CN1218954C - Human hematopoietic cell relating gene-1 - Google Patents

Abstract

The present invention relates to a new gene which is successfully separated by comparing the gene expression difference of four-month human fetal's liver tissues and adult's liver tissues. The name of the new gene is hematopoietic cell differentiation relevant gene-1(Hemagene-1). The gene mRNA has the total length of 2166 bp nucleotides and comprises a 110 bp5'-non translation sequence, a 602 bp3'-non translation sequence and Hemagene-1 gene encoding 484 amino acids, and the molecular weight is 55.3 kd proteins. The gene has converting activity, is expressed in leukemia cells with high abundance, is tightly relevant to hematopoietic cell differentiation and can be used for diagnosing and treating leukemia.

Description

Human hematopoietic cell differentiation associated gene-1

One, research field

This research relates to molecular cloning and the functional study of human hematopoietic cell differentiation associated gene-1 (to call Hemagene-1 in the following text) full length gene cDNA.Obtain marking protein by making up prokaryotic expression carrier and transforming suitable prokaryotic host cell; By making up carrier for expression of eukaryon and transfection proper host cell, after screening, obtain the cell strain of stably express Hemagene-1, pernicious transformation takes place in the cell of stably express Hemagene-1, shows that this gene has activity of conversion; This gene may be applied to leukemia cell's diagnosis at leukemia cell's high expression level, or becomes the novel targets of screening anti-leukemia medicine.

Two, correlative study both at home and abroad

As everyone knows, the most basic biological phenomenas such as differentiation, growth, propagation and death that determined cell are expressed in the selection of gene, the expression of research embryo stage gene Selection be gene when specific opening or closing of phase be the key of understanding the regulation and control of different developmental phases physiological function.In the vertebrates embryo development procedure, liver has very special course.Early stage fetal liver has very vigorous propagation and differentiation capability, is again the hemocytopoietic organ that body weight for humans is wanted simultaneously, just transfers marrow hemopoiesis to by liver about human body in utero greatly.Therefore, the hepatic tissue in this stage is the hematopoiesis regulatory gene of expressed in abundance both, also expresses the regulatory gene of liver growth.Studies show that from age in April in the past can be found people's tire hepatic tissue and separate many important gene as augmenter of liver regenration (1), serine/threonine kinases (2) and other unknown gene (3) etc.For the foregoing reasons, relatively embryonic liver and adult's liver gene differential expression are expected to find important controlling gene.

Three, the present invention sums up

The present invention is by representative difference display analysis technology (Represention DifferentialAnalysis, RDA), compared people's tire hepatic tissue and the difference of normal adult hepatic tissue genetic expression in age in April, successfully isolate people's tire liver specific expression gene first, called after Hemagene-1, full-length cDNA, Hemagene-1 full length gene 2166bp, comprise 110bp5 '-non-translational region sequence, 602bp3 '-non-translational region sequence.The protein that these genes encoding 484 amino acid are formed, molecular weight is 55.3kd.This gene has activity of conversion, and high abundance is expressed in the leukemia cell, may be applied to diagnosis and treatment leukemia.

The present invention has found a kind of new gene, utilizes Protocols in Molecular Biology that full length cDNA sequence and the biological function thereof of Hemagene-1 are provided first.

The invention provides Hemagene-1 encoded protein matter aminoacid sequence.

The invention provides the method for a kind of recombinant production people Hemagene-1, a kind of Hemagene-of containing Prokaryotic Expression carrier is provided, utilize it can obtain Hemagene-1 albumen in a large number.

The invention provides a kind of carrier for expression of eukaryon of the Hemagene-1 of containing gene and the cell strain of acquisition stably express Hemagene-1, utilize it can obtain the Hemagene-1 polypeptide.

Four. main caption

In order to understand this research contents better, below main accompanying drawing is illustrated:

Fig. 1 shows the genome structure of Hemagene-1

Fig. 2 shows the external translation carrier of structure;

Fig. 3 display body is translated the result outward

Fig. 4 shows that PCR detects the expression of Hemagene-1mRNA in different tissues

Table 5 shows that PCR detects the expression of Hemagene-1 in leukaemic's peripheral blood cells

Fig. 6 shows that Northern hybridization detects the expression of Hemagene-1mRNA in different tumor cell lines

Fig. 7 shows the structure of GFP-Hema fusion expression vector

Fig. 8 shows the expression of GFP-Hema fusion rotein in the COS-7 cell

Fig. 9 shows the Hemagene-1 Construction of eukaryotic

Figure 10 shows Hemagene-1 antisense Construction of eukaryotic

Figure 11 shows Hemagene-1 deletant Construction of eukaryotic

After Figure 12 shows detection Hemagene-1 expression vector rotaring copolymering NIH 3 T 3 cell, Hemagene-1mRNA

Expression in different cell strains

Figure 13 shows the phenotype variation that obtains stable expression cell strain through the G418 screening;

Figure 14 shows that stably express Hemagene-1 cell strain aggressive increases

Figure 15 shows the structure of fusion expression vector

Figure 16 shows the amalgamation and expression result of Hemagene-1 in intestinal bacteria

Five. describe in detail

1.RNA extract

With Trizol (Life Technologies, Inc.) extract respectively age in April people's tire hepatic tissue and the total RNA of normal adult hepatic tissue as follows, the 50-100mg hepatic tissue adds 1mlTrizol, place 5min at 25-30 after the homogenate, add 0.2ml chloroform/1mlTrizol, concuss 15min gets upper water and moves into a new EP pipe mutually, add the 0.5ml/1mlTrizol Virahol, 15-30 precipitates 10min, 2-8 then, the centrifugal 10min of 1200g, adding 1ml75% ethanol/Trizol washs once, 2-8, the centrifugal 5min of 7500g, partial desiccation RNA, adding 50ul does not have the RNA enzyme and removes Gao Zishui, and ultraviolet spectrophotometer is counted OD260/OD280 down near 2.0.

