CN114958914A - Efficient human and mammal cell attachment expression vector, construction method and application - Google Patents

Abstract

The invention belongs to the technical field of gene engineering and gene therapy, and particularly relates to a high-efficiency human and mammal cell attachment expression vector, and a construction method and application of the attachment expression vector. The invention inserts a first nuclear matrix binding region sequence in the upstream reverse direction of a promoter, inserts an intron sequence in the downstream forward direction of the promoter, inserts a second nuclear matrix binding sequence in the downstream forward direction of the intron sequence, and further preferably constructs an adhesion body expression vector by using the promoter sequence as an EF-1 alpha sequence, the first nuclear matrix binding region sequence as MAR X-29, the intron sequence as an hCMV intron sequence and the second nuclear matrix binding region sequence as a MAR characteristic sequence. Experiments prove that by using the expression vector constructed by the invention, the CHO cell or the human colon cancer cell HCT116 as the host cell can obviously improve the transgene expression level and the expression stability, and the expressed target protein has biological functions.

Description

Efficient human and mammal cell attachment expression vector, construction method and application

Technical Field

The invention belongs to the technical field of gene engineering and gene therapy, and particularly relates to a high-efficiency human and mammal cell attachment expression vector, and a construction method and application of the attachment expression vector.

Background

Gene therapy brings new therapeutic possibilities for many areas of modern medicine. Gene therapy refers to the introduction of a normal gene or a therapeutic DNA sequence into a target cell in a specific manner to correct a defective gene, thereby curing a disease. Since the first successful gene therapy clinical trials in the early 90 s of the 20 th century, thousands of gene therapy clinical trials have been completed. The success of gene therapy depends to a large extent on the selection of suitable gene delivery vectors, including mainly viral vectors and plasmid vectors. Although viral vectors have the advantage of high gene transfer efficiency, the production of viral proteins may cause pathological damage to host cells. Plasmid vectors do not produce viral proteins, but their transfection efficiency is low. Vectors can be classified into integration vectors and attachment vectors according to their existence state after entering host cells. The exogenous gene is integrated on the chromosome of the host cell, so that the insertion mutation is easily induced, and the safety is low. Unlike integration vectors, the episomal vector does not integrate into the genome but attaches to the chromosome, which reduces the risk of insertional mutagenesis and is safer.

The application publication number CN102703503A of Chinese invention discloses a human and mammal cell attachment expression vector and application thereof, which cuts and splices sequences of 2200 bp human interferon-beta nuclear matrix binding region (MAR) into MAR characteristic sequences with the size of 367 bp to construct a non-viral attachment vector pEGFP-C1-M, wherein pEGFP-C1-MAR is a novel mammal cell expression vector, can be attached to but not integrated into chromosomes of Chinese Hamster Ovary (CHO) cells, overcomes side effects caused by the integration effect of the traditional vector, and can drive transgene expression. However, in the research, the CMV promoter of the vector drives the expression copy number of the foreign gene, and the expression level of the gene is low and unstable. In order to solve the technical problem, the invention patent of China, with the application publication number of CN105802997A, replaces the CMV promoter of pEGFP-C1-MAR with a human Elongation factor 1 alpha (EF-1 alpha) promoter to construct a pEME attachment vector, and finds that the EF-1 alpha promoter can drive the efficient and stable expression of a non-viral attachment vector, and although a better promoter is screened, the transgenic expression of the vector still has the problems of instability and low copy number (6-12 copies/cell).

The regulatory elements are DNA sequences which regulate the expression of transgenes and do not encode any protein, and researches show that the regulatory elements such as introns, UCOE, STAR 40, MAR and the like have the function of improving the expression level of the transgenes of the integration vector, but the function is related to the type, direction and position of the regulatory elements. How to combine and use these regulatory elements, select the appropriate direction and position, further improve the stability and copy number of the transgene expression of the attached expression vector, have become the technical problem that needs to be solved at present.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a high-efficiency expression vector for the cell attachment surface of human and mammals, which has higher stability and further improves the expression quantity of target genes compared with the prior vectors of the same type.

The second purpose of the present invention is to provide a method for constructing a high-efficiency expression vector for human and mammalian cell attachments.

