CN1966685A - Upstream transcription control element for human oesophagus cancer cell ezrin gene - Google Patents

Abstract

The invention involves gene expression regulatory, and discloses the upstream transcription regulatory domain of human esophageal carcinoma cells. The invention constructs a series of eukaryotic expression vectors by inserting ezrin gene upstream sequence into a vector containing reporter gene, transfecting mammalian cells for transient expression, identifying the important transcription regulatory elements of human ezrin gene upstream sequence in esophageal carcinoma cells concluding negative regulatory elements and enhancer sequences. The invention can be used for studying the expression regulatory mechanism of Ezrin in tumor cells and clinical therapy with Ezrin as the target, conduces to the study of pathogenesis of esophageal carcinoma on molecular level, and provides theoretical basis for preventing and treating esophageal carcinoma.

Description

The thin chest ezrin of human esophagus cancer upstream region of gene transcriptional regulatory element

Technical field

The present invention relates to gene expression regulation, specifically, relate to the transcriptional regulatory element of human esophagus cancer cell ezrin upstream region of gene.

Background technology

The esophageal carcinoma is China's common cancer, and its pathogenesis is still indeterminate.We show that the ezrin gene is one of esophagus cancer invasion metastasis related gene at nearest result of study.The eznn gene claims VIL2 again, be positioned karyomit(e) 6q25.2～q26, human ezrin gene DNA length is about 24kb, contain 13 exons, cDNA length is 1761bp, its expression product Ezrin albumen is one of ERM (ezrin-radixin-moesin) family member, mainly participates in being connected between the cytoskeleton and after birth in the cell, have the cellular form of keeping with motion, be connected functions such as adhesion molecule and conditioning signal transduction.More and more evidences shows the development of Ezrin modulate tumor.Shift with non-metastatic tumour clone and tissue sample in the comparing result of genetic expression show, significantly improve from the expression of Ezrin in the metastasis of cancer cell of rodent mammary gland, human pancreas and colon; Simultaneously, Ezrin is strong expression in multiple human tumor, and these tumours comprise osteosarcoma, melanoma, astrocytoma, carcinoma of the pancreas, lung cancer, carcinoma of endometrium etc.; Studies show that further suppressing Ezrin albumen can eliminate murine rhabdosarcoma and osteosarcoma cell, Ezrin is likely the key adjusting molecule of malignant tumour.Present research mainly concentrates in the proteic function of Ezrin, the cell molecule and the mechanism of action thereof with the Ezrin protein-interacting, and about the research of the upstream transcription expression and regulation mechanism of ezrin gene, does not appear in the newspapers as yet.

In eukaryote, a typical gene, its upstream is the dna sequence dna of one section core promoter, comprise apart from the about dna sequence dna in-40 to+50 scopes of transcription initiation site, core promoter is in conjunction with rna plymerase ii (Pol II) and cofactor (basic transcription mechanism) thereof, the location transcription initiation site is also controlled transcriptional orientation, and is adjoining or incitant and supressor are made and being replied at a distance in the pair cell.In most cases, core promoter is not directly regulated and is transcribed.Upstream near core promoter is one section control promotor, it be positioned at around the core promoter, a transcription initiation site hundreds of base pair with the DNA zone.Be arranged in upstream region of gene or downstream or be positioned at intron, be enhancer sequence or negative regulatory element apart from far away one section regulation and control of transcription initiation site zone.SDBP is being controlled the mechanism of transcribing with combining of control promotor, enhanser and negative regulatory element.

Summary of the invention

The object of the present invention is to provide a kind of human esophagus cancer cell ezrin upstream region of gene transcription regulatory region, determine the enhanser and the negative regulatory element of people ezrin gene.

Another object of the present invention is to provide the carrier that contains the said gene transcriptional regulatory element.

Further purpose of the present invention is to provide the host cell that contains above-mentioned carrier.

To achieve these goals, the present invention utilizes molecular biology software that the preceding upstream sequence (1759～+ 134) of human esophagus cancer cell ezrin gene translation initiation site (translation initiation site ATG be positioned at+135～+ 137) is analyzed, the average GC content of finding this zone is 70.15%, there are CpG island and more than 20 potential transcriptional regulatory element, there is multiple factor in prompting ezrin gene expression regulation in human esophagus cancer, therefrom find out crucial transcriptional control zone or controlling element, significant to illustrating in the human esophagus cancer cell ezrin expression of gene regulatory mechanism.

