CN1532281A

CN1532281A - RFecombination of carcino-emhyyonic antigen gene fragment and gene vaccine

Info

Publication number: CN1532281A
Application number: CNA031112528A
Authority: CN
Inventors: 岩谭; 谭岩; 熊伟; 方艳秋; 段秀梅; 许淑芬; 姜艳芳; 宋艳
Original assignee: First Hospital Jinlin University
Current assignee: First Hospital Jinlin University
Priority date: 2003-03-21
Filing date: 2003-03-21
Publication date: 2004-09-29

Abstract

The present invention belongs to the field of biotechnology, and aims at providing one 1.1 Keyboard recombinant CEA gene segment, which is prepared by means of gene recombining technology and contains CEA signal peptide coding region, region III sequence and no functional areas I and II of CEA gene, and the CEA gene vaccine. During the expression, the CEA gene vaccine can eliminate the interference between the repeated sequences to strengthen immunogeenicity, activate effectively T cell and introduce specific anti-tumor immune response. The prepared gene segment is relatively short, and is easy to recombine with other immune strengthening gene and tumor gene to prepare united or multivalent vaccine with ever high antitumor effect.

Description

Segmental reorganization of carcinoembryonic antigen and gene vaccine

Technical field:

The present invention be a reorganization the CEA gene fragment and a kind of be the CEA naked DNA gene vaccine that foreign gene makes up with this fragment, belong to biological technical field.

Background technology:

(Carcinoembryonic antigen is by the tumour in human body entoderm source expressed important tumor associated antigen and generally acknowledged in the world tumor markers CEA) to carcinomebryonic antigen.The CEA positive tumor comprises 90% digestive tract cancer, 70% lung cancer and 50% mammary cancer etc., is in first of the malignant tumour M ﹠ M, and the serious threat mankind's life and health is badly in need of seeking a kind of effective ways of thorough radical cure clinically.

Tumor gene vaccine combines immunotherapy of tumors and gene therapy two aspect contents, is one of the most potential current oncotherapy means.

But in its development, should be noted that and solve following key issue: 1. screen and identify tumour antigen; 2. the immunogenicity of enhancement antigen; 3. effective activated T cell is removed immunosuppression; 4. improve the security of vaccine.At present in the world development be that the gene vaccine of target spot mainly contains vector-viral vaccine and naked DNA vaccine with CEA.Though virus vector can strengthen the immunogenicity of CEA, and the angtigen presentation effect is arranged; But naked DNA vaccine is because use eukaryon expression plasmid as carrier, with the genome conformity of host cell the time, inserts sudden change and possibility of infection, is more suitable in preparation human body vaccine than virus vector.But up to now, what use in the CEA naked DNA vaccine is the full cDNA sequence of CEA, and fragment is longer, and immunocompetence is not high.

Find according to result of study, in the structure of dna vaccination, remove the partial function encoding sequence, can strengthen the antigenicity of vaccine some viruses, bacterium, protozoon and tumour immunity.The about 3.1Kb of known CEAcDNA total length mainly is made of similar repeating unit (I, II, III district) on signal peptide encoding gene and three structures, encodes and contains 686 amino acid whose CEA molecules.By three sequential analyses that repeat function unit carries out are shown that wherein 70% aminoacid sequence is guarded, this disappearance that just means one or two repeating unit will can not influence the antigenicity of proteins encoded.

Technology contents:

The object of the present invention is to provide one section utilization gene recombination technology I, II functional zone preparation, that do not comprise the CEA gene, and only contain the reorganization of 1.1Kb CEA gene fragment and the CEA gene vaccine of CEA signal peptide coding region and III region sequence.

Carcinoembryonic antigen fragment of the present invention comprises the 1.1Kb CEA portion gene of CEA signal peptide and III district encoding sequence.

