containing PE38KDEL genes; the upstream and downstream primers add NcoI and XbaI restriction enzyme recognition sites, respectively, and add a protecting base at the 5' end. In the specific implementation process of the invention, the upstream primer and the downstream primer are synthesized by the company of Biotechnology engineering (Shanghai).

In the invention, the reaction system for PCR amplification of the PE38KDEL gene is preferably as follows: 16.2 μ L of sterilized water, 2.5 μ L of 10 XBuffer, 4 μ L of 2mM dNTP, 0.5 μ L of upstream primer, 0.5 μ g of downstream primer 0.5 μ L, PE38 template plasmid, 0.3 μ L of FastPfu DNA polymerase, and 25 μ L of total amount; the reaction program for PCR amplification of the PE38KDEL gene is preferably as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; extending for 10min after 72 ℃; and terminated at 4 ℃.

The invention provides a recombinant plasmid containing the PE38KDEL gene in the scheme, wherein pGL3-Basic is taken as an original plasmid of the recombinant plasmid, a tumor-specific promoter is inserted into a multiple cloning site of pGL3-Basic, and the PE38KDEL gene is used for replacing an original luciferase gene; the tumor specific promoter is a SERPINB3 promoter or a CEACAM6 promoter. The original plasmid of the recombinant plasmid is pGL 3-Basic; the pGL3-Basic was inserted in sequence with a tumor-specific promoter and a PE38KDEL gene.

In the invention, pGL3-Basic is used as an original plasmid of the recombinant plasmid, because pGL3-Basic does not contain a eukaryotic cell promoter, the construction of the plasmid is convenient, and the luciferase expression gene is contained, so that the detection of the activity of the promoter is convenient.

In the invention, the PE38KDEL gene is inserted between NcoI and XbaI enzyme cutting sites on an original plasmid pGL 3-Basic; the tumor-specific promoter is inserted between NheI and NcoI cleavage sites on the original plasmid pGL 3-Basic; the tumor specific promoter is an SERPINB3 promoter or a CEACAM6 promoter; the SERPINB3 promoter has a nucleotide sequence shown as SEQ ID No. 4; the CEACAM6 promoter has a nucleotide sequence shown as SEQ ID No. 5.

In the invention, the amplification template of the tumor specific promoter which is an SERPINB3 promoter or a CEACAM6 promoter is a TCA8113 cell genome; the TCA8113 cell genome was obtained by extraction using a TCA8113 cell genome DNA extraction kit (centrifugal column type).

In the invention, the sequences of the SERPINB3 promoter and the CEACAM6 promoter are predicted through UCSC (http:// genome. UCSC. edu /), and a software primer 5.0 is used for designing primers aiming at the promoter regions of the SERPINB3 promoter and the CEACAM6 promoter; an upstream primer of the PCR for amplifying the SERPINB3 promoter has a nucleotide sequence shown as SEQ ID No.6 in a sequence table; the downstream primer of the PCR for amplifying the SERPINB3 promoter has a nucleotide sequence shown as SEQ ID No.7 in the sequence table; the upstream primer of PCR for amplifying the CEACAM6 promoter has a nucleotide sequence shown as SEQ ID No.8 in the sequence table; the downstream primer of PCR for amplifying the CEACAM6 promoter has a nucleotide sequence shown as SEQ ID No.9 in the sequence table.

In the invention, an upstream primer and a downstream primer of the SERPINB3 promoter or CEACAM6 promoter are respectively added with NheI and NcoI restriction endonuclease recognition sites, and a protection base is added at the 5' end. In the specific implementation process of the invention, the upstream primer and the downstream primer are synthesized by Shanghai.

In the invention, the PCR reaction system of the SERPINB3 promoter is as follows: 12.2 muL of sterilized water, 2.5 muL of 10 XBuffer, 4 muL of L2mM dNTP, 0.5 muL of upstream primer, 0.5 mu L, TCA8113 genome 0.1 mug of downstream primer, 0.3 muL of FastPfu DNA polymerase, and 25 muL of total amount; the PCR reaction conditions of the SERPINB3 promoter or CEACAM6 promoter are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 3min, and 35 cycles; extending for 10min after 72 ℃; and terminated at 4 ℃.

In the invention, the PCR reaction system of the CEACAM6 promoter is as follows: 12.2 muL of sterilized water, 2.5 muL of 10 XBuffer, 4 muL of L2mM dNTP, 0.5 muL of upstream primer, 0.5 mu L, TCA8113 genome 0.1 mug of downstream primer, 0.3 muL of FastPfu DNA polymerase, and 25 muL of total amount; the PCR reaction conditions of the SERPINB3 promoter or CEACAM6 promoter are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min after 72 ℃; and terminated at 4 ℃.

After obtaining PCR products of an SERPINB3 promoter or a CEACAM6 promoter, carrying out gel recovery on the PCR products of the SERPINB3 promoter or the CEACAM6 promoter; the gel recovery is preferably recovered using an Axygen multifunctional DNA purification recovery kit (spin column type).

2) carrying out double digestion recovery on the pSERPINB3-Basic plasmid or pCEACAM6-Basic plasmid and PE38KDEL gene obtained in the step 1) respectively, and then connecting to obtain a recombinant plasmid pSERPINB3-PE38KDEL or pCEACAM6-PE38 KDEL.

The invention respectively carries out double enzyme digestion and recovery on pGL3-Basic and a SERPINB3 promoter or a CEACAM6 promoter, and then connects to obtain a pSERPINB3-Basic plasmid or a pCEACAM6-Basic plasmid.

In the invention, the double enzyme digestion reaction system is as follows: pGL3-Basic plasmid 1 mug, SERPINB3 promoter or CEACAM6 promoter 1 mug, NheI 1 mug L, NcoI 1 mug, 10 x CutSmart1 mug and the balance of water, the system size is 10 mug; the NheI and NcoI were purchased from NEB; the temperature of the double enzyme digestion reaction is preferably 37 ℃; the time of the double enzyme digestion reaction is preferably 4 hours.

In the present invention, the double enzymatic digestion recovery is performed by using the gel recovery method of the PCR product of the SERPINB3 promoter or CEACAM6 promoter in the above-mentioned scheme, which is not described herein again.