The invention also aims to provide the application of the efficient expression vector for the cell attachment surface of human and mammals.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a high-efficiency expression vector for human and mammal cell attachment body is composed of promoter sequence, the first nuclear matrix binding region sequence inserted in upstream and reverse direction of promoter sequence, the intron sequence inserted in downstream and forward direction of intron sequence, and the second nuclear matrix binding region sequence inserted in downstream and forward direction of intron sequence.

Preferably, the promoter sequence is an EF-1 alpha sequence shown in SEQ ID NO 1.

Further, the first nuclear matrix binding domain sequence is a MAR X-29 sequence as set forth in SEQ ID NO:2, respectively.

Further, the intron sequence is an hCMV intron sequence, as shown in SEQ ID NO:3, respectively.

Optionally, a reporter gene sequence is further included between the intron sequence and the second nuclear matrix binding region sequence; the second nuclear matrix binding domain sequence is a MAR characteristic sequence as shown in SEQ ID NO: 4, respectively. In some embodiments of the invention, the reporter gene sequence is EGFP.

Alternatively, any human or mammalian cell expression vector may be used as the starting vector, and in some embodiments of the invention, the starting vector is pEGFP-C1, or pEGFP-C1-MAR constructed according to the method of patent CN102703503A, or pEME constructed according to patent CN105802997A, for example.

The expression vector for the above-mentioned high-efficiency human and mammalian cell adhesion body can be constructed according to a conventional method in the field of genetic engineering, and as an illustration, in some embodiments of the present invention, the construction method comprises the following steps:

1) artificially synthesizing a promoter sequence, performing double enzyme digestion on the promoter sequence and a starting vector, connecting, transforming and identifying to construct an intermediate vector 1;

2) artificially synthesizing a nuclear matrix binding region sequence, performing double enzyme digestion on the nuclear matrix binding region sequence and the intermediate vector 1, connecting, transforming and identifying to construct an intermediate vector 2;

3) artificially synthesizing an intron sequence, performing double enzyme digestion on the intron sequence and the intermediate vector 2, connecting, transforming and identifying, and constructing to form the vector.

The high-efficiency human and mammal cell attachment expression vector is applied to the aspect of serving as a foreign gene transfer vector, a foreign gene is cloned to the attachment expression vector to form a foreign gene expression vector, and the foreign gene expression vector transfects host cells to express target proteins.

By way of illustration, the foreign gene is the HSV-TK gene (GenBank accession number: JX 392980.1).

By way of illustration, the host cell is a human colon cancer cell HCT 116.

The invention has the beneficial effects that:

according to the invention, a nuclear matrix binding region sequence, an intron sequence and a promoter sequence are combined to form an element of an expression vector of a human or mammal attachment, a first nuclear matrix binding region sequence is reversely inserted into the upstream of a promoter, an intron sequence is forwardly inserted into the downstream of the promoter, a second nuclear matrix binding sequence is forwardly inserted into the downstream of the intron sequence, and further, the promoter sequence is preferably an EF-1 alpha sequence, the first nuclear matrix binding region sequence is MAR X-29, the intron sequence is an hCMV intron sequence, and the second nuclear matrix binding region sequence is a MAR characteristic sequence, so that the continuous, efficient and stable expression of an exogenous target gene by the attachment expression vector is improved. Experiments prove that by using the expression vector constructed by the invention, the CHO cell or the human colon cancer cell HCT116 as the host cell can obviously improve the transgene expression level and the expression stability, and the expressed target protein has biological functions.

Drawings

FIG. 1 is a schematic structural diagram of recombinant plasmid pEME;

FIG. 2 is a schematic structural diagram of a recombinant plasmid pEME-hCMVI (F);

FIG. 3 is a schematic structural diagram of recombinant plasmid pMAR (R) -hCMVI (F);

FIG. 4 is a schematic structural diagram of the recombinant plasmid pEME-hCMVI (R);

FIG. 5 is a schematic structural diagram of the recombinant plasmid pEME-MAR (F);

FIG. 6 is a schematic structural diagram of the recombinant plasmid pEME-MAR (R);

FIG. 7 is a graph showing the comparison of EGFP transient expression levels in pEME, pEME-MAR (F), pEME-MAR (R), pEME-hCMVI (F), pEME-hCMVI (R) transfected CHO cells;

