CN101514340B - Human autophagy gene Beclin 1 promoter sequence and its application - Google Patents

Abstract

The invention belongs to the field of molecular biology and discloses a promoter derived from the promoter region of autophagy gene beclin 1 and its application. The invention also discloses an expression vector and a host cell containing the promoter. The invention also discloses a method for highly expressing the target gene by using the promoter. The promoter of the present invention has a wide range of activities and can direct the high-level expression of the target gene in different types of host cells.

Description

Human autophagy gene Beclin 1 promoter sequence and its application

technical field

The invention belongs to the field of molecular biology; more specifically, the invention relates to a human autophagy gene promoter and the application of the promoter.

Background technique

Autophagy is a widespread physiological mechanism, which mainly combines macromolecular substances in the cytoplasm (such as proteins, glycogen, etc.) Organelles) are degraded in large quantities in vesicles wrapped in unit membranes to achieve recycling to maintain the process of cell self-stabilization. Autophagy has a variety of physiological functions, such as: ①Provide nutrients for cells when nutrients are scarce; ②Cell differentiation; ③Cell death and aging; ④Prevent the occurrence of cancer. Autophagy is also related to the pathological process of various diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease and heart failure.

The human autophagy gene beclin 1 is the first gene involved in autophagy found in mammals. Studies have shown that the gene has many important biological functions such as inducing autophagy, anti-virus, inhibiting tumor growth and participating in early embryonic development. However, there are few studies on the expression regulation of this gene.

Contents of the invention

The object of the present invention is to provide a promoter derived from the promoter region of the autophagy gene beclin 1 and its application.

Another object of the present invention is to provide expression vectors and host cells containing the promoter.

Another object of the present invention is to provide a method for high expression of a target gene using the promoter.

In a first aspect of the present invention, an isolated nucleic acid (promoter) is provided, the nucleic acid sequence being selected from the group consisting of:

(1) a nucleic acid sequence having the sequence shown in (368±50)-(816±25) in SEQ ID NO: 1;

or

(2) A nucleic acid sequence that can hybridize with the nucleic acid sequence defined in (1) under stringent conditions and has the function of directing high-level expression of the target gene;

(3) A nucleic acid sequence that has more than 95% homology with the nucleic acid sequence defined in (1) and has the function of directing the high-level expression of the target gene.

(4) A nucleic acid sequence that is complementary (preferably fully complementary) to the nucleic acid sequence defined in any one of (1)-(3).

In another preferred example, the "high-level expression" refers to: using the nucleic acid of the present invention (promoter of the present invention) as a promoter element to guide the expression of a target gene and utilizing the sequence of SEQ ID NO: 1 Compared with the promoter directing the expression of the target gene, the expression level obtained by using the nucleic acid of the present invention is 50% higher, preferably 100% higher in expression level, more preferably 200% higher in expression level, further preferably 300% higher in expression level or more high.

In another preferred example, in item (1), there is a nucleic acid sequence shown in (368±20)-(831±10) in SEQ ID NO:1; preferably, it has SEQ ID NO:1 Nucleic acid sequence of the sequence shown at position (368±10)-(836±5). More preferably, it has the nucleic acid sequence shown in the 368-841 sequence in SEQ ID NO:1.

In the second aspect of the present invention, a vector is provided, which contains the nucleic acid as a promoter element.

In another preferred example, the vector is a eukaryotic expression vector.

In another preferred example, the vector does not contain the nucleic acid sequence shown in SEQ ID NO: 1.

In another preferred example, the vector further contains a target gene operably linked to the nucleic acid.

In another preferred example, the target gene is a foreign gene.

In another preferred example, the target gene is a structural gene.

In another preferred example, the target gene sequence is located downstream of the nucleic acid sequence and is a gene-coding sequence directly adjacent to the nucleic acid sequence. Usually, the distance between the nucleic acid sequence and the target gene sequence is less than 500bp (preferably, less than 200bp; more preferably, less than 100bp; most preferably, less than 50bp).