In the invention, the connection system of the SERPINB3 promoter or the enzyme digestion fragment of CEACAM6 and pGL3-Basic is as follows: 0.1pmol of pGL3-Basic double restriction enzyme fragment, 0.3pmol of SERPINB3 promoter or CEACAM6 promoter double restriction enzyme fragment, 1 muL of T4 DNA Ligase, 1 muL of 10 XT 4 DNA Ligase Buffer and the balance of water, wherein the total amount is 10 muL; the temperature of the connection is preferably 16 ℃; the connection time is preferably 10-14 h, and more preferably 12 h.

After obtaining pSERPINB3-Basic plasmid or pCEACAM6-Basic plasmid, the invention respectively carries out double enzyme digestion and recovery on pSERPINB3-Basic plasmid or pCEACAM6-Basic plasmid and PE38KDEL gene, and then carries out connection to obtain recombinant plasmid pSERPINB3-PE38KDEL or pCEACAM6-PE38 KDEL.

In the invention, the double-enzyme digestion reaction system of the pSERPINB3-Basic plasmid or the pCEACAM6-Basic plasmid is as follows: 1 mu g of SERPINB3 promoter or CEACAM6 promoter, 1 mu g of pGL3-Basic plasmid, 1 mu L of NcoI, 1 mu L, XbaI 1 mu L of NcoI, 10 XCutSmart 1 mu L and the balance of water, wherein the system size is 10 mu L; the NcoI and XbaI were purchased from NEB; the temperature of the double enzyme digestion reaction is preferably 37 ℃; the time of the double enzyme digestion reaction is preferably 4 hours.

In the invention, the double enzyme digestion reaction system of the PE38KDEL gene is as follows: PE38KDEL fragment 1. mu.g, NcoI 1. mu. L, XbaI 1. mu.L, 10 × CutSmart 1. mu.L and balance water, system size 10. mu.L; the temperature of the double enzyme digestion reaction is preferably 37 ℃; the time of the double enzyme digestion reaction is preferably 4 hours.

In the invention, the connection system of the pSERPINB3-Basic plasmid or the pCEACAM6-Basic plasmid and the PE38KDEL gene is as follows: pSERPINB3-Basic or pCEACAM6-Basic double restriction enzyme fragment 0.1pmol, PE38KDEL double restriction enzyme fragment 0.3pmol, T4 DNA Ligase1 uL, 10 XT 4 DNA Ligase Buffer 1 uL and the balance of water, the total amount is 10 uL; the temperature of the connection is preferably 16 ℃; the connection time is preferably 10-14 h, and more preferably 12 h.

The enzyme digestion and the connection reaction are preferably carried out in a microcentrifuge tube.

After the recombinant plasmid pSERPINB3-PE38KDEL plasmid or pCEACAM6-PE38KDEL plasmid is prepared, the recombinant plasmid pSERPINB3-PE38KDEL plasmid or pCEACAM6-PE38KDEL plasmid is used for transfecting escherichia coli DH5 alpha, and escherichia coli liquid is obtained by culture.

After obtaining escherichia coli liquid, recombinant plasmid extraction is carried out; the recombinant plasmid extraction procedure used a BioTeke high purity plasmid miniprep kit (spin column type).

After the recombinant plasmid is obtained, the plasmid is subjected to double enzyme digestion according to the double enzyme digestion method, 1% agarose gel is used for electrophoresis, and the plasmid with the correct double enzyme digestion result is selected and sent to Shanghai Meiji biological medicine science and technology company Limited for gene sequencing.

The invention provides the application of the PE38KDEL gene or the recombinant plasmid in the scheme in the preparation of medicines for targeted inhibition and killing of tumor cells; the tumor cell is preferably a human squamous carcinoma cell of the tongue.

The invention provides a recombinant strain comprising the recombinant plasmid in the scheme; the original strain of the recombinant strain is preferably Escherichia coli. In the invention, the recombinant bacteria can realize the rapid proliferation of the recombinant plasmid.

The construction method and application of the PE38KDEL gene expression plasmid mediated by the tumor specific promoter provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

EXAMPLE 1 construction of recombinant plasmids pSERPINB3-PE38KDEL and pCEACAM6-PE38KDEL

The construction process is shown in FIG. 2, wherein FIG. 2-a is a construction process of the recombinant plasmid pSERPINB3-PE38 KDEL; FIG. 2-b is a flow chart of construction of recombinant plasmid pCEACAM6-PE38 KDEL. The method specifically comprises the following steps:

extraction of TCA8113 cell genome

Extracting the genome of the SERPINB3 promoter by using a TCA8113 cell genome DNA extraction kit (centrifugal column type), and specifically comprising the following steps:

(1) digesting with trypsin, beating, and collecting 10⁵～10⁶TCA8113 cells in a 1.5mL centrifuge tube;

(2) centrifuging at 13,000rpm for 1min to precipitate the cells, discarding the supernatant and leaving the cell pellet and about 10-20. mu.L of residual liquid;

(3) adding 200. mu.L of buffer TB to resuspend the washed cells, centrifuging at 13,000rpm for 1min, discarding the supernatant, and then resuspending the cells again with 200. mu.L of buffer TB;

(4) adding 200 μ L of binding solution CB, immediately turning upside down and shaking gently to mix thoroughly, adding 200 μ L of proteinase K (200mg/mL) solution, mixing thoroughly, and standing in 70 deg.C water bath for 10 min;

(5) after cooling, 100. mu.L of isopropanol is added, and the mixture is inverted and shaken gently to be fully mixed, wherein flocculent precipitates can appear;

(6) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column AC (the adsorption column is placed into a collecting pipe), centrifuging at 10,000rpm for 1min, and pouring off the waste liquid in the collecting pipe;

(7) adding 500 μ L inhibitor removing solution IR at 12,000rpm, centrifuging for 1min, and discarding the waste solution;

(8) adding 700 μ L of rinsing liquid WB (added with absolute ethanol), centrifuging at 12,000rpm for 1min, and discarding the waste liquid;

(9) adding 500 μ L of the rinse solution WB added with absolute ethanol at 12,000rpm, centrifuging for 1min, and discarding the waste solution;

(10) putting the adsorption column AC back into an empty collection pipe, centrifuging at 13,000rpm for 2min, and removing the rinsing liquid as much as possible to prevent ethanol remained in the rinsing liquid from inhibiting downstream reaction;

(11) taking out the adsorption column AC, putting the adsorption column AC into a clean 1.5mL centrifuge tube, adding 100 mu L of elution buffer EB which is heated in water bath at 65-70 ℃ in advance into the middle part of the adsorption membrane, standing at room temperature for 3-5 min at 12,000rpm, and centrifuging for 1 min; the resulting solution was re-loaded into the centrifugal adsorption column, and left at room temperature for 2min at 12,000rpm, and centrifuged for 1 min.