2. the branch of differential fragment is high

Utilize RDA (4) (Clontech Laboratories, Inc.) enrichment tire liver specific gene fragment.Detailed process as:, with cDNA synthetic primer 5 '-TTTTGTACAAGTTNN-3 ' the tire liver is become cDNA with normal adult hepatic tissue mRNA reverse transcription, with Rsa1 restriction enzyme 37 digestion 1.5 hours, through two-wheeled hybridization, hybridization conditions was 98 sex change 1.5min.68 hybridization 8 hours or spends the night.Get hybridization product 1ul then and carry out the nest-type PRC amplification, amplification system is 25ml, and reaction conditions is: 94 ℃ of 5min, 94 ℃ of 10sec, 68 ℃ of 30sec, 72 1.5min, totally 27 circulations.2.0% agarose gel electrophoresis is analyzed the PCR product, with enrichment tire liver specific gene fragment transformed competence colibacillus bacillus coli DH 5, picking 53 strain clones at random behind the coated plate, use T7, SP6 primer order-checking (dna sequencing instrument), carry out sequential analysis then, GeneBank Blast and EST library searching find that it is the partial sequence (Fig. 1) of this research Hemagene-1 gene that the cloned sequence of an about 300bp is wherein arranged, its nucleotide sequence and any known array do not have homology, subsequently it have been carried out Full Length cDNA Cloning.

1 GGATAGTGGT GGGAACAGAC AAATAAGGAA GCGGGGAGGA CTGGATAATT

51 GGTTTTCCCC CCTAAGAACA TTTATTTACG TCTTAAGAGC AGATAAGTGA

101 CTAAGACTGA ACACATACAT TTTGTGGAGT ATATAGTTTT CTTGTAAATG

151 CTGTTCAATT ATTAATGTAA CAGTAGCATC AAAATTTTAT TCAGGCTTTA

201 GTTGACTCTT TTGGTCAGTT TTAACAATTC TCCTTAAAAG ATATTTTGGA

251 GTGATGAATG TAGTTTACTT

The partial sequence of Fig. 1 Hemagene-1 gene

3. the separation of full-length cDNA

A. screen the cDNA library

Hemagene-1cDNA (300bp) fragment that obtains with RDA is a probe, 32P random priming (Promega) mark, bacterium colony in situ hybridization screening people embryonic liver in April cDNA library (5), the positive colony of acquisition carries out the nucleotide sequence (Fig. 2) that sequencing obtains an about 1.6kb with T7, SP6.

