CN101358199A

CN101358199A - Expression of human CDC25 B3 plasmid and construction method thereof

Info

Publication number: CN101358199A
Application number: CNA2008100207139A
Authority: CN
Inventors: 石欣; 卫文俊; 孔波; 张齐; 杨正平; 肖志
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2008-02-22
Filing date: 2008-02-22
Publication date: 2009-02-04

Abstract

The present invention provides a plasmid expressing the human CDC25 B3, which is a vector pcDNA3.1(plus) containing CDC25B3, and wherein, the CDC25B3 is provided with a nucleotide sequence shown by SEQ ID NO:1, which can be expressed by the PCMV promoter in the pcDNA3.1(plus). The present invention provides a plasmid expressing the human CDC25 B3 and a method for constructing the plasmid expressing the human CDC25 B3. The plasmid transfects tumour cells weakly expressing CDC25B protein, so that the CDC25B protein can be highly expressed, and the plasmid can be used for the research of the potential carcinogenic mechanism of CDC25B3 gene and medically has an important application value. The present invention also provides a method for constructing the plasmid.

Description

A kind of expression human CDC 25 B 3 plasmid and construction process thereof

One, technical field

The invention belongs to gene engineering technology field in the biological chemistry, particularly a kind of expression human CDC 25 B 3 plasmid and construction process thereof.

Two, background technology

Prior art: CDC25 family is the one group of Soviet Union that plays a great role in cell cycle regulating/tyrosine bifunctional enzyme, and CDC25B (cell division cycle 25 homolog B) is one of difunctional phosphatase family important member of CDC25.The CDC25B gene is positioned at human chromosomal 20p13, is made of M phase inducement structure territory and rhodanese structural domain.Different cut modes can produce different spliced bodies, finds the mRNA of 5 kinds of CDC25B up to now altogether, but only finds 3 kinds of CDC25B albumen (CDC25B1, CDC25B2, CDC25B3), and have an appointment 95.5% sequence of three kinds of spliced bodies is consistent.The normal operation of CDC25B cell cycle plays important biological action, and the whole cell cycle can both monitor the transcribing of mRNA of CDC25B.It not only the S phase of cell cycle play prograding, also the phase transition plays " trigger " effect to G2/M, and the G2/M phase is played the check position effect.Overexpression CDC25B in the tire mouse liver, the female mice that knocks out CDC25B has been lost Fertility, and prompting CDC25B may be relevant with the growth of fetal development and ovum.

At present existing suitable studies show that, in multiple human malignancies, all there is the overexpression of different ratios CDC25B as prostate cancer, oesophagus Squamous Cell Carcinoma, mammary cancer, ovarian cancer, carcinoma of endometrium, carcinoma of the pancreas, tumor of head and neck, cancer of the stomach, lung non-small cell carcinoma, colorectal carcinoma, non-Hodgkin lymphoma, neuroblastoma, thyroid carcinoma etc.Wherein find in the research of mammary cancer, ovarian cancer (30%) and colorectal cancer (43%): the CDC25B of overexpression and the grade of malignancy of tumour, transfer and patient's prognosis is relevant.In the research of non-Hodgkin knurl, CDC25B2 is reported in overexpression in 56% the sample, though find relevant with aggressive and patient's prognosis of tumour, relevant with mortality.Find that in the research of Tiroidina malignant lymphoma CDC25B overexpression rate is 63.8%, be higher than chronic thyroiditis far away, the big emphatically effect of prompting performance in CDC25B Tiroidina malignant lymphoma transforms.In the research of esophagus Squamous Cell Carcinoma, the investigator finds that the patient of high CDC25B is extremely sensitive to radiotherapy, and whether high expression level CDC25B is relevant with poor prognosis at present also less than final conclusion.Find that in the research of esophageal cancer cell strain overexpression CDC25B can strengthen ray and induce tricky effect of dying.Bibliographical information in cancer of the stomach up to the CDC25B overexpression being arranged in 49% the sample and being proportionate with the degree of depth, the nodus lymphoideus transferring rate of neoplasm staging, infiltration.Excessive CDC25B (90%) is relevant with ER-α (65%) in the carcinoma of endometrium in early days, and late in the carcinoma of endometrium CDC25B to express only be 42%, ER-α then reduces to 17%.As seen the early stage CDC25B of Endometrial Carcinomas generation and ER-α have the collaborative effect that transforms that stimulates.CDC25B overexpression rate is up to 97% in prostate cancer, and the CDC25B of overexpression can strengthen androgenic transcriptional activation, helps prostate cancer to transform to non-androgen-dependent growth.Expression rate at the CDC25B of lung non-small cell carcinoma, tumor of head and neck, neuroblastoma is 40%, 50%, 85%.These show that all the CDC25B overexpression has key effect for generation, the development of tumour, may be potential diagnostic flag and treatment target spot,