(12) The obtained solution is TCA8113 cell genome, and is preserved at the temperature of minus 20 ℃ for a long time.

SERPINB3 promoter and CEACAM6 promoter primer design

The SERPINB3 promoter and CEACAM6 promoter sequences are predicted through UCSC (http:// genome. UCSC. edu /), a software primer 5.0 is used for designing primers aiming at promoter regions of the SERPINB3 promoter and the CEACAM6 promoter (an upstream primer amplified by the SERPINB3 promoter through PCR has a nucleotide sequence shown as SEQ ID No.6 in a sequence table, a downstream primer amplified by the SERPINB3 promoter through PCR has a nucleotide sequence shown as SEQ ID No.7 in the sequence table, an upstream primer amplified by the CEACAM6 promoter through PCR has a nucleotide sequence shown as SEQ ID No.8 in the sequence table, a downstream primer amplified by the CEAM 6 promoter through PCR has a nucleotide sequence shown as SEQ ID No.9 in the sequence table), NheI restriction endonuclease recognition sites and NcoI restriction endonuclease recognition sites are added to the upstream primer and the downstream primer respectively, a protective base is added to the 5' end, and the primers are synthesized from Shanghai.

PE38KDEL fragment primer design

The method is characterized in that a plasmid containing PE38 toxin existing in a laboratory is used as a template, Primer 5.0 is used for Primer design of PE38 toxin (an upstream Primer amplified by the PCR of the PE38KDEL gene has a nucleotide sequence shown as SEQ ID No.2 in a sequence table, and a downstream Primer amplified by the PCR of the PE38KDEL gene has a nucleotide sequence shown as SEQ ID No.3 in the sequence table). The upstream primer and the downstream primer are added with NcoI and XbaI restriction enzyme recognition sites respectively and protective bases are added at the 5' end, and the primers are synthesized by Shanghai.

PCR amplification of the SERPINB3 promoter and CEACAM6 promoter fragment.

The PCR reaction system is as follows: preparing the following solution in a microcentrifuge tube, 10 multiplied by Buffer 2.5 mu L, L2mM dNTP 4 mu L, upstream primer 0.5 mu L, downstream primer 0.5 mu L, TCA8113 genome 0.1 mu g, Fastpfu DNA polymerase 0.3 mu L and the balance of water, wherein the total amount is 25 mu L;

the reaction conditions for PCR amplification of the SERPINB3 promoter were as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 3min, and 35 cycles; extending for 10min after 72 ℃; 4 ℃ forever;

the reaction conditions for PCR amplification of CEACAM6 promoter were: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 1min, and 35 cycles; extending for 10min after 72 ℃; and terminated at 4 ℃.

PCR amplification of PE38KDEL fragment

The PCR reaction system is as follows: in a microcentrifuge tube, 10 XBuffer 2.5. mu.L, 2mM dNTP 4. mu.L, upstream primer 0.5. mu.L, downstream primer 0.5. mu. L, PE38 template plasmid 0.05. mu.g, FastPfu DNA polymerase 0.3. mu.L and the balance water were prepared, and the total amount was 25. mu.L.

The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, extension at 72 ℃ for 90s, and 35 cycles; extending for 10min after 72 ℃; and terminated at 4 ℃.

Gel recovery of DNA fragments

And (3) recovering the product obtained by the PCR by using an Axygen multifunctional DNA purification and recovery kit (a centrifugal column type), and specifically comprising the following steps:

(1) under a long-wave ultraviolet lamp, cutting off the DNA agarose gel needing to be recovered by a clean blade, wherein the smaller the volume of the obtained gel is, the better the gel is;

(2) putting the gel containing the target DNA band into a 1.5mL centrifugal tube, and weighing (weighing an empty 1.5mL centrifugal tube firstly, then putting the gel block into the centrifugal tube and weighing the gel block again, subtracting the weights of the two times to obtain the weight of the gel);

(3) three gel volumes of Buffer DE-A were added (if the gel weight was 0.1g, the volume could be regarded as 100. mu.L, 300. mu.L of the sol solution/conjugate DB was added);

(4) the liquid was placed in a 75 ℃ water bath for 10min (or until the gel was completely dissolved). Vortex and shake every 2-3min to help accelerate gel dissolution;

(5) adding 0.5 Buffer DE-B with the volume of the Buffer DE-A, and uniformly mixing;

(6) adding the solution obtained in the previous step into a DNA preparation tube (an adsorption column is placed into a collection tube), centrifuging at 12,000rpm for 60s, and pouring off waste liquid in the collection tube (if the total volume exceeds 700 mu L, adding the solution into the same adsorption column AC twice);

(7) adding 700 μ L of the absolute ethyl alcohol rinsing liquid WB, centrifuging at 12,000rpm for 1min, and discarding the waste liquid;

(8) adding 500 μ L of the absolute ethanol rinsing liquid WB, centrifuging at 12,000rpm for 1min, and discarding the waste liquid;

(9) placing the preparation tube back into an empty collection tube, centrifuging at 12,000rpm for 2min, and removing rinsing liquid as much as possible to prevent residual ethanol in the rinsing liquid from inhibiting downstream reaction;

(10) the preparation tube was placed in a clean 1.5mL centrifuge tube, 30. mu.L of Eluent or deionized water previously heated in a 70 ℃ water bath was added to the middle of the adsorption membrane, the mixture was left at room temperature for 1min, and the mixture was centrifuged at 12,000rpm for 1 min. If a larger amount of DNA is required, the resulting solution may be re-introduced into the preparation tube and the above operation repeated. The obtained solution is the SERPINB3 promoter DNA fragment.

Double digestion of the PE38KDEL fragment

A double digestion reaction system of 1. mu.g of PE38KDEL fragment, 1. mu.L of NcoI (NEB) 1. mu. L, XbaI (NEB), 10. mu.L of 10 XCutSmart 1. mu.L and the balance of water was prepared in a microcentrifuge tube, and the size of the system was 10. mu.L. The reaction is carried out for 4h at 37 ℃. DNA was recovered as described in 6 by electrophoresis on a 1% agarose gel at 120V and 300mA for 30min.