In another preferred example, the target gene includes (but not limited to): autophagy gene (beclin1), luciferase gene, green fluorescent protein.

In the third aspect of the present invention, there is provided a genetically engineered host cell, which: contains the vector; or has the exogenous nucleic acid integrated in its genome and is operably linked to the nucleic acid the target gene.

In another preferred example, the cells are selected from (but not limited to): Escherichia coli cells, human immortalized normal liver cells L02, liver cancer cells HepG2, liver cancer cells BEL-7404, human embryonic kidney cells HEK-293T, or Human breast cancer cell MCF-7.

In the fourth aspect of the present invention, the use of the nucleic acid described above is provided as a promoter element to direct the high expression of the target gene.

In a fifth aspect of the present invention, a method for highly expressing a gene of interest is provided, said method comprising:

(a) cultivating said host cell under conditions suitable for expression; and

(b) Isolating the protein of interest (ie, the protein expressed by the gene of interest) from the culture.

Other aspects of the invention will be apparent to those skilled in the art from the technical disclosure herein.

Description of drawings

Figure 1 shows the activity assays of beclin 1 promoter fragments of different lengths in L02 cells, and the -277/+197 region of the 5' upstream of the beclin 1 gene was identified to have the greatest activity, and the -58/+197 region of the 5' upstream of the beclin 1 gene contained the most basic promoter region.

Figure 2 shows the activity assays of beclin 1 promoter fragments of different lengths in BEL-7404 (A) and HepG2 (B) cells, proving that the beclin 1 promoter is active in liver cancer cells, and the 5' upstream of beclin 1 gene -277 The /+197 region has the highest activity.

Figure 3 shows the activity assay of different lengths of beclin 1 promoter in HEK-293T cells, which proves that the beclin 1 promoter is active in human embryonic kidney cells, and the -277/+197 region of the 5' upstream of the beclin 1 gene has the highest active.

Figure 4 shows the activity assay of beclin 1 promoters of different lengths in MCF7 cells, which proves that the beclin 1 promoter is active in breast cancer cells, and the -277/+197 region of the 5' upstream of the beclin 1 gene has the highest activity.

Figure 5 shows the beclin 1 promoter sequence and potential transcription factor binding sites therein. Arrows indicate transcription start sites.

Detailed ways

Through extensive and in-depth research, the inventors unexpectedly found a nucleic acid, which can be used as a promoter element to guide the high-level expression of a target gene. The promoter of the present invention is derived from the promoter region of the autophagy gene Beclin1. The promoter of the present invention has a wide range of activities, and can guide the high-level expression of the target gene in different types of host cells, without the specificity of cell types. The present invention has been accomplished on this basis.

As used herein, the "promoter" or "promoter region (domain)" refers to a nucleic acid sequence, which usually exists upstream (5' end) of the coding sequence of the gene of interest, and can guide the transcription of the nucleic acid sequence into mRNA . Generally, a promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription. Herein, the promoter or promoter region includes variants of the promoter, which are obtained by inserting or deleting regulatory regions, performing random or site-directed mutations, and the like.

As used herein, "isolated" means that the material is separated from its original environment (if the material is native, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances that exist together in the natural state .

As used herein, the "operably linked" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example: the promoter region is placed at a specific position relative to the nucleic acid sequence of the target gene, so that the transcription of the nucleic acid sequence is guided by the promoter region, thus, the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, the "high-level expression" refers to: using the promoter of the present invention to guide the expression of a gene of interest (such as luciferase gene) and utilizing the promoter with SEQ ID NO: 1 sequence to guide the gene of interest Compared with expression, the expression obtained by using the promoter of the present invention is 50% higher, preferably 100% higher, more preferably 200% higher, and further preferably 300% or higher. The detection of protein expression is well known to those skilled in the art.

Promoters and the gene expression they direct

During the research process, the inventors found that the activity of the promoter of the full-length human beclin 1 gene (sequence shown in Figure 5) was not high enough, and unexpectedly found that the human beclin 1 gene promoter sequence (368 ± 50) -(816±25) (preferably (368±20)-(831±10), more preferably 368-841) eukaryotic expression vector constructed in the region, which can activate exogenous Gene expression.

