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

Abstract

The invention belongs to the technical field of genetic engineering and gene therapy, in particular to an efficient human and mammalian cell attachment expression vector, and also relates to a construction method and application of the attachment expression vector. In the present invention, the first nuclear matrix binding region sequence is inserted in the upstream of the promoter, the intron sequence is inserted in the downstream of the promoter, and the second nuclear matrix binding sequence is inserted in the downstream of the intron sequence, and the promoter sequence is further preferably: The EF-1α sequence, the first nuclear matrix binding region sequence is MAR X-29, the intron sequence is the hCMV intron sequence, and the second nuclear matrix binding region sequence is the MAR characteristic sequence to construct the attachment body expression vector. It is verified by experiments that using the expression vector constructed by the present invention, using CHO cells or human colon cancer cells HCT116 as host cells, can significantly improve the expression level and expression stability of the transgene, and the expressed target protein has biological functions.

Description

A high-efficiency human and mammalian cell attachment expression vector, construction method and application

technical field

The invention belongs to the technical field of genetic engineering and gene therapy, in particular to an efficient human and mammalian cell attachment expression vector, and also relates to a construction method and application of the attachment expression vector.

Background technique

Gene therapy has brought new therapeutic possibilities to many areas of modern medicine. Gene therapy is the introduction of normal genes or therapeutic DNA sequences into target cells in a specific way to correct the defective gene and thereby cure the disease. Since the first successful gene therapy clinical trials in the early 1990s, thousands of gene therapy clinical trials have been completed. The success of gene therapy depends to a large extent on the selection of appropriate gene delivery vectors, mainly including 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 relatively low. According to the state of existence of the vector after entering the host cell, the vector can be divided into the integration vector and the attachment vector. Integrating the foreign gene into the host cell chromosome is easy to induce insertion mutation, and the safety is low. Unlike integrating vectors, episome vectors do not integrate into the genome, but attach to the chromosome, which reduces the risk of insertional mutagenesis and has higher safety.

The Chinese invention patent application with application publication number CN102703503A discloses a human and mammalian cell attachment expression vector and its application, in which the 2200 bp human beta-interferon nuclear matrix binding region (matrix attachment region, MAR) sequence is cut and spliced The non-viral attachment vector pEGFP-C1-M was constructed into a 367 bp characteristic sequence of MAR. pEGFP-C1-MAR is a novel mammalian cell expression vector that can attach but not integrate into Chinese hamster ovary (Chinese hamster ovary). , CHO) cell chromosomes, overcome the side effects of traditional vector integration effects, and can drive transgene expression. However, in the study, it was found that the CMV promoter of the vector itself drives foreign gene expression with low copy number, low gene expression level and instability. In order to solve this technical problem, the Chinese invention patent application publication number CN105802997A replaced the CMV promoter of pEGFP-C1-MAR with the human elongation factor-1α (EF-1α) promoter to construct a pEME attachment vector, and found that The EF-1α promoter can drive the high-efficiency and stable expression of non-viral attachment vectors. Although a better promoter has been screened, the transgene expression of the vector still has problems of instability and low copy number (6-12 copies/cell). .

Regulatory elements are DNA sequences that regulate the expression of transgenes without encoding any protein. Studies have found that regulatory elements such as introns, UCOE, STAR 40, and MAR can improve the expression level of transgenes in integrated vectors. and location. How to use these regulatory elements in combination, select the appropriate direction and position, and further improve the stability and copy number of the transgene expression of the attached expression vector, has become a technical problem that needs to be solved at present.

SUMMARY OF THE INVENTION

In order to overcome the defects of the prior art, one of the purposes of the present invention is to provide a highly efficient human and mammalian cell attachment expression vector, which has higher stability compared with the existing vectors of the same type, and further improves the expression of the target gene quantity.

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

The third object of the present invention is to provide the application of high-efficiency human and mammalian cell attachment expression vectors.

In order to realize the above-mentioned purpose of the invention, the technical scheme adopted in the present invention is as follows:

A high-efficiency human and mammalian cell attachment expression vector, comprising a promoter sequence, a first nuclear matrix binding region sequence is inserted upstream of the promoter sequence in a reverse direction, an intron sequence is inserted downstream of the promoter sequence in a forward direction, and the downstream of the intron sequence is inserted. A second nuclear matrix binding region sequence was inserted in the forward direction.