Construction of pSERPINB3-Basic and pCEACAM6-Basic plasmids

(1) Double digestion of SERPINB3 promoter fragment, CEACAM6 promoter fragment and pGL3-Basic vector

A double-enzyme digestion reaction system is prepared in a microcentrifuge tube, wherein the size of the system is 10 mu L, and the double-enzyme digestion reaction system comprises 1 mu g of pGL3-Basic plasmid, 1 mu g of SERPINB3 promoter or CEACAM6 promoter, 1 mu L of NheI (NEB)1 mu L, NcoI (NEB), 10 XCutSmart 1 mu L and the balance of water. The reaction is carried out for 4h at 37 ℃. DNA was recovered as described in 6 by electrophoresis on a 1% agarose gel at 120V and 300mA for 30min.

(2) Ligation of the cleaved fragment to the vector

In a microcentrifuge tube, 0.1pmol of pGL3-Basic double digested fragment, 0.3pmol of SERPINB3 promoter or CEACAM6 promoter double digested fragment, T4 DNA Ligase 1. mu.L, 10 XT 4 DNA Ligase Buffer 1. mu.L and the balance of water were prepared, and the total amount was 10. mu.L. Mix well and ligate overnight at 16 ℃.

Construction of pSERPINB3-PE38KDEL plasmid and pCEACAM6-PE38KDEL plasmid

(1) Double digestion of pSERPINB3-Basic plasmid and pCEACAM6-Basic plasmid

The following double restriction reaction system, pSERPINB3-Basic plasmid or pCEACAM6-Basic plasmid 1. mu.g, NcoI (NEB) 1. mu. L, XbaI (NEB) 1. mu.L, 10 XCutSmart 1. mu.L and the balance of water, were prepared in a microcentrifuge tube, and the system size was 10. mu.L. The reaction is carried out for 4h at 37 ℃. DNA was recovered as described in 6 by electrophoresis on a 1% agarose gel at 120V and 300mA for 30min.

(2) Double digestion of PE38KDEL fragment

A double digestion reaction system of 1. mu.g of PE38KDEL fragment, 1. mu.L of NcoI (NEB) 1. mu. L, XbaI (NEB), 1. mu.L of 10 XCutSmart 1. mu.L and the balance of water was prepared in a microcentrifuge tube, and the system size was 10. mu.L. The reaction is carried out for 4h at 37 ℃. DNA was recovered as described in 6 by electrophoresis on a 1% agarose gel at 120V and 300mA for 30min.

(3) Ligation of the cleaved fragment to the vector

In a microcentrifuge tube, 0.1pmol of pSERPINB3-Basic or pCEACAM6-Basic double cleavage fragment, 0.3pmol of PE38KDEL double cleavage fragment, 1. mu.L of T4 DNA Ligase, 1. mu.L of 10 XT 4 DNA Ligase Buffer and the balance of water were prepared, and the total amount was 10. mu.L. Mix well and ligate overnight at 16 ℃.

10. Preparation of E.coli DH5 alpha competence

(1) Using LB plate culture medium, picking out Escherichia coli DH5 alpha (glycerol at minus 80 ℃) by using an inoculating needle, and carrying out graded streaking on the plate culture medium, wherein the streaking is suitable for single colony to appear;

(2) inverting the streaked plate culture medium in a constant temperature incubator at 37 deg.c for overnight culture;

(3) selecting a newly activated E.coli DH5 alpha single colony from an LB plate, inoculating the single colony in 5mL of liquid LB culture medium, carrying out shake culture at 37 ℃ for about 12h until the late logarithmic growth phase, inoculating the bacterial liquid in a 100mLLB culture medium in a ratio of 1:100, and carrying out shake culture at 37 ℃ for 2-3 h until OD600 is about 0.5;

(4) transferring the culture solution into a 50mL centrifuge tube, standing on ice for 10min, and centrifuging at 3000g and 4 ℃ for 10 min;

(5) discard the supernatant and use pre-cooled 0.1M CaCl₂Gently suspending cells with 10mL of the solution, standing on ice for 30min, 3000g, 4 ℃, and centrifuging for 10 min;

(6) discarding the supernatant, adding 4mL of precooled 0.1M CaCl2 solution containing 15% glycerol, gently suspending the cells, and standing on ice for 10min to obtain competent cell suspension;

(7) the competent cell suspension was divided into 100. mu.L aliquots and stored at-80 ℃.

11. Transformation of E.coli DH5 alpha

(1) Taking 100 mu L of escherichia coli DH5 alpha competent cells, placing the cells on ice to be completely dissolved, and then gently and uniformly suspending the cells;

(2) adding 10 μ L of ligation reaction solution, mixing, and standing on ice for 30 min;

(3) heat-shocking at 42 deg.C for 90s, and standing on ice for 2 min;

(4) adding 400 μ LLB culture medium, and culturing at 37 deg.C under shaking at 120rpm for 1 h;

(5) during this period, LB plates containing 100. mu.g/mL ampicillin were prepared;

(6) the shake culture (100. mu.L of competent, 10. mu.L of ligation medium, 400. mu.L of LB medium) was centrifuged at 4000g for 5min at room temperature, 400. mu.L of supernatant was aspirated off, and the cells were suspended with the remaining liquid;

(7) plates were plated and plates were grown overnight (16-18h) in 37 ℃ incubator with inversion.