The relation of CDC25B and carcinoma of the pancreas research at present is also fewer, Guo J etc. discovers that CDC25B is respectively 48.6 ± 16.3%, 71.7 ± 3.1%, 8.3 ± 1.8% in the expression of carcinoma of the pancreas primary tumor and metastasis and Normal Pancreas, obviously CDC25B is overexpression in the carcinoma of the pancreas tissue, and the expression of metastasis is apparently higher than primary tumor.The CDC25B specific inhibitor acts on the human pancreas cancer cell strain of high expression level CDC25B and finds, inhibitor can obviously suppress the growth and the generation G2/M phase of this cell strain and block.

In our research, by the oligonucleotide gene chip scientific discovery, the CDC25B gene has 20 examples (83.3%) up-regulated in 24 routine carcinoma of the pancreas tissues, compare with healthy tissues, and it is expressed in carcinoma of the pancreas and obviously increases; Find that by Western blot all normal pancreatic tissues have only very weak even do not have the protein expression of CDC25B, and stronger CDC25B protein expression is then arranged in the carcinoma of the pancreas tissue.Through quantitative analysis behind the computer scanning, normal pancreatic tissue CDC25B protein expression is 1.22 ± 0.33, and carcinoma of the pancreas is organized as 5.56 ± 1.57, raises 4.56 times.In the cell experiment, we are by shearing discovering of isomer to three kinds of CDC25B in four kinds of pancreas cancer cell strains, at transcriptional level, and the expression intensity no significant difference of CDC25B1 and CDC25B2 among the Panc-1; The expression of the CDC25B2 of CFPAC-1 and SW-1990 is higher than CDC25B1; And the expression intensity of the CDC25B1 of Bxpc-3 is higher than CDC25B2, and CDC25B3 expresses all not remarkable in four kinds of pancreas cancer cell strains.Western blot results suggest CDC25B albumen is in Panc-1 and CFPAC-1 overexpression, and the expression intensity of SW-1990 is medium, Bxpc-3 weak expression or do not express CDC25B albumen.The above results shows that CDC25B1 and CDC25B2 cross expression in four kinds of pancreas cancer cell strains, and CDC25B3 and carcinoma of the pancreas relation are not still understood.

Three, summary of the invention

Technical problem: the invention provides a kind of expression human CDC 25 B 3 plasmid and construction process thereof, the low proteic tumour cell of CDC25B of expressing of this plasmid transfection can make CDC25B albumen high expression level, can be used to inquire into the potential carcinogenic mechanism of CDC25B3 gene, medically have important use value; The present invention simultaneously also provides the construction process of this plasmid.We wish by making up the recombinant plasmid of CDC25B3, carry out experiment in vitro subsequently, with the low proteic pancreas cancer cell strain of CDC25B of expressing of plasmid transfection, observe the variation that the cytobiology behavior of transfection front and back takes place, inquire into CDC25B3 potential mechanism in the carcinoma of the pancreas pathogenic process.In addition, because there is overexpression in CDC25B in multiple human tumor, we can utilize aforesaid method, and this type of plasmid is used for the research of other human tumors, with further investigation with illustrate the role that the CDC25B gene is served as in human tumor develops.Therefore, application of the present invention is not limited to carcinoma of the pancreas, can be widely used in CDC25B in the research and inquirement of human tumor.

Technical scheme: technical solution of the present invention is a kind of plasmid of expressing human CDC 25 B 3, this plasmid is the carrier pcDNA3.1 (+) that contains CDC25B3, wherein CDC25B3 has the nucleotide sequence shown in the SEQ ID NO:1, can be by the PCMV promoter expression among the pcDNA3.1 (+).A kind of method that makes up the described plasmid of claim 1, construction step is: the cDNA full-length clone IRATp970A0662D with CDC25B1 is a template, the PCR directed expansion is sheared a fragment and the b fragment of isomer C DC25B3; Design two pairs of primers:

Primer 1:5-AGG GGC CCT CTA GAC TCG AGA TGG AGG TGC CCC AGC

CG-3

Primer 2: 5-TTG GTA CCG AGC TCG GAT CCT CAC TTA TCG TCG TCA

TCC TTG TAA TCC TGG TCC TGC AGC CGG CTA C-3

Primer 3:5-CAT GGC ATC TTG AAG ACA AAT CCA TCC GGC ATA GAC

TGG AAG CGT C-3

Primer 4:5-GAC GCT TCC AGT CTA TGC CGG ATG GAT TTG TCT TCA

AGA TGC CAT C-3

With IRATp970A0662D is template, uses PCR method, with a fragment of

primer

1 and 3 clone CDC25B3, and the b fragment of

primer

2 and 4 clone CDC25B3; Obtain the CDS district total length of CDC25B3 gene: with the method for pcr amplification, fragment a and b are pieced together knot, obtain the CDS district total length of CDC25B3 gene with primer 1 and primer 2; Obtain recombinant plasmid pcDNA3.1-CDC25B3: at first plasmid pcDNA3.1 (+) is transformed, with BamH I and Xho I digested plasmid pcDNA3.1 (+), the linearization plasmid fragment that obtains transforming, the CDC25B3 gene that pcr amplification is produced is connected with improved linearization plasmid fragment with the T4 ligase enzyme again, obtains recombinant plasmid pcDNA3.1-CDC25B3.

Beneficial effect: the preparation approach of gene has following several mode: 1. prepare genome dna library, obtain the special genes fragment by screening by hybridization; 2. prepare the cDNA library, obtain the special genes fragment by screening by hybridization; 3. directly cut target gene fragment from genomic dna or other recombinant plasmids with restriction enzyme; 4. use round pcr amplification in vitro relative dna fragment; 5. with the synthetic target gene fragment of chemical process.Among the present invention, we have directly bought the cDNA full-length clone of CDC25B1 and have obtained the purpose fragment in a large number with the method for PCR from RZPD company, and can introduce different restriction enzyme sites and realize that directed cloning obtains its clever two shearing isomer at 5 of upstream and downstream primer ' end.We think that this is a kind of segmental method of purpose that obtains fast, efficiently, in a large number, and can effectively avoid the base mispairing that causes owing to reverse transcription.Simultaneously, used pcDNA3.1 (+) plasmid of the present invention is a kind of mammalian cell expression vector, belong to the amicillin resistance selective system, have ampicillin resistance gene Ampr, under the PCMV promoters driven, can in eukaryotic cell, express, its coded product can make neomycin analog G418 inactivation, so can obtain to import the positive cell clone of expression vector with the G418 screening; In the multiple clone site upstream of pcDNA3.1 (+) plasmid a PCMV promotor is arranged, the allogenic gene that is inserted into multiple clone site can obtain effective expression under the driving of this promotor; The carboxyl terminal of target gene merges underlined peptide FLAG in addition, very conveniently is used for immunoreactive detection and detects the yeast two-hybrid result as bait protein.Therefore, utilize this carrier conveniently to be used for screening and evaluation.

Four, description of drawings

Fig. 1 is pcDNA3.1 (+) plasmid map

Fig. 2 identifies the pcDNA3.1-CDC25B3 positive colony for PCR, and the band of Marker is 2kb, 1kb, 750bp, 500bp, 250bp, 100bp successively.

Fig. 3 is CDC25B3 heterogenous expression Western blot figure, and the plasmid amount of 1,4 transfections is 0.5 μ g, and the plasmid amount of 2,3 transfections is 3 μ g, and 5 and 6 is control group.Western blot detected result shows, the overexpression of CDC25B3 in the 293T cell of transient transfection pcDNA3.1-CDC25B3 carrier, and it is successful pointing out this construction of recombinant plasmid, can be used for further experiment research.

Fig. 4 is the sequencing result of recombinant plasmid pcDNA3.1-CDC25B3.

The normal SW-1990 of Fig. 5

Fig. 6 transfection empty plasmid pcDNA3.1

Fig. 7 stable transfection pcDNA3.1-CDC25B3

Fig. 8 microchip electrophoresis mimic diagram shows the differential expression of CDC25B3mRNA

The differential expression of CDC25B albumen in SW-1990 before and after Fig. 9 transfection

Five, embodiment

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Joe Sambrook, David Russell, condition described in " Molecular Cloning:ALaboratory Manual " 3rd ed (Cold Spring Harbor Laboratory Press 2001), or the condition of advising according to manufacturer.