Among the present invention, the sequence called after EZ1.759 that contains human esophagus cancer cell ezrin upstream region of gene, its length is 1893bp, nucleotide sequence is shown in SEQ ID NO:1, kept the fragment between ezrin upstream region of gene-1759～+ 134, for one section sequence before the translation initiation site, in SEQ ID NO:1 ,+1 is transcription initiation site.

One of human esophagus cancer cell ezrin upstream region of gene negative regulatory element provided by the invention, its length is 212bp, nucleotide sequence has kept the fragment between ezrin upstream region of gene-1102～-891 shown in SEQ ID NO:2.

Another negative regulatory element of human esophagus cancer cell ezrin gene provided by the invention, its length is 329bp, nucleotide sequence has kept the fragment between ezrin upstream region of gene-1102～-774 shown in SEQ ID NO:5.

One of human esophagus cancer cell ezrin upstream region of gene enhanser provided by the invention, its length is 117bp, nucleotide sequence has kept the fragment between ezrin upstream region of gene-890～-774 shown in SEQ ID NO:3.

Another enhanser of human esophagus cancer cell ezrin gene provided by the invention, its length is 77bp, nucleotide sequence has kept the fragment between ezrin upstream region of gene-773～-697 shown in SEQ ID NO:4.

In order to differentiate ezrin gene transcription controlling element, need carry out deletion analysis to people ezrin upstream area of gene.In a deletion analysis, utilize the nested deletion technology, the digestion time of control exonuclease I II (ExoIII), make the ezrin upstream region of gene since 5 ' end-1759 places brachymemma gradually, obtaining a series of is the recombinant mammalian expressing vector of promotor with ezrin upstream region of gene different lengths fragment, utilize the Photinus pyralis LUC reporter gene, detect the transcriptional activity of these plasmids in human esophagus cancer cell EC109.For reducing the error that the cell cultures microenvironment is caused, with the plasmid pRL-TK that expresses renilla luciferase as internal reference, with above-mentioned nested deletion plasmid transfection simultaneously EC109 cell, measure the enzymic activity of Photinus pyralis LUC and renilla luciferase, with the ratio of the two, promptly the plain enzymic activity of relative fluorescence is represented the transcriptional activity of aim sequence.The result shows: the zone between ezrin upstream region of gene-1324～-696 is an important transcriptional activity control region, and the zone between-146～-32 is another important transcriptional activity control region.

In order further to differentiate the transcriptional control effect in ezrin upstream region of gene-1324～-696 zones, on the recombinant mammalian expressing vector that with ezrin upstream region of gene-1324～+ 134 sequences is promotor between right-1102～-774 the 329bp fragment delete, the plain enzymic activity of the relative fluorescence of plasmid improves as a result, show that-1102～-774 fragments have the effect of the transcriptional activity of inhibition, exist and transcribe negative regulatory element.In addition, the segmentation pcr amplification is carried out in right-1102～-697 zones, be connected on the plasmid that contains the SV40 promotor, the plain enzymic activity detected result of relative fluorescence shows, ezrin upstream region of gene-1102～-891 and-1102～-774 fragments have transcripting suppressioning action, and-890～-774 and-773～-697 fragments are transcribed enhancement.

Compared with prior art, the present invention has following beneficial effect: the present invention has identified Ezrin gene promoter for human esophagus cancer cell first, a series of eukaryon expression plasmids that are used to detect ezrin gene upstream sequence transcriptional regulatory element have been made up, the transfection mammalian cell transient expression has been determined the important transcriptional regulatory element of ezrin gene upstream sequence in human esophagus cancer cell.The present invention to research Ezrin in tumour cell expression and regulation mechanism and be that the clinical treatment of target spot is significant with Ezrin.To help to explore the pathogenesis of the esophageal carcinoma, for prevention of esophageal cancer provides new theoretical foundation from molecular level.

Description of drawings

Fig. 1 is for being the construction of recombinant plasmid schema of promotor with the ezrin gene upstream sequence;

Fig. 2 is the structure schema of ezrin gene upstream sequence 5 ' end nested deletion plasmid;

Fig. 3 is the PCR qualification result of ezrin gene upstream sequence 5 ' end nested deletion plasmid;

Fig. 4 is for being the construction of recombinant plasmid schema of enhanser or negative regulatory element with the ezrin gene upstream sequence;

Fig. 5 is for being the recombinant plasmid double digestion qualification result of enhanser with the ezrin gene upstream sequence;

Fig. 6 is the plain enzyme activity assay of the ezrin upstream region of gene relative fluorescence of 5 ' end nested deletion plasmid in the EC109 cell;

Fig. 7 is the plain enzyme activity assay of the relative fluorescence of the segmental recombinant plasmid of ezrin upstream region of gene deletion in the EC109 cell;

Fig. 8 is for the ezrin gene upstream sequence being the plain enzyme activity assay of the relative fluorescence of recombinant plasmid in the EC109 cell of enhanser or negative regulatory element;

Wherein, among Fig. 3, M1 is DNA Marker DL2000; M2 is DNA Marker100bp ladder; 1～12 is the nested deletion plasmid; 13 is control plasmid pGL3-Basic.