12. Plasmid extraction

Plasmids were extracted using a BioTeke high purity plasmid miniprep kit (spin column type) with the following specific steps:

(1) taking 3mL of overnight cultured bacterial liquid, centrifuging at 9,000rpm for 30s, pouring out supernatant as much as possible, and collecting thalli;

(2) resuspending the thallus precipitate with 250 μ L of solution P1, and vortex oscillating until the thallus is thoroughly mixed;

(3) adding 250 mu L of solution P2, and gently turning over the mixture up and down for 6-10 times to fully crack the thalli until the solution becomes clear;

(4) adding 400 mu L of solution P3, immediately and gently turning up and down for 6-10 times, standing at room temperature for 5min, centrifuging at room temperature at 13,000rpm for 10min, carefully sucking supernatant without sucking floccules;

(5) placing the adsorption column on a collecting tube, adding the supernatant obtained in the previous step into an adsorption column AC (the adsorption column is placed in the collecting tube, and the solution can be added twice), centrifuging at 13,000rpm for 1min, and discarding the filtrate;

(6) adding 500 mu L of deproteinized liquid PE, centrifuging at 13,000rpm for 30-60 s, and discarding the filtrate;

(7) adding 500 mu L of rinsing liquid WB added with absolute ethyl alcohol, centrifuging at 13,000rpm for 30-60 s, and discarding the filtrate;

(8) repeating the step (7) once; the empty column was centrifuged at 13,000rpm for 2 min. Standing at room temperature for 3-5 min, and removing residual ethanol;

(9) taking out the adsorption column AC, placing into a clean centrifuge tube, adding elution buffer EB heated in 70 deg.C water bath in advance into the middle part of the adsorption membrane, standing at room temperature for 1min, centrifuging at 13,000rpm for 1min to elute DNA, and storing at-20 deg.C.

13. Double enzyme digestion identification and sequencing identification of plasmid

And carrying out double enzyme digestion on the plasmids according to the double enzyme digestion method, carrying out electrophoresis by using 1% agarose gel, selecting the plasmids with correct double enzyme digestion results, and sending the plasmids to Shanghai Meiji biological medicine science and technology limited company for gene sequencing. Through double enzyme digestion and sequencing identification, the invention successfully constructs pSERPINB3-Basic, pSERPINB3-PE38KDEL, pCEACAM6-Basic and pCEACAM6-PE38KDEL plasmids. The sequence of the SERPINB3 promoter is shown as the nucleotide sequence shown in SEQ ID No.4, the sequence of the CEACAM6 promoter is shown as the nucleotide sequence shown in SEQ ID No.5, and the DNA sequence of the PE38KDEL toxin is shown as the nucleotide sequence shown in SEQ ID No. 1. The sequencing results were aligned with the predicted SERPINB3 promoter sequence using NCBI Blast, and the alignment results are shown in fig. 3. The sequence alignment result shows that the sequence obtained by PCR amplification is consistent with the sequence of the SERPINB3 promoter predicted according to USCC and NCBI, namely the SERPINB3 promoter sequence is successfully obtained.

Example 2: SERPINB3 promoter and CEACAM6 promoter activity assay, comprising the following steps:

(1) detection of luciferase

a. Cell transfection:

one day before transfection, 1.5X 10 per well of six-well plate was added⁵Individual cells and 2mL of antibiotic-free DMEM medium (10% FBS) to achieve 60% -80% cell confluence as possible at transfection;

② adding 2 mug DNA into 250 mug serum-free culture medium, mixing lightly;

③ adding 2.7 microliter of 1 microgram/microliter PEI solution into 250 microliter serum-free culture medium, and gently mixing;

standing at room temperature for 5min, slowly adding the diluted PEI into the plasmid solution, uniformly mixing while adding the diluted PEI into the plasmid solution, and then incubating at 37 ℃ for 30 min;

fifthly, replacing the cell culture solution in the six-hole plate with 1.5mL of DMEM medium without FBS;

sixthly, forming a complex of the PEI obtained in the fourth step and the plasmidSlowly and uniformly adding the mixture into a six-hole plate, and performing reaction at 37 ℃ and 5% CO₂Incubating for 4 hr at a concentration, replacing with 10% FBS-containing complete medium, continuing to incubate at 37 deg.C and 5% CO₂And (4) incubating under concentration conditions.

b. Cell lysis: after 48h of cell transfection, the cell culture was aspirated, and 500. mu.L of reporter cell lysate (six well plate) was added to lyse the cells sufficiently.

c. After the cells are fully lysed, 10,000-150,000 g of the cells are centrifuged for 3-5 minutes, and the supernatant is taken for detection.

d. The luciferase assay reagent was thawed and brought to room temperature.

e. The fluorescence measuring instrument was started according to the instrument instructions, and the measurement interval was set to 2 seconds and the measurement time was set to 10 seconds.

f. When each sample is measured, 10 microliter of sample is taken, 100 microliter of luciferase detection reagent is added, a pipette is used for evenly stirring, and reporter gene lysate is used as a blank control.

(2) Protein content determination by BCA method

Divalent copper ions can be reduced by proteins to monovalent copper ions under alkaline conditions, and the monovalent copper ions interact with a unique BCA Solution A (containing BCA) to produce a sensitive color reaction. Two molecules of BCA chelate a copper ion to form a purple colored reaction complex. The water-soluble compound shows strong light absorption at 562nm, and the light absorption and the protein concentration have good linear relation in a wide range, so the protein concentration can be calculated according to the light absorption value, and the specific result is shown in figure 4. The specific formula is that y is 0.03478x +0.1398(y is expressed as absorbance value, x is expressed as protein concentration, unit is μ g/μ L), and the calculation formula of the protein concentration is obtained by deduction:

the method comprises the following specific steps:

a. when in use, the solution A is shaken and mixed evenly, and according to the number of samples, a proper amount of BCA working solution is prepared by adding 50 volumes of solution A and 1 volume of solution B (50:1), and the mixture is mixed evenly. The BCA working solution is stable at room temperature for 24 h.

b. The BSA standard was diluted to a gradient as shown in table 1. (7 well BCA working solution requires 1.4mL solution A + 28. mu.L solution B to mix to prepare BCA working solution)

TABLE 1 protein standards BSA (1mg/mL) with deionized Water dH₂Proportion of O

c. The diluted standards are added into a 96-well plate respectively, and the standard dilution is added to make up all the standards to 10 mu L.

d. The samples for the experiment were added at 10. mu.L per well.

e. Add 200. mu.L of LBCA media to each well, gently blow the wells with a sample gun (care should not be taken to disturb the air bubbles to affect the reading), and incubate at 37 ℃ for 30min.

f. And (4) measuring A562 by using a microplate reader, and drawing a standard curve by using the absorbance as a vertical coordinate and the corresponding protein content as a horizontal coordinate.

g. Diluting a sample to be detected to a proper concentration to enable the total volume of a sample diluent to be 10 mu L, adding 200 mu L of BCA working solution, fully and uniformly mixing, standing at 37 ℃ for 30min, measuring A562 by an enzyme-labeling instrument, taking the standard curve No. 0 as a reference, and recording a light absorption value. And substituting the standard curve according to the light absorption value of the measured sample to calculate the corresponding protein content (mu g), dividing by the total volume (10 mu L) of the sample diluent, and multiplying by the sample dilution factor to obtain the actual concentration (unit mu g/mu L) of the sample.