Embodiment 1:

1. the structure of recombinant plasmid pcDNA3.1-CDC25B3

1.1 the cDNA (IRATp970A0662D) of the CDC25B1 that buys from RZPD company that preserves with this laboratory is a template;

1.2 with IRATp970A0662D is template, utilizes primer 1 and the 3 a fragments from amplification CDC25B3, the b fragment of 2 and 4 amplification CDC25B3.

A. reaction system

The a fragment

Reagent
Reagent		Primer(-)P3 Primer(+)P1 ddH ₂O 10 * buffer DNTPs (2.5mM) pfu polymerase template (10ng/ul)	0.4μl 0.4μl 13.6μl 2μl 0.8μl 0.2μl 1μl
Total	20μl		0.4μl 0.4μl 13.6μl 2μl 0.8μl 0.2μl 1μl

The b fragment

Reagent
Reagent		Primer(-)P4 Primer(+)P2 ddH ₂O 10 * buffer DNTPs (2.5mM) pfu polymerase template (10ng/ul)	0.4μl 0.4μl 13.6μl 2μl 0.8μl 0.2μl 1μl
Total	20μl		0.4μl 0.4μl 13.6μl 2μl 0.8μl 0.2μl 1μl

B. reaction conditions

The PCR instrument reacts, and presses following cycling condition:

94℃ 30sec

72℃ 30sec

72℃ 6min

1.3 separation and purification a fragment and b fragment

Above-mentioned blended reactant is placed 37 ℃, and behind the 1h, electrophoresis identification reaction product reclaims with PCR product purification test kit, obtains a fragment and the b fragment of purifying, measures its concentration and purity at the uv-spectrophotometric instrument, and-20 ℃ of preservations are standby.

1.4 the segmental splicing of a fragment and b

For obtaining the total length of CDC25B3, need to adopt the method for pcr amplification that fragment a and b are spliced, template is the PCR product of last round of recovery.Pcr amplification, reaction system are 50 μ L:

Pfu DNA Polymerase	0.5μl
Pfu DNA Polymerase	0.5μl	10 * Pfu DNA polymerase Buffer 10mM 4xdNTP Primer P1 Primer P2 reclaims a fragment and reclaims b fragment ddH ₂O	5μl 1μl 1μl 1μl 1μl 1μl 39.5μl
Total	50μl		5μl 1μl 1μl 1μl 1μl 1μl 39.5μl

The PCR cycling condition:

94℃ 2min

72℃ 5min

1.5 enzyme is cut PCR fragment and carrier

Use BamH I and Xho I to carry out enzyme and cut digestion last round of PCR product and carrier, enzyme is cut product with DNA purification kit purifying, after the uv-spectrophotometric instrument is measured its concentration and purity, and-20 ℃ of preservations.

1. endonuclease reaction

A. reaction system: in 0.5ml Eppendorf pipe, add following reagent:

Enzyme is cut carrier:

The DNA plasmid of purifying (1ug/ μ l)

2μl

10×buffer 100×BSA BamH I(10U/μl) Xho I(10U/μl) H ₂O	5μl 0.5μl 1μl 1μl 40.5μl
10×buffer 100×BSA BamH I(10U/μl) Xho I(10U/μl) H ₂O	5μl 0.5μl 1μl 1μl 40.5μl	Total	50μl

Enzyme is cut the PCR product:

The DNA plasmid of purifying (1ug/ μ l)	2μl
The DNA plasmid of purifying (1ug/ μ l)	2μl	10×buffer 100×BSA BamH I(10U/μl) Xho I(10U/μl) H ₂O	5μl 0.5μl 1μl 1μl 40.5μl
Total	50μl	10×buffer 100×BSA BamH I(10U/μl) Xho I(10U/μl) H ₂O	5μl 0.5μl 1μl 1μl 40.5μl

B. reaction conditions: behind 37 ℃ of effect 1h, electrophoresis conclusive evidence enzyme is cut product.

Enzyme is cut product with DNA purification kit purifying, after the uv-spectrophotometric instrument is measured its concentration and purity, and-20 ℃ of preservations.