Among Fig. 5, M1 is DNA Marker DL2000; M2 is DNA Marker 100bpladder; 1 is pGL3-Promoter; 2 is pGLP/-1102～-891; 3 is pGLP/-890～-774; 4 is pGLP/-773～-697; 5 is pGLP/-1102～-774.

Among Fig. 6, (a) be the mode chart of ezrin gene upstream sequence (1759～-32) series disappearance recombinant plasmid (on the pGL3-Basic carrier); (b) be the plain enzymic activity of relative fluorescence of corresponding disappearance.Experiment is repeated 3 times, representational data in this shows from 3 experiments, and error line is represented standard deviation.

Among Fig. 7, (a) be the mode chart of the recombinant plasmid (on the pGL3-Basic carrier) of deletion ezrin upstream region of gene partial sequence; (b) be the plain enzymic activity of relative fluorescence of corresponding plasmid.Experiment is repeated 3 times, representational data in this shows from 3 experiments, and error line is represented standard deviation.

Among Fig. 8, ezrin gene upstream sequence segmentation amplified fragments is structured on the carrier pGL3-Promoter, and the plain enzyme assay experiment of the relative fluorescence of plasmid is repeated 3 times, representational data in this shows from 3 experiments, and error line is represented standard deviation.

Embodiment

Embodiment 1 contains the structure of the eukaryon expression plasmid of ezrin gene upstream sequence

1, the clone of people ezrin gene upstream sequence (1759～+ 134)

The people ezrin gene order translation initiation site ATG that document is delivered is positioned at+and 135～+ 137, designed and synthesized in view of the above and be used to increase segmental a pair of primers F 1 of people ezrin gene translation upstream from start codon 1893bp and R1, described primer sequence is as follows:

F1：

5 '-CGG GGTACC ^-1759AGTGAATGCTGTTGCTGCTCGTCTGGAAG-3 ' (underscore is Kpn I site)

R1：

5 '-CCC AAGC ^{+ 134} TTTCGGTTTCTGGTGAGTATCCTCGATCCC-3 ' (underscore is Hind III site)

Use this to primer, from human esophagus cancer cell EC109 genomic dna, go out the dna fragmentation of the about 1.9kb consistent with expection through pcr amplification.Reclaim the purpose fragment increased, behind the Kpn I/Hind III double digestion, and be connected through the carrier pGL3-Basic of Kpn I/Hind III double digestion (Promega company).PGL3-Basic is the expression vector that does not contain promotor before the Photinus pyralis LUC reporter gene.Sequence between sequencing result reference ezrin upstream region of gene-1759～+ 134 directed cloning to the upstream of firefly luciferase gene, this sequence called after EZ1.759, recombinant plasmid called after pEZ1.759.The construction of recombinant plasmid flow process as shown in Figure 1.

The sequencing result of human esophagus cancer cell ezrin gene upstream sequence EZ1.759 is seen SEQ IDNO:1.Compare with the GenBank known array, the Nucleotide homogeny is 99.79%.

2, hold the structure of nested deletion carrier based on the ezrin gene upstream sequence 5 ' of pEZ1.759

StuI is positioned at ezrin upstream region of gene-1447 place, and this restriction endonuclease can make DNA form smooth end, is further degraded by exonuclease ExoIII; Kpn I goes up restriction enzyme site for carrier pGL3-Basic, and this restriction endonuclease can make DNA form 3 ' terminal outstanding fragment, is not degraded by ExoIII.Plasmid pEZ1.759 through StuI/Kpn I double digestion, is cut through the ExoIII enzyme again.Get the enzyme of differential responses time and cut after product, with S1 nuclease excision single stranded DNA, Klenow mends flat terminal, the T4 dna ligase makes fragment from connecting, connect product transformed into escherichia coli JM109, carry out resistance screening and order-checking, obtain a series of ezrin gene upstream sequences and hold the eukaryon expression plasmid of brachymemma gradually from 5 ', the construction of recombinant plasmid flow process is referring to Fig. 2.By the upstream and downstream sequencing primer RVprimer3 of plasmid pGL3-Basic and the GLprimer2 primer that partners, recombinant plasmid is carried out PCR identify, the results are shown in Figure 3.The recombinant plasmid of all structures passes through sequence verification.