The nucleotide sequence and the open reading frame of 1091 bp CEA gene fragments

1 CAGAGGAATT?CAGAGCAGAC?AGCAGAGACC? ATG?GAGTCTC?CCTCGGCCCC

TCCCCACAGA

[ORF]?M E S P S A P P H R

61?TGGTGCATCC?CCTGGCAGAG?GCTCCTGCTC?ACAGCCTCAC?TTCTAACCTT?CTGGAACCCG

W C I P W Q R L L L T A S L L T F

W N P

121?CCCACCACTG?CCAAGCTCAC?TATTGAATCC?ACGCCGTTCA?ATGTCGCAGA?GGGGAAGGAG

P T T A K L T I E S T P F N V A E

G K E

181?GTGCTTCTAC?TTGTCCACAA?TCTGCCCCAG?CATCTTTTTG?GCTACAGCTG?GTACAAAGGT

V L L L V H N L P Q H L F G Y S W

Y K G

241?GAAAGAGTGG?ATGGCAACCG?TCAAATTATA?GGATATGTAA?TAGGAACTCA?ACAAGCTACC

E R V D G N R Q I I G Y V I G T Q

Q A T

301?CCAGGGCCCG?CATACAGTGG?TCGAGAGATA?ATATACCCCA?ATGCATCCCT?GCTGATCCAG

P G P A Y S G R E I I Y P N A S L

L I Q

361?AACATCATCC?AGAATGACAC?AGGATTCTAC?ACCCTACACG?TCATAAAGTC?AGATCTTGTG

N I I Q N D T G F Y T L H V I K S

D L V

421?AATGAAGAAG?CAACTGGCCA?GTTCCGGGTA?TACTCGAGGC?TGCCCAAGCC?CTCCATCTCC

N E E A T G Q F R V Y S R L P K P

S I S

481?AGCAACAACT?CCAAACCCGT?GGAGGACAAG?GATGCTGTGG?CCTTCACCTG?TGAACCTGAG

S N N S K P V E D K D A V A F T C E

P E

541?GCTCAGAACA?CAACCTACCT?GTGGTGGGTA?AATGGTCAGA?GCCTCCCAGT?CAGTCCCAGG

A Q N T T Y L W W V N G Q S L P V S

P R

601?CTGCAGCTGT?CCAATGGCAA?CAGGACCCTC?ACTCTATTCA?ATGTCACAAG?AAATGACGCA

L Q L S N G N R T L T L F N V T R N

D A

661?AGAGCCTATG?TATGTGGAAT?CCAGAACTCA?GTGAGTGCAA?ACCGCAGTGA?CCCAGTCACC

R A Y V C G I Q N S V S A N R S D P

V T

721?CTGGATGTCC?TCTATGGGCC?GGACACCCCC?ATCATTTCCC?CCCCAGACTC?GTCTTACCTT

L D V L Y G P D T P I I S P P D S

S Y L

781?TCGGGAGCGA?ACCTCAACCT?CTCCTGCCAC?TCGGCCTCTA?ACCCATCCCC?GCAGTATTCT

S G A N L N L S C H S A S N P S

P Q Y S

841?TGGCGTATCA?ATGGGATACC?GCAGCAACAC?ACACAAGTTC?TCTTTATCGC?CAAAATCACG

W R I N G I P Q Q H T Q V L F I A K

I T

901?CCAAATAATA?ACGGGACCTA?TGCCTGTTTT?GTCTCTAACT?TGGCTACTGG?CCGCAATAAT

P N N N G T Y A C F V S N L A T G

R N N

961?TCCATAGTCA?AGAGCATCAC?AGTCTCTGCA?TCTGGAACTT?CTCCTGGTCT?CTGAGCTGGG

S I V K S I T V S A S G T S P G

L S A G

1021?GCGACTGTCG?GCATCATGAT?TGGAGTGCTG?GTTGGGGTTG?CTCTGATA? TA?G?CAGCCCTGGG

A T V G I M I G V L V G V A L I

1081?TGTGGACCTT?C

The segmental production method of carcinoembryonic antigen is the restriction enzyme site (CTCGAG) that has added XhoI in 3 ' end PCR primer of 5 ' end of CEA signal peptide coding region and III region sequence, make two kinds of gene fragments that RT-PCR amplifies after restriction enzyme XhoI effect, has identical sticky end, and can be connected synthetic target gene fragment by dna ligase.