1.6 ligation

A. reaction system: in 0.5ml Eppendorf pipe, add following reagent:

PCR product 100ng/ μ l 10 * T4 phage DNA ligase enzyme damping fluid T4 phage DNA ligase enzyme dd H that the carrier DNA 100ng/ μ l enzyme that enzyme cuts back to close cuts back to close ₂O	1μl 1μl 1μl 1μl 6μl
	1μl 1μl 1μl 1μl 6μl	Total	10μl

B. reaction conditions: 4 ℃ of effect 12h obtain reorganization pcDNA3.1-CDC25B3 expression plasmid.

Embodiment 2: the recombinant plasmid transformed intestinal bacteria

2.1 competence bacterium preparation (summary)

2.2 the screening of positive colony and evaluation

(1) screening of positive colony (summary)

(2) PCR of positive colony identifies

The recombinant clone performing PCR is identified, PCR product electrophoresis in sepharose has 535bp left and right sides length to insert the fragment person of discharging and is considered as positive recombinant chou.

(3) the positive colony nucleic acid sequencing is identified

Optional 2 clones from the clone that aforesaid method is identified are inoculated in the fresh bacterium liquid of preparation on the substratum, and order-checking is identified and inserted segmental exactness.

Embodiment 3: cell cultures and recombinant plasmid cotransfection

3.1 the cultivation of 293T cell and cover plant

3.2 cell transient transfection

3.3 the limiting dilution assay screening efficiently expresses the positive cell clone of CDC25B3

The detection of embodiment 4:CDC25B3 protein expression

Western blot detects the expression of CDC25B3 albumen in the 293T cell

Embodiment 5: for illustrating that CDC25B3 in the application aspect the tumor research, is that example is carried out following experiment with the carcinoma of the pancreas:

1. cell transfecting

1.1G418 concentration gradient test

To do the concentration gradient test before the G418 screening positive clone and determine best screening concentration.

1) trypsin digestion cell becomes cell suspension, is diluted to 1,000cell/mL.

2) get 20 holes in 24 orifice plates, every hole adds cell suspension and the unparalleled anti-substratum 900 μ L of 100 μ L.

3) with 10 ranks such as the G418 concentration dilution to 0,200,300,400,500,600,700,800,900 in every hole, 1,000 μ g/mL.

4) changed liquid in per 3～5 days, cultivated 10 days, the minimum concentration that the record cell is all dead.Adding a rank with all dead minimum concentration of cell is screening concentration.

1.2 transfectional cell

1) gets the SW-1990 cell that is in logarithmic phase, discard enchylema, add 2ml PBS and wash once.

2) add 1ml trysinization 1～2min.

3) discard pancreatin, add 3-4ml DMEM (not containing serum) piping and druming evenly.

4) cell is dripped in 6 well culture plates (every hole 2ml), make cultivate 24h after, the cytogamy degree is controlled at 70～80%.

5) for each hole of 6 orifice plates, the nutrient solution volume is adjusted into 1ml, mixes with 240 μ l Opti-MEM I with 2 μ g CDC25B3 DNA plasmids.

6) LipofectAMINE 2000 reagent are softly shaken up, get 5 μ l LipofectAMINE, 2000 reagent and mix, at room temperature incubation 5min at another Guan Zhongyu 245 μ l Opti-MEM I.

7) DNA after the dilution is mixed with Lipofectamine 2000 after the dilution, put upside down mixing lightly, vibrate.Must within 5min, mix.

8) mix after, incubation 20min at room temperature is so that form the transfection composite of plasmid DNA and LipofectAMINE 2000 diluents.

9) mixed solution of plasmid DNA and LipofectAMINE 2000 is added culturing cell, soft front and back push away shakes culture plate with mixing liquid.Then culture plate is sent into cell culture incubator.

10) behind the full 8h of transfection, need to change the fresh nutrient solution that contains serum.

11) after the transfection behind the 36h, then 1: the 2-3 ratio goes down to posterity, and adds the G418 screening simultaneously, and concentration is 500 μ g/ml.

12) treat after two weeks that the monoclonal cell island forms after, trypsin digestion cell is bed board and change G418 to keep concentration be that stable cultivation of 100 μ g/ml gone down to posterity again.

1.3 detect the expression of CDC25B3 in SW-1990 with RT-PCR and Western blot

100 μ g/ml G418 keep and cultivate transfectional cell two weeks of SW-1990, and going down to posterity, row RT-PCR and Western blot detect (method is the same) after 5 times.

Embodiment 6:G418 concentration gradient test-results

G418 concentration with 400 μ g/mL is cultivated, and the SW-1990 cell is all dead at l0 days time cells.500 μ g/mL should be the suitable screening concentration of SW-1990 cell.