3, be used to identify the construction of recombinant plasmid of ezrin genetic enhancer or negative regulatory element

Restriction enzyme Pst I has two restriction enzyme sites to lay respectively at-1102 and-773 of ezrin upstream region of gene, plasmid pEZ1.324 cuts through Pst I enzyme, reclaim big segment, the T4 dna ligase makes fragment from connecting, and promptly obtains removing the segmental recombinant plasmid pEZ1.324D of 329bp between eznn upstream region of gene-1102～-773.The sequence of being deleted is seen SEQ ID NO:5.

The design primer carries out the segmentation amplification to the sequence between ezrin upstream region of gene-1102～-697, with the amplified fragments of different lengths BamH I/Sal I double digestion, be connected again with through the carrier pGL3-Promoter of BamH I/SalI double digestion (Promega company), make up the recombinant plasmid (seeing Table 1) that is used to detect ezrin genetic enhancer or negative regulatory element.PGL3-Promoter is the carrier for expression of eukaryon that contains the SV40 promotor before the Photinus pyralis LUC reporter gene.The whole structure flow process of this recombinant plasmid as shown in Figure 4, recombinant plasmid carries out double digestion with BamH I/Sal I to be identified, the results are shown in Figure 5.Described primer sequence is as follows:

The F2:(underscore is BamH I site)

5’-CGC GGATCC ^-1102CTGCAGGCGCCGGGTGAGGCGTGC-3’

The F3:(underscore is BamH I site)

5’-CGC GGATCC ^-890TCCCCTCAGGTCTCTCCCGAAGGAAACGCG-3’

The F4:(underscore is BamH I site)

5’-CGC GGATCC ^-773CTGCAGCCGCGGGGCAACGGTTGCT-3’

The R2:(underscore is Sal I site)

5’-ACGC GTCGAC ^-891CCCGGCGCCGAGGGGAAGGTCGC-3’

The R3:(underscore is Sal I site)

5’-ACGC GTCGAC ^-774TCACAACCGTCAAGCCTTTGAGAAACTCTTTCAAAAACTGCAACCGC-3’

The R4:(underscore is Sal I site)

5’-ACGC GTCGAC ^-697GCTGCCAGGAAGCCCGTGAGAAGCCGAG-3’

Table 1 is the construction of recombinant plasmid of enhanser or negative regulatory element with the ezrin gene upstream sequence

The plasmid title	Primer	Expanding fragment length (bp)	The fragment position
				pGLP/-1102～-891 pGLP/-890～-774 pGLP/-773～-697 pGLP/-1102～-774	F2/R2 F3/R3 F4/R4 F2/R3	212 117 77 329	-1102～-891 -890～-774 -773～-697 -1102～-774

Embodiment 2 cell cultures, DNA transfection and the activation analysis of human esophagus cancer cell ezrin gene upstream sequence

Extract experiment plasmid, control plasmid pGL3-Basic and the internal reference plasmid pRL-TK that needs transfection with QIAprep Spin Miniprep Kit, measure its content and purity.Experiment plasmid and the control plasmid of getting above-mentioned expression Photinus pyralis LUC are diluted to 100ng/ μ l with Buffer EB (10mmol/LTrisCl, pH 8.5), and the internal reference plasmid pRL-TK that expresses renilla luciferase is diluted to 20ng/ μ l with Buffer EB.To test plasmid and mix by 50: 1, be i.e. 1 μ g: 20ng (10 μ l: 1 μ l) with the internal reference plasmid.

The routine of the carrying out human esophagus cancer cell EC109 cultivation of going down to posterity is inoculated in 24 well culture plates every hole 0.5ml.The cell full scale reaches at 50%～60% o'clock and promptly can be used for transfection behind the 24h.

Transfection method is as follows: in Bechtop, get some of 7ml glass centrifuge tubes, carry out mark, add 199 basic culture solutions and 4.5 μ l Fugene6 transfection reagents (Roche company) that 70.5 μ l contain 12.5mmol/L HEPES, vortex 1sec places 5min under the room temperature.Add corresponding plasmid mixed solution 16.5 μ l again, add 16.5 μ l Buffer EB in the blank pipe, vortex 1sec places 15min under the room temperature.24 orifice plates are carried out mark, and each sample is established 3 parallel holes, and every hole adds 28 μ l transfection liquid, shakes up gently, in 37 ℃ of 5% CO ₂Continue to cultivate 48h in the incubator, harvested cell carries out the detection of two luciferase reporter genes.Experiment is repeated 3 times.