FIG. 8 is a graph showing comparison results of EGFP stable expression levels in pEME, pEME-MAR (F), pEME-MAR (R), pEME-hCMVI (F), pEME-hCMVI (R) transfected CHO cells;

FIG. 9 is a diagram showing the comparison of EGFP stable expression levels of vector-transfected CHO cells pEME, pEME-MAR (R), pEME-hCMVI (F), pMAR (R) -hCMV (F);

FIG. 10 shows Western Blot to detect the expression level of HSV-TK gene in transfected HCT116 cells;

FIG. 11 shows the functional experiment of HSV-TK, and the detection result of bystander effect.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; the culture medium, reagent, cell line reagent and the like used in the examples and test examples are commercially available.

Escherichia coli (Escherichia coli) JM109 used in the examples of the present invention, and the cell line, reagents and instrument enzymes used therein were commercially available. The following examples all use pEME as starting vector, which is constructed according to the construction method disclosed in the patent CN105802997A, and specifically, the eukaryotic expression vector containing EF-1 alpha promoter, EGFP reporter gene and MAR characteristic sequence, which is constructed on the basis of pEGFP-C1 vector, is kanamycin-resistant (as shown in figure 1).

Example 1

This example provides a high efficiency expression vector for human and mammalian cell adherends comprising a nuclear matrix binding domain sequence (3387 bp MAR X-29 sequence shown in SEQ ID NO: 2) located upstream of a promoter in the reverse direction and an hCMV intron sequence (821 bp hCMV intron shown in SEQ ID NO: 3) located downstream of the promoter in the forward direction; the construction method comprises the following specific operation steps:

1. construction of pEME-hCMVI (F)

S1: synthesis of the forward sequence of the hCMV intron (hCMV intron, hCMV I), the two ends of the sequence being introduced separatelyNhe Ⅰ、Eco47 III enzyme cutting site; (synthesized by general biosystems (Anhui) Inc.);

s2: enzyme digestion reaction

Utilizing a forward hCMV intron sequence and a pEME plasmid synthesized by double enzyme digestion of Nhe I/Eco 47 III, after the enzyme digestion is finished, identifying the enzyme digestion condition by 1.5% agarose gel electrophoresis, and recovering a forward hCMV intron fragment and a linear pEME plasmid after the enzyme digestion by gel;

the cleavage system of the hCMV intron sequence is as follows: hCMV intron (1. mu.g/. mu.L) 10. mu.L, 20 XNEB buffer 1. mu.L,Nhe0.5 mu L of I enzyme (10U/. mu.L), adding three distilled water to 20 mu L, mixing well, performing enzyme digestion in water bath at 37 ℃ for 6h, performing electrophoresis on 1.5% agarose gel after enzyme digestion, recovering the intron fragment after enzyme digestion by gel, and performingEco47 III enzyme digestion; the recovered hCMV intron (1. mu.g/. mu.L) was contained in 10. mu.L, 10 XO buffer 2. mu.L,Eco adding 0.5 mu L of 47 III enzyme (10U/mu L), adding three distilled water to 20 mu L, fully mixing, and performing enzyme digestion in water bath at 37 ℃ for 6 h;

the restriction system of pEME plasmid is as follows: firstly, the methodNhe I Single restriction, 10. mu.L of pEME plasmid (1. mu.g/. mu.L), 1. mu.L of 20 XNEB buffer,Nhe0.5 mu L of I enzyme (10U/. mu.L), adding three distilled water to 20 mu L, mixing well, performing enzyme digestion in water bath at 37 ℃ for 6h, performing electrophoresis on 1.5% agarose gel after enzyme digestion, recovering the fragment after enzyme digestion by gel, and performingEco47 III enzyme digestion; recovered pEME (1. mu.g/. mu.L) was 10. mu.L, 10 XO buffer 2. mu.L,Ecoadding 0.5 mu L of 47 III enzyme (10U/mu L), adding three distilled water to 20 mu L, fully mixing, and performing enzyme digestion in water bath at 37 ℃ for 6 h;

s3: connection of

5 mu L of linear pEME plasmid (1 mu g/. mu.L), 1 mu L of positive hCMV intron fragment (1 mu g/. mu.L) after enzyme digestion, 1 mu L of T4 ligase buffer, 0.5 mu L of T4 ligase (350U/. mu.L), and adding triple distilled water to 20 mu L, fully mixing uniformly, and connecting overnight at 16 ℃;