The carcinoembryonic antigen vaccine, the multiple clone site with CEA fragment insertion carrier for expression of eukaryon pIRES1neo makes up CEA naked DNA gene vaccine.The foreign gene that is cloned into eucaryon plasmid pIRES1neo is a CEA portion gene fragment.

The CEA vaccine can be got rid of the interference between tumor-necrosis factor glycoproteins in the expression process, enhancing immunity originality, and more effectively activated T cell induces specificity antineoplastic immunity and replys.Simultaneously,, therefore be easy to make associating or polyvalent vaccine, further improve the antitumous effect of gene vaccine with other immunostimulant gene and oncogene reorganization because freshly prepd gene fragment is shorter.

Embodiment:

At first, design and synthesize specific PCR primer (primer 1:5 ' the C AGAG of amplification CEA signal peptide coding region respectively according to the gene order of CEA GAATTCAGAGCAGACAGCAGAGACCATGGA 3 ' contains EcoRI restriction enzyme site and initiator codon; Primer 2: 5 ' AGC CTCGAGTATACCCGGAACTGGCCAGTTG 3 ' contains the XhoI restriction enzyme site) and the primer (primer 3:5 ' AGTCTC of amplification III sequence CTCGAGGCTGCCCAAGCCCTCCATC 3 ' contains the XhoI restriction enzyme site; Primer 4:5 ' GAA GGATCCACACCAGGGCTGCTATATCAGA 3 ' contains BamHIRestriction enzyme site and terminator codon), be template to carry again, use the synthetic corresponding gene fragment of RT-PCR technology from total RNA of large bowel cancer tissue, with two PCR products respectively the XhoI enzyme cut, the sticky end that produces connects with dna ligase, and preparation is about the CEA recombination of 1.1Kb.Then, with EcoRI and BamHI respectively enzyme cut recombination and carrier for expression of eukaryon pIRES1neo, through the DNA ligation, make up the pIRES-CEA recombinant plasmid.This plasmid is the CEA naked DNA vaccine, and it not only can be expressed in mammalian cell, and has colibacillary replicon and ampicillin resistance gene, can massive duplication production in intestinal bacteria.

Characteristics of the present invention and positively effect are the two big key element-foreign genes that constitute gene vaccine and the optimum combination of expression vector:

1, the reorganization of foreign gene

What participate in the antitumor immunity of organism reaction mainly is the T cell, the T cell recognition be not whole C EA antigen molecule, but CTL antigen peptide epi-position wherein.Therefore, we produce the III district fragment of CEA CTL epi-position-YLSGANLNL and TYACFVSNL at preferred can the coding from CEAcDNA, be connected in after the CEA signal peptide coding region, be used for the preparation of CEA gene vaccine, to get rid of the interference between tumor-necrosis factor glycoproteins, improve antigenic submission, improve the immunogenicity of gene vaccine, and make the effect of vaccine more single-minded, accurately and safety.

2. the selection of carrier for expression of eukaryon

The expression vector that gene therapy is in the past used places foreign gene and screening-gene respectively under the control of different promoters, so the expression of the two do not have inevitable dependency, has only 5-30% to express goal gene in the positive colony that screening obtains.(internal ribosome entry site IRES), inserts the CEA portion gene and the screening-gene Neo of multiple clone site and the eukaryon expression plasmid pIRES1neo of this experimental selection contains internal ribosome entry site ^rLay respectively at the upstream and downstream of IRES, be in together under the regulation and control of the main immediate early promoter of the human cytomegalic inclusion disease virus that can in eukaryotic cell, efficiently express/enhanser CMV, behind the transfection target cell, can transcribe and translate CEA albumen and neomycin phosphotransferase II from a mRNA, make transfection after the resistance clone that the G418 screening obtains all is the cell strain of high expression level CEA gene, improved working efficiency.What also contain two copies among the pIRES1neo is core with CpG, have immunostimulation dna sequence dna (the immunostimulatory DNA quence of (5 ' AACGTT 3 ') of palindrome, ISS), can guide immune response to tilt, and significantly strengthen the antigenic immunne response of external source genes encoding to TH1.