1 GAACACTTTT CTGAAATATG TCAAGAAAGT AACATATATC AGGAGAATTT

51 TTCTGAGTAC CAAGAAATAG CAGTACAAAA CCATTCTTCT GAAACATGCC

101 AACATGTGTC TGAACCTGAA GACCTCTCTC CTAAAATGTA CCAAGAAATA

151 TCTGTACTTC AAGACAATTC TTCCAAAATA TGCCAAGACA TGAAGGAACC

201 TGAAGACAAC TCTCCTAACA CATGCCAAGT AATATCTGTA ATTCAAGACC

251 ATCCTTTCAA AATGTACCAA GATATGGCTA AACGAGAAGA TCTGGCTCCT

301 AAAATGTGCC AAGAAGCTGC TGTACCCAAA ATCCTTCCTT GTCCAACATC

351 TGAAGACACA GCTGATCTGG CAGGATGCTC TCTTCAAGCA TATCCAAAAC

401 CAGATGTGCC TAAAGGCTAT ATTCTTGACA CAGACCAAAA TCCAGCAGAA

451 CCAGAGGAAT ACAATGAAAC AGATCAAGGA ATAGCTGAGA CAGAAGGCCT

501 TTTTCCTAAA ATACAAGAAA TAGCTGAGCC TAAAGACCTT TCTACAAAAA

551 CACACCAAGA ATCAGCTGAA CCTAAATACC TTCCTCATAA AACATGTAAC

601 GAAATTATTG TGCCTAAAGC CCCCTCTCAT AAAACAATCC AAGAAACACC

651 TCATTCTGAA GACTATTCAA TTGAAATAAA CCAAGAAACT CCTGGGTCTG

701 AAAAATATTC ACCTGAAACG TATCAAGAAA TACCTGGGCT TGAAGAATAT

751 TCACCTGAAA TATACCAAGA AACATCCCAG CTTGAAGAAT ATTCACCTGA

801 AATATACCAA GAAACACCGG GGCCTGAAGA CCTCTCTACT GAGACATATA

851 AAAATAAGGA TGTGCCTAAA GAATGCTTTC CAGAACCACA CCAAGAAACA

901 GGTGGGCCCC AAGGCCAGGA TCCTAAAGCA CACCAGGAAG ATGCTAAAGA

951 TGCTTATACT TTTCCTCAAG AAATGAAAGA AAAACCCAAA GAAGAGCCAG

1001 GAATACCAGC AATTCTGAAT GAGAGTCATC CAGAAAATGA TGTCTATAGT

1051 TATGTTTTGT TTTAACAATT CTCAACCATA AAGTTGTGGT CCAATGGAAC

1101 ATACAGCTTA ATAGTTTATG CGTGATTTTC TCAAAATATT GTAAAACTTT

1151 TGACAATGCT CATTAATATT ATTTTTTCTA TTTGTAGACC ATATCTGAAA

1201 GAAATAACAT TTTTTAAGGC TCTACCACAT AGACAATATC ATGCTAGAAT

1251 GTGTGTGTGT GTGTGTGTGT GTGTGTGTAT GTATGTATAG GTCGGGGAGA

1301 GGATAGTGGT GGGAACAGAC AAATAAGGAA GCGGGGAGGA CTGGATAATT

1351 GGTTTTCCCC CCTAAGAACA TTTATTTACG TCTTAAGAGC AGATAAGTGA

1401 CTAAGACTGA ACACATACAT TTTGTGGAGT ATATAGTTTT CTTGTAAATG

1451 CTGTTCAATT ATTAATGTAA CAGTAGCATC AAAATTTTAT TCAGGCTTTA

1501 GTTGACTCTT TTGGTCAGTT TTAACAATTC TCCTTAAAAG ATATTTTGGA

1551 GTGATGAATG TAGTTTACTT TTGTATTTGA ATTTTGATTT TCTATTTTTA

1601 TTTTTTAAAT ATTGTATTTG TGCACAATGT ACATTAAATC ATTATTACAT

1651 GAAAAAAAAA AAAAAA

Fig. 2 shows that screening the cDNA library obtains the Hemagene-1 Partial cDNA Sequence;

B.5′-RACE

According to sequence synthesized primer thing 5 '-GGT CTT CAG GTT CAG ACA CAT GTT GGC-3 ', adopting the Clontech Marathon-ready cDNA of company is template, carry out 5 ' RACE (6) pcr amplification (ClontechLaborateries, Inc), the first set reaction volume is 50ul, reaction conditions is: 94 ℃ of 30sec, 94 ℃ of 5sec, 72 ℃ of 4min totally 5 circulations, 94 ℃ of 5sec, 70 ℃ of 4sec totally 5 circulations, 94 5sec68 4sec are totally 20 circulations.The nest-type PRC reaction volume is 25ul, and reaction conditions is 94 ℃ of 10sec, 68 ℃ of 30sec, 72 ℃ of 30sec, totally 25 circulations.The PCR product is electrophoretic separation in 1% agarose, and subclone pGEM-T carrier, T7, SP6 carry out the nucleotide sequence (Fig. 3) that sequencing obtains an about 0.6kb, and electronic splicing obtains Hemagene-1 full-length cDNA (Fig. 4).

