CN102558331A - tRNA (transfer ribose nucleic acid) binding protein participated in cell cycle regulation and coding gene and application thereof - Google Patents

Abstract

The invention provides a novel tRNA (transfer ribose nucleic acid) binding protein participated in the formation and maintenance of Artemia resting eggs and coding gene of the tRNA binding protein. The protein is located through binding with tRNA and stayed in a cell nucleus, participates in cell cycle regulation through signaling pathways such as MAPK (Mitogen Activated Protein Kinase), PI3K (Phosphatidyl Inositol 3-kinase)-AKT and FKHR/FoxO to cyclin D/CDK6 (Cyclin Dependent Kinase 6)/Rb and the like, so that cells (normal cells and tumour cells) stay in G1/S stage. The mechanism of the invention can provides a new direction for researching antitumor drugs.

Description

The tRNA that participates in cell cycle regulating is conjugated protein, encoding sox and application

(1) technical field

The present invention relates to that a kind of tRNA that participates in cell cycle regulating is conjugated protein, encoding sox and application.

(2) background technology

Genetic expression transcribe with post-transcriptional level on receive complicacy and accurate regulation and control.The various factors that relate on the transcriptional level and mechanism have obtained explaination to a great extent, and the regulation and control on the post-transcriptional level are then understood seldom.Be similar to through research and set forth the mechanism of transcribing, then need inquire into the effect of various rna binding proteins for the research of post-transcriptional level regulation and control various transcription factors.Similar with the effect of transcription factor; Rna binding protein is mainly concerned with the regulation and control of this process from the rna transcription to the protein expression; It can decide the destiny of bonded RNA (comprising mRNA or noncoding RNA) with it through number of mechanisms, for example, and the change of the shearing of RNA mechanism; The increase of rna stability or reduce, the change of translation efficiency and RNA are in intracellular different location etc.In all RNA, and noncoding RNA (noncoding RNA, ncRNA) number is numerous, and its transcriptional level alters a great deal under different environment, and has many evidences to show that these variations are often relevant with the generation of tumour.Play an important role in the stress response that rna binding protein carries out when organism is in unfavourable condition the regulation and control on the post-transcriptional level, many rna binding proteins help its mRNA target of discerning them to realize the regulation and control to transcribing through the regulation and control non-coding RNA.When receiving genetoxic or other environmental stress, the tRNA of yeast cell accumulates in nucleus, causes cellular proteins to synthesize and stops, and cell is stuck in the G1 phase, has influence on the cell cycle.How these noncoding RNA participate in the attention that regulate gene expression is causing more and more investigators.

Biological intravital non-coding RNA is mainly from the rna plymerase iii transcription product, and the corresponding conjugated protein La of family albumen plays an important role in regulation and control these proteic maturations, location and transhipment.TRNA is as a kind of important RNA polymerase III transcription product, and nearest its maturation of research demonstration and location possibility cell cycle and the cell adaptation of environment to external world play regulating and controlling effect.

The halogen worm has very strong adaptive capacity to environment, under different environment, adopts different mode of reproduction.Both can under the suitable condition of environment, directly give birth to nauplius larva, also can when environmental degradation, give birth to the winter egg that encapsulates rigid shell, resist severe environment through this unique dormancy embryo through the oviparity approach through approach ovoviviparous.Be used to study the model animals of cell cycle in the past and compared, the winter egg of halogen worm is stagnated the G1 phase in the cell cycle for a long time, for the cell cycle regulating Study on Mechanism provides a fine experiment material.

(3) summary of the invention

The object of the invention provides a kind of a kind of novel conjugated protein and encoding sox of tRNA that halogen worm winter egg forms and keeps of participating in, and this albumen is positioned nucleus through combine to make it with tRNA, thereby participates in the regulation and control of cell cycle.

The technical scheme that the present invention adopts is:

A kind of tRNA conjugated protein (Afu albumen) that participates in cell cycle regulating, its aminoacid sequence is shown in SEQ ID No.1.This albumen has tRNA and combines activity, is positioned nucleus, participates in regulation and control tRNA transhipment and also causes cell cycle arrest.

The invention still further relates to the protein-bonded encoding sox of a kind of said tRNA.This cDNA total length 1425bp, corresponding encoded protein has 404aa.

Concrete, said gene order is shown in SEQ ID No.2.

The invention still further relates to the conjugated protein application in the medicine in preparation modulate tumor cell cycle of described tRNA.

Beneficial effect of the present invention is mainly reflected in: a kind of a kind of novel conjugated protein and encoding sox of tRNA that halogen worm winter egg forms and keeps of participating in is provided; This albumen is identified with the function of regulation and control kinds of tumor cells cell cycle through the transportation of control tRNA; Find that this albumen has tRNA and combines active; Be positioned nucleus; Participate in regulation and control tRNA transhipment and cause cell cycle arrest, can be used for preparing the medicine in modulate tumor cell cycle, for tumour medicine research provides new direction.

(4) description of drawings

Fig. 1 is the electrophorogram of Afu gene and proteins encoded; A is an Afu gene coding region PCR product, and c is the Afu albumen that the GST of purifying merges, and b is the contrast of GST albumen;

Fig. 2 is the influence that the interference of Afu gene is grown the halogen worm; , a changes for the proteic expression amount of Afu after interfering for the variation of the Afu mRNA of the Afu double-stranded RNAs interference back detection of the different amounts of use, b, and c explains at the 800ng double-stranded RNA and interferes the dormant state of the winter egg of back halogen worm generation to be broken;

It is active that to be Afu albumen combine with tRNA external Fig. 3; A detects for using GoldView dyeing, and b is for using the result of different tRNA probe in detecting;

The Afu albumen that Fig. 4 merges for GFP is appraised and decided the position in different tumour cells; Scale, 50 μ m;

Fig. 5 is Afu albumen and the interior activity that combines of the body of tRNA; To be Afu albumen combine actively in the halogen worm cell of different development stage with tRNA a, and to be the albumen that merges of GFP combine activity with tRNA to b in the HeLa cell;

After the Afu albumen that Fig. 6 merges for GFP is expressed in the HeLa cell, the variation that tRNA distributes in the cell; A is for using normal tRNA probe (can detect the tRNA that contains intron and do not contain intron simultaneously), and b is the intron region probe; Scale, 100 μ m;

After the Afu albumen that Fig. 7 merges for use BrdU detection GFP is expressed in different tumour cells, the variation of cell cycle; In the time detecting the BrdU signal in the cell, explain that cell can pass through the S phase, otherwise the explanation cell is stuck in the G1/S phase; Scale, 100 μ m.

After the Afu albumen that Fig. 8 merges for use flow cytometry detection GFP is expressed in the HeLa cell, the variation of cell cycle; A is the variation of dna content in the cell of handling, b be the HeLa cell at 18h after the transfection to G1 between 24h, the variation of S and G2/M phase cell proportion;

Fig. 9 is the influence of Afu albumen to HeLa cell proliferation;

The expression of the Afu albumen that Figure 10 merges for GFP in Xenopus laevis and zebra fish young cell and the stagnation of cell cycle of causing thereof; What arrow was indicated is the detection signal of BrdU, only in cellular control unit, occurs;

Figure 11 expresses 24h for the Afu albumen of halogen worm different development stage and GFP fusion in the HeLa cell, behind 36h and the 48h, and the expression of cell within a cell cycle GAP-associated protein GAP and the variation of phosphorylation level;

Figure 12 participates in the signal path of cell cycle regulation for the Afu albumen of prediction;

Figure 13 is growth-inhibiting and the cell cycle regulating of Afu albumen to the nude mice Subcutaneous tumor.A, Afu albumen have suppressed HeLa and two kinds of growth of tumor of HT1080.B, after use BrdU detection Afu albumen is expressed in different tumour cells, the variation of cell cycle; In the time detecting the BrdU signal in the cell, explain that cell can pass through the S phase, otherwise the explanation cell is stuck in the G1/S phase; Scale, 50 μ m.C, Afu albumen in tumour cell after, the expression of cell within a cell cycle GAP-associated protein GAP and the variation of phosphorylation level.

(5) embodiment

Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:

The acquisition of embodiment 1:Afu gene and proteins encoded

1. obtain material, method and the result of Afu gene

1.1. the method for utilizing Trizol reagent to provide is extracted total RNA of oviparity approach halogen worm, and utilizes ThermoScript II to synthesize the first chain cDNA as template.

1.2. obtain in the est sequence DB of the difference expression gene that obtains according to the inhibition subtractive hybridization a kind of in the artemia nauplii growth course partial sequence of the gene of expression amount downward modulation design a pair of Auele Specific Primer rbpF1:TAG TCT CCC TTC CAA ATACAAA and rbpR1:TAC CTA AAC TGA GTG CGG ACA T.

1.3. with the first chain cDNA is template, rbpF1 and rbpR1 are that primer is accomplished the PCR reaction.Reaction system is: cDNA template 1 μ L, and each 1 μ L of primer, 10 * PCR Buffer, 2.5 μ L, Mcgl2 1.5 μ L, dNTPs 1 μ L, TaqE 0.25 μ L, deionized water is supplied 25 μ L.The PCR program is: 95C sex change 5min, then 30 amplification cycles (95C sex change 30sec, 58C anneals 30sec, 72C extends 30sec), the 10min of 72C extension at last.

Sequence is connected to the pMD18-T carrier, order-checking.

1.4., design a pair of Auele Specific Primer rbpF2:ACG AGG GACATA TAA AGG TAA T and rbpR2:CGG ACA TCC TCT TTT GTGACT T according to the sequence that order-checking obtains.Adopt PCR system and program amplification part fragment in 1.3, and the method for utilizing DIG High prime labeling test kit to provide is marked as the screening probe.

1.5. the method for utilizing

synthesis Kit test kit and providing, the cDNA library of structure oviparity approach halogen worm.

1.6. use the probe screening from the library that 1.5 make up in 1.4 to obtain positive signal and order-checking, obtain complete Afu gene order:

5’-GGCACGAGGGCAAATGCGAGTTTCCTTACAAGAAGAAAAA

GAAATGGCAGAAAATATCCAACATGATGAACGCATACTTCGAA

AAGTTAGGAAGCAAATTGAATTTTACTTAAGTGATGCAAACTTG

TCTAAAGACCGGGTAACTTGCTGTACTTACTCTGAGGCCATCTC

AAGTGGAGGTATTCCCGCAGATTTCTTCTTGAAATGCAATAGAG

TAAAAGAACGCATTACAACAGTTGCTGAAATTGAAGAAGCGCT

GAAGACATCTAAATATTTGCAATTTGCAGATGGAAAAGTCTCCA

GAAAATTTCCTTTTCAACCTCGAACAAACCAGGATAGATGCAC

CATTTATGTGGAAAATATTCCCACGTATGCAACGCAAGAGAAAG

TTCGTAAATGGTTCCGACCTTACGGAAAGGTAGTTTATGTCTCC

CTTCCTAAATCAAAAGTAGGGACAATTAAAGGATATGCTTTTGT

TGAGTTCAATACAGAAGAAGAAGCAGAAATCTGCATGTTATCC

TATCAAGAATCTGGGAGATATATTGAAAACCGTGACCCAGCGG

AGCTGTTATCAGTTAAAACGTTTGAAGGATTTGAAGATGCTGA

GGTTGAAACTGAAACAGCGGAACCTGAATATCAGCCCTTTGAG

AGCGCTGAAGAGGCCCATCCTCAGGAAGTCACAAAAGAGGAT

GTCACCACTCAGTTTAGGATTCTTAGTAGGAACGATTGGAAAA

AGGAGAGAAACAAGTACCTGAATAACTGGAGGAAAGAAGGAA

AGGCACGTAGGCAAGAAATTTGGAGGCAGCAGATGAAAATTA

ACCGAAGGAGGAAAATAGCCGAGGAGGAAAAAGCTTTACAGC

CTAAAACGGAACTAAACTTCCAAAGCGGCTTGATTGTCAAGAT

TTCTTTGAAGGAAGAAATTCATGATCCAAAATCTATAAAGGAAC

AATTAAAAGCTATTGGCGGCATAAAATATGTTGAAGCCATGCAA

GGTGCAGTGGAAGCAATTGTCCGTACAGAAAGCCCAGATGTGG

CCAGCTCACTTATCAAAAATCCATTAGGAAAGGGTGAAGTCCTT

ACTGGTGCTGAGGAACTAGAATATTGGTTTAAAATACTTTCAGG

CAAGGAAGCGAAGCTTTCGGCATCAAAAGAAGTCAATAAAGT

AAAGGTGAAGAAGAAGAAGAAAAAGGCTCAGCATATTCGGTT

TGATGATGATGAAAATACAAAATCGGAAATAGAAACAACAGAC

GTGATTTGATAAGCTGTTTTCCGAAAAGCGTAGTTTATTGACGT

GCATTATTTTTTTATGTATTTTACTTTTTTACTTTTTACAATATTTA

TTTTTTACTTTCTTGTAAGATTACATTTATACGTCAATACATTACA

TGATTAATAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

(SEQ ID No.2)

This full length gene is 1425bp, and (5 '-UTR), (3 '-UTR) forms with the ORFs of 1215bp the 3 ' non-translational region of 167-bp by the 5 ' non-translational region of 43-bp.The ORFs protein that 404 amino acid are formed of can encoding wherein, the molecular weight of supposition is 46.81kDa, and iso-electric point is 8.6, and its aminoacid sequence is:

MAENIQHDERILRKVRKQIEFYLSDANLSKDRVTCCTYSEAISSGG

IPADFFLKCNRVKERITTVAEIEEALKTSKYLQFADGKVSRKFPFQP

RTNQDRCTIYVENIPTYATQEKVRKWFRPYGKVVYVSLPKSKVG

TIKGYAFVEFNTEEEAEICMLSYQESGRYIENRDPAELLSVKTFEG

FEDAEVETETAEPEYQPFESAEEAHPQEVTKEDVTTQFRILSRND

WKKERNKYLNNWRKEGKARRQEIWRQQMKINRRRKIAEEEKAL

QPKTELNFQSGLIVKISLKEEIHDPKSIKEQLKAIGGIKYVEAMQG

AVEAIVRTESPDVASSLIKNPLGKGEVLTGAEELEYWFKILSGKEA

KLSASKEVNKVKVKKKKKKAQHIRFDDDENTKSEIETTDVI*

(SEQ ID No.1)

2.Afu proteic expression and purification

2.1. the Afu gene order according to obtaining designs a pair of primer rbpeF:CCG GGA TCC TAG CGA GAA ATA TAC CAA CTA and the rbpeR:CCG CTC GAG TTA TCAATA CAC GTC TGT TGT TTC that contains restriction enzyme site.With the order-checking plasmid is template, and rbpeF and rbpeR are that primer carries out pcr amplification.Be connected among the prokaryotic expression carrier pGEX-4T-1 that contains the GST label behind amplified production process BamHI and the XhoI double digestion.Recombinant plasmid transformed is in E.coli BL21 (DE3) cell of the plasmid pSJS1240 that contains ileX and argU gene, and order-checking is identified.

2.2. utilize final concentration under 25C, to induce the DE3 cell that contains recombinant plasmid, express and have the Afu albumen that active GST merges for the IPTG of 0.1mM.

2.3. expression product utilizes glutathione S eperose 4B purifying, its electrophoresis result is seen Fig. 1.

Embodiment 2:RNA interferes the checking to the Afu function

1. according to the Afu gene order that obtains, design a pair of primed RNA if:CGT CTA GAA CCT CGA ACA AAC CAG GTA AGA and the RNAir:GGA TAT CTA TTT TTA CGC CGC ATAACG TT that contains restriction enzyme site.With the order-checking plasmid is template, and RNAif and RNAir are that primer carries out pcr amplification.Be connected into the pET-T7 carrier behind amplified production XbaI and the EcoRI double digestion, construct dsRNA expression plasmid to the tRBP gene.Control plasmid is the GFP gene order.

2. the plasmid that makes up is transformed into E.coli HT115 bacterial strain.In LB substratum (containing 100ug/ml Amp, 10ug/ml Ter), be cultured to OD ₆₀₀Be 0.4, it is 0.4mM that adding IPTG makes its final concentration, induces 4h for 37 ℃.

3. use phenol (pH4.5): chloroform extracting and purifying double-stranded RNA, be dissolved in the 50ul DEPC treating water, electrophoresis detection is measured concentration, is kept at-20 ℃.

4. use microinjection instrument UltraMicroPump II equipped with the Micro4TM MicroSyringe Pump Controller that the halogen worm individuality that occurs before the egg capsule is carried out microinjection.Injection volume is every 800ng double-stranded RNA or contrast double-stranded RNA.Halogen worm after the injection is cultivated under 8% artificial seawater, 28 ℃, short periodicity of illumination (illumination 4h, dark 20h).Observe it and grow and the situation of laying eggs, the result sees Fig. 2.

Conclusion: visible by Fig. 2, the 800ng double-stranded RNA interferes the amount of Afu mRNA in the winter egg that back halogen worm produces to have only 15% of control group, its expressing quantity through western blotting almost detect less than.The dormant state of output ovum is broken, and can directly hatch into larva.The result shows that Afu albumen plays a key effect in the dormant state of keeping halogen worm winter egg.

The external activity research (gel retardation assasy and co-immunoprecipitation experiment) that combines of embodiment 3:Afu albumen and tRNA

1. the Afu albumen that GST is merged mixes in following system with tRNA: Yeast tRNAs20ug; GST-Afu/GST contrast body protein 0-10ug; 20 * RNA Binding Buffer (RBB) 1ul; RiboLockTM RNase inhibitor 0.5ul uses the DEPC treating water that system is adjusted to 20ul.

2. with the soft mixing of articulated system, after room temperature combined 30min, last appearance was to the polyacrylamide gel of non-sex change, the about 2h of electrophoresis under the 80V.

3. the gel behind the electrophoresis carries out respectively: the 1.GoldView staining analysis; 2. forward nylon membrane to and use tRNA specific probe tRNA ^ArgACG (gR-ACG:TAT AGA AGT CAGACG CGT TCG CTA TAC CGC ACG CG), tRNA ^Ile _ATA(gI-TAT:TTA CGA CGG TCG CTT AAC CAA CTG GCG CAA GAG AC), tRNA ^Leu _CAA(gL-CAA:CTA AGA GTA TCG AAC TCT TCG TACTTA CGA TAC CT), tRNA ^Lys _TTT(gK-TTT:AAA CGC GAA CCGTCT ACC AAC TGA CGT AAC AAG GA), tRNA ^Tyr _GTA(gY-GTA:CAG TCT TCG CGC TTAAAC CAACTT GCG TAC CGAGA), and tRNA ^Ser _AGA(gS-AGA:TCG AGT CTC TCG CCT TAA CCA CTCGCG CTAAGT CG) carries out Northern blotting and analyzes, and the result sees Fig. 3.

Conclusion: visible by Fig. 3, after hatching jointly with Afu albumen, the electrophoretic velocity of tRNA is slower than tRNA freely, shows that Afu albumen and tRNA combine to have formed mixture external, and Afu albumen has tRNA and combines activity.

Embodiment 4:Afu gene transfection is to tumour cell and the proteic position of appraising and deciding of Afu

1. according to the Afu gene order that obtains, design a pair of primer GFPz-rbpeF:CCG CTC GAG AAA TGG CAG AAA TAA TCC AACTA and the GFPz-rbpeR:CCG GGA TCC TTA TCA ATA CAC GTC TGTTGT TTC that contains restriction enzyme site.With the order-checking plasmid is template, and GFPz-rbpeF and GFPz-rbpeR are that primer carries out pcr amplification.Amplified production is connected into the pEGFP-C1 carrier after using XhoI and BamHI double digestion, constructs and can in tumour cell, express the proteic plasmid of Afu that GFP merges, and order-checking is identified.

2. when tumour cell degree of converging reaches 90% left and right sides; Use Lipfectamine 2000 transfection reagents to carry out cell transfecting,, get the 4ug DNA for every hole (6 orifice plate) cell; 10ulLipfectamine 2000 transfection reagents are diluted in respectively in the substratum of 250ul serum-free, gently mixing; Room temperature is mixed after placing 5min, joins in the cell culture medium of normal cultured after leaving standstill 20min.Cell is placed 37 ℃, 5%CO ₂Use fluorescent microscope to observe after cultivating 18-48h in the incubator, the result sees Fig. 4.

Conclusion: the Afu albumen of in tumour cell, expressing is positioned in the nucleus.

Embodiment 5:Afu albumen and the interior activity research (co-immunoprecipitation experiment) that combines of the body of tRNA

1. under the condition that keeps protein interaction, the use diverse ways obtains the cell pyrolysis liquid of the HeLa cell after halogen worm winter egg and the transfection respectively.To halogen worm winter egg, at first use 50% Youxiaolin shelling, under the lysate effect that contains 0.1% Triton X-100 and 1% Sodium desoxycholate, grind the collecting cell lysate then; Then use 0.5% NP40 to carry out cracking for the HeLa cell.All add an amount of proteinase inhibitor and RNA enzyme inhibitors in the cracking system.

2. add 50 μ l proteinA sepharose and under 4 ℃, shake 2h, the pair cell lysate carries out pre precipitation.

3. in lysate, add 15 μ g Anti-Afu rabbit source antibody, 4 ℃ are shaken and spend the night.

4. add 200 μ l proteinA sepharose, 4 ℃ are shaken 4h and collect complex body.

5. extracting RNA is used the tRNA probe in detecting behind the commentaries on classics film, and the result sees Fig. 5.

Conclusion: the co-immunoprecipitation result shows, Afu albumen is that the form with complex body exists in active somatic cell nuclear, and this species complex possibly participated in the regulation and control that tRNA transports between the cell caryoplasm.

Embodiment 6: study the regulation and control that Afu albumen is transported between the cell caryoplasm tRNA through fluorescence in situ hybridization

1. the deckglass of bacterium of will going out is put into the culture hole of 6 orifice plates;

2. cell is gone down to posterity on the deckglass in normal ratio, cell is placed 37 ℃, 5%CO ₂Cultivate 24h in the incubator and reach about 90% to cell degree of converging, carry out transfection;

3. behind expressing fusion protein (about 18-24h) discards substratum, with PBS rinsing 1 time;

4. with the fixing 1h of 4% Paraformaldehyde 96 room temperature, with the PBS rinsing of precooling 2 times, each 15min;

5. the acetone that adds the 2ml precooling is handled 3min for-20 ℃; Use the PBS rinsing subsequently 2 times, each 5min;

6. add the 2ml prehybridization solution, 39 ℃ of effect 1h;

7.75 ℃ processing 10min places on ice immediately;

8. tRNA probe (the tRNA Leu CAA (hL-CAA:TTG AGTCTG CGG CCT TAG ACC ACT CGG CCA TCC TGA C) that adds the DIG mark; TRNA TyrGTA (hY-GTA:CTA CAG TCC TCC CGT CTA CCG CTG ACG TTACGA AGG); TRNA Gly CGC (hG-CGC:TCG TAT GCG CGG GAATCG AAC CCG GG); TRNA Phe GAA (hF-GAA:CCG TCT CCCAAC TGA CGT TAT TCG CG); TRNA Ser AGA (hS-AGA:CGC TTAACC ACT CGG CCA CGA CTA C); TRNA Lys CTT (hK-CTT:TCGTCT ACC GAC TGA CGT ACG CGG CG); And precursor tRNA LeuCAA (hL-CAA-pre:CAG ATA CCC CAC CCG GGA GGA ACG TTACGT TGA), precursor tRNA Tyr GTA (hY-GTA-pre:AAG GTA GCG CACGGG CCG CGG CCT ACA G)), 39 ℃ of hybridization are spent the night;

9. use 2 * SSC rinsing 3 times respectively, 45 ℃, each 10min; With 1 * SSC rinsing 3 times, room temperature, each 10min; With the 4 * SSC rinsing that contains 1%Triton X-100 1 time;

10. add the 4 * SSC sealing 2h that contains 1%BSA;

11. add the anti-DIG antibody that contains the rhodamine mark, lucifuge (following steps are all wanted lucifuge) effect 2h;

12. use 4 * SSC rinsing respectively 2 times, room temperature, each 10min; With the 4 * SSC rinsing that contains 1% Triton X-100 2 times, each 10min; After 4 * SSC rinsing 1 time, add 100ul DAPI effect 15min transfect cell nuclear;

13. once, on slide glass, drip anti-fluorescent quenching mounting liquid, cover and have with the PBS rinsing

The deckglass of cell is observed under fluorescent microscