CN1216321A

CN1216321A - Cloning method, sequence and application of carcinoembryonic antigen promoter DNA

Info

Publication number: CN1216321A
Application number: CN 97114280
Authority: CN
Inventors: 王晓怀; 谢波; 江悦华
Original assignee: Department Of Medical Training Guangzhou General Hospital Guangzhou Military Region
Current assignee: Department Of Medical Training Guangzhou General Hospital Guangzhou Military Region
Priority date: 1997-10-31
Filing date: 1997-10-31
Publication date: 1999-05-12

Abstract

The present invention features that the DNA sequence is dissimilar from the carcinoembryonic antigen (CEA) promoter of colon cancer in that the +93 place is C rather than G base. In CEA secretary tumor gene therapy, the gene segment is recombined with therapeutic gene, and this makes it possible to achieve the effect of killing tumor cells selectively and keeping normal cells unaffected.

Description

The cloning process of carcinoembryonic antigen promoter DNA, sequence and uses thereof

The present invention relates to the cloning process of human carcinoembryonic antigen (CEA) gene promoter DNA, and nucleotidesequence, the purposes of this CEA specific promoter DNA.Belong to the cma gene field of engineering technology.

Using suicide gene (HSV-TK gene), antioncogene, apoptosis gene treatment tumour is the new development of current oncotherapy research, though obtained certain curative effect (CulverRW et al:Gene therapy for the treament of malignantbrain tumors with in vivo tumor transduction with the herpessiwplexthymidine kinase gene/gancidovir system.Hunan Genefherapy 1994 clinically; 5:3431), but the application of simple said gene, the specificity of shortage tumour has limited gene therapy range of application and result of treatment clinically.

Carcinomebryonic antigen (CEA) is people embryo a kind of serum protein in period, is mainly produced by the big intestinal epithelial cells secretion of embryo.Lost the proteic function of secretion carcinomebryonic antigen at big intestinal epithelial cells of ripening stage, but at tumour patients such as part large bowel cancer, lung cancer, carcinoma of the pancreas, mammary cancer, carcinomebryonic antigen albumen generation unconventionality expression (Boring C S, et al:Cancer stati-stics.CA-Cancer Journal-for Clinians, 42:19-38,1992).(Gold P and Fredman S O:Specific carcino-embryonic antigen of the human digestive systen since nineteen sixty-five is found CEA, J Exp Ked, 122:467,1965), CEA has become the tumor associated antigen the most widely of research in the world, its not only conventional diagnosis, differential diagnosis and curative effect and evaluating prognosis that is used for clinical CEA secretion property tumour, data shows that also CEA is a most promising target antigen in the human tumor specificity active immunity treatment recently.Determine that at present the expression of carcinoembryonic antigen is subjected to the regulation and control of the promotor of its upstream, believe that the unconventionality expression of expressing some relevant factors with CEA has activated the promotor of CEA gene again, so the unconventionality expression of CEA is arranged in CEA secretion property tumour.For carrying out the gene therapy of CEA secretion property tomour specific, clone CEA gene promoter DMA from colon cancer cell abroad, it is connected with therapeutic gene, make therapeutic gene localization and expression in tumour cell only specifically, reach the selective killing tumour cell, and the impregnable result of treatment of normal cell.Be connected with the HSV-TK gene as the CEA promoter gene, the TK gene is only expressed in CEA secretion property lung cancer (gland cancer) cell, do not express non-CEA secretion sexual cell, make lung carcinoma cell improve 1000 times of (Oski T et al:Gene therapy for carcinoembr-yoincantigen-producing human lungcacer cells by cell type-specificexpression of herpes siwplex virus thymidine kinasegene.CancerRes.54 to the susceptibility of medicine GCV, 5258,1994), bring new prospect for therapy of tumor.

The nucleotidesequence that the purpose of this invention is to provide a kind of people's cancer of the stomach CEA gene promoter dna clone method, cancer of the stomach CEA specific promoter DNA.Be characterized in: this dna sequence dna has the active necessary sequence of carcinoembryonic antigen promoter, and is simultaneously different with colorectal carcinoma CEA gene promoter, is C rather than G base (Schreme H et al.Nol Cell Biol, 1990 at+93; 10:2738; Richards CA et al.Hum Gene Ther 1995; 6:881-93); This dna clone is as a kind of escherichia coli plasmid, can increase in a large number intestinal bacteria,, use the different restriction enzyme site enzymes that specially design and be connected this gene clone DNA two ends to cut through simple purification, it can be downcut, and the new gene therapy carrier of orientable insertion.Under these segment DNA regulation and control, foreign gene can only just be expressed at the tumour cell of expressing CEA, and treating for the specific gene of tumours such as CEA secretion property cancer of the stomach provides a new gene element.