Genetic vaccine combines the advantage of three generations's vaccine, has the validity of attenuated live vaccine, the security of subunit vaccine and the accuracy of gene vaccine concurrently.It is the simulating nature course of infection in vivo, continues to produce recombinant antigen protein, stimulates body immune system, excites the ideal anti-tumor immune response; The quality of vaccine is easy to control and estimates simultaneously, and convenient the storage and transportation, is suitable for scale operation, is with a wide range of applications and the commercialization potentiality.The present invention is according to the therapeutic purpose CEA gene fragment of having recombinated, and the DNA and the aminoacid sequence of vaccine are clear, and antigenicity is stronger, and has reduced the interference between the sequence, makes the effect of genetic immunization more single-minded, accurately and safety.Prove that through sequential analysis the opening code-reading frame shelf structure of CEA gene fragment is correct in the recon, and is consistent with experimental design, can in eukaryotic cell, obtain correct the expression and shearing, and be discerned by specific T-cells to newly-built pIRES-CEA plasmid,

Implementation method of the present invention is as follows:

One, the reorganization of CEA gene fragment

1, the PCR design of primers is with synthetic: the two pairs of PCR primers all design voluntarily and are synthesized by dna synthesizer with terminal cessation method.Primer sequence is as follows.

CEA signal peptide coding region primer:

Primer 15 ' CAGAG GAATTCAGAGCAGACAGCAGAGACCATGGA3 '

(containing EcoRI restriction enzyme site and initiator codon)

Primer 25 ' AGCCTCGAGTATACCCGGAACTGGCCAGTTG 3 '

(contain XhoIRestriction enzyme site)

III district encoding sequence primer:

Primer 35 ' AGTCTCCTCGAGGCTGCCCAAGCCCTCCATC 3 '

(contain XhoIRestriction enzyme site)

Primer 45 ' GAAGGATCCACACCAGGGCTGCTATATCAGA 3 '

(contain BamHIRestriction enzyme site and terminator codon)

2, extract the total RNA of Colorectal Carcinoma:

(1) gets fresh Colorectal Carcinoma, the per 0.1 gram tissue in back that is cut into small pieces adds the sex change lysate (4.0M guanidine thiocyanate, 25mM Trisodium Citrate pH7.0,0.5% dodecyl creatine sodium, 0.72% beta-mercaptoethanol) of 1.2ml precooling, in homogenizer, pulverize lysing cell.

(2) add 2M acetate and receive (pH4.0) 0.12ml, mix.

(3) add phenol: chloroform: primary isoamyl alcohol (25: 24: 1) 1.2ml, after firmly mixing, put 15min on ice.

(4) 4 ℃, the centrifugal 20min of 12000rpm.

(5) carefully shift the upper strata water, add the equal-volume Virahol, more than-20 ℃ of precipitation 30min.

(6) 4 ℃, the centrifugal 20min of 12000rpm.

(7) discard supernatant, add the resuspended RNA throw out of sex change liquid 1ml again.

(8) add the equal-volume Virahol, more than-20 ℃ of precipitation 30min.

(9) 4 ℃, the centrifugal 20min of 12000rpm.

(10) abandon clean supernatant, centrifugal 2 times of 75% washing with alcohol of usefulness precooling.

(11) after precipitation is dried, with 50 μ lDEPC water dissolution.

(12) ultraviolet spectrophotometer quantitatively after ,-20 ℃ of preservations are standby.

3, cDNA is synthetic

In reaction system, add RNA template 1-2 μ g, oligo (dT) successively ₁₂1 μ l (100 μ g/ml), dNTP (each 10mmol/L) 2 μ l, RNA enzyme inhibitors 20U, AMV ThermoScript II 20U, 10 * AMV reaction buffer, 2 μ l, add DEPC water to 20 μ l, 42 ℃ of water-bath 30min, 95 ℃ of 5min.