1 GGTTATGAAG ATAGGTACTG TGGGTGTTAG AAAGATTCAC GGCAAAACAG

51 GGAAGCATCT AGGCTGCTTG TGGAAGTCAG ACCAAAATAG CAGGAAGGTA

101 TTGCAGCAAG ATGGATTTGG GAAAGGACCA ATCTCATTTG AAGCACCATC

151 AGACACCTGA CCCTCATCAA GAAGAGAACC ATTCTCCAGA AGTCATTGGA

201 ACCTGGAGTT TGAGAAACAG AGAACTACTT AGAAAAAGAA AAGCTGAAGT

251 GCATGAAAAG GAAACATCAC AATGGCTATT TGGAGAACAG AAAAAACGCA

301 AGCAGCAGAG AACAGGAAAA GGAAATCGAA GAGGCAGAAA GAAACAACAA

351 AACACAGAAT TGAAGGTGGA GCCTCAGCCA CAGATAGAAA AGGAAATAGT

401 GGAGAAAGCA CTGGCACCTA TAGAGAAAAA AACTGAGCCA CCTGGGAGCA

451 TAACCAAAGT ATTTCCTTCA GTAGCCTCCC CGCAAAAAGT TGTGCCTGAG

501 GAACACTTTT CTGAAATATG TCAAGAAAGT AACATATATC AGGAGAATTT

551 TTCTGAGTAC CAAGAAATAG CAGTACAAAA

Fig. 3 shows that 5 '-RACE obtains dna sequence dna

1 GGTTATGAAG ATAGGTACTG TGGGTGTTAG AAAGATTCAC GGCAAAACAG

51 GGAAGCATCT AGGCTGCTTG TGGAAGTCAG ACCAAAATAG CAGGAAGGTA

101 TTGCAGCAAG ATGGATTTGG GAAAGGACCA ATCTCATTTG AAGCACCATC

151 AGACACCTGA CCCTCATCAA GAAGAGAACC ATTCTCCAGA AGTCATTGGA

201 ACCTGGAGTT TGAGAAACAG AGAACTACTT AGAAAAAGAA AAGCTGAAGT

251 GCATGAAAAG GAAACATCAC AATGGCTATT TGGAGAACAG AAAAAACGCA

301 AGCAGCAGAG AACAGGAAAA GGAAATCGAA GAGGCAGAAA GAAACAACAA

351 AACACAGAAT TGAAGGTGGA GCCTCAGCCA CAGATAGAAA AGGAAATAGT

401 GGAGAAAGCA CTGGCACCTA TAGAGAAAAA AACTGAGCCA CCTGGGAGCA

451 TAACCAAAGT ATTTCCTTCA GTAGCCTCCC CGCAAAAAGT TGTGCCTGAG

501 GAACACTTTT CTGAAATATG TCAAGAAAGT AACATATATC AGGAGAATTT

551 TTCTGAGTAC CAAGAAATAG CAGTACAAAA CCATTCTTCT GAAACATGCC

601 AACATGTGTC TGAACCTGAA GACCTCTCTC CTAAAATGTA CCAAGAAATA

651 TCTGTACTTC AAGACAATTC TTCCAAAATA TGCCAAGACA TGAAGGAACC

701 TGAAGACAAC TCTCCTAACA CATGCCAAGT AATATCTGTA ATTCAAGACC

751 ATCCTTTCAA AATGTACCAA GATATGGCTA AACGAGAAGA TCTGGCTCCT

801 AAAATGTGCC AAGAAGCTGC TGTACCCAAA ATCCTTCCTT GTCCAACATC

851 TGAAGACACA GCTGATCTGG CAGGATGCTC TCTTCAAGCA TATCCAAAAC

901 CAGATGTGCC TAAAGGCTAT ATTCTTGACA CAGACCAAAA TCCAGCAGAA

951 CCAGAGGAAT ACAATGAAAC AGATCAAGGA ATAGCTGAGA CAGAAGGCCT

1001 TTTTCCTAAA ATACAAGAAA TAGCTGAGCC TAAAGACCTT TCTACAAAAA

1051 CACACCAAGA ATCAGCTGAA CCTAAATACC TTCCTCATAA AACATGTAAC

1101 GAAATTATTG TGCCTAAAGC CCCCTCTCAT AAAACAATCC AAGAAACACC

1151 TCATTCTGAA GACTATTCAA TTGAAATAAA CCAAGAAACT CCTGGGTCTG

1201 AAAAATATTC ACCTGAAACG TATCAAGAAA TACCTGGGCT TGAAGAATAT

1251 TCACCTGAAA TATACCAAGA AACATCCCAG CTTGAAGAAT ATTCACCTGA

1301 AATATACCAA GAAACACCGG GGCCTGAAGA CCTCTCTACT GAGACATATA

1351 AAAATAAGGA TGTGCCTAAA GAATGCTTTC CAGAACCACA CCAAGAAACA

1401 GGTGGGCCCC AAGGCCAGGA TCCTAAAGCA CACCAGGAAG ATGCTAAAGA

1451 TGCTTATACT TTTCCTCAAG AAATGAAAGA AAAACCCAAA GAAGAGCCAG

1501 GAATACCAGC AATTCTGAAT GAGAGTCATC CAGAAAATGA TGTCTATAGT

1551 TATGTTTTGT TTTAACAATT CTCAACCATA AAGTTGTGGT CCAATGGAAC

1601 ATACAGCTTA ATAGTTTATG CGTGATTTTC TCAAAATATT GTAAAACTTT

1651 TGACAATGCT CATTAATATT ATTTTTTCTA TTTGTAGACC ATATCTGAAA

1701 GAAATAACAT TTTTTAAGGC TCTACCACAT AGACAATATC ATGCTAGAAT

1751 GTGTGTGTGT GTGTGTGTGT GTGTGTGTAT GTATGTATAG GTCGGGGAGA

1801 GGATAGTGGT GGGAACAGAC AAATAAGGAA GCGGGGAGGA CTGGATAATT

1851 GGTTTTCCCC CCTAAGAACA TTTATTTACG TCTTAAGAGC AGATAAGTGA

1901 CTAAGACTGA ACACATACAT TTTGTGGAGT ATATAGTTTT CTTGTAAATG

1951 CTGTTCAATT ATTAATGTAA CAGTAGCATC AAAATTTTAT TCAGGCTTTA

2001 GTTGACTCTT TTGGTCAGTT TTAACAATTC TCCTTAAAAG ATATTTTGGA

2051 GTGATGAATG TAGTTTACTT TTGTATTTGA ATTTTGATTT TCTATTTTTA

2101 TTTTTTAAAT ATTGTATTTG TGCACAATGT ACATTAAATC ATTATTACAT

2151 GAAAAAAAAA AAAAAA

Fig. 4 shows the Hemagene-1 full length cDNA sequence

C. analyze the cDNA clone

Use DNA Tools, softwares such as BLAST carry out bioinformatic analysis, and sequential analysis discloses the open reading frame that this gene contains 484 the amino acid constitutive proteins of encoding, and the complete nucleotide sequence and the aminoacid sequence of supposition are seen Fig. 5.Codon ATG is positioned at 111-113 Nucleotide, initial ATG plays-3 and is A, + 4 is G, belong to typical Kozak sequence, the same encoder block in its upstream has 2 termination codons, 3 ' sequence contains 2 copy ATTTA sequences, and this element shows that this mRNA has the short transformation period (6), and 3 ' does not find the conservative tailer sequence that adds of AATAAA '.BLAST retrieval show this assignment of genes gene mapping in No. 9 chromosomal long-armed, its gene contains four introns (accompanying drawing 1).484 amino acid constitutive protein matter of coding do not contain known functional domain and see Fig. 5.