Embodiment 7:RT-PCR product microchip electrophoresis qualification result

The expression abundance of the mRNA of CDC25B3 is 33.21 ± 5.67, and the mRNA of normal cell and commentaries on classics empty plasmid group CDC25B3 expression abundance has only 1.957 ± 0.112 and 1.645 ± 0.242.The expression of transfection group CDC25B3mRNA is about 20 times (P＜0.05) of changeing the empty plasmid group, as shown in Figure 5.The illustrative in nature grain can successfully be transcribed in pancreatic cancer cell, lays a good foundation for carrying out next step Cell Biology Experiment, can be used to inquire into CDC25B3 and in carcinoma of the pancreas developing effect takes place.

Embodiment 8:

100 μ g/ml G418 compare with normal cell with the transfection empty plasmid after keeping and cultivating for two weeks, and Westernblot detects CDC25 B3 protein expression rising in the transfection group, and apparently higher than changeing empty plasmid group and normal cell group (Fig. 6).The illustrative in nature grain can successfully be expressed in pancreatic cancer cell, lays a good foundation for carrying out next step Cell Biology Experiment, can be used to inquire into CDC25B3 and in carcinoma of the pancreas developing effect takes place.

Sequence table

＜110〉Southeast China University

＜120〉a kind of expression human CDC 25 B 3 plasmid and construction process thereof

<160>5

<210>1

<211>3578

<212>DNA

＜213〉artificial sequence

<220>

<221>CDS

<222>(1)...(3578)