s4: transformation and characterization

Taking 200 μ L of freshly prepared E.coli JM109 competent bacteria under sterile conditions into a sterilized 1.5mL EP tube, adding 10 μ L of the above ligated reaction solution for transformation, then inoculating on an agar plate containing kanamycin, culturing overnight at 37 ℃, picking positive transformants from the cultured plate, placing in 3mL tubes of LK liquid medium, and shake culturing overnight at 37 ℃; extracting recombinant plasmid, performing double enzyme digestion verification of the recombinant plasmid, selecting the plasmid with correct enzyme digestion verification, performing sequencing verification, and naming the carrier with completely correct target gene sequence as pEME-hCMVI (F) (as shown in figure 2);

2. construction of pMAR (R) -hCMV (F)

S1: the reverse (R, Revers) sequence was synthesized with reference to the MAR X-29 DNA fragment sequence (GenBank: EF694970.1), and gene synthesis was performed by general biosystems (Anhui) Inc.;

s2: inserting MAR X-29 reverse fragment into the upstream of EF-1 alpha promoter expression cassette of pEME-hCMVI (F) vector (figure 2) by adopting a seamless cloning method, transforming competent cell E.coli JM109 by the constructed recombinant vector, then inoculating the competent cell E.coli JM109 into a culture medium containing kanamycin for overnight at 37 ℃, selecting a single colony, continuously culturing the single colony in the culture medium containing kanamycin, and shaking the single colony at 37 ℃ and 300r/min for overnight; extracting recombinant plasmids; 10 μ L of the recombinant DNA plasmid was taken forKpn I/ApaI, double enzyme digestion identification, namely selecting a plasmid with correct enzyme digestion verification, carrying out sequencing verification, and naming the vector with correct sequencing as pMAR (R) -hCMV (F) (shown in figure 3).

Comparative example 1

This comparative example A hCMVI reverse sequence was synthesized artificially in the same manner as described above for pEME-hCMVI (F) in example 1 to construct pEME-hCMVI (R), as shown in FIG. 4.

Comparative example 2

This comparative example constructs pEME-MAR (F):

by adopting a seamless cloning method, the MAR X-29 forward fragment is inserted into the pEME vector (figure 1), and the attachment body surface of the MAR X-29 is inserted upstream of the EF-1 alpha promoter expression cassette of the MAR X-29To a carrier. Transforming competent cells E.coli JM109 with the constructed recombinant vector, inoculating the competent cells in a kanamycin-containing culture medium at 37 ℃ overnight, selecting a single colony, continuously culturing the single colony in the kanamycin-containing culture medium, and shaking the colony at 37 ℃ at 300r/min overnight; extracting recombinant plasmid by SDS alkaline lysis method; taking 10 mu L of recombinant DNA plasmid for enzyme digestion identificationNhe I/Kpn I, double enzyme digestion, selecting a plasmid with correct enzyme digestion, carrying out sequencing verification, and verifying that the correct vector is named as pEME-MAR (F), as shown in figure 5.

Comparative example 3

This comparative example constructs pEME-MAR (R):

the MAR X-29 reverse fragment was inserted upstream of the EF-1. alpha. promoter expression cassette of the pEME vector (FIG. 1) to construct an expression vector for the attachment containing MAR X-29 by the method of seamless cloning. Transforming competent cell E.coli JM109 with the constructed recombinant vector, inoculating in a kanamycin-containing culture medium at 37 ℃ overnight, selecting a single colony, continuously culturing in the kanamycin-containing culture medium, and shaking at 37 ℃ at 300r/min overnight; extracting recombinant plasmid by SDS alkaline lysis method; taking 10 mu L of recombinant DNA plasmid for enzyme digestion identificationSpe I/ApaI, double enzyme digestion, selecting a plasmid with correct enzyme digestion, carrying out sequencing verification, and verifying that the correct vector is named as pEME-MAR (R), as shown in figure 6.