4, pcr amplification and product reclaim:

Add Auele Specific Primer 1+2 (or 3+4) 4 μ l (20pmol), dNTP8 μ l, Tag (Ex) polysaccharase 2.5U, 10 * Tag enzyme reaction buffer solution, 10 μ l in the above-mentioned reaction system again, add distilled water to 100 μ l.The cyclic amplification program is: pre-95 ℃ of 5min of sex change, then 95 ℃ of 1min of sex change, 58 ℃ of 1min of renaturation, extend 72 ℃ of 1.5min, 72 ℃ of 10min are fully extended in totally 30 circulations.Agarose gel electrophoresis reclaims with the DNA purification kit after identifying the PCR product.

5, the digestion with restriction enzyme of DNA:

The endonuclease reaction of DNA all carries out according to this condition, and promptly every μ g dna fragmentation adds the restriction enzyme reaction damping fluid of 1/10 volume with 5U digestion with restriction enzyme (being XhoI) herein, adds distilled water 10 times to the enzyme volume again.Provide according to restriction enzyme supplier thermotonus 2-3 hour.

6, DNA ligation:

Reaction system comprises CEA signal peptide coding region (sticky end that 3 ' end has the XhoI enzyme to cut) and III district encoding sequence (sticky end that 5 ' end has the XhoI enzyme to cut) each 0.5 μ g, T ₄Dna ligase 1 μ l, 10 * T ₄Dna ligase damping fluid 2 μ l, add distilled water to 20 μ l.16 ℃, connect 12-16 hour.

Two, make up eukaryon expression plasmid pIRES-CEA

1, double digestion reaction:

The CEA recombination and the linear carrier pIRES1neo that prepare the toughness end with EcoRI and BamH I respectively.

2, the connection of dna fragmentation:

The volume ratio of CEA recombination and linear carrier pIRES1neo is 1 in the reaction system: 2-4.16 ℃, connect 12-16 hour.

Three, the mass production of recombinant plasmid pIRES-CEA

1, preparation competence intestinal bacteria (Calcium Chloride Method):

(1) scrape with the aseptic inoculation ring and get frozen intestinal bacteria bacterial classification in liquid nitrogen, streak inoculation is cultivated 12-16h for 37 ℃ in the LB agar plate.

(2) picking list bacterium colony is inoculated in the 5ml LB liquid nutrient medium, 37 ℃, 200rpm/min jolting cultivation 12h.

(3) get 1% nutrient solution, be inoculated in the 50ml LB liquid nutrient medium, 37 ℃, 250rpm/min jolting cultivation 2-3h are to OD ₆₀₀=0.4-0.6.

(4) take out after ice bath 10min immediately.

Below all need aseptic technique.

(5) nutrient solution is transferred in the ice-cold 50ml centrifuge tube 4 ℃, the centrifugal 10min of 4000rpm.

(6) abandon clean supernatant liquor, ice-cold 0.1mol CaCl ₂The resuspended thalline of 10ml, ice-water bath 30min.

(7) 4 ℃, the centrifugal 10min of 4000rpm abandon clean supernatant liquor.

(8) ice-cold 0.1mol CaCl ₂The resuspended thalline of 2ml is placed 12-24h for 4 ℃, the competence bacterium.Can be packed as 200 μ l/ parts, add 30% glycerine ,-70 ℃ of preservations are standby.

2, bacterium transforms

(1) gets the 50ng plasmid and add and to contain in the aseptic glass test tube of 0.2ml competence bacterium, mixing gently, ice-water bath 30min.

(2) 42 ℃ of heat-shocked 90s.

(3) add 0.8 μ l LB liquid nutrient medium, 37 ℃, the gentle jolting 45mi of 120rpm/min.

(4) with glass elbow shop bacterium device 200 μ l bacterium liquid are applied in the agar culture dish that contains penbritin 50 μ g/ml, 37 ℃ keep flat absorption 20min after, be inverted and cultivate 10-14h.

3, the Rapid identification of recombinant plasmid

(1) will transform bacterium colony and transfer to successively in the agar culture dish that contains Amp, after the numbering, 37 ℃ of incubated overnight.

(2) an amount of bacterium colony of picking adds 25 μ l solution A, mixing.

(3) add 25 μ l solution B, mixing, behind 70 ℃ of insulation 5min, 4 ℃, the centrifugal 10min of 12000rpm.

(4) get supernatant, row agarose gel electrophoresis (comparing with empty plasmid simultaneously) determines whether to be recombinant plasmid according to the molecular weight size.

4, a large amount of extractions (alkaline lysis) of plasmid:

(1) 1% transformed bacteria is inoculated in the 100ml LB substratum (containing Amp50 μ g/ml), 37 ℃, 250rpm/min jolting cultivation 12-16h are to OD ₆₀₀=0.6-0.8.

(2) take out after ice bath 10min immediately, transfer in the ice-cold 50ml centrifuge tube 4 ℃, the centrifugal 10min of 4000rpm.

(3) abandon clean supernatant liquor, add solution I (40mmol/L glucose, 2540mmol/L Tris-CI pH8.0, the 10mmol/L EDTA pH8.0) 4ml of ice precooling, the concuss mixing.

(4) add freshly prepared solution II (0.2mol/L NaCI, 1%SDS) 8ml, slowly put upside down mixing, place 5-10min.

(5) add ice-cold solution III (5mol/L potassium acetate 3.6ml, 3mol/L glacial acetic acid 0.69ml, distilled water 1.71ml) 6ml, put upside down mixing, ice-water bath 10min.4 ℃, the centrifugal 10min of 8000rpm.

(6) get supernatant liquor, add 0.6 volume primary isoamyl alcohol, mixing ,-20 ℃ of precipitation 20min.4 ℃, the centrifugal 10min of 12000rpm.

(7) abandon clean supernatant liquor, wash precipitation, dry, be dissolved in 1.2mlTE (pH8.0) solution with 70% ethanol.

(8) add Rnase A 30 μ l, 37 ℃ of water-bath 30min.

(9) add the equal-volume balance phenol, mix.The centrifugal 10min of 5000rpm.

(10) carefully draw the upper strata water, balance phenol extracting and chloroform-primary isoamyl alcohol (24: 1) extracting.

(11) the centrifugal 10min of 5000rpm carefully draws the upper strata water.

(12) add 1/10 volume NaAc (pH5.3), the dehydrated alcohol of 2.5 times of volumes, mixing ,-20 ℃ of precipitation 20min.

(13) the centrifugal 10min of 12000rpm abandons clean supernatant, and 70% washes 2 times.

(14) precipitation is dried, and is dissolved in 200 μ l TE (pH8.0) solution.

(15) get 10 μ l and measure OD by 1: 100 dilution back ₂₆₀, calculate plasmid DNA concentration.-20 ℃ of preservations are standby.

Claims

1, a kind of carcinoembryonic antigen fragment, it comprises the 1.1Kb CEA portion gene of CEA signal peptide and III district encoding sequence;

The nucleotide sequence and the open reading frame of 1091bp CEA gene fragment

1?CAGAGGAATT?CAGAGCAGAC?AGCAGAGACC? ATG?GAGTCTCTCCCCACAGA

[ORF] M E S P S A P P H R

61 TGGTGCATCC?CCTGGCAGAG?GCTCCTGCTC?ACAGCCTCAC?TTCTAACCTT?CTGGAACCCG

W C I P W Q R L L L T A S L L T F

W N P

121 CCCACCACTG?CCAAGCTCAC?TATTGAATCC?ACGCCGTTCA?ATGTCGCAGA?GGGGAAGGAG

P T T A K L T I E S T P F N V A E

G K E

181 GTGCTTCTAC?TTGTCCACAA?TCTGCCCCAG?CATCTTTTTG?GCTACAGCTG?GTACAAAGGT

V L L L V H N L P Q H L F G Y S W

Y K G

241 GAAAGAGTGG?ATGGCAACCG?TCAAATTATA?GGATATGTAA?TAGGAACTCA?ACAAGCTACC

E R V D G N R Q I I G Y V I G T Q

Q A T

301 CCAGGGCCCG?CATACAGTGG?TCGAGAGATA?ATATACCCCA?ATGCATCCCT?GCTGATCCAG

P G P A Y S G R E I I Y P N A S L

L I Q

361 AACATCATCC?AGAATGACAC?AGGATTCTAC?ACCCTACACG?TCATAAAGTC?AGATCTTGTG

N I I Q N D T G F Y T L H V I K S

D L V

421 AATGAAGAAG?CAACTGGCCA?GTTCCGGGTA?TACTCGAGGC?TGCCCAAGCC?CTCCATCTCC

N E E A T G Q F R V Y S R L P K P

S I S

481 AGCAACAAGT?CCAAACCCGT?GGAGGACAAG?GATGCTGTGG?CCTTCACCTA?TGAACCTGAG

S N N S K P V E D K D A V A F T C E

P E

541 GCTCAGAACA?CAACCTACCT?GTGGTGGGTA?AATGGTCAGA?GCCTCCCAGT?CAGTCCCAGG

A Q N T T Y L W W V N G Q S L P V S

P R

601 CTGCAGCTGT?CCAATGGCAA?CAGGACCCTC?ACTCTATTCA?ATGTCACAAG?AAATGACGCA

L Q L S N G N R T L T L F N V T R N

D A

661 AGAGCCTATG?TATGTGGAAT?CCAGAACTCA?GTGAGTGCAA?ACCGCAGTGA?CCCAGTCACC

R A Y V C G I Q N S V S A N R S D P

V T

721 CTGGATGTCC?TCTATGGGCC?GGACACCCCC?ATCATTTCCC?CCCCAGACTC?GTCTTACCTT

L D V L Y G P D T P I I S P P D S

S Y L

781 TCGGGAGCGA?ACCTCAACCT?CTCCTGCCAC?TCGGCCTCTA?ACCCATCCCC?GCAGATTCT

S G A N L N L S C H S A S N P S

P Q Y S

841 TGGCGTATCA?ATGGGATACC?GCACGAACAC?ACACAAGTTC?TCTTTATCGC?CAAAATCTCG

W R I N G I P Q Q H T Q V L F I A K

I T

901 CCAAATAATA?ACGGGACCTA?TGCCTGTTTT?GTCTCTAACT?TGGCTACTGG?CCGCAATAAT

P N N N G T Y A C F V S N L A T G

R N N

961 TCCATAGTCA?AGAGCATCAC?AGTCTCTGCA?TCTGGAACTT?CTCCTGGTCT?CTCAGCTGGG

S I V K S I T V S A S G T S P G

L S A G

1021?GCCACTGTCG?GCATCATGAT?TGGAGTGCTG?GTTGGGGTTG?CTCTGATA? TA?G?CAGCCCTGG

A T V G I M I G V L V G V A L I

1081?TGTGGACCTT?C

2, the segmental production method of carcinoembryonic antigen according to claim 1, it is characterized in that: hold the restriction enzyme site (CTCGAG) that has added Xho I in the PCR primer at 3 ' of 5 ' end of CEA signal peptide coding region and III region sequence, make two kinds of gene fragments that RT-PCR amplifies after restriction enzyme Xho I effect, has identical sticky end, and can be connected synthetic target gene fragment by dna ligase.

3, carcinoembryonic antigen vaccine according to claim 1 is characterized in that: the multiple clone site with CEA fragment insertion carrier for expression of eukaryon pIRES1neo makes up CEA naked DNA gene vaccine.

4, according to the described carcinoembryonic antigen vaccine of right claim 3, it is characterized in that: the foreign gene that is cloned into eucaryon plasmid pIRES1neo is a CEA portion gene fragment.