The detection of two luciferase reporter genes adopts the Dual-LuciferaseReporter Assay System of Promega company to analyze.Transfection is had the attached cell of target DNA wash once with 1 * PBS, remove most raffinate, add the freshly prepared 1 * PLB of 100 μ l, under the room temperature, thermal agitation 15min is with lysing cell on the shaking table.Getting the above-mentioned cell pyrolysis liquid adding of 20 μ l contains in the 1.5ml centrifuge tube of 100 μ l LARII solution, mensuration firefly luciferase activity, in same centrifuge tube, add the freshly prepared 1 * STOP liquid of 100 μ l again, measure renilla luciferase (internal reference) activity, computed in software according to the preparation of TD20/20 type luxmeter goes out respectively to organize relative intensity of fluorescence (Photinus pyralis LUC/renilla luciferase), represents the plain enzymic activity of relative fluorescence with this.Each organizes equal calculating mean value of experimental data and standard deviation, with x ± s.Use the SPSS14.0 statistical software to whether having significant difference to carry out the t check between each group experimental data.

1, the evaluation of ezrin gene upstream sequence transcription regulatory region

Ezrin gene upstream sequence (1759～-32) is seen Fig. 6 from the mode chart of 5 ' end nested deletion plasmid (on the pGL3-Basic carrier) and the plain enzymic activity of relative fluorescence of corresponding disappearance, and the group difference significance analysis sees Table 2.

The plain enzymic activity of relative fluorescence of table 2 ezrin gene upstream sequence 5 ' end nested deletion plasmid

Numbering	Plasmid	The plain enzymic activity of relative fluorescence	The P value
				1 2 3 4 5 6 7	pEZ1.759 pEZ1.324 pEZ0.696 pEZ0.213 pEZ0.146 pEZ0.032 pGL3-Basic	130.8±13.56 152.3±17.97 92.73±12.23 89.54±12.43 82.09±8.411 4.916±1.504 0.254±0.018	0.174(1vs2) 0.009(2vs3) 0.768(3vs4) 0.439(4vs5) 0.282(3vs5) 0.003(5vs6)

In human esophagus cancer cell, the 1893bp sequence of ezrin gene translation upstream from start codon has tangible transcriptional activity.The plain enzymic activity difference of relative fluorescence does not have significance (P＞0.05) between plasmid pEZ1.759 and pEZ1.324 group, the plain enzymic activity difference of relative fluorescence has highly significant (P＜0.01) between pEZ1.324 and pEZ0.696 group, there are no significant (P＞0.05) for the plain enzymic activity difference of relative fluorescence between pEZ0.696 and pEZ0.213 group, between pEZ0.696 and pEZ0.146 group and between pEZ0.213 and pEZ0.146 group, and the plain enzymic activity difference of relative fluorescence has highly significant (P＜0.01) between pEZ0.146 and pEZ0.032 group.When the ezrin gene upstream sequence is truncated to-1324 and when-696 are truncated to-146 from-1759, transcriptional activity does not have considerable change, illustrate the ezrin upstream region of gene between-1759～-1324 and the dna sequence dna between-696～-146 transcriptional activity is not had obvious influence, be non-transcribed activity regulation district.When the ezrin gene upstream sequence when-1324 are truncated to-696, transcriptional activity descends 39.1%, but the transcriptional activity of comparing with control plasmid pGL3-Basic is still very high, be presented at the important transcriptional regulatory element of this regional influential transcriptional activity, have ezrin genetic enhancer sequence probably.When the ezrin gene upstream sequence when-146 are truncated to-32, transcriptional activity descends 94.0%, near control plasmid pGL3-Basic, the crucial transcriptional regulatory element that the remarkably influenced transcriptional activity is arranged in this zone is described, is ezrin gene regulating promoter region.

2, the evaluation of ezrin upstream region of gene enhanser or negative regulatory element

The mode chart of the recombinant plasmid (on the pGL3-Basic carrier) of deletion ezrin upstream region of gene partial sequence and the corresponding plain enzymic activity of relative fluorescence are seen Fig. 7, and the group difference significance analysis sees Table 3.