4. external translation

With the SP6 rna polymerase promoter downstream (accompanying drawing 2) of Hemagene-1 coding region cDNA subclone to pSP70 plasmid (Promega), transformed into escherichia coli JM109, extract plasmid (Promega Kit), carry out the external translation of coupled reticulocyte lysate system (Promega) according to the test kit operational manual then, with L-35Smethionine (Amersham) mark, the SDS-PAGE electrophoresis, the radioautograph display result.Luciferase SP6 control DNA (Promega) compares.The protein that the true coding molecule amount of this gene of experiment confirm is 55.3KD, molecular weight with infer albumen conform to (accompanying drawing 3).

Fig. 5 shows the nucleotide sequence of Hemagene-1 and the aminoacid sequence of supposition

5.PCR detect the distribution of Hemagene-1 gene in different tissues

Be the distribution situation of research Hemagene-1 at different tissues, the Multiple tissue cDNA (Clontech) that employing is demarcated through multiple house-keeping gene makes template, detect Hemagene-1 mRNA with PCR, pcr amplification carries out on pcr amplification instrument GeneAmp PCR System 9700, and reaction volume is 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations under identical conditions, are made internal reference with G3PDH.The PCR product is electrophoresis in 1.5% agarose, gel imaging instrument scanning imagery.As seen the results are shown in accompanying drawing 4, expression amount is extremely low or do not express in multiple tissues such as the brain of normal adult, the heart, kidney, liver, lung, muscle, tonsilla, peripheral blood cells, expresses and be high abundance in marrow, embryonic liver tissue.Detect the expression of 10 routine Patients with Acute Leukemia and 1 routine lymphoma patient Hemagene-1, found that the expression that high abundance Hemagene-1 is all arranged in 7 routine acute lymphoblastic leukemia patients' wherein marrow and the peripheral blood, then do not detect the expression (table 1) of Hemagene-1 in the anxious non-pouring leukemia patient peripheral bloods of 3 examples.

Table 1 leukaemic peripheral blood Hemagene-1 expresses

Numbering	Diagnosis	Hemagene-1 expresses
			1 2 3 4 5 6 7 8 9 10 11 12	Suddenly drench non-suddenly pouring of the anxious pouring of the non-anxious pouring of suddenly pouring and suddenly drench the anxious pouring of the non-anxious pouring of suddenly pouring normal person normal person normal person	++ + - +++ - ++ ++ - +++ - - -

6.Northern hybridization detects the expressing K 562 of Hemagene-1 in different tumor cell lines, HL60 and MO7e (human leukemia cell line) cell suspension culture is in the RPMI1640 nutrient solution that contains 10% new-born calf serum (GiBco), and MO7e adds G-CSF (5ng/ml) in addition; KBV cell (strain of people's laryngeal cancer cell), HTC cell (mouse hepatoma cell strain), HepG2 cell (human hepatoma cell strain), HLE cell (human hepatoma cell strain), Hep2 cell (strain of people's laryngeal cancer cell), NIH3T3 cell (l cell strain), the equal adherent culture of 7402 cells (human hepatoma cell strain) is in the DMEM substratum that contains 10% new-born calf serum.Nutrient solution all contains penbritin and the Streptomycin sulphate of each 100mg/L, 37 ℃, 5%CO ₂Cultivate under the saturated humidity fully.

Extract various cell total rnas with Trizol TM test kit.1mlTrizol cracking 1 * 10 ⁷Cell adds chloroform 200ml extracting once then, and the centrifugal 15min of 1200g4 gets supernatant, and with 0.5ml dehydrated alcohol precipitation, 75% ethanol is washed 2 times, the aseptic deionized water dissolving RNA that handle with DEPC dry back.The uv-spectrophotometric instrument is measured its concentration.

Get total RNA20 μ g and carry out 1% denaturing formaldehyde gel electrophoresis, the transfer printing nitrocellulose filter, film adds 6 * SSC at 80 ℃ of oven dry 2h, 5 * Denhardt ' s, 0.5%SDS, 100 μ g/ml salmon sperm DNAs, at 68 ℃ of prehybridization 1h, add the sex change probe, 68 ℃ of hybridization are spent the night; To wash film 2 times under the 2 * SSC room temperature that contains 0.5%SDS, wash film 2 times for 50 ℃ ,-70 ℃ of following radioautograph after the drying at room temperature with the 0.5% * SSC that contains 0.5%SDS again.Found that only high expression level in K562 and MO7e cell of Hemagene-1, and in all the other various kinds of cell strains, do not express (accompanying drawing 5).