People's cancer of the stomach carcinoembryonic antigen promoter DNA cloning vector pCEAp of the present invention (is deposited at intestinal bacteria JN109 strain, the bacterial classification called after Eschertchia coli JN109/pCEAp that contains this plasmid, be stored in China Microbial Culture Preservation Commission common micro-organisms center on September 11st, 1997, the number of registering on the books is CCNCC No:0324) see and form Fig. 1 by following DNA element:

1, specific promoter--the carcinoembryonic antigen upstream regulatory sequence of CEA secretion property tumor cells expression;

2, escherichia coli plasmid skeleton (comprising acyl ammonia enzyme gene in intestinal bacteria replicon and the amicillin resistance β)--plasmid pGEM-3zf (+).

Main cell strain and original plasmid that the present invention is used to make up people's cancer of the stomach carcinoembryonic antigen promoter DNA clone are:

PGEN-3zf (+): be derived from magnificent company.

Human stomach cancer cell line GN803 cell is derived from Shanghai cell biological institute of Chinese Academy of Sciences cell bank.

Aforementioned people's cancer of the stomach carcinoembryonic antigen promoter DNA cloning process is as follows:

1. at first design primer.

Utilize Priwer software in computer, to design a pair of suitable primer,, introduced Sac I restriction enzyme site sequence (GAGCTC) at 5 of primer I ' end respectively, introduced Hind III restriction enzyme site sequence (AAGCTT) at 5 of primer II ' end for the ease of the clone,

The primer I: 5 '-AATTGAGCTCTCCATCCACCTTGCCGAA

The primer II: 5 '-GCTCAAGCTTACTCCATGGTCTCTGCTG

Primer is synthetic by Sangon Shanghai bio-engineering corporation.

2, from the conventional preparation dna profiling that separates of human stomach cancer cell line GN803.

3, pcr amplification: with extractive 803 cell DNAs is template, designs the synthetic primer with us and does pcr amplification, obtains single CEA promoter dna fragment and sees Fig. 2 (3).

4, CEA promotor clone: with pGEN-3zf (+) be cloning vector, with carrier with Sac I Hind III double digestion after, electrophoresis reclaims the enzyme section and breaks.The promoter dna fragment that pcr amplification obtains is used ethanol sedimentation behind same double digestion; In dna fragmentation: the ratio of carrier=3: 1, under the effect of T4 ligase enzyme in 14 ℃ of connections of spending the night, transfection bacterium JN109 host bacterium and bed board; With penbritin and indigo plant, the white dual screening of bacterium colony, obtain the CEA promoter DNA plasmid clone of purifying.

5, CEA promoter DNA sequencing analysis: by the gene promoter DNA that above-mentioned cloning process obtains, its sequence is as follows: ↓-156AAGATTTGTCT GAGGAACTGA AAATAGAAGG GAAAAAAGAG-116GAGGGACAAA AGAGGCAGAA ATGAGAGGGG AGGGGACAGA-76GGACACCTGA ATAAAGACCA CACCCATGAC CCACGTGATG-36CTGAGAAGTA CTCCTGCCCT AGGAAGAGAC TCAGGGCAGA+5GGGAGGAAGG ACAGCAGACC AGACAGTCAC AGCAGCCTTG+45ACAAAACGTT CCTGGAACTC AAGCTCTTCT CCACAGAGGA+85GGACAGACCA GACAGCAGAG ACCATGGAGT+115

↑+93

(base residing sequence numbering in carcinoembryonic antigen promoter DNA of the numeral arrow indication behind the arrow, the sequence numbering of its left side base of the numeral of no arrow.)

The main application of gene promoter DNA of the present invention is as follows:

The gene therapy carrier (as virus or plasmid vector) that orientable insertion is new, under these segment DNA regulation and control, exogenous therapeutic gene (as tumour suicide gene, cancer suppressor gene, apoptosis gene, cytokine, antisense nucleotide etc. such as TK) is only just expressed at the tumour cell of expressing CEA, can be used for the gene therapy of CEA secretion property tumour, particularly cancer of the stomach.

PCEAp plasmid provided by the invention is deposited with intestinal bacteria JN109, contains bacterial strain called after e. coli jm109/pCEAp (Eschertchia coli JN109/pCEApl of pCEAp plasmid.This bacterial classification can (1% tryptone.0.5% yeast extract, 1% NaCl) be cultivated amplification in containing the LB substratum of 50-100ug/ml penbritin.