(3) SERPINB3 promoter Activity assay

LO2 (human liver cell), Hela (human cervical cancer cell), TCA8113 (human tongue squamous carcinoma cell), ECA-109 (human esophageal carcinoma cell), and MG-63 (human osteosarcoma cell) were mixed at 2X 10⁵The amount of each well was inoculated in 6-well plates at 37 ℃ with 5% CO₂Culturing for 16h under the condition to ensure that the confluence degree reaches 70-80%. According to the nitrogen-phosphorus ratio of the plasmid to the PEI, 2 mu g of plasmid is transfected in each hole, the luciferase expression level is determined after 48 hours, normalization treatment is carried out according to the determined protein content, and the calculation formula is as follows.

(4) Analysis of CEACAM6 promoter Activity

LO2 (human liver cell), Hela (human cervical cancer cell), TCA8113 (human tongue squamous carcinoma cell), SGC-7901 (human gastric adenocarcinoma cell), SW480 (human colorectal adenocarcinoma cell) were mixed at 2X 10⁵The amount of each well was inoculated in a 6-well plate, and transfection, measurement and data processing were performed according to the method in (1) above.

The above analysis proves that the SERPINB3 promoter and the CEACAM6 promoter are specifically highly expressed in human tongue squamous cell carcinoma cell TCA8113 cell and are lowly expressed in human normal liver cell LO 2. Specific results are shown in FIG. 5, wherein FIG. 5-a shows luciferase assay of SERPINB3 promoter expression activity in different cells; FIG. 5-b shows luciferase assay of CEACAM6 promoter expression activity in different cells.

Example 3: the research of the inhibition effect of pSERPINB3-PE38KDE and pCEACAM6-PE38KDEL on the protein synthesis of oral squamous cell carcinoma cells comprises the following steps:

pGL3-Control (containing SV40 promoter and luciferase cDNA) was co-transfected with pSERPINB3-PE38KDEL plasmid and pCEACAM6-PE38KDEL plasmid of different qualities, respectively, according to the transfection method in example 2, and the expression level of luciferase in the cells was measured after 48h and normalized according to the measured protein content, thereby measuring the inhibition effect of pSERPINB3-PE38KDEL plasmid and pCEACAM6-PE38KDEL plasmid on cell protein synthesis. The results show that pSERPINB3-PE38KDEL and pCEACAM6-PE38KDEL can specifically inhibit the protein synthesis of human tongue squamous cell carcinoma cell TCA 8113. Specific results are shown in FIG. 6, in which FIG. 6-a shows luciferase assays for inhibition of protein synthesis in different cells using different doses of pSERPINB3-PE38 KDEL; FIG. 6-b shows the luciferase assay for inhibition of protein synthesis in different cells using different doses of pCEACAM6-PE38 KDEL.

Example 4: flow cytometry is used for detecting the influence of pSERPINB3-PE38KDEL plasmid and pCEACAM6-PE38KDEL plasmid on cell cycle distribution of oral squamous cell carcinoma

The method comprises the following specific steps:

1. grouping experiments: experimental group pSERPINB3-PE38KDEL/pCEACAM6-PE38 KDEL; negative group pGL 3-Basic; PEI alone was added to the transfection reagent group; blank group Control.

2. Cells were plated for 12h, serum-free starvation was performed for 48h after complete adherence, then plasmid transfection was performed as in example 2, and cell division cycle was determined 48h later:

a, precooling by PBS, preparing 2 groups of 1.5mLEP tubes, marking, adding 300 mu L of absolute ethyl alcohol into each tube B, and precooling at-20 ℃.

b. The medium was discarded, 500. mu.L of pancreatin was added to each well, and digested at 37 ℃ for 2 min.

c. The pancreatin was discarded, 1mL of precooled PBS was added to each well, the cells were washed off and transferred to 1.5mLEP tubes (group A)

d.2000r/min for 5min, discarding the supernatant, adding 500 μ L of precooled PBS, and centrifuging again.

e. The supernatant was discarded, and the mixture was aspirated by a gun, followed by addition of 100. mu.L of precooled PBS, addition of 20. mu.L of LRNaseA, and water bath at 37 ℃ for 30min.

f. Adding 300 mu L of PI, mixing the mixture evenly and gently, keeping out of the sun at 4 ℃, and incubating for 30-60 min.

The results show that pSERPINB3-PE38KDEL can specifically block the cycle of human tongue squamous cell carcinoma cell TCA8113 in S phase, and has little influence on the cell cycle distribution of normal cell LO 2. Specific results are shown in FIG. 7, and FIG. 7-a is a flow cytometry detection graph of the periodic distribution of human tongue squamous cell carcinoma cells TCA8113 by pSERPINB3-PE38KDEL, wherein Control is a Control group, PEI is a PEI group only added with a transfection reagent, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group; FIG. 7-b is a histogram of the periodic distribution of pSERPINB3-PE38KDEL to human tongue squamous cell carcinoma cells TCA8113, wherein Control is a Control group, PEI is a PEI group added with a transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group; FIG. 7-c is a flow cytometry examination of pSERPINB3-PE38KDEL on human hepatocyte LO2 cycle distribution, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group; FIG. 7-d is a histogram of the periodic distribution of pSERPINB3-PE38KDEL on human hepatocytes LO2, in which Control is a Control group, PEI is a PEI group supplemented with only a transfection reagent, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group. Wherein FIG. 7-a and FIG. 7-b show the effect of pSERPINB3-PE38KDEL on TCA8113 cycle distribution of human tongue squamous cell carcinoma cells; FIGS. 7-c and 7-d show the effect of pSERPINB3-PE38KDEL on the LO2 cycle distribution in human normal hepatocytes.

Example 5: flow cytometry is used for detecting the influence of pSERPINB3-PE38KDEL plasmid and pCEACAM6-PE38KDEL plasmid on oral squamous cell carcinoma cell apoptosis

The method comprises the following specific steps:

1. grouping experiments: experimental group pSERPINB3-PE38 KDEL; negative group pGL 3-Basic; positive group pcDNA3.0-PE38 KDEL; PEI alone was added to the transfection reagent group; BLANK group BLANK.