7.Hemagene-1 cellular localization pull with the pGEM-Hemagene-1 plasmid mould that makes up, utilize pcr amplification Hemgene-1 coding region cDNA, the PCR primer is P1:5 ' CGG AAT TCA TGG ATT TGGGAA AGG AC-3 '; P2:5 '-GGG GTA CCT AAA ACA AAA CAT AAC TAT AG-3; The PCR condition is 94 ℃ of 30sec, 50 ℃ of 60sec, 72 ℃ of 90sec, totally 25 circulations.The PCR product is through EcoRI, and Kpen I enzyme is cut the back subclone to pGFP-C1 plasmid (accompanying drawing 6), and plasmid is after the sequential analysis affirmation is errorless, and with liposome transfection cos-7 cell, cell is containing 10%FCS DMEM, 5%CO ₂Cultivated 24-48 hour under 37 ℃ of conditions, fluorescent microscope is observed the fluorescent protein expression situation down.Simultaneously, the empty pGFP-C1 carrier of transfection transcription factor ETS GFP expression vector and lotus in contrast.The expression product of finding green-emitting fluorescence mainly concentrates on nucleus, be expressed in nuclear transcription factor ETS-1 consistent (7) with known, and GFP transfectional cell fluorescence is diffusivity (accompanying drawing 7), and prompting Hemagene-1 gene expression product is positioned in the nucleus.

8. the structure of expression plasmid utilizes pcr amplification Hemagene-1 coding region cDNA, condition is the same, it is subcloned on pcDNA3.1 (+) CMV promotor downstream, be built into carrier for expression of eukaryon (accompanying drawing 8), simultaneously cDNA is oppositely inserted pcDNA3.1 (+) CMV promotor downstream, be built into reverse carrier for expression of eukaryon (accompanying drawing 9); In addition, utilize PCR to be built into N end disappearance 175 amino acid whose deletant expression vectors (Hemagene-) (accompanying drawing 10).Above-mentioned plasmid all is used for experiment after sequential analysis is confirmed.The H-ras-V12-pBabe expression vector is so kind as to give by Huibin doctor Sun ⁽⁷⁾

9. cell cultures and stable expression cell strain are set up the different carriers (each 20 μ g) that will make up with liposome method rotaring copolymering NIH 3 T 3 cell respectively, and cell is containing the DMEM of 10%FCS, 5%CO ₂Cultivated under 37 ℃ of conditions 24 hours, and, continued to cultivate containing under G418 (the 200 μ g/ml) condition with 2 week of G418 (400 μ g/ml) screening back picking mono-clonals.

10. the RNA of the different plasmid cells of transfection is extracted in the evaluation of stable expression cell strain, detects the Hemagene-1mRNA expression with RT-PCR.With Oligo (dT) is initial primers, adds total RNA 1ug, behind 72 ℃ of 10mins, adds no RNase water, 10 * RT buffer, 25mM MgCl successively ₂, 250uM dNTPs, RNasin and AMV reversed transcriptive enzyme, reaction system is 20ul, behind 42 ℃ of insulation 15mins, adds deionized water to 100ul.And carry out PCR as template.The PCR primer is P1 5 '-ATG TAC CAA GAA ATA TCTGTA-3; P2:5 '-TAA AAC AAA ACA TAA CTA TAG-3 '.The PCR reaction volume is 25ul, and cycling condition is 94 ℃ of 1min, 50 ℃ of 1min, and 72 ℃ of 1min, totally 30 circulations, the PCR product is through 1.5% agarose electrophoresis, gel imaging instrument scanning imagery.Specific amplified band molecular weight is the Hemagene-1 forward that will success makes up of 1.0kb., oppositely reaches the eukaryotic expression plasmid rotaring copolymering NIH 3 T 3 cell of 175 aminoacid deletion bodies of N-end disappearance, after G418 screened for 4 weeks, the PCR method detects the expression of different cell strain Hemagene-1 mRNA, find that NIH3T3 cell itself do not express Hemagene-1, but after the transfection certain plasmid cell expressing Hemagene-1 mRNA (accompanying drawing 11).Cellular form is observed the cell strain (two strains) of finding transfection forward expression vector tangible form change (accompanying drawing 12) is taken place, cell surface becomes level and smooth and the refractivity increase, and the trend and the contact inhibition phenomenon of the parallel growth of normal fibroblast have been lost, intersect mutually between the cell and the formation multilayer, and transfection oppositely, the cell strain form of deletant and lotus empty carrier do not have obvious variation, prompting Hemagene-1 may have the activity of the cell transformation of making.

11. the soft agar colony forms experiment with the NIH3 cell trysinization of the different plasmids of transfection respectively of G418 screening back, the aseptic injection syringe needle by 25G 5/8 is dispersed into individual cells, plants 1 * 10 with every 35mm diameter culture dish ³The density of individual cell is uniformly distributed in cell in the DMEM nutrient solution that contains 20%FCS, 0.3% ultrapure agar of 1ml as top layer, evenly be layered on subsequently and completed on the DMEM that contains 0.6% ultrapure agar, 20%FCS of 1ml as bottom, all contain G418 (400ug/ml) in the two-layer agar, every group of each three ware.Add once new top-agar in every 4-5 days, surpass clone's number of 50 cells after two weeks at microscopically statistics colony.With the different cell strain inoculation soft agars of build, cultivate to count after 14 days and surpass 50 colony number of cell, the results are shown in Table 2, two strain forward clonal cell lines, soft agar forms independent cloning efficient and is respectively 32.5% and 33.6%, transfection oppositely, the cell strain of deletant, lotus empty carrier then do not have the ability that grappling independently is grown, and can not form independently and clone.