Test example 1

The test method comprises the following steps: after the expression vectors constructed by the starting vector, the example 1 and the comparative examples 1-3 are respectively transfected into CHO cells, the expression condition of a reporter group EGFP is detected:

CHO-K1 cells with good growth status were selected and inoculated onto 6-well plates and transfected until the plating density reached about 80%. Transfection was divided into 6 groups in total according to experimental design: untransfected group, pEME-MAR (F), pEME-MAR (R), pEME-hCMVI (F), pEME-hCMVI (R), pMAR (R) -hCMV (F) vector group. Adding 4 μ g of the above carrier into 250 μ L of DMEM-F2 culture medium without serum and antibiotics, mixing gently, diluting 10 μ L of liposome 2000 reagent in 250 μ L of DMEM-F2 culture medium without serum and antibiotics, mixing gently, and standing at room temperature for 5 min; the liposome 2000 diluent is added into the plasmid DNA diluent dropwise and mixed evenly at the same time, incubated at room temperatureIncubating for 20min, adding liposome 2000/DNA complex to each air and mixing by gentle shaking, adding 5% CO ₂ The serum-free medium was changed to complete DMEM after 6h at 37 ℃ in the cell culture chamber. 48h after transfection, the Mean Fluorescence Intensity (MFI) of EGFP was measured by flow cytometry for each group of cells.

And (3) test results:

transient expression: the transient expression level of EGFP was increased by the remaining recombinant vectors except for pEME-hCMVI (R) compared to the control group, which set the MFI of EGFP at 1, pEME-MAR (F), pEME-MAR (R) and pEME-hCMVI (F) at 1.15, 1.50 and 1.34 times, respectively, as shown in FIG. 7;

screening of stable cell lines: and (3) pressurizing and screening the cells 48h after transfection, replacing the culture solution every 3d, changing the culture solution after 7-10 days when untransfected group cells die, changing the G418 culture solution to a maintenance concentration of 400 mug/ml, continuously screening for 2 weeks, continuously culturing after stably transformed cell colonies are formed, and collecting the cells and detecting the expression quantity of the stable EGFP by flow cytometry when the cell density reaches 80% -90%. EGFP expression level of control pEME was 3.05X 10 ⁵ The pEME MAR (R) group is 4.88X 10 ⁵ pEME-hCMV (F) group 4.27X 10 ⁵ Similar to transient expression, the stable expression levels of EGFP were increased by 1.29, 1.67 and 1.46 times in the pEME-mar (f), pEME-mar (r) and pEME-hcmvi (f) groups, respectively, compared to the control group pEME, while the expression level of EGFP was not increased in the pEME-hcmvi (r) group, as shown in fig. 8;

pMAR (R) -hCMV (F) was 9.49X 10 ⁵ . The pMAR (R) -hCMV (F) group increased the expression level of EGFP by 3.11-fold compared to the pEME group, 1.95-fold compared to the pEME MAR (R) group, and 2.22-fold compared to the pEME-hCMV (F) group, as shown in FIG. 9, indicating that the combination of reverse insertion of MAR X-29 upstream of the promoter and forward insertion of the hCMV intron downstream of the promoter significantly increased the stable expression level of the transgene.

Test example 2

The test method comprises the following steps: the expression vector constructed in example 1 was used to construct an attached expression system containing the gene of interest HSV-TK:

synthesis of 1 HSV-TK sequence

HSV-TK sequence (GenBank: JX 392980.1), synthesized by general biosystems (Anhui) Inc., was introduced at both ends of the sequence for directional cloningNhe Ⅰ、Kpn I enzyme cutting site.

2 constructing expression vector containing HSV-TK target gene

By usingNhe I/KpnI HSV-TK sequence synthesized by double enzyme digestion and simultaneously usedNhe I/KpnI, double enzyme digestion of pMAR (R) -hCMV (F) plasmid, and enzyme digestion at 37 ℃ for 6h by adopting a conventional enzyme digestion method; agarose gel electrophoresis is carried out to identify the digestion result, and HSV-TK sequence segment and pMAR (R) -hCMV (F) linear plasmid are recovered from gel; after the digestion, the recovered pMAR (R) -hCMV (F) linear plasmid DNA and HSV-TK fragment are subjected to the following steps of (1) in a molar ratio of 5: 1 at 16 ℃ overnight; then, the ligation product is transformed into E.coliJM109 strain competent cell suspension, and the bacterial liquid is inoculated on an LB plate containing kanamycin and cultured overnight at 37 ℃; selecting a single colony for culturing, and extracting recombinant plasmids; extracted plasmid is carried outNhe I/KpnI, double enzyme digestion verification, namely selecting a plasmid with correct enzyme digestion verification to perform sequencing verification, and naming the vector with correct sequencing as pMAR (R) -hCMV (F) -HSV-TK;