Therefore, the present invention provides a kind of isolated nucleic acid, described nucleic acid has: the nucleotide sequence shown in (368±50)-(816±25) in SEQ ID NO:1, described nucleic acid can be used as A promoter element that directs the expression of a gene of interest.

In addition, the present invention also includes some variants of the above nucleic acids with the same function. include:

The sequence can hybridize with the sequence shown in (368±50)-(816±25) of SEQ ID NO: 1 under stringent conditions and has the function of directing the high-level expression of the target gene; or

A nucleic acid having more than 95% homology with the sequence shown in (368±50)-(816±25) of SEQ ID NO: 1 and having the function of directing the high-level expression of the target gene; or

A nucleic acid whose sequence is complementary (preferably fully complementary) to the polynucleotide sequence shown in positions (368±50)-(816±25) of SEQ ID NO:1.

Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization properties of a particular pair of nucleic acids indicate their similarity or identity. Therefore, the present invention also relates to hybridization with the nucleic acid sequence shown in the (368±50)-(816±25) positions in SEQ ID NO: 1 and there is at least 50%, preferably at least 70%, between the two sequences, More preferably nucleic acids that are at least 80% (eg, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical. The invention particularly relates to polynucleotides hybridizable under stringent conditions to the nucleic acids of the invention.

In the present invention, "stringent conditions" refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%. Moreover, the hybridizable nucleic acid also has the function of directing the high-level expression of the target gene.

In the example of the present invention, under the guidance of the nucleic acid of the present invention (the promoter of the present invention), the luciferase gene can be expressed at a high level. Therefore, it can be seen that the promoter of the present invention is a kind of promoter which is particularly suitable for directing the expression of the target gene, and has important application value in the study of gene expression.

The promoter of the present invention can be operably linked to a gene of interest, which can be foreign (heterologous) with respect to the promoter. The target gene can generally be any nucleic acid sequence (such as a structural nucleic acid sequence), and the target gene preferably encodes a protein with specific functions, such as some proteins with important properties or functions.

For example, the target gene includes but not limited to: autophagy gene (beclin 1), luciferase gene, etc. As a representative tool for indicating gene expression status, luciferase can well indicate the gene expression status directed by the promoter. From the example that the promoter of the present invention can direct the high expression of luciferase, it can be clearly known that the promoter of the present invention, as a gene regulatory element, can direct the high expression of any other suitable gene.

As a preferred mode of the present invention, the target gene may be a gene that is missing or underexpressed for a certain mammal to be treated, and the target gene may be operably linked to the promoter of the present invention, Or the target gene and the promoter of the present invention are operably linked into a suitable vector, introduced into a suitable cell in an appropriate manner, and delivered to the mammal to be treated, thereby expressing the target gene at a high level.

The promoter of the present invention can also be operably linked to an improved gene sequence of interest which is foreign (heterologous) to the promoter. The gene of interest can be modified to produce various desired properties. For example, the target gene can be improved to increase the content of essential amino acids, improve the translation of amino acid sequences, change post-translational modifications (such as phosphorylation sites), transport translation products outside the cell, improve protein stability, insertion or deletion cell signaling, etc.

Additionally, promoters and genes of interest can be engineered to downregulate specific genes. This is generally accomplished by linking the promoter to the gene sequence of interest directed in antisense reverse. Those of ordinary skill in the art are familiar with such antisense technology. Any nucleic acid sequence can be modulated in this manner.

The promoter and target gene sequence of the present invention can be contained in a recombinant vector.

The recombinant vector generally includes (from 5' to 3' direction): a promoter that guides the transcription of the target gene, and the target gene. If necessary, the recombinant vector may also include a 3' transcription terminator, a 3' polynucleotide signal, other non-translated nucleic acid sequences, transport and targeting nucleic acid sequences, resistance selectable markers, enhancers or operators.