The plain enzymic activity of relative fluorescence of the recombinant plasmid of table 3. deletion ezrin upstream region of gene partial sequence

Numbering	Plasmid	The plain enzymic activity of relative fluorescence	The P value
				1 2 3 4	pEZ1.324 pEZ1.324D pEZ0.890 pEZ0.696	77.13±6.835 93.35±2.519 69.56±6.135 44.43±4.419	0.224(1vs3) 0.018(1vs2) 0.005(3vs4) 0.002(1vs4)

The plain enzymic activity difference of relative fluorescence does not have significance (P＞0.05) between plasmid pEZ1.324 and pEZ0.890 group.The plain enzymic activity difference of relative fluorescence between pEZ1.324 and pEZ1.324D group has significance (P＜0.05), in the sequence between the deletion-1102～-774 on the pEZ1.324 basis transcriptional activity is improved, sequence may be to having transcribed the negative regulation effect between-1102～-774 in prompting.The plain enzymic activity difference of relative fluorescence between pEZ0.890 and pEZ0.696 group has highly significant (P＜0.01), and the transcriptional activity that makes of sequence descends between the deletion-890～-697, and sequence has enhancement to transcriptional activity between the prompting-890～-697.

In order further to analyze the influence of ezrin upstream region of gene fragment-1102～-891 ,-890～-774 ,-773～-697 and-1102～-774 pairs of transcriptional activities, utilize PCR method to clone above-mentioned fragment, the purpose fragment is connected to plasmid pGL3-Promoter goes up construction recombination plasmid.Recombinant plasmid pGLP/-1102～-891 are for being connected with ezrin upstream region of gene fragment-1102～-891 on plasmid pGL3-Promoter, other plasmid name similarly.Utilize the plain enzymic activity of relative fluorescence to analyze inhibition or the enhancement of these dna fragmentations to transcriptional activity.Experimental result is seen Fig. 8 and table 4.

Table 4 ezrin gene upstream sequence is to the regulating and controlling effect of transcriptional activity

Numbering	Plasmid	The plain enzymic activity of relative fluorescence	The P value
				1 2 3 4 5	pGL3-Promoter pGLP/-1102～-891 pGLP/-890～-774 pGLP/-773～-697 pGLP/-1102～-774	22.07±0.822 14.68±1.562 24.96±0.820 36.18±1.056 18.84±1.129	0.002(1vs2) 0.013(1vs3) 0.000(1vs4) 0.016(1vs5)

Be connected with the segmental plasmid of ezrin upstream region of gene (pGLP/-1102～-891, pGLP/-890～-774, pGLP/-773～-697, pGLP/-1102～-774) compare with control plasmid pGL3-Promoter, the plain enzymic activity difference of the relative fluorescence between group has significance or highly significant (P＜0.05 or P＜0.01).As can be seen, the dna sequence dna between-1102～-697 is divided into 3 sections, the fragment between-1102～-891 makes the plain enzymic activity of relative fluorescence reduce by 33.5%, and transcriptional activity is had restraining effect; Fragment between-890～-774 makes the plain enzymic activity of relative fluorescence raise 13.1%, and transcriptional activity is had weak enhancement; Fragment between-773～-697 makes the plain enzymic activity of relative fluorescence raise 63.9%, and transcriptional activity is had enhancement.And-1102～-774 fragments make the plain enzymic activity of relative fluorescence reduce by 14.6%, show restraining effect to transcriptional activity, this experimental result with the front is consistent, and promptly plasmid pEZ1.324 plain enzymic activity of relative fluorescence when the sequence of deleting between-1102～-774 raises.

To sum up, the dna sequence dna that can determine long 406bp between ezrin upstream region of gene-1102～-697 is important transcriptional activity control region.The 212bp fragment is an ezrin negative gene controlling element between-1102～-891; The 117bp fragment is the weak enhancer sequence of ezrin gene between-890～-774; The 77bp fragment is an ezrin genetic enhancer sequence between-773～-697.

The upstream transcription control element for human oesophagus cancer cell ezrin gene sequence table

SEQUENCE LISTING

＜110〉Medical College of Shantou University

＜120〉upstream transcription control element for human oesophagus cancer cell ezrin gene

<130>

<160>1

<170>Patent In version 3.2

<210>1

<211>1893

<212>DNA

＜213〉human esophagus cancer cell (HUMAN ESOPHAGEAL CANCER CELLS)