2. Cells were plated and transfected as in example 2 and apoptosis was determined after 48 h:

a. the DMEM suspension in the six-well plate was transferred to 4mL EP tubes.

b. 1mL PBS per well was added to the six well plate, and after washing, PBS was added to 4mL EP tubes (one-to-one, labeled), followed by 1mL pancreatin in the six well plate. Then 1mL PBS was added to each well, cells were blown (note: gentle motion to avoid cell disruption), the cell suspension was transferred to 4mL EP tube at 2000r/min and centrifuged for 5 min.

c. Discard the supernatant, add 1mL PBS in 4mL EP tube, transfer the cell suspension to 1.5mL EP tube, 2000r/min, centrifuge for 5min, discard the supernatant.

d. Adding the binding solution into a 1.5mL EP tube, wherein the total volume of the binding solution and the dye is 50 mu L, and specifically adding 50 mu L of the binding solution into a non-dyed control group; FITC singly-dyed control group, 2 μ L FITC and 48 μ L binding solution are added; adding 2 mu L of PI and 48 mu L of binding solution into a PI singly-dyed control group; FITC and PI double staining control group, adding 2 μ L FITC, 2 μ L PI and 46 μ L binding solution; for the double-stained experimental group, 2. mu.L of FITC, 2. mu.L of PI and 46. mu.L of the binding solution were added.

e. The cells were transferred to the dark or in a refrigerator at 4 ℃ and left to stand for 30min.

f. The cells were removed and 450. mu.L of binding solution was added.

g. Filtering with double-layer flow filter cloth, adding into flow tube, and performing on-machine detection.

The results show that: pSERPINB3-PE38KDEL and pCEACAM6-PE38KDEL can specifically induce the apoptosis of human tongue squamous cell carcinoma cells TCA8113, but have almost no influence on human normal liver cells LO2, and specific results are shown in FIG. 8, wherein FIG. 8-a is the influence of pSERPINB3-PE38KDEL on the apoptosis of human tongue squamous cell carcinoma cells TCA8113, wherein Control is a Control group, PEI is a PEI group only added with a transfection reagent, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group; FIG. 8-b shows the effect of pSERPINB3-PE38KDEL on apoptosis of human hepatocytes LO2, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group; FIG. 8-c shows the effect of pCEACAM6-PE38KDEL on human tongue squamous cell carcinoma cell TCA8113 apoptosis, wherein Control is a Control group, PEI is a PEI group added with transfection reagent only, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group; FIG. 8-d shows the effect of pCEACAM6-PE38KDEL on apoptosis of human normal cells LO2, in which Control is a Control group, PEI is a group to which only a transfection reagent PEI was added, pGL3-Basic is an empty plasmid vector transfection group, and pSERPINB3-PE38KDEL is a target plasmid transfection group. Wherein FIGS. 8-a and 8-b show the effect of pSERPINB3-PE38KDEL on apoptosis in human tongue squamous cell carcinoma cells TCA8113 and human normal cells LO 2; FIGS. 8-c and 8-d show the effect of pCEACAM6-PE38KDEL on apoptosis in human squamous cell carcinoma of the tongue TCA8113 and in human normal cell LO 2.

The embodiments show that the recombinant plasmid for regulating and controlling the PE38KDEL gene by using the tumor cell specific expression gene promoter provided by the invention can inhibit and kill tumor cells in a targeted manner, so that the recombinant plasmid can be applied to preparation of tumor targeted gene therapy medicines, and a new means is provided for tumor targeted gene therapy.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Jilin university

<120> construction method and application of PE38KDEL gene expression plasmid mediated by tumor specific promoter

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1041

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccggaaggtg gctccctggc agctctgacc gcacatcagg catgccacct gccgctggaa 60

accttcacgc gtcatcgtca gccgcgcggc tgggaacagc tggaacaatg tggttatccg 120

gttcagcgcc tggtcgccct gtacctggca gcacgtctga gctggaacca ggtcgatcaa 180

gtgattcgta atgcactggc atcaccgggt tcgggtggcg acctgggtga agcaatccgc 240

gaacagccgg aacaagctcg tctggcactg accctggcag ctgcagaaag tgaacgcttt 300

gtgcgtcagg gtacgggtaa cgatgaagca ggtgcagcaa atggtccggc agattccggt 360

gacgcactgc tggaacgcaa ctatccgacc ggcgccgaat ttctgggtga tggtggcgac 420

gtgtcgttca gcacccgcgg cacgcagaat tggaccgttg aacgtctgct gcaggcgcat 480

cgccaactgg aagaacgtgg ttatgttttt gtcggctacc acggtacctt cctggaagct 540

gcgcagagca ttgtgtttgg tggcgttcgt gcccgctctc aagatctgga cgcaatttgg 600

cgcggcttct atatcgcagg tgatccggct ctggcgtatg gctacgctca ggatcaagaa 660

ccggacgcgc gtggccgtat ccgtaacggt gcactgctgc gtgtgtatgt tccgcgttcc 720

tcactgccgg gtttttaccg tacctctctg acgctggcag caccggaagc tgcaggcgaa 780

gtggaacgcc tgattggtca cccgctgccg ctgcgtctgg atgcaatcac cggtccggaa 840

gaagaaggcg gccgtctgga aacgattctg ggttggccgc tggctgaacg taccgtggtt 900

attccgagcg cgatcccgac ggatccgcgc aatgttggtg gcgatctgga cccgtcgagc 960

attccggata aagaacaggc catctcagca ctgccggact atgcgtcgca accgggtaaa 1020

ccgccgaagg acgagctgta a 1041

<210> 2

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

catgccatgg caccggaagg tggctccc 28

<210> 3

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ctagtctaga ttacagctcg tccttcggcg gtttacccgg ttg 43