Following embodiment explains preparation, evaluation and the characteristics of gastric carcinoma cells carcinoembryonic antigen promoter DAN clone pCEAp of the present invention, but does not mean that restriction content of the present invention.In these examples, human stomach cancer cell line is available from biological study institute of Chinese Academy of Sciences cell bank.Plasmid pGEN-3zf (+) is available from magnificent company.

Fig. 1 is the structure synoptic diagram of plasmid pCEAp.

Fig. 2 is restriction enzyme digestion and electrophoresis figure.

Fig. 3 is that the order-checking fluorogram is general.

Embodiment 1: the amplification and the preparation of cancer of the stomach CEA gene promoter dna fragmentation.

Utilize Primer software in computer, to design a pair of suitable primer,, add Sac I restriction enzyme site, Hind III restriction enzyme site respectively at two primers, 5 ' end for the ease of the clone.

The primer I: 5 '-AATTGAGCTCTCCATCCACCTTGCCGAA

The primer II: 5 '-GCTCAAGCTTACTCCATGGTCTCTGCTG

Human stomach cancer cell line GN803 cell, is cultivated in the 5%CO2 incubator at 37 ℃ through containing 1640 substratum of 10% calf serum, presses Stafford modification method (Blin W and O.W.Stafford:Nacleic Acid Res 1996; 3:2303) from well-grown 803 cells (about 1.3 * 10 of cultured continuously ⁷) the middle preparation dna profiling that separates, cut through the Hpal enzyme, with above design synthetic primer, adopt pcr amplification method (the Sally WE et al.Wature.1989:341:544. and the O ' Aquila K T et al Nucleic acid Res.1992:20 of warm start; 1717), carry out 30 circulations by following condition; 94.5 ℃ 1 minute → 60 ℃ 1 minute → 72 ℃ 2 minutes, the last circulation was extended 5 minutes for 72 ℃, promptly obtained CEA gene promoter DNA cloning fragment.Get a little and do gel electrophoresis, present the single band (Fig. 2 (3)) of 317bp.

Embodiment 2: cancer of the stomach CEA gene promoter dna clone sequencing analysis.

The order-checking of pcr amplification product: behind the promoter dna fragment purifying as template, press Sanger sequencing (Sanger F et al:Proc Natl Acad Sci 1977,74:5463) add fluorescein-labeled ddWTP, under the guiding of primer I, carry out pcr amplification, get in the amplified production adding automatic sequencer and check order.In reading the preface instrument automatically, can detect A, T, the different fluorescein wave mode of four kinds of colors of C, G, whole pattern is clear, be easy to read, once read 270 bases except that primer, with the CEA gene promoter of abroad having reported that clones from colon cancer cell with section (CEA gene promoter-156 to+115 sequence) sequence (Schrewe H et al.Cloningof the complet gene conveying cell typesp specific expression.NolCell Biol, 1990; 10:2738. and Richards CA et al.Tranwscrip-tionl regulatory seqwences of carcinoeabryonic antigen:Iden-tification and use with cytosine deauinase for tunor-specificgene therapy Hum Gene Ther 1995; 6:881) compare, + 93 external sequences is G, this sequence is C, homology is 99.63%, its order-checking fluorescence pattern is seen Fig. 3, and this promoter DNA sequencing result is as follows: ↓-156AAGATTTGTCT GAGGAACTGA AAATAGAAGG GAAAAAAGAG-116GAGGGACAAA AGAGGCAGAA ATGAGAGGGG AGGGGACAGA-76GGACACCTGA ATAAAGACCA CACCCATGAC CCACGTGATG-36CTGAGAAGTA CTCCTGCCCT AGGAAGAGAC TCAGGGCAGA+5GGGAGGAAGG ACAGCAGACC AGACAGTCAC AGCAGCCTTG+45ACAAAACGTT CCTGGAACTC AACCTCTTCT CCACAGAGGA+85GGACAGACCA GACAGCAGAG ACCATGGAGT+115

↑+93

Embodiment 3, the structure of CEA gene promoter DNA plasmid clone.