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

Further, the sequence of the first nuclear matrix binding region is a MAR X-29 sequence, as shown in SEQ ID NO: 2.

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

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 region sequence is a MAR characteristic sequence, as shown in SEQ ID NO: 4. In some embodiments of the present invention, the reporter gene sequence is EGFP.

Optionally, the starting vector can use any human or mammalian cell expression vector. As an example, in some embodiments of the present invention, the starting vector is pEGFP-C1, or pEGFP-C1 constructed according to the method of patent CN102703503A. C1-MAR, or pEME constructed according to patent CN105802997A.

The above-mentioned high-efficiency human and mammalian cell attachment expression vectors can be constructed according to conventional methods in the field of genetic engineering. As an example, in some embodiments of the present invention, the construction method includes the following steps:

1) Synthesize the promoter sequence artificially, connect, transform and identify the promoter sequence and the starting vector after double digestion, and construct the intermediate vector 1;

2) Artificially synthesize the sequence of the nuclear matrix binding region, connect, transform and identify the sequence of the nuclear matrix binding region and the intermediate vector 1 after double digestion, and construct the intermediate vector 2;

3) Synthesize the intron sequence artificially, connect the intron sequence and the intermediate vector 2 after double digestion, transform and identify, and construct to form the vector.

The application of the above-mentioned high-efficiency human and mammalian cell attachment expression vectors as exogenous gene transfer vectors, the exogenous gene is cloned into the attachment expression vector to form an exogenous gene expression vector, and the exogenous gene expression vector is transfected into host cells to express the target protein .

As an example, the exogenous gene is the HSV-TK gene (GenBank accession number: JX392980.1).

By way of illustration, the host cell is human colon cancer cell HCT116.

Beneficial effects of the present invention:

In the present invention, the sequence of the nuclear matrix binding region, the intron sequence and the promoter sequence are combined as the elements of the human or mammalian attachment body expression vector, the first nuclear matrix binding region sequence is inserted in the upstream of the promoter in the reverse direction, and the downstream of the promoter is in the forward direction. Insert an intron sequence, and insert the second nuclear matrix binding sequence downstream of the intron sequence in the forward direction, and further preferably the promoter sequence is EF-1α sequence, the first nuclear matrix binding region sequence is MAR X-29, and the intron sequence is The intron sequence of hCMV and the sequence of the second nuclear matrix binding region are characteristic sequences of MAR, which can improve the continuous, efficient and stable expression of exogenous target genes of the attachment body expression vector. It is verified by experiments that using the expression vector constructed by the present invention, using CHO cells or human colon cancer cells HCT116 as host cells, can significantly improve the expression level and expression stability of the transgene, and the expressed target protein has biological functions.

Description of drawings

Fig. 1 is the structural representation of recombinant plasmid pEME;

Fig. 2 is the structural representation of recombinant plasmid pEME-hCMVI (F);

Fig. 3 is a schematic diagram of the structure of recombinant plasmid pMAR(R)-hCMVI(F);

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

Fig. 5 is the structural representation of recombinant plasmid pEME-MAR (F);

Fig. 6 is the structural representation of recombinant plasmid pEME-MAR (R);

Figure 7 is a schematic diagram showing the comparison results of transient expression levels of EGFP in CHO cells transfected with pEME, pEME-MAR (F), pEME-MAR (R), pEME-hCMVI (F), and pEME-hCMVI (R);

Figure 8 is a schematic diagram showing the comparison results of the stable expression levels of EGFP in CHO cells transfected with pEME, pEME-MAR (F), pEME-MAR (R), pEME-hCMVI (F), and pEME-hCMVI (R);

Figure 9 is a schematic diagram showing the comparison results of the stable expression levels of EGFP in CHO cells transfected with pEME, pEME-MAR(R), pEME-hCMVI(F), and pMAR(R)-hCMV(F) vectors;

Figure 10 shows the expression level of HSV-TK gene in transfected HCT116 cells detected by Western Blot;

Figure 11 is the functional experiment of HSV-TK, and the results of the bystander effect detection.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited to this; the culture medium, reagents, cell line reagents, etc. used in the examples and test examples are all commercially available commodities.