Methods for preparing recombinant vectors are well known to those skilled in the art. The term "expression vector" refers to bacterial plasmid, bacteriophage, yeast plasmid, mammalian cell virus or other vectors well known in the art. In conclusion, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. Preferably, the expression vector is a eukaryotic expression vector.

Methods well known to those skilled in the art can be used to construct an expression vector containing the promoter and/or target gene sequence of the present invention. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein (GFP), etc.

In addition to the promoter of the present invention, the recombinant vector may also contain one or more other promoters. Said other promoters are, for example: tissue-specific, constitutive or inducible.

As an example of the present invention, the vector is pGL3-Basic, which itself contains the sequence encoding the luciferase gene. Through transformation, that is, using the multiple cloning site on pGL3-Basic, the promoter region of the present invention can be constructed in front of the luciferase coding gene, and transformed into host cells, the promoter will activate the expression of the luciferase coding gene, so The above-mentioned initiation is regulated by various cis-acting elements in the promoter region, simulating the situation that the gene is activated and transcribed in vivo.

The vector containing the above-mentioned appropriate promoter and the gene of interest can be used to transform appropriate host cells so that they can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include Escherichia coli, yeast, animal tissue cells, plant cells, and the like.

When the polynucleotide of the present invention is expressed in higher eukaryotic cells, if an enhancer sequence is inserted into the vector, the transcription will be enhanced. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs in length, that act on promoters to enhance gene transcription.

Those of ordinary skill in the art will know how to choose an appropriate vector, promoter, enhancer or host cell.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

Experiments of the present invention have also demonstrated the specificity of the promoter cell-free species of the present invention, in BEL-7404 cells (human liver cancer cells), HepG2 cells (human liver cancer cells), HEK-293T cells (human embryonic kidney cells) and MCF7 Cells (human breast cancer cells) have a wide range of activities. Those skilled in the art can understand that a promoter is a gene regulatory element, and its scope of application is related to the cell where the promoter is located, but has little to do with the expression of the foreign gene it directs or the gene itself. The activity of the promoter of the present invention does not depend on the type of cell and thus can be widely used.

In an example of the present invention, the fragment containing the nucleotide sequence shown in the 368-841 position in SEQ ID NO: 1 is cloned into the 5' upstream of the eukaryotic expression vector pGL3-Basic plasmid reporter gene, and constructed into p (-277/+197) expression vector, which also contains a luciferase reporter gene. The vector is introduced into eukaryotic cells, and the expression level of the exogenous gene is determined by detecting the activity of luciferase. The p(-277/+197) clone was transiently transfected into HepG2 cells, and it was found that the beclin 1 promoter (-277/+197) fragment of the present invention can enhance the expression of luciferase gene by more than 80 times.

The main advantages of the present invention are:

(1) A promoter that can direct the high-level expression of the target gene is disclosed, and the large-scale expression of the target gene can be promoted with high intensity by using the promoter of the present invention.

(2) The promoter has a wide range of activities, and can guide the high-level expression of the target gene in different types of host cells, without the specificity of the cell type. Therefore, the promoter sequence has the advantage of a wide range of applications.

(3) The promoter of the present invention is a human-derived promoter, which can be constructed in a eukaryotic expression vector and applied in human cells, providing a new approach for the treatment of human diseases.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions described in the manufacture conditions recommended by the manufacturer.

Example 1

PCR amplification of promoter sequence of human beclin 1 gene

First, the genomic DNA of the cells was extracted from the commercially available human normal liver cell line L02 (purchased from the Cell Bank of the Chinese Academy of Sciences), that is, in the collected cultured cell samples (5×10 ⁷ ), 10 times the volume of extraction buffer (10 mM Tris-HCl pH 8.0, 0.1M EDTA pH 8.0, 20 μg/mL RNase, 0.5% SDS), react at 37°C for 1 hour, add proteinase K to a final concentration of 100 μg/ml, and bathe in 50°C water for 3 hours, stirring from time to time. Cool to room temperature, add an equal volume of Tris saturated phenol, mix well and centrifuge at 12,000 rpm for 5 minutes, collect the supernatant, extract once more with phenol: chloroform and chloroform, and precipitate genomic DNA with 2 times the volume of absolute ethanol , the resulting genomic DNA was fully dissolved with an appropriate amount of TE and stored at 4°C.

After the genomic DNA of L02 cells was obtained, the PCR method was used to add 5'- AGA TCT AGA CGG GATTTA ACC AT-3' (SEQ ID NO: 2, the underlined part is the added BglII restriction site) and 5' -GGG AAG GGA CTC CAA TA-3' (SEQ ID NO: 3) are respectively the upstream and downstream primers to amplify the -644/+197 fragment of the beclin1 promoter.

The PCR reaction was carried out in a 25 μl system, including 1 μl L02 genomic DNA solution (100ng/ul), 1 μl dNTP mixture (10 mM for each dNTP), 1 μl each of upstream and downstream primers (10 μM), 2.5 μl 10×PCR buffer, 0.125 μl Taq enzyme. The PCR reaction conditions were: 94°C for 2 minutes; followed by 32 cycles, each cycle including 94°C for 1 minute, 55°C for 40 seconds, 72°C for 1 minute; 72°C for 10 minutes.

The fragment of beclin 1 promoter -644/+197 amplified by PCR was sequenced by the biological company, and its sequence was consistent with the result of human genome sequencing.

Example 2

Construction of human beclin 1 gene promoter eukaryotic expression vector

The beclin 1 promoter -644/+197 fragment amplified by PCR was forwardly or reversely inserted into the pMD18-T vector (purchased from TaKaRa Company) to obtain the plasmid pMD18-T-beclin1 containing appropriate restriction sites (-644/+197) and pMD18-T-beclin1-rev (+197/-644).

Use BglII and HindIII to digest pMD18-T-beclin1 (-644/+197) to obtain a fragment containing 841bp promoter sequence (SEQ ID NO: 1), and then insert the fragment into eukaryotic through BglII and HindIII restriction sites Expression vector pGL3-Basic (purchased from Promega Company) of the luciferase reporter gene 5' end upstream, thus completing the p(-644/+197) plasmid (i.e. containing the positively inserted SEQ ID NO: 1 sequence construction of the plasmid).

To complete the construction of the p(+197/-644) plasmid (i.e., the plasmid containing the sequence of SEQ ID NO: 1 inserted in reverse), the pMD18-T-beclin1-rev(+197/-644 ) to obtain a fragment containing 841bp promoter sequence, and then insert the fragment into pGL3-Basic through BglII and KpnI restriction sites.

For the construction of the p(-277/+197) plasmid (i.e., the plasmid containing the 368-841st position in the SEQ ID NO: 1 sequence), the p(-644/+197) plasmid was first digested with SmaI and Eco72I to obtain The vector can be blunt-ended by self-ligation.

Digest the p(-644/+197) plasmid with SacI, and the obtained vector can obtain the p(-58/+197) plasmid (that is, the plasmid containing positions 587-841 in the sequence of SEQ ID NO: 1) by self-ligation.

In addition, the fragment obtained by digesting the p(-644/+197) plasmid with SacI can also be inserted into the SacI restriction site on the pGL3-Basic vector, and the p(-644/-59) plasmid (ie Containing the plasmid of position 1-586 in the sequence of SEQ ID NO:1).

After the above operations, the beclin 1 promoter sequences of different lengths were cloned into the upstream of the 5' end of the luciferase reporter gene. The restriction endonucleases used in the above operations were all purchased from Dalian Takara Company. To ensure the complete digestion reaction, each 1 μg of plasmid DNA was treated with 10 units of restriction endonuclease at 37°C for more than 2 hours. The DNA restriction endonuclease reaction system contains 1-5 μg DNA, 1/10 of the total volume of 10× enzyme digestion buffer and an appropriate amount of DNA restriction endonuclease, and the reaction volume is generally 20 μl. The digested product was electrophoresed on a 1% agarose gel to separate the fragment and the carrier, and the target fragment and/or carrier was cut out from the gel under ultraviolet light, and purified using an agarose gel DNA recovery kit (Shanghai TIANGEN Biological Company) Get fragments and vectors.

The purified fragment and the vector were ligated with T4 ligase (Dalian Takara Company) at 16°C for more than 8 hours, and the ligation product was transformed into competent E. coli at 42°C for 90 seconds, and the transformed E. coli was spread on LB containing 100 μg/ml ampicillin Plates were incubated upside down at 37°C overnight. Pick a single clone the next day, expand and culture it in 3ml liquid LB containing 100μg/ml ampicillin at 37°C for 16 hours, take 1.5ml bacterial solution, centrifuge at 12,000rpm for 30 seconds, remove the supernatant, and replace it with 100μl pre-cooled solution P1 suspended bacteria. Add 200 μl of solution P2, mix gently, add 150 μl of pre-cooled solution P3 (see the QIAGEN Plasmid Midi Kit manual for the preparation of solutions P1, P2, and P3), mix well, and centrifuge at 12,000 rpm for 10 minutes. Take the supernatant, add an equal volume of phenol:chloroform:isoamyl alcohol solution (25:24:1 by volume), mix vigorously, and centrifuge at 12,000 rpm for 5 minutes. Take the upper aqueous phase and add twice the volume of 95% ethanol to precipitate DNA. After the precipitate was dried, it was dissolved in 20 μl of TE (pH 8.0) solution containing 20 μg/ml RNase A, and the RNA was digested in a 37°C water bath for 30 minutes, and then stored at -20°C.

Example 3

Activity identification of human beclin 1 gene promoter in L02 human immortalized normal liver cells

The preparation of ultrapure plasmids for cell transfection uses QIAGEN Plasmid Midi Kit and carries out plasmid column purification according to the method provided by the supplier:

1. Take 25-50ml of the cultured bacteria solution overnight (that is, the aforementioned transformed E. coli), centrifuge at 6,000rpm for 10 minutes, and collect the cells;

2. Resuspend the pellet in 4ml Buffer P1 (provided by QIAGEN Plasmid Midi Kit), add 4ml Buffer P2, mix by inverting gently, let stand at room temperature for 5 minutes, add 4ml pre-cooled BufferP3 (provided by QIAGEN Plasmid Midi Kit), and invert to mix Evenly, place on ice for 15 minutes;

Centrifuge at 20,000g for 30 minutes at 3.4°C, and immediately remove the supernatant containing plasmid DNA;

Centrifuge at 20,000g for 15 minutes at 4.4°C, and immediately remove the supernatant containing plasmid DNA;

5. Equilibrate the QIAGEN-tip100 column with 4ml Buffer QBT (provided by QIAGEN Plasmid Midi Kit), and the liquid flows out under the action of gravity;

6. Add the supernatant in step 4 to make it flow into the medium by gravity. After the liquid flows out, wash twice with 10ml Buffer QC (provided by QIAGEN Plasmid Midi Kit);

7. Add 5ml Buffer QF (provided by QIAGEN Plasmid Midi Kit) to elute DNA and collect it in a new centrifuge tube;

8. Add 0.7 volume (3.5ml) of isopropanol, mix well, centrifuge at 20,000g at 4°C for 30 minutes, carefully discard the supernatant, wash the precipitate with 75% ethanol, centrifuge at 15,000g for 10 minutes, and discard the supernatant;

9. After air-drying, dissolve the DNA in an appropriate amount of sterile deionized water, detect it by agarose gel electrophoresis, and measure the O.D. value for quantification.

Seed L02 cells in a 6-well plate at a density of 2×10 ⁵ /well. After 24 hours, purify p(-644/+197), p(-277/+197), p (-58/+197), p(-644/-59), p(+197/-644) plasmids (1 μg/well) and pGL3-Basic empty vector (1 μg/well) as a control and internal reference plasmid pRL respectively -null (0.5 μg/well) (purchased from Promega) to transfect cells together. Twenty-four hours after transfection, the cells were replaced with fresh medium. Luciferase activity was detected 48 hours after transfection.

The luciferase activity was measured using the Dual Reporter assay system kit (purchased from Promega), and carried out with reference to the method provided by the supplier. The luciferase substrate in the kit was dissolved in 10ml luciferase reaction buffer II, and after aliquoting Freeze at -80°C. Equilibrate at room temperature for more than half an hour before use. The stop reagent (Stop&Glo) was prepared immediately before use, and the Stop&Glo substrate was diluted 1/50 with Stop&Glo buffer. After the cells were rinsed with PBS, 100 μl of 1× lysis buffer was added to each well and left at room temperature for 10 minutes. After the suspension was aspirated, shake and mix well, centrifuge at 13,200 rpm for 2 minutes, and aspirate the supernatant to detect luciferase activity. 5 μl of supernatant was mixed with 25 μl of luciferase substrate solution, read by fluorescence counter (L ₁ ), added 25 μl of stop reagent to the measured mixture, and read by fluorescence counter after mixing (L ₂ ). The value of L ₁ /L ₂ is the luciferase activity of the corresponding reporter plasmid. The formulations of the materials or solutions used above can be found in the instructions provided in the kit.

The experimental results are shown in Figure 1, p(-277/+197) has the strongest activity, which is more than 30 times higher than the luciferase relative activity of the control empty vector.

The activity of p(-644/+197) was second, indicating that there may be an attenuator in the -644/-277 region of the beclin 1 promoter sequence.

The activity of p(-58/+197) was even lower, but there was still a significant difference compared to the empty vector.

There is no significant difference between p(-644/-59) and the empty vector, so the -58/+197 region contains the core region of the beclin 1 promoter.

p(-644/+197) has luciferase activity but p(+197/-644) has no luciferase activity, indicating that the beclin 1 promoter sequence conforms to the general rule that it has directionality with the promoter.

Example 4

The beclin 1 promoter is active in HepG2 and BEL-7404 liver cancer cells

Plasmids such as p(-644/+197) obtained in Example 2 (1 μg/well) and pGL3-Basic empty vector (1 μg/well) as a control were respectively together with the internal reference plasmid pRL-null (0.5 μg/well) BEL-7404 cells (human liver cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences) and HepG2 cells (human liver cancer cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were transfected as described in Example 3, and the luciferase activity was determined.

The experimental results are shown in Figure 2, and the results show that the beclin 1 promoter is active in both HepG2 and BEL-7404 liver cancer cells. The activity of p(-277/+197) was still the strongest compared with other expression vectors containing beclin 1 promoters of different lengths. In BEL-7404 cells (Fig. 2A), the relative luciferase activity of p(-277/+197) was more than 15 times higher than that of the control empty vector, and in HepG2 cells (Fig. 2B), p(-277/+ 197) the relative activity of luciferase was more than 80 times higher than that of the control empty vector.

In addition, through the conventional PCR method, the present inventors also obtained promoter fragments corresponding to the sequences at positions 365-839 and 372-840 in the sequence of SEQ ID NO: 1, and cloned the fragments forward into pGL3- In the SacI restriction site on the Basic vector, the recombinant vector was transformed into HepG2 and BEL-7404 liver cancer cells, and the expression activity of the promoter fragment directing luciferase was detected. As a result, it was found that the ability of the promoter fragment to direct the expression of luciferase was equivalent to that of the promoter fragment having the sequence at positions 368-841 in SEQ ID NO:1.

Example 5

The beclin 1 promoter is active in HEK-293T cells

Plasmids such as p(-644/+197) obtained in Example 2 (1 μg/well) and pGL3-Basic empty vector (1 μg/well) as a control were respectively together with the internal reference plasmid pRL-null (0.5 μg/well) HEK-293T cells (human embryonic kidney cells, purchased from the Cell Bank of Chinese Academy of Sciences) were transfected as described in Example 3, and luciferase activity was measured.

The experimental results are shown in Figure 3, and the results show that the beclin 1 promoter is also active in HEK-293T human embryonic kidney cells. The activity of p(-277/+197) was still the strongest compared with other expression vectors containing beclin 1 promoters of different lengths, and it was more than 20 times higher than the luciferase relative activity of the control empty vector.

In addition, through the conventional site-directed mutagenesis technique, the inventors also obtained a promoter fragment corresponding to the 368-841th sequence in the sequence of SEQ ID NO: 1, and wherein the 369th position is changed from g to c, and the fragment Forward cloning into the SacI restriction site on the pGL3-Basic vector, transforming the recombinant vector into HEK-293T cells, and detecting the expression activity of the luciferase directed by the promoter fragment. As a result, it was found that the ability of the promoter fragment to direct the expression of luciferase was equivalent to that of the promoter fragment having the sequence at positions 368-841 in SEQ ID NO:1.

Example 6

The beclin 1 promoter is active in MCF7 cells

Plasmids such as p(-644/+197) obtained in Example 2 (1 μg/well) and pGL3-Basic empty vector (1 μg/well) as a control were respectively together with the internal reference plasmid pRL-null (0.5 μg/well) MCF7 cells (human breast cancer cells, purchased from the Cell Bank of Chinese Academy of Sciences) were transfected as described in Example 3, and the luciferase activity was measured.

The experimental results are shown in Figure 4, and the results show that the beclin 1 promoter is also active in MCF7 human breast cancer cells. The activity of p(-277/+197) was still the strongest compared with other expression vectors containing beclin 1 promoters of different lengths, and was more than 50 times higher than the luciferase relative activity of the control empty vector.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> Human autophagy gene beclin 1 promoter sequence and its application

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gttgctgttg ttgttctgag atggagcctt gccctgtcgc ccaggctgga gtgcagtggc 180

ccgatctcgg ctcactgcaa cctccacctc ccaggttcaa gcgattctcc tgcctcagcc 240

tcccgagtag ctgggattaa gctgggatta taggcgtgcc ccaccacgcc cggctagttt 300

ttgtattttt agtagagacg gggtttcact gtgttggcca ggctggtctc gaactcctga 360

cctcacgtga tccgccctcc tcggcctccc caagtgctga gattacaggc gtgagccacc 420

gcgcccgcct ccccctgaat ttagagaata gcggagcctc cccattctct gcggccttgg 480

ctcctacact tcccgtggta accttgttca tccgctgaag cccgctgctt ttcccagccc 540

ggcctctggg ggccgctgcc gggcctgtga gcctgtggac caggagctcc tgctgccgtc 600

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Claims (9)

1. An isolated nucleic acid, characterized in that the nucleic acid sequence is selected from the group consisting of: The nucleic acid sequence of the sequence shown in positions 368-841, 365-839 or 372-840 in SEQ ID NO:1. 2. nucleic acid as claimed in claim 1, is characterized in that, described nucleic acid is the nucleic acid of sequence shown in No. 368-841 in SEQ ID NO:1. 3. A vector, characterized in that the vector contains the nucleic acid according to claim 1 as a promoter element, and the vector is a eukaryotic expression vector. 4. The vector according to claim 3, further comprising a target gene operably linked to the nucleic acid. 5. The carrier according to claim 4, wherein the target gene comprises: autophagy gene, luciferase gene, green fluorescent protein. 6. A genetically engineered host cell, characterized in that the cell is a eukaryotic cell, and: Containing the carrier of claim 3; or The exogenous nucleic acid of claim 1 and the target gene operably linked to the nucleic acid are integrated in its genome. 7. The host cell according to claim 6, wherein the cell is selected from the group consisting of: human immortalized normal liver cell L02, liver cancer cell HepG2, liver cancer cell BEL-7404, human embryonic kidney cell HEK-293T, or Human breast cancer cell MCF-7. 8. The use of the nucleic acid according to claim 1, characterized in that the nucleic acid is used as a promoter element to guide the high expression of the target gene. the 9. A method for highly expressing a gene of interest, characterized in that, the method comprises: (a) cultivating the host cell of claim 6 under conditions suitable for expression; and (b) Isolating the protein of interest from the culture. the

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