3 establishment of vector transfection HCT16 cell expression system

HCT16 cells in good growth state were seeded in 6-well plates and transfected when the plating density reached about 80% -90%. The specific operation steps are as follows: add 10. mu.L lipofectamine2000 to 250. mu.L serum-free RPMI-1640 medium, while mixing 250. mu.L serum-free RPMI-1640 medium with 4. mu.g expression plasmid; then the liposome 2000 diluted solution is dripped into the plasmid DNA diluted solution, the mixture is uniformly mixed while being dripped, and the mixed solution of the liposome 2000/DNA compound is dripped into the hole after the incubation for 20min at room temperature; adding 5% CO ₂ After the culture is carried out for 6 hours at 37 ℃ in the cell culture box, the serum-free RPMI-1640 culture medium is replaced by a complete RPMI-1640 culture medium, and the cell culture box is put into the cell culture box for continuous culture;

expression analysis of 4-purpose gene HSV-TK

After transfection for 48h, PBS washes cultured cells, trypsinizes, adds some cells into RPMI-1640 cell culture medium to continue culturing; another portion of the cells were collected in a 1.5mL EP tube and centrifuged at 1000 rpm for 5min to collect cell pellets; adding RIPA lysate into the cell sediment, centrifuging at 13000 rpm for 15 min, and collecting the supernatant; western bolt detects HSV-TK transient expression.

Selection of stable strains was performed 48h after transfection: culturing the cells in a complete culture medium containing 800 ug/mL G418), dying a blank control cell after about 1 w, further culturing by using an RPMI-1640 complete culture medium containing 400 ug/mL G418, stably screening for about 5 w to obtain a stable cell strain, collecting the precipitate of the stable cell strain, adding RIPA lysate, centrifuging at 13000 rpm for 15 min to collect the cell supernatant, and detecting the stable expression of HSV-TK by Western bolt. Western Blot results show that HSV-TK expression can be detected in both transiently and stably screened HCT116 cells, and the stable expression level is improved by 2.15 times compared with the transient expression level through gray scale analysis, as shown in FIG. 10;

functional verification of 5 HSV-TK protein

The prodrug, Ganciclovir (GCV), is a nucleotide analog that forms Ganciclovir monophosphate under the catalytic action of HSV-TK, and then converts to Ganciclovir triphosphate, interfering with DNA synthesis during cell division, and ultimately leading to apoptosis. Not only are transgenic tumor cells killed after application of GCV, but a large number of surrounding untransfected cells are also killed, a phenomenon known as bystander effect. The bystander effect can be used for identifying the function of the target gene HSV-TK, and more than 60% of cells can be killed by less than 50% of HSV-TK positive cells to consider that the suicide gene is effective. The specific operation steps of the paracellular effect are as follows: stably transfected HCT116 cell line and untransfected cells were mixed at different concentrations (0: 10, 1: 9, 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3, 8: 2, 9: 1, 10: 0) and seeded in 96-well plates, 5000 cells per well, at 37 ℃ and 5% CO ₂ Culturing in the incubator; after the cells are attached to the wall, replacing the culture medium with a culture medium containing GCV 80 ug/mL; after 48h of incubation, 20 uL of MTT solution (5 mg/mL) was added to each well and the cells were incubated for 4 h on tinfoil; the well broth was discarded, 150 uL DMSO was added to each well and the mixture was incubated on a shaker at low speedShaking for 10 min to completely dissolve the crystals; and measuring the absorbance value of each hole in an enzyme-linked immunosorbent assay instrument, and calculating the cell growth inhibition rate. As a result, it was found that the cell inhibition rate reached 33.25% when the proportion of the stably transfected HCT116 cell line was 10%, 75.87% when the proportion reached 40%, and 82.67% when the proportion reached 50%, as shown in FIG. 11, indicating that a small amount of the stably transfected HCT116 cell line caused most of apoptosis, i.e., the bystander effect was significant and the suicide gene was functional.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, and not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Sequence listing