The different cell soft agars of table 2 are cultivated the clone and are formed number

Cell	The clone forms (%)
		pcDNA3.1 Hemagene-1 Hemagene-2 Hemagene-as Hemagene- H-RasV12-pBabe	0 32.5 33.6 0 0 50.2

12. mouse becomes the knurl experiment respectively transfection to be had the NIH3T3 cell (1 * 10 of different plasmids ⁶) be expelled to 4 all female nude mices the back subcutaneous, three every group, observe twice weekly, to inoculation 20 weeks of back.Put to death after becoming the knurl mouse to take a picture, get knurl and do the pathology histological examination.The nude mice of back all around six injection high expression level Hemagene-1 NIH3T3 cell strains all forms tumour (accompanying drawing 13), and grows up rapidly.Histopathologic examination shows that this knurl has fibrocellular tumor sample characteristic, the NIH3T3 cell of pointing out this tumour to come from transfection.Other each group is observed and 20 weeks was not seen tumor growth (table 3).

Table 3 nude mice tumour forms

Cell	Tumour forms
		pcDNA Hemagene-1 Hemagene-2 Hemagene-as Hemagene-	0/3 3/3 3/3 0/3 0/3

13. invasion and attack experiment (8) respectively with the NIH3T3 cell of the different plasmids of transfection with 1.0 * 10 ⁵The density of/ml is seeded in the 0.8um aperture that contains 0.1%BSA DMEM and wraps by in the cultivation cup (available from BectonDickinsono) of matrigel, and lower floor is serum-free DMEM, 5%CO ₂, 37 ℃ of conditions cultivate after 12 hours, scrape off interior confluent monolayer cells, outerly are inverted and cultivate cup with the methyl alcohol dyeing that contains 1% Viola crystallina, light microscopic is observed cell and the counting that passes through counterdie down.The different N IH3T3 cell strain inoculation 0.8um aperture of setting up is wrapped by the cultivation cup of matrigel, the saturating film ability of observation of cell after 12 hours of cultivating is to estimate the aggressive of cell, the result shows that stably express Hemagene-1 cell strain strengthens about about 10 times than the cell strain aggressive of lotus empty carrier, and transfection oppositely, aggressive there was no significant difference (accompanying drawing 14) between the cell strain of deletant and lotus empty carrier.

14.Hemagene-1 the expression in intestinal bacteria utilizes pcr amplification Hemagene-1 coding region DNA, with KpnI and EcoRI site (accompanying drawing 15) of its subclone to the PGEX-4T-2 plasmid, Transformed E coli DH5 α, utilize enzyme to cut, PCR identifies recombinant plasmid, and its sequence is determined with nucleotide sequence analysis.The bacterium that will contain recombinant plasmid crosses liquid at the LB nutrient solution that contains the 100Ug/ml penbritin and cultivates, be cultured to OD600=0.8 by dilution in 1: 10 with the LB nutrient solution of the fresh 1OOUg/ml of containing penbritin next day, add IPTG to final concentration be 1.OmM, continue to cultivate after 4 hours, collect bacterium, with SDS-PAGE electrophoresis detection expressing protein, the result shows that expressing protein accounts for 42% (Figure 16) of total bacterial protein.

In a word, the present invention has cloned a kind of new gene order, and confirms that it has activity of conversion.Obtain marking protein by making up prokaryotic expression carrier and transforming suitable prokaryotic host cell; By making up carrier for expression of eukaryon and transfection proper host cell, after screening, obtained the cell strain of stably express Hemagene-1.Pernicious transformation takes place in the NIH3T3 cell of stably express Hemagene-1, shows that this gene has activity of conversion; This gene may be applied to leukemia cell's diagnosis at leukemia cell's high expression level, or becomes the novel targets of screening anti-leukemia medicine.

Reference

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Claims (3)

1. one kind has the activity of conversion gene---and human hematopoietic cell differentiation associated gene-1 has following nucleotide sequence:

1 GGTTATGAAG ATAGGTACTG TGGGTGTTAG AAAGATTCAC GGCAAAACAG

51 GGAAGCATCT AGGCTGCTTG TGGAAGTCAG ACCAAAATAG CAGGAAGGTA

101 TTGCAGCAAG ATGGATTTGG GAAAGGACCA ATCTCATTTG AAGCACCATC

151 AGACACCTGA CCCTCATCAA GAAGAGAACC ATTCTCCAGA AGTCATTGGA

201 ACCTGGAGTT TGAGAAACAG AGAACTACTT AGAAAAAGAA AAGCTGAAGT

251 GCATGAAAAG GAAACATCAC AATGGCTATT TGGAGAACAG AAAAAACGCA

301 AGCAGCAGAG AACAGGAAAA GGAAATCGAA GAGGCAGAAA GAAACAACAA

351 AACACAGAAT TGAAGGTGGA GCCTCAGCCA CAGATAGAAA AGGAAATAGT

401 GGAGAAAGCA CTGGCACCTA TAGAGAAAAA AACTGAGCCA CCTGGGAGCA

451 TAACCAAAGT ATTTCCTTCA GTAGCCTCCC CGCAAAAAGT TGTGCCTGAG

501 GAACACTTTT CTGAAATATG TCAAGAAAGT AACATATATC AGGAGAATTT

551 TTCTGAGTAC CAAGAAATAG CAGTACAAAA CCATTCTTCT GAAACATGCC

601 AACATGTGTC TGAACCTGAA GACCTCTCTC CTAAAATGTA CCAAGAAATA

651 TCTGTACTTC AAGACAATTC TTCCAAAATA TGCCAAGACA TGAAGGAACC

701 TGAAGACAAC TCTCCTAACA CATGCCAAGT AATATCTGTA ATTCAAGACC

751 ATCCTTTCAA AATGTACCAA GATATGGCTA AACGAGAAGA TCTGGCTCCT

801 AAAATGTGCC AAGAAGCTGC TGTACCCAAA ATCCTTCCTT GTCCAACATC

851 TGAAGACACA GCTGATCTGG CAGGATGCTC TCTTCAAGCA TATCCAAAAC

901 CAGATGTGCC TAAAGGCTAT ATTCTTGACA CAGACCAAAA TCCAGCAGAA

951 CCAGAGGAAT ACAATGAAAC AGATCAAGGA ATAGCTGAGA CAGAAGGCCT

1001 TTTTCCTAAA ATACAAGAAA TAGCTGAGCC TAAAGACCTT TCTACAAAAA

1051 CACACCAAGA ATCAGCTGAA CCTAAATACC TTCCTCATAA AACATGTAAC

1101 GAAATTATTG TGCCTAAAGC CCCCTCTCAT AAAACAATCC AAGAAACACC

1151 TCATTCTGAA GACTATTCAA TTGAAATAAA CCAAGAAACT CCTGGGTCTG

1201 AAAAATATTC ACCTGAAACG TATCAAGAAA TACCTGGGCT TGAAGAATAT

1251 TCACCTGAAA TATACCAAGA AACATCCCAG CTTGAAGAAT ATTCACCTGA

1301 AATATACCAA GAAACACCGG GGCCTGAAGA CCTCTCTACT GAGACATATA

1351 AAAATAAGGA TGTGCCTAAA GAATGCTTTC CAGAACCACA CCAAGAAACA

1401 GGTGGGCCCC AAGGCCAGGA TCCTAAAGCA CACCAGGAAG ATGCTAAAGA

1451 TGCTTATACT TTTCCTCAAG AAATGAAAGA AAAACCCAAA GAAGAGCCAG

1501 GAATACCAGC AATTCTGAAT GAGAGTCATC CAGAAAATGA TGTCTATAGT

1551 TATGTTTTGT TTTAACAATT CTCAACCATA AAGTTGTGGT CCAATGGAAC

1601 ATACAGCTTA ATAGTTTATG CGTGATTTTC TCAAAATATT GTAAAACTTT

1651 TGACAATGCT CATTAATATT ATTTTTTCTA TTTGTAGACC ATATCTGAAA

1701 GAAATAACAT TTTTTAAGGC TCTACCACAT AGACAATATC ATGCTAGAAT

1751 GTGTGTGTGT GTGTGTGTGT GTGTGTGTAT GTATGTATAG GTCGGGGAGA

1801 GGATAGTGGT GGGAACAGAC AAATAAGGAA GCGGGGAGGA CTGGATAATT

1851 GGTTTTCCCC CCTAAGAACA TTTATTTACG TCTTAAGAGC AGATAAGTGA

1901 CTAAGACTGA ACACATACAT TTTGTGGAGT ATATAGTTTT CTTGTAAATG

1951 CTGTTCAATT ATTAATGTAA CAGTAGCATC AAAATTTTAT TCAGGCTTTA

2001 GTTGACTCTT TTGGTCAGTT TTAACAATTC TCCTTAAAAG ATATTTTGGA

2051 GTGATGAATG TAGTTTACTT TTGTATTTGA ATTTTGATTT TCTATTTTTA

2101 TTTTTTAAAT ATTGTATTTG TGCACAATGT ACATTAAATC ATTATTACAT

2151 GAAAAAAAAA AAAAAA

2. the human hematopoietic cell by the described dna sequence encoding of claim 1 breaks up related protein-1, has following aminoacid sequence:

1 MDLGKDQSHL KHHQTPDPHQ EENHSPEVIG TWSLRNRELL RKRKAEVHEK

51 ETSQWLFGEQ KKRKQQRTGK GNRRGRKKQQ NTELKVEPQP QIEKEIVEKA

101 LAPIEKKTEP PGSITKVFPS VASPQKVVPE EHFSEICQES NIYQENFSEY

151 QEIAVQNHSS ETCQHVSEPE DLSPKMYQEI SVLQDNSSKI CQDMKEPEDN

201 SPNTCQVISV IQDHPFKMYQ DMAKREDLAP KMCQEAAVPK ILPCPTSEDT

251 ADLAGCSLQA YPKPDVPKGY ILDTDQNPAE PEEYNETDQG IAETEGLFPK

301 IQEIAEPKDL STKTHQESAE PKYLPHKTCN EIIVPKAPSH KTIQETPHSE

351 DYSIEINQET PGSEKYSPET YQEIPGLEEY SPEIYQETSQ LEEYSPEIYQ

401 ETPGPEDLST ETYKNKDVPK ECFPEPHQET GGPQGQDPKA HQEDAKDAYT

451 FPQEMKEKPK EEPGIPAILN ESHPENDVYS YVLF*

3. the described proteic method of recombinant production claim 2 is characterized in that the described dna sequence dna of claim 1 is placed suitable carrier, transforms protokaryon or eukaryotic host cell, obtains aforementioned polypeptides.

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