<400>1

-1759 AGTGAATGCT GTTGCTGCTC GTCTGGAAGC CAGACGTTGA GAACCCCTTC

-1709 TAGAGTGAGC TCTCCCGCAG CAAATTCTAC TGGCCCCCAA AGTATGTGTT

-1659 TTGTGTGTCT TAAAAATTTG TTGAGAACCA TTAGCAAAAA AACAAACAAA

-1609 AAAACTTAAT TCCTAGAATT CCAGAGAAAT CCCATGGAGC TTTTTGCCAG

-1559 TCACGTCAAG AGAGGCCACA AACGTGCCAC TTAACCAGAG CTTCGGAAAG

-1509 GCGGCGGCTG GGCCGGCCAC GTGCACCGAG ACTCGGGGCC AGGTGCAGCC

-1459 GCCCCAGGGC CGAGGCCTCG GAACTGGCCC CCGGTCCCGG CCCCAAGCGG

-1409 TCCAGCGATT CCCCCAAGCC GTCCGCCCCT CCAGATTTAT TTACGTTTTC

-1359 CTGACTTCCC CCTGCCCGCT GTGGGACAAA CAGCCTCCCC ACTTGCATCT

-1309 GCGAGGGGAG TAGCGCGCAC TTCCGCCAAG TTCCGCCCCC ACCCAGCCCG

-1259 AGGCCCGGCT GCCGCCATCT TGCGGGGGGC GCACCTCACA GGTCGGGAGC

-1209 TGGGCGGGAA GGGGCGTGGT CCCGGGACCC GCCCCGCCGG GGCTTTTGGG

-1159 AGCGCGGGCA GCGAGCGCAC TCGGCGGACG CAAGGGCGGC GGGGAGCACA

-1109 CGGAGCACTG CAGGCGCCGG GTGAGGCGTG CGGCGGCCGG GGTCGGGACG

-1059 GGGGTTCTGG GCGGGGGGTT CCTGGTGGAG GGCCCGGGCG GGCGGCGGGG

-1009 TTCGGCGGCA GGTGCGGCGG GCAGCCTAGG GGGCGCGGCG CGGGGTTCTC

-959 GCCCGGCACC CCCGGGGCAG GTGGAGCTGA GCCGGCCCGC GGCCCCGCGA

-909 CCTTCCCCTC GGCGCCGGGT CCCCTCAGGT CTCTCCCGAA GGAAACGCGG

-859 AGCCTGGGTG CCTGGGCGCC GTCCCTCGGC GGCTCCCGAG CGGTTGCAGT

-809 TTTTGAAAGA GTTTCTCAAA GGCTTGACGG TTGTGACTGC AGCCGCGGGG

-759 CAACGGTTGC TACACAAAGT GAAACTTGCC GAGTGCTCGG CTTCTCACGG

-709 GCTTCCTGGC AGCCCCGGGA AGTTCCTCGG CGGACCCCGA GCCCGCGCCC

-659 CCTCTCCACG GATCCCTCCC CAGCGAGTGC CCCCCCGCCC GCCCTGTGCC

-609 CCCTCTCCCC TGACCCCTCC CTGTCGGGTG CCCCGCGGGC TCGCGCTGGC

-559 TGTCCTGGGA CTCCTTCCTC CTAGGTGTTC CTCCTGCCCC TCGCCCTCTC

-509 TCTCCCAGGC GCGCGCTCCC TCTCCCCGGG CCTTTCCCCG CCGGGTATCC

-459 CTGGGCCCGC GCCCCGTCTT CTCCGCCTCT CTCCGCTGGG TGCACCTCGA

-409 GTGTCCCCCA GACCCCTCCC CGCCCGGCCG GCGCTCTCTC CCCTGACCCT

-359 CCTGGCCGAG TGTTCCCCGG GGCCCGCGCC CCCTCCCCCC GATCCTCCCC

-309 ACTGAGTGTT CCCCCTGCCC TCTCTCTCCC GGGCCTGCGC CCCCCACCAG

-259 CCCCTTCATG CTGGGGGTCC CCTGGGTGCG CACCCCCTCT CCTCGGACCC

-209 ACCCCCAACT GGGGGGCACC TCCAGTGCCC GCCGGCTGCC CCTTGGGCGC

-159 GCGCCCCCGC TCTCGGGCGC CTCCTCGCCG GGGGCCCGGC CCGGCCCCGC

-109 CCCGCCCGTG CCCCCTCCCC ATGCCCGCAG TGCTGGGCGG GGCGCTGACT

-59 CACCCGGGCC CGGGCTGGCC GGTTCTTAAG CGGCAGCGCG CTGCGGGCGC

-9 CGAGTGTCGG GCGCGGCAGG AGGACGAGGC AGGGCGGGCG GGCGCTCTAA

+42 GGGTTCTGCT CTGACTCCAG GTTGGGACAG CGTCTTCGCT GCTGCTGGAT

+92 AGTCGTGTTT TCGGGGATCG AGGATACTCA CCAGAAACCG AAA

<210>2

<211>212

<212>DNA

＜213〉human esophagus cancer cell (HUMAN ESOPHAGEAL CANCER CELLS)

<400>2

-1102 CTGCAGGCGC CGGGTGAGGC GTGCGGCGGC CGGGGTCGGG ACGGGGGTTC

-1052 TGGGCGGGGG GTTCCTGGTG GAGGGCCCGG GCGGGCGGCG GGGTTCGGCG

-1002 GCAGGTGCGG CGGGCAGCCT AGGGGGCGCG GCGCGGGGTT CTCGCCCGGC

-952 ACCCCCGGGG CAGGTGGAGC TGAGCCGGCC CGCGGCCCCG CGACCTTCCC

-902 CTCGGCGCCG GG

<210>3

<211>117

<212>DNA

＜213〉human esophagus cancer cell (HUMAN ESOPHAGEAL CANCER CELLS)

The upstream transcription control element for human oesophagus cancer cell ezrin gene sequence table

<400>3

-890 TCCCCTCAGG TCTCTCCCGA AGGAAACGCG GAGCCTGGGT GCCTGGGCGC

-840 CGTCCCTCGG CGGCTCCCGA GCGGTTGCAG TTTTTGAAAG AGTTTCTCAA

-790 AGGCTTGACG GTTGTGA

<210>4

<211>77

<212>DNA

＜213〉human esophagus cancer cell (HUMAN ESOPHAGEAL CANCER CELLS)

<400>4

-773 CTGCAGCCGC GGGGCAACGG TTGCTACACA AAGTGAAACT TGCCGAGTGC

-723 TCGGCTTCTC ACGGGCTTCC TGGCAGC

<210>5

<211>329

<212>DNA

＜213〉human esophagus cancer cell (HUMAN ESOPHAGEAL CANCER CELLS)

<400>5

-1102 CTGCAGGCGC CGGGTGAGGC GTGCGGCGGC CGGGGTCGGG ACGGGGGTTC

-1052 TGGGCGGGGG GTTCCTGGTG GAGGGCCCGG GCGGGCGGCG GGGTTCGGCG

-1002 GCAGGTGCGG CGGGCAGCCT AGGGGGCGCG GCGCGGGGTT CTCGCCCGGC

-952 ACCCCCGGGG CAGGTGGAGC TGAGCCGGCC CGCGGCCCCG CGACCTTCCC

-902 CTCGGCGCCG GGTCCCCTCA GGTCTCTCCC GAAGGAAACG CGGAGCCTGG

-852 GTGCCTGGGC GCCGTCCCTC GGCGGCTCCC GAGCGGTTGC AGTTTTTGAA

-802 AGAGTTTCTC AAAGGCTTGA CGGTTGTGA

<210>6

<211>

<212>DNA

＜213〉primer

<400>6

F1：5’-CGGGGTACCAGTGAATGCTGTTGCTGCTCGTCTGGAAG-3’

R1：5’-CCCAAGCTTTCGGTTTCTGGTGAGTATCCTCGATCCC-3’

<210>7

<211>

<212>DNA

＜213〉primer

<400>7

F2：5’-CGCGGATCCCTGCAGGCGCCGGGTGAGGCGTGC-3’

F3：5’-CGCGGATCCTCCCCTCAGGTCTCTCCCGAAGGAAACGCG-3’

F4：5’-CGCGGATCCCTGCAGCCGCGGGGCAACGGTTGCT-3’

R2：5’-ACGCGTCGACCCCGGCGCCGAGGGGAAGGTCGC-3’

R3：5’-ACGCGTCGACTCACAACCGTCAAGCCTTTGAGAAACTCTTTCAAAAACTGCAACCGC-3’

R4：5’-ACGCGTCGACGCTGCCAGGAAGCCCGTGAGAAGCCGAG-3’

Claims (8)

1, a kind of upstream transcription control element for human oesophagus cancer cell ezrin gene, its nucleotide sequence is selected from down group:

(1) nucleotide sequence shown in the SEQ ID NO:2,

(2) nucleotide sequence shown in the SEQ ID NO:3, or

(3) nucleotide sequence shown in the SEQ ID NO:4.

2, upstream transcription control element for human oesophagus cancer cell ezrin gene as claimed in claim 1 is characterized in that containing the controlling element that at least one controls ezrin genetic expression.

3, a kind of carrier, it contains the described upstream transcription control element for human oesophagus cancer cell ezrin gene of claim 1.

4, carrier as claimed in claim 3 is characterized in that, it also contains the foreign gene that described upstream transcription control element for human oesophagus cancer cell ezrin gene is operably connected.

5, carrier as claimed in claim 4 is characterized in that, described foreign gene is a reporter gene.

6, a kind of host cell is characterized in that, it contains the described carrier of claim 3.

7, host cell as claimed in claim 6 is characterized in that, described host cell is a mammalian cell.

8, host cell as claimed in claim 7 is characterized in that, described mammalian cell is people's a cell.

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