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gatatgcagt tcaggttaaa tgtcagttga gttaccaaag cacatacata ctcaccaccc 1440

tatccaaatc tacaagcctc ccagtttgtc ttcactattt tggttaaatt aatatgaatt 1500

cctagatgaa aatttcactg atccaaatga aataaaaaat atattacaaa actcacacct 1560

gtaatctcaa cattttggga ggccaaggca ggtagatcac ttgaggccag gagttcaaga 1620

ccagcctgat caacatggtg aaaccctgtc tctactaaaa atacaaaaat tagccaggtg 1680

tggtggcatg tgcctgtagt cctacctact cgggaggctg aggcacaaga atcgcttgaa 1740

tgtgggaggt ggaggttgca gtgacctgag atcgtgccac tgcactccag cctaggcaac 1800

agagtgagat catgtgtcat atatatatat atatatatat atatatatat atatatatac 1860

acacacacac acatatatat atacacatat atatacgtat atatatatat gtatatatat 1920

acatatatat acatatatat atatacgtat atatatacgt atatatatat caatgtaaat 1980

tatttgggaa atttggtatg aatagtcttc cctgtgaaca cagatcataa aatcatatat 2040

caagcagaca aataagtagt agtcacttat atgcttatac ttgtaactta aagtaaaaga 2100

attacaaaag catatgacaa agactaattt taagatatcc taatttaaat tgttttctaa 2160

aagtgtgtat accattttac ctatcatatg aataatttag aaacatgttt ataaaattaa 2220

tgtccaaatc cattcaaaag ttttgtaatg cagatcaccc acaacaacaa agaatcctag 2280

cctattaaaa aagcaacacc acctacatat aatgaaatat tagcagcatc tatgtaacca 2340

aagttacaca gtgaatttgg gccatccaac actttgagca aagtgttgaa ttcatcaaat 2400

gaatgtgtaa tcatttactt actaatgcca atacacttta aggtaatctt aagtagaaga 2460

gatagagttt agaatttttt aaatttatct cttgttgtaa agcaatagac ttgaataaat 2520

aaattagaag aatcagtcat tcaagccacc agagtatttg atcgagattt cacaaactct 2580

aactttctga tacccattct cccaaaaacg tgtaacctcc tgtcgatagg aacaacccac 2640

tgcagggatg tttctcgtgg aaaaaggaaa tttcttttgc attggtttca gacctaactg 2700

gttacaagaa aaaccaaagg ccattgcaca atgctgaagt acttttttca aatttaaaat 2760

ttgaaagttg ttcttaaaat ctatcattta ttttaaaata cggatgaatg agaaagcata 2820

gatttgataa agtgaattct tttctgcaat ctacagacac ttccaaaaat cactacagac 2880

actacagaca ctacagaaaa tcataaataa acaagtgcta gtatcaatat ttttaccaaa 2940

aaatggcatt cttagaattt tttataggct agaaggtttg tacaaactaa tctgccacgg 3000

attttaaaat atgagtgaat aaattatatt gcaaaaaaaa tcaggttaca gagaactggc 3060

aaggaagact cttatgtaaa acacagaaaa catacaaaac gtatttttaa gacaaataaa 3120

aacagaactt gtacctcaga tgatactgga gattgtgttg acatattagc attatcactg 3180

tcttgctaaa acataaaaat aaaaagatgg aagatgaaat tacaatacaa atgatgattt 3240

aaacatataa aaggaaaata aaaattgttc tgaccaacta ctaaaggaag acctactaaa 3300

gatatgccat ccagcacatt gccactctac atgtggtctg taaaccagca gcatagggat 3360

ccatcggccg ggcgcgccgt cgac 3384

<210> 3

<211> 821

<212> DNA

<213> Artificial Sequence

<400> 3

gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg 60

tttttggctt ggggcctata cacccccgct ccttatgcta taggtgatgg tatagcttag 120

cctataggtg tgggttattg accattattg accactcccc tattggtgac gatactttcc 180

attactaatc cataacatgg ctctttgcca caactatctc tattggctat atgccaatac 240

tctgtccttc agagactgac acggactctg tatttttaca ggatggggtc ccatttatta 300

tttacaaatt cacatataca acaacgccgt cccccgtgcc cgcagttttt attaaacata 360

gcgtgggatc tccacgcgaa tctcgggtac gtgttccgga catgggctct tctccggtag 420

cggcggagct tccacatccg agccctggtc ccatgcctcc agcggctcat ggtcgctcgg 480

cagctccttg ctcctaacag tggaggccag acttaggcac agcacaatgc ccaccaccac 540

cagtgtgccg cacaaggccg tggcggtagg gtatgtgtct gaaaatgagc tcggagattg 600

ggctcgcacc gtgacgcaga tggaagactt aaggcagcgg cagaagaaga tgcaggcagc 660

tgagttgttg tattctgata agagtcagag gtaactcccg ttgcggtgct gttaacggtg 720

gagggcagtg tagtctgagc agtactcgtt gctgccgcgc gcgccaccag acataatagc 780

tgacagacta acagactgtt cctttccatg ggtcatcgag c 821

<210> 4

<211> 367

<212> DNA