<210> 4

<211> 1994

<212> DNA

<213> human

<400> 4

tggccttgga caacaaccct ctccctggcc acagacattc ttcagattac aagatattcc 60

agaggaaaca ctggaatgag tctgaagcca ggtgctaaat ggaaggacca ccaagaaacg 120

ttgtgatcct gacaggtcaa gcaacttctt tttctgctta atttttaaat gaaaaattag 180

aaagctgaca ttcaaaatgg cccgtctgtt tcaattgctc ttctcagtgt cagcctgtta 240

actcaatgtg ttagtctgtt ttcatgctgc tgataaagac atacctgaga ttaggaagta 300

aaagaggttt aattggactt agagttccac gtgattgggg aggcctcaga atcacggtag 360

gaggcaaaag ttattcttac atggtggctg caagagaaaa tgaggaagaa gcaaaagaag 420

aaacccctaa taaacccatt ggatctcctg agacttatta actatcatga gaatagcaca 480

agaaagaccg gcccccatga ttcaattacc tctacctggg tccctccaac aacatgtgga 540

aattctggta gatacaattc aagttgagat ttgggtggga acacagccaa accatatcac 600

tcagcaaggc agataacttt ctcactgagc ctatgcaaca gaaaaccatc tgggatggtt 660

gtaaggggca caggaagtga ctggtaggat cactgccaaa gctgagcatt caggagaagg 720

caatagaatc ctattctcca tagtatgcta taagatactg aagtacactt cttcactatc 780

tctttggact tagaattagc actatattcc ttgttataca gaaaaattac taaggaaatt 840

cataggatga caaaaacttt cagaactgaa aaacaggaaa tgtaagcttt ttagttcttt 900

ggtattcgaa gtatgcctaa aagacaatgc aaaatccaag aaaagaatgg tggggttttt 960

gtttgtttgt ttttgttttt gttttacagc tggagtagaa tacaaaggga tggagttgaa 1020

acaaatgaga ggaaattgga attctaaact tattctcatt ggcattagaa aggcacctac 1080

atgtatttca catgagccgg tgactgctga cttgcattct tattttttcc ctatagatta 1140

aaaaggaggt acaatggtag aactgtaatc ctgtcctttg tcataaattt tcgtattcat 1200

aaaggtgagt gttagcccgc ttgtgaaatc tgaagttgag taacttcaaa tactaaccac 1260

agagggagag gcagcaagag gagaggcata aattcaggat ctcacccttc attccacaga 1320

cacacatagc ctctctgccc acctctgctt cctctaggaa cacaggtaag agcttcaagc 1380

ctctccagct taataacatg aattattttt gagaataata atgatactgt gttctatatc 1440

atgcatctcc tgcattctgt ctgattatat tttacttatt ctgccagagc aaaattaaaa 1500

tacctatttc atctgatttg tcctttatct aaattgctta gttccaagta aaccaaggca 1560

cttttaggaa cacagaggga gagtgccttg cagccagaga gtcttgaagg agatgtcagg 1620

gacgcatctt aacagctggt tggatgtgat ccacagaggt ctcctgttag cattcattgt 1680

aaagccttcc tacctagccc tagtgtagcc agcaatgaag gaaagagggt ctattactta 1740

tttacagtag tctttaaaaa cactaatttt gtgaggcttc taattaagac attaatatat 1800

ttaatatatg cacattgtag aaagattgaa acgttaaaaa taagatgagg aaaactttaa 1860

atgtcaaaat ctcacaacac agatatataa tttctttaag aaaattgtac tacaaaatac 1920

cattccattt attaaagtca ttctgacagg aatctgatgc ttttccagga gttccagatc 1980

acatcgagtt cacc 1994

<210> 5

<211> 413

<212> DNA

<213> human

<400> 5

agagaaaata acaccaggtt tgaggacccc agggactctc tgtgtggtgc tgacagaccc 60

aaggcccaga cacagcagag gtccgtgctg gggagggcgg gtcgtcctgt tatggaacag 120

gggtccaaac aagcttgctt ctcagagcat cttctgggga actgaatata aacagaaagg 180

gaagaggagg agggacaaaa gagacagaaa tgagagggga ggggatagag gattcctgaa 240

cagagaccgc acccatgacc cacgtgaccc tgggaaatgc ttctatccct gagaggaggc 300

tcagcacaga aggaggaagg acagcagggc caacagtcac agcagccctg accagagcat 360

tcctggagct caagctcctc tacaaagagg tggacagaga agacagcaga gac 413

<210> 6

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cctagctagc gattaaatgg ccttggacaa caacc 35

<210> 7

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

catgccatgg tggcggtgaa ctcgatgtga tctggaactc c 41

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cctagctagc agagaaaata acaccaggtt tgaggac 37

<210> 9

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

catgccatgg tctctgctgt cttctctgtc cac 33

Claims

1. A recombinant plasmid containing a PE38KDEL gene, wherein the recombinant plasmid takes pGL3-Basic as an original plasmid, a tumor-specific promoter is inserted into a multiple cloning site of pGL3-Basic, and the PE38KDEL gene is used for replacing the original luciferase gene; the tumor specific promoter is an SERPINB3 promoter or a CEACAM6 promoter; the nucleotide sequence of the PE38KDEL gene is shown as SEQ ID No. 1.

2. The recombinant plasmid according to claim 1, wherein the SERPINB3 promoter has a nucleotide sequence shown in SEQ ID No. 4.

3. The recombinant plasmid of claim 1, wherein the CEACAM6 promoter has the nucleotide sequence shown in SEQ ID No. 5.

4. The recombinant plasmid according to claim 1, wherein the tumor-specific promoter is inserted between the NheI and NcoI cleavage sites on the original plasmid pGL 3-Basic.

5. The recombinant plasmid according to claim 1, characterized in that the PE38KDEL gene is inserted between the NcoI and XbaI cleavage sites on the original plasmid pGL 3-Basic.

6. The method for constructing a recombinant plasmid according to any one of claims 1 to 5, comprising the steps of:

after being respectively subjected to double enzyme digestion and recovery, pGL3-Basic and a SERPINB3 promoter or a CEACAM6 promoter are connected to obtain a pSERPINB3-Basic plasmid or a pCEACAM6-Basic plasmid;

carrying out double digestion recovery on the pSERPINB3-Basic plasmid or the pCEACAM6-Basic plasmid obtained in the step 1) and the PE38KDEL gene respectively, and then connecting to obtain a recombinant plasmid pSERPINB3-PE38KDEL plasmid or pCEACAM6-PE38KDEL plasmid.

7. Use of the recombinant plasmid according to any one of claims 1 to 5 or the recombinant plasmid obtained by the construction method according to claim 6 in the preparation of a medicament for targeted inhibition and killing of oral squamous cell carcinoma cells.

8. A recombinant strain comprising the recombinant plasmid of any one of claims 1 to 5.