The Escherichia coli JM109 used in the examples of the present invention, the used cell lines, reagents, and tool enzymes are all commercially available products. The following examples all use pEME as the starting vector, and pEME is constructed and obtained according to the construction method disclosed in the patent CN105802997A, and is specifically constructed on the basis of the pEGFP-C1 vector, containing EF-1α promoter, EGFP reporter gene, MAR characteristic sequence. The eukaryotic expression vector for kanamycin resistance (shown in Figure 1).

Example 1

This embodiment provides a high-efficiency human and mammalian cell attachment expression vector, which includes a nuclear matrix binding region sequence (3387 bp MAR X-29 sequence, shown as SEQ ID NO: 2) reversed upstream of the promoter. and the hCMV intron sequence (821bp hCMV intron, as shown in SEQ ID NO: 3) downstream of the promoter; the specific operation steps of its construction method are:

1. Construction of pEME-hCMVI(F)

S1: The forward sequence of hCMV intron (hCMV intron, hCMVI) is synthesized, and Nhe I and Eco 47 III restriction sites are introduced at both ends of the sequence; (synthesized by General Biosystems (Anhui) Co., Ltd.);

S2: Enzymatic cleavage reaction

The synthesized forward hCMV intron sequence and pEME plasmid were digested with NheⅠ/Eco47Ⅲ double enzyme. After the digestion, the digestion status was identified by 1.5% agarose gel electrophoresis, and the digested forward hCMV was recovered from the gel. Intron fragments and linear pEME plasmids;

The digestion system of hCMV intron sequence is as follows: hCMV intron (1μg/μL) 10μL, 20×NEB buffer 1μL, Nhe Ⅰ enzyme (10U/μL) 0.5μL, add three distilled water to 20μL, mix well, Digestion in a water bath at 37°C for 6 h. After the digestion, electrophoresis was carried out on a 1.5% agarose gel. The intron fragments after digestion were recovered from the gel and then digested with Eco 47 III; the recovered hCMV introns (1 μg/ μL) 10 μL, 10× O buffer 2 μL, Eco 47 Ⅲ enzyme (10U/μL) 0.5 μL, add three distilled water to 20 μL, mix well, and digest in 37°C water bath for 6 h;

The digestion system of pEME plasmid is as follows: First, Nhe I single digestion, pEME plasmid (1μg/μL) 10μL, 20×NEBbuffer 1μL, Nhe I enzyme (10U/μL) 0.5μL, add three distilled water to 20μL, mix well , digested in a water bath at 37°C for 6 h, after the digested, electrophoresed on a 1.5% agarose gel, and the digested fragments were recovered from the gel and then digested with Eco 47 III; the recovered pEME (1 μg/μL) 10 μL, 10 × O buffer 2μL, Eco 47 Ⅲ enzyme (10U/μL) 0.5μL, add three distilled water to 20μL, mix well, and digest with 37℃ water bath for 6h;

S3: connect

Linear pEME plasmid (1μg/μL) 5μL, digested forward hCMV intron fragment (1μg/μL) 1μL, T4 ligase buffer 1μL, T4 ligase (350U/μL) 0.5μL, add three distilled water to 20μL, Mix well and connect overnight at 16°C;

S4: Transformation and Identification

Take 200μL of freshly prepared E.coli JM109 competent bacteria into a sterile 1.5mL EP tube under aseptic conditions, add 10μL of the above ligated reaction solution for transformation, and then inoculate it on an agar plate containing kanamycin , cultured at 37°C overnight, picked positive transformants from the cultured plate and placed them in a test tube of 3 mL LK liquid medium, and cultured overnight at 37°C on a shaker; extract the recombinant plasmid, and verify the recombinant plasmid by double-enzyme digestion, select the enzyme The correct plasmid was cut and verified by sequencing, and the vector with completely correct target gene sequence was named pEME-hCMVI(F) (as shown in Figure 2);

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

S1: Referring to the MAR X-29 DNA fragment sequence (GenBank: EF694970.1), the reverse (Revers, R) sequence was synthesized, and the gene synthesis was completed by General Biosystems (Anhui) Co., Ltd.;

S2: Using the seamless cloning method, insert the MAR X-29 reverse fragment into the pEME-hCMVI(F) vector (Figure 2) upstream of the EF-1α promoter expression cassette, and transform the constructed recombinant vector into competent cells E.coli JM 109, then inoculated in kanamycin-containing medium overnight at 37 °C, pick a single colony and continue to cultivate in kanamycin-containing medium, shake the bacteria overnight at 37 °C, 300 r/min; extract Recombinant plasmid; take 10 μL of recombinant DNA plasmid for Kpn I/ Apa I double digestion identification, select the correct plasmid for digestion verification, perform sequencing verification, and name the correctly sequenced vector pMAR(R)-hCMV(F) (Figure 3 shown).

Comparative Example 1

In this comparative example, pEME-hCMVI(R) was constructed by artificially synthesizing the reverse sequence of hCMVI according to the same method for constructing pEME-hCMVI(F) in Example 1 above, as shown in FIG. 4 .

Comparative Example 2

This comparative example constructs pEME-MAR (F):

Using the method of seamless cloning, the forward fragment of MAR X-29 was inserted into the pEME vector (Fig. 1). The upstream of the EF-1α promoter expression cassette was inserted into the MAR X-29 attachment expression vector. The constructed recombinant vector was transformed into competent cells E.coliJM 109, then inoculated in kanamycin-containing medium overnight at 37°C, and a single colony was picked and continued to culture in kanamycin-containing medium at 37°C The bacteria were shaken at 300 r/min overnight; the recombinant plasmid was extracted by SDS alkaline lysis method; 10 μL of the recombinant DNA plasmid was taken for restriction enzyme digestion identification, and double-enzyme digestion with Nhe I/ Kpn I was used to select the plasmid with the correct restriction enzyme digestion, and sequenced to verify that it was correct. The vector was named pEME-MAR (F), as shown in Figure 5.

Comparative Example 3

This comparative example constructs pEME-MAR(R):

Using the seamless cloning method, the MAR X-29 reverse fragment was inserted into the pEME vector (Fig. 1) upstream of the EF-1α promoter expression cassette to construct an attachment expression vector containing MAR X-29. The constructed recombinant vector was transformed into competent cells E.coliJM 109, then inoculated in kanamycin-containing medium overnight at 37°C, and a single colony was picked and continued to culture in kanamycin-containing medium at 37°C Shake bacteria overnight at 300 r/min; extract the recombinant plasmid by SDS alkaline lysis method; take 10 μL of the recombinant DNA plasmid for restriction digestion identification, double restriction digestion with Spe I/ Apa I, select the plasmid with the correct restriction digestion, and perform sequencing to verify that it is correct The vector was named pEME-MAR(R), as shown in Figure 6.

Test Example 1

Test method: After transfecting the starting vector, the expression vectors constructed in Example 1 and Comparative Examples 1 to 3 into CHO cells, respectively, the expression of the reporter group EGFP was detected:

Select CHO-K1 cells with good growth status and inoculate them on 6-well culture plates, and transfect them when the plating density reaches about 80%. According to the experimental design, the transfection was divided into 6 groups: untransfected group, transfected with pEME, pEME-MAR (F), pEME-MAR (R), pEME-hCMVI(F), pEME-hCMVI(R), pMAR ( R) - hCMV (F) vector set. Take 4 μg of the above vector and add it to 250 μL DMEM-F2 medium without serum and antibiotics, and mix gently. The liposome 2000 dilution was added dropwise to the plasmid DNA diluent, and the mixture was evenly mixed. Incubate at room temperature for 20 minutes, and then add the liposome 2000/DNA complex to each air and gently Shake to mix, put into a 5% CO ₂ cell incubator, and after culturing for 6 h at 37 °C, change the serum-free medium to complete DMEM medium. 48h after transfection, the mean fluorescence intensity (MFI) of EGFP in each group was detected by flow cytometry.

test results:

Transient expression: Compared with the control group pEME, except for pEME-hCMVI(R), the rest of the recombinant vectors increased the transient expression level of EGFP. The MFI of the control group EGFP was set to 1, pEME-MAR (F), pEME-MAR (R) and pEME-hCMVI (F) groups increased by 1.15, 1.50 and 1.34 times, respectively, as shown in Figure 7;

Screening of stable cell lines: 48h after cell transfection, pressurize and screen with G418 medium with a final concentration of 800 μg/ml, change the medium every 3 days, after 7-10 days of cell death in the untransfected group, change the G418 medium to The concentration was maintained at 400 μg/ml and screened for 2 weeks. After stably transformed cell colonies were formed, the culture was continued. When the cell density reached 80%-90%, the cells were collected and the expression of stable EGFP was detected by flow cytometry. The expression level of EGFP in the control group pEME was 3.05×10 ⁵ , the pEME MAR(R) group was 4.88×10 ⁵ , and the pEME-hCMV(F) group was 4.27×10 ⁵ , similar to the transient expression. Compared with the control group pEME, pEME -MAR (F), pEME-MAR (R) and pEME-hCMVI(F) groups increased the stable expression level of EGFP by 1.29, 1.67 and 1.46 times, respectively, while pEME-hCMVI(R) group did not increase the expression level of EGFP, As shown in Figure 8;

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

Test Example 2

Test method: Use the expression vector constructed in Example 1 to construct an attachment expression system containing the target gene HSV-TK:

1 Synthesis of HSV-TK sequences

The HSV-TK sequence (GenBank: JX392980.1) was synthesized by General Biosystems (Anhui) Co., Ltd. In order to achieve directional cloning, Nhe I and Kpn I restriction sites were introduced at both ends of the sequence.

2 Construction of expression vector containing HSV-TK target gene

The synthesized HSV-TK sequence was digested with Nhe I/ Kpn I double enzyme, and the pMAR(R)-hCMV(F) plasmid was digested with Nhe I/ Kpn I double enzyme, and the conventional enzyme digestion method was used for 6 hours at 37°C; Agarose gel electrophoresis was used to identify the digestion results, and the HSV-TK sequence fragment and pMAR(R)-hCMV(F) linear plasmid were recovered from the gel; the pMAR(R)-hCMV(F) linear plasmid DNA and HSV recovered by enzyme digestion -TK fragment was ligated overnight at 16°C in a molar ratio of 5:1; the ligation product was then transformed into a suspension of competent cells of E. coli JM109 strain, and the bacterial solution was inoculated on LB plates containing kanamycin, 37 Cultivate overnight at ℃; pick a single colony for culture, and extract the recombinant plasmid; the extracted plasmid is verified by Nhe I/ Kpn I double-enzyme digestion, and the correct plasmid is selected for sequencing verification, and the sequenced vector is named as pMAR(R)-hCMV(F)-HSV-TK;

3. Establishment of expression system of vector transfected HCT16 cells

Well-grown HCT16 cells were seeded into 6-well culture plates, and transfection was performed when the plating density reached about 80%-90%. The specific operation steps are as follows: add 10 μL lipofectamine2000 to 250 μL serum-free RPMI-1640 medium, and at the same time mix 250 μL serum-free RPMI-1640 medium with 4 μg expression plasmid; then add lipofectamine 2000 dilution dropwise to the plasmid DNA In the diluent, add dropwise and mix well, incubate at room temperature for 20 min, and gently drop the mixture of liposome 2000/DNA complex into the well; put it in a 5% CO ₂ cell incubator at 37 °C for 6 h after incubation , replace the serum-free RPMI-1640 medium with RPMI-1640 complete medium, and put it into the cell incubator to continue culturing;

4. Expression analysis of target gene HSV-TK

After 48 hours of transfection, the cultured cells were washed with PBS, digested with trypsin, and a part of the cells were added to RPMI-1640 cell culture medium to continue the culture; another part of the cells were collected into a 1.5 mL EP tube and centrifuged at 1000 rpm for 5 min to collect the cell pellet; RIPA lysate was added to the pellet, and the supernatant was collected by centrifugation at 13,000 rpm for 15 min; the transient expression of HSV-TK was detected by Western bolt.

Screening of stable strains 48h after transfection: cells were continued to culture in complete medium containing 800 ug/mL G418), and the blank control cells died after about 1 w, and were replaced with RPMI-1640 complete medium containing 400 ug/mL G418 for further The cells were cultured and screened for about 5 weeks to obtain stable cell lines. The stable cell line pellets were collected, RIPA lysate was added, and the cell supernatants were collected by centrifugation at 13,000 rpm for 15 min. The stable expression of HSV-TK was detected by Western bolt. Western Blot results showed that the expression of HSV-TK could be detected in both transient and stably screened HCT116 cells, and the stable expression level was 2.15 times higher than that of transient expression by grayscale analysis, as shown in Figure 10;

5 Functional verification of HSV-TK protein

The prodrug Ganciclovir (GCV) is a nucleotide analog that is catalyzed by HSV-TK to form glycine monophosphate, which is then converted to glycine triphosphate, interfering with cells DNA synthesis during division, ultimately leading to apoptosis. After the application of GCV, not only the transgenic tumor cells are killed, but also a large number of untransfected cells around them, which is called the bystander effect. The bystander effect can be used to identify the function of the target gene HSV-TK. Less than 50% of HSV-TK positive cells can kill more than 60% of the cells, and the suicide gene is considered effective. The specific operation steps of the paracellular effect are as follows: Mix the stably transfected HCT116 cell line and the untransfected cells 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 with 5000 cells per well, cultured at 37°C and 5% CO in an incubator After the cells adhered, the medium was replaced with a medium containing 80 ug/mL of GCV; after 48 hours of culture, 20 uL of MTT solution (5 mg/mL) was added to each well, and the culture was continued for 4 hours by wrapping in tin foil; Discard the culture medium in the wells, add 150uL DMSO to each well, and shake at low speed on a shaker for 10 min to completely dissolve the crystals; measure the absorbance value of each well in an enzyme-linked immunosorbent assay, and calculate the cell growth inhibition rate. The results showed that when the proportion of stably transfected HCT116 cell line was 10%, the cell inhibition rate reached 33.25%, when the proportion reached 40%, the cell inhibition rate was 75.87%, and when the proportion reached 50%, the cell inhibition rate reached 50%. The percentage of cells was 82.67%, as shown in Figure 11, indicating that a small amount of stably transfected HCT116 cell lines can cause most cells to undergo apoptosis, that is, the bystander effect is obvious, and the suicide gene is functional.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be The technical solutions described in the foregoing embodiments are modified, or some technical features thereof are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

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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 atatatac 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

gatagagtttt 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

ttttttggctt 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 human and mammalian cell attachment body expression vector, comprising a promoter sequence, it is characterized in that, the upstream of the promoter sequence inserts the first nuclear matrix binding region sequence in the reverse direction, and the downstream of the promoter sequence inserts the intron sequence in a forward direction , a second nuclear matrix binding region sequence is inserted downstream of the intron sequence. 2 . The high-efficiency human and mammalian cell attachment body expression vector according to claim 1 , wherein the promoter sequence is an EF-1α sequence, as shown in SEQ ID NO: 1. 3 . 3 . The high-efficiency human and mammalian cell attachment body expression vector according to claim 2 , wherein the sequence of the first nuclear matrix binding region is a MAR X-29 sequence, as shown in SEQ ID NO: 2. 4 . 4 . The high-efficiency human and mammalian cell attachment body expression vector according to claim 3 , wherein the intron sequence is an hCMV intron sequence, as shown in SEQ ID NO: 3. 5 . 5. The high-efficiency human and mammalian cell attachment body expression vector according to claim 4, wherein the intron sequence and the second nuclear matrix binding region sequence also comprise a reporter gene sequence; the second The nuclear matrix binding region sequence is a characteristic sequence of MAR, as shown in SEQ ID NO:4. 6 . The high-efficiency human and mammalian cell attachment expression vector according to claim 5 , wherein the starting vector is pEGFP-C1 or pEGFP-C1-MAR. 7 . 7. a construction method of efficient human and mammalian cell attachment body expression vector as claimed in claim 6, is characterized in that, comprises the following steps: 1) Synthesize the promoter sequence artificially, connect, transform and identify the promoter sequence and the starting vector after double digestion, and construct the intermediate vector 1; 2) Artificially synthesize the sequence of the nuclear matrix binding region, connect, transform and identify the sequence of the nuclear matrix binding region and the intermediate vector 1 after double digestion, and construct the intermediate vector 2; 3) Synthesize the intron sequence artificially, connect the intron sequence and the intermediate vector 2 after double digestion, transform and identify, and construct to form the vector. 8. a high-efficiency human and mammalian cell attachment body expression vector as described in any one of claim 1～6 is used as the application aspect of exogenous gene delivery carrier, it is characterized in that, exogenous gene is cloned into attachment body expression vector An exogenous gene expression vector is formed, and the exogenous gene expression vector is transfected and attached to the host cell to express the target protein. 9 . The application of the high-efficiency human and mammalian cell attachment expression vector as claimed in claim 8 as an exogenous gene delivery vector, wherein the exogenous gene is HSV-TK gene. 10 . 10 . The application of the highly efficient human and mammalian cell attachment expression vector as claimed in claim 8 as an exogenous gene delivery vector, wherein the host cell is human colon cancer cell HCT116. 11 .

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