<400>1

1 GCAGCCAGTC GCGGAGGCGG GGAGGCTGCG CGGTCAGAGG CGCCTGGAGC GAGCGAATCC

61 TGGCCCACCG CCTGCCCAAC CGCGTGACCT TGATTGAGTT AATGAACTTC ACGCCTCAGC

121 GTCCAGGTCT GTAAAATGGG GTGTCTAACG CAGACCGTAC AGCCCAGCTG GGTTTAGCAA

181 ACTTCCGGGA GCCAGTTGGA GCCTCTCCCC ATCCCTAGCG GTGATCCCAG GTGACGACAT

241 GCCGCGGGGG GTCCTGCGGA GGCCACCCTA GGGCGTTGCT GCTGCCTTTG GGAGTGTGGA

301 GCTCCAAACC ATGTCGCGAG AGGCGGATTT TGGGAGGCCG GGATCCTCGC GCCAGGGGGA

361 TGTGCGAGGG TGTGGGATAA ATCTTAATTC CTCCGGCCCA CCCAAAGCCT GGAAATCCAG

421 CCTCCGCGCC TCTTGCCCTG CGGGCCCCGC CCTCAGTCCC GCCCTCATCT AACCCGCTAC

481 CCCATTGGTG GCGTCCGGCG GCGCGGCTGC TGTTATTTTT CGAATATATA AGGAGGTGGA

541 AGTGGCAGCT GCAACTAGAG GCTTCCCTGG CTGGTGCCTG AGCCCGGCGT CCCTCGCCCC

601 CCGCCCTCCC CGCATCCCTC TCCTCCCTCG CGCCTGGCCC TGTGGCTCTT CCTCCCTCCC

661 TCCTTCCCCC CCCCCCCACC CCTCGCCCGC TGCCTCCCTC GGCCCAGCCA GCTGTGCCGG

721 CGTTTGTTGG CTGCCCTGCG CCCGGCCCTC CAGCCAGCCT TCTGCCGGCC CCGCCGCGAT

781 GGAGGTGCCC CAGCCGGAGC CCGCGCCAGG CTCGGCTCTC AGTCCAGCAG GCGTGTGCGG

841 TGGCGCCCAG CGTCCGGGCC ACCTCCCGGG CCTCCTGCTG GGATCTCATG GCCTCCTGGG

901 GTCCCCGGTG CGGGCGGCCG CTTCCTCGCC GGTCACCACC CTCACCCAGA CCATGCACGA

961 CCTCGCCGGG CTCGGCAGCG AAACCCCAAA GAGTCAGGTA GGGACCCTGC TCTTCCGCAG

1021 CCGCAGCCGC CTGACGCACC TATCCCTGTC TCGACGGGCA TCCGAATCCT CCCTGTCGTC

1081 TGAATCCTCC GAATCTTCTG ATGCAGGTCT CTGCATGGAT TCCCCCAGCC CTATGGACCC

1141 CCACATGGCG GAGCAGACGT TTGAACAGGC CATCCAGGCA GCCAGCCGGA TCATTCGAAA

1201 CGAGCAGTTT GCCATCAGAC GCTTCCAGTC TATGCCGGAT GGATTTGTCT TCAAGATGCC

1261 ATGGAAGCCC ACACATCCCA GCTCCACCCA TGCTCTGGCA GAGTGGGCCA GCCGCAGGGA

1321 AGCCTTTGCC CAGAGACCCA GCTCGGCCCC CGACCTGATG TGTCTCAGTC CTGACCGGAA

1381 GATGGAAGTG GAGGAGCTCA GCCCCCTGGC CCTAGGTCGC TTCTCTCTGA CCCCTGCAGA

1441 GGGGGATACT GAGGAAGATG ATGGATTTGT GGACATCCTA GAGAGTGACT TAAAGGATGA

1501 TGATGCAGTT CCCCCAGGCA TGGAGAGTCT CATTAGTGCC CCACTGGTCA AGACCTTGGA

1561 AAAGGAAGAG GAAAAGGACC TCGTCATGTA CAGCAAGTGC CAGCGGCTCT TCCGCTCTCC

1621 GTCCATGCCC TGCAGCGTGA TCCGGCCCAT CCTCAAGAGG CTGGAGCGGC CCCAGGACAG

1681 GGACACGCCC GTGCAGAATA AGCGGAGGCG GAGCGTGACC CCTCCTGAGG AGCAGCAGGA

1741 GGCTGAGGAA CCTAAAGCCC GCGTCCTCCG CTCAAAATCA CTGTGTCACG ATGAGATCGA

1801 GAACCTCCTG GACAGTGACC ACCGAGAGCT GATTGGAGAT TACTCTAAGG CCTTCCTCCT

1861 ACAGACAGTA GACGGAAAGC ACCAAGACCT CAAGTACATC TCACCAGAAA CGATGGTGGC

1921 CCTATTGACG GGCAAGTTCA GCAACATCGT GGATAAGTTT GTGATTGTAG ACTGCAGATA

1981 CCCCTATGAA TATGAAGGCG GGCACATCAA GACTGCGGTG AACTTGCCCC TGGAACGCGA

2041 CGCCGAGAGC TTCCTACTGA AGAGCCCCAT CGCGCCCTGT AGCCTGGACA AGAGAGTCAT

2101 CCTCATTTTC CACTGTGAAT TCTCATCTGA GCGTGGGCCC CGCATGTGCC GTTTCATCAG

2161 GGAACGAGAC CGTGCTGTCA ACGACTACCC CAGCCTCTAC TACCCTGAGA TGTATATCCT

2221 GAAAGGCGGC TACAAGGAGT TCTTCCCTCA GCACCCGAAC TTCTGTGAAC CCCAGGACTA

2281 CCGGCCCATG AACCACGAGG CCTTCAAGGA TGAGCTAAAG ACCTTCCGCC TCAAGACTCG

2341 CAGCTGGGCT GGGGAGCGGA GCCGGCGGGA GCTCTGTAGC CGGCTGCAGG ACCAGTGAGG

2401 GGCCTGCGCC AGTCCTGCTA CCTCCCTTGC CTTTCGAGGC CTGAAGCCAG CTGCCCTATG

2461 GGCCTGCCGG GCTGAGGGCC TGCTGGAGGC CTCAGGTGCT GTCCATGGGA AAGATGGTGT

2521 GGGTGTCCTG CCTGTCTGCC CCAGCCCAGA TTCCCCTGTG TCATCCCATC ATTTTCCATA

2581 TCCTGGTGCC CCCCACCCCT GGAAGAGCCC AGTCTGTTGA GTTAGTTAAG TTGGGTTAAT

2641 ACCAGCTTAA AGGCAGTATT TTGTGTCCTC CAGGAGCTTC TTGTTTCCTT GTTAGGGTTA