With pGEN → 3zf (+) is cloning vector, and with carrier Sacl, behind the Hind III double digestion, it is disconnected that electrophoresis reclaims the enzyme section.The promoter dna fragment (seeing embodiment 1) that pcr amplification obtains is used ethanol sedimentation behind same double digestion.In dna fragmentation: the ratio of carrier=3: 1, in 14 ℃ of connections of spending the night, transfection JN109 host bacterium also is laid on and contains penbritin and X-Gal LB agarose plate overnight incubation under the effect of T4 ligase enzyme, next day the picking white colony.With LB substratum (1% tryptone that contains penbritin, 0.5% yeast extract, 1%NaCl), cultivate amplification for 37 ℃, the extracting plasmid is used the Sac I, and Hind III enzyme is two to be cut, cut out nearly 300bp (containing a part primer) fragment (Fig. 2 (2)), and do not see this 300bp fragment (Fig. 2 (4) behind the contrast empty carrier double digestion! Confirm to clone successfully (Fig. 1).The present invention compared with prior art has following advantage and characteristics:

1, this clone have the CEA gene promoter activity must sequence area (90 to+60) and abroad from the isolating CEA gene promoter of colon cancer cell (Schrene H et al, Wol CellBiol, 1990; 10:2738; Richards Ca et al. Hum Gene Ther 1995; 6; 881-93) comparing, is G+93 external sequences, and this sequence is C.

2, the PCR primer of design has CEA promoter DNA high conservative and special sequence voluntarily, contains the Sac I again respectively, Hind III restriction enzyme site sequence, the amplification, order-checking and the clone that just do the CEA promoter DNA; And the amplification of specific band nothing but.

3, this dna clone is as a kind of escherichia coli plasmid, can increase in a large number intestinal bacteria, through simple purification, use the different restriction enzyme site enzymes that specially design and be connected this gene clone DNA two ends to cut, it can be downcut, and the new gene therapy carrier of orientable insertion, under these DNA regulation and control, foreign gene is only just expressed at the tumour cell of expressing CEA.Thereby reach the effect of specific killing CEA secretion property tumour.

Claims

1. cloning vector that contains people's cancer of the stomach carcinoembryonic antigen promoter DNA is characterized in that: be made up of following DNA element:

(1), specific promoter--the carcinoembryonic antigen upstream regulatory sequence of carcinomebryonic antigen (CEA) secretion property tumor cells expression;

(2), escherichia coli plasmid skeleton (comprising acyl ammonia enzyme gene in intestinal bacteria replicon and the amicillin resistance β).

2. the cloning process of DNA as claimed in claim 1 is characterized in that: created a pair of suitable substance P CR primer,

The primer I: 5 '-AATTGAGCTCTCCATCCACCTTGCCGAA,

The primer II: 5 '-GCTCAAGCTTACTCCATGGTCTCTGCTG),

This not only has carcinomebryonic antigen promoter DNA high conservative and special sequence to primer, and contains Sacl respectively, Hind III restriction enzyme site sequence.

3. as the sequence of claim 2 method cloned genes promoter DNA, it is characterized in that: have the active necessary sequence area (90 to+60) of carcinoembryonic antigen promoter, but compare from the isolating CEA gene promoter of colon cancer cell with prior art, at+93 is not G, but C: ↓-156AAGATTGTCT GAGGAACTGA AAATAGAAGG GAAAAAAGAG-116GAGGGACAAA AGAGGCAGAA ATGAGAGGGG AGGGGACAGA-76GGACACCTGA ATAAAGACCA CACCCATGAC CCACGTGATG-36CTGAGAAGTA CTCCTGCCCT AGGAAGAGAC TCAGGGCAGA+5GGGAGGAAGG ACAGCAGACC AGACAGTCAC AGCAGCCTTG+45ACAAAACGTT CCTGGAACTC AAGCTCTTCT CCACAGAGGA+85GGACAGACCA GACAGCAGAG ACCATGGAGT+115

↑+93

4. according to the purposes of claim 2 method clone's gene promoter with claim 3 sequence, it is characterized in that: as a kind of escherichia coli plasmid, can increase in a large number intestinal bacteria, through simple purification, use special design and be connected in and cut at the different restriction enzyme site enzymes at this gene clone DNA two ends, it can be downcut, and the new gene therapy carrier (as virus or plasmid vector etc.) of orientable insertion, under these segment DNA regulation and control, exogenous therapeutic gene is (as tumour suicide genes such as TK, cancer suppressor gene, apoptosis gene, cytokine, 1 tumour cell at expression CEA such as antisense nucleotide is just expressed, thereby can be used for CEA secretion property tumour, as cancer of the stomach, large bowel cancer, lung cancer, carcinoma of the pancreas, the gene therapy of malignant tumours such as mammary cancer, with purposes such as prevention of recurrence.