<213> Artificial Sequence

<400> 4

atttagttta tatacatcta cagataaata catatcatat atttgaattc taatctccct 60

ctcaacccta cagtcaccca tttggtatat taaagatgtg ttgtctactg tctagtatcc 120

ctcaagcagt gtcaggaatt agtcatttaa atagtctgca agccaggagt ggtggctcat 180

gtctgtaatt ccagcacttg agaggtagaa gtgggaggac tgcttgagct caagagtttg 240

atattatcct ggacaacata gcaagacctc gtctctactt aaaaaaaaaa aattagccag 300

gcatgtgatg tacacctgta gtcccagcta ctcaggaggc cgaaatggga ggatcagatc 360

tggatcc 367

Claims (10)

1. A high-efficiency expression vector for human and mammal cell attachment bodies comprises a promoter sequence, and is characterized in that a first nuclear matrix binding region sequence is inserted into the upstream of the promoter sequence in a reverse direction, an intron sequence is inserted into the downstream of the promoter sequence in a forward direction, and a second nuclear matrix binding region sequence is inserted into the downstream of the intron sequence in a forward direction.

2. The vector according to claim 1, wherein the promoter sequence is an EF-1 α sequence, as shown in SEQ ID NO:1 is shown.

3. The high-efficiency human and mammalian cell adhesion body expression vector of claim 2, wherein the first matrix binding domain sequence is a MAR X-29 sequence as set forth in SEQ ID NO:2, respectively.

4. The vector for expressing the cell adhesion promoter of claim 3, wherein the intron sequence is hCMV intron sequence as set forth in SEQ ID NO:3, respectively.

5. The vector according to claim 4, further comprising a reporter gene sequence between the intron sequence and the second nuclear matrix binding domain sequence; the second nuclear matrix binding region sequence is a MAR characteristic sequence, and is shown as SEQ ID NO: 4, respectively.

6. The vector for expressing the high-potency human or mammalian cell adhesion body of claim 5, wherein the starting vector is pEGFP-C1 or pEGFP-C1-MAR.

7. The method for constructing the expression vector of claim 6, comprising the steps of:

1) artificially synthesizing a promoter sequence, carrying out double enzyme digestion on the promoter sequence and a starting vector, then connecting, transforming and identifying to construct an intermediate vector 1;

2) artificially synthesizing a nuclear matrix binding region sequence, performing double enzyme digestion on the nuclear matrix binding region sequence and the intermediate vector 1, connecting, transforming and identifying to construct an intermediate vector 2;

3) artificially synthesizing an intron sequence, performing double enzyme digestion on the intron sequence and the intermediate vector 2, connecting, transforming and identifying, and constructing the vector.

8. The use of the high efficiency expression vector of human and mammalian cell attachments as claimed in any one of claims 1 to 6 as a foreign gene delivery vector, wherein a foreign gene is cloned into the attachment expression vector to form a foreign gene expression vector, and the foreign gene expression vector transfects the attachment host cell to express a target protein.

9. The use of the high efficiency human and mammalian cell attachment expression vector of claim 8 as a foreign gene delivery vector wherein the foreign gene is the HSV-TK gene.

10. The use of the high expression vector of human and mammalian cell attachments as a vector for foreign gene delivery according to claim 8, wherein the host cell is human colon cancer HCT 116.

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