2701 ACCCTTCATC TTCCTGTGTC CTGAAACGCT CCTTTGTGTG TGTGTCAGCT GAGGCTGGGG

2761 GAGAGCCGTG GTCCCTGAGG ATGGGTCAGA GCTAAACTCC TTCCTGGCCT GAGAGTCAGC

2821 TCTCTGCCCT GTGTACTTCC CGGGCCAGGG CTGCCCCTAA TCTCTGTAGG AACCGTGGTA

2881 TGTCTGCCAT GTTGCCCCTT TCTCTTTTCC CCTTTCCTGT CCCACCATAC GAGCACCTCC

2941 AGCCTGAACA GAAGCTCTTA CTCTTTCCTA TTTCAGTGTT ACCTGTGTGC TTGGTCTGTT

3001 TGACTTTACG CCCATCTCAG GACACTTCCG TAGACTGTTT AGGTTCCCCT GTCAAATATC

3061 AGTTACCCAC TCGGTCCCAG TTTTGTTGCC CCAGAAAGGG ATGTTATTAT CCTTGGGGGC

3121 TCCCAGGGCA AGGGTTAAGG CCTGAATCAT GAGCCTGCTG GAAGCCCAGC CCCTACTGCT

3181 GTGAACCCTG GGGCCTGACT GCTCAGAACT TGCTGCTGTC TTGTTGCGGA TGGATGGAAG

3241 GTTGGATGGA TGGGTGGATG GCCGTGGATG GCCGTGGATG CGCAGTGCCT TGCATACCCA

3301 AACCAGGTGG GAGCGTTTTG TTGAGCATGA CAGCCTGCAG CAGGAATATA TGTGTGCCTA

3361 TTTGTGTGGA CAAAAATATT TACACTTAGG GTTTGGAGCT ATTCAAGAGG AAATGTCACA

3421 GAAGCAGCTA AACCAAGGAC TGAGCACCCT CTGGATTCTG AATCTCAACA TCCCGGCAGG

3481 GCTGTGCTTG AAGGCCCTGC TGAGTCATCT GTTAGGGCCT TGGTTCAATA AAGCACTGAG

3541 CAAGTTGAGA AAAAAAAAAA AAAAAAAAAA AAAAAAAA

<210>2

<211>38

<212>DNA

＜213〉synthetic primer

<400>2

1 AGGGGCCCTC TAGACTCGAG ATGGAGGTGC CCCAGCCG

<210>3

<211>67

<212>DNA

＜213〉synthetic primer

<400>3

1 TTGGTACCGA GCTCGGATCC TCACTTATCG TCGTCATCCT TGTAATCCTG GTCCTGCAGC

61 CGGCTAC

<210>4

<211>46

<212>DNA

＜213〉synthetic primer

<400>4

1 CATGGCATCT TGAAGACAAA TCCATCCGGC ATAGACTGGA AGCGTC

<210>5

<211>46

<212>DNA

＜213〉synthetic primer

<400>5

1 GACGCTTCCA GTCTATGCCG GATGGATTTG TCTTCAAGAT GCCATC

Claims

1. a plasmid of expressing human CDC 25 B 3 is characterized in that this plasmid is the carrier pcDNA3.1 (+) that contains CDC25B3, and wherein CDC25B3 has the nucleotide sequence shown in the SEQ ID NO:1, can be by the PCMV promoter expression among the pcDNA3.1 (+).

2. method that makes up the described plasmid of claim 1 is characterized in that construction step is:

A. the cDNA full-length clone IRATp970A0662D with CDC25B1 is a template, and the PCR directed expansion is sheared a fragment and the b fragment of isomer C DC25B3;

B. design two pairs of primers:

Primer 1:5-AGG GGC CCT CTA GAC TCG AGA TGG AGG TGC CCC

AGC CG-3

Primer 2: 5-TTG GTA CCG AGC TCG GAT CCT CAC TTA TCG TCG

TCA TCC TTG TAA TCC TGG TCC TGC AGC CGG CTA

C-3

Primer 3:5-CAT GGC ATC TTG AAG ACA AAT CCA TCC GGC ATA

GAC TGG AAG CGT C-3

Primer 4:5-GAC GCT TCC AGT CTA TGC CGG ATG GAT TTG TCT

TCA AGA TGC CAT C-3

With IRATp970A0662D is template, uses PCR method, with a fragment of primer 1 and 3 clone CDC25B3, and the b fragment of primer 2 and 4 clone CDC25B3;

C. obtain the CDS district total length of CDC25B3 gene: with the method for pcr amplification, fragment a and b are pieced together knot, obtain the CDS district total length of CDC25B3 gene with primer 1 and primer 2;

D. obtain recombinant plasmid pcDNA3.1-CDC25B3: at first plasmid pcDNA3.1 (+) is transformed, with BamH I and Xho I digested plasmid pcDNA3.1 (+), the linearization plasmid fragment that obtains transforming, the CDC25B3 gene that pcr amplification is produced is connected with improved linearization plasmid fragment with the T4 ligase enzyme again, obtains recombinant plasmid pcDNA3.1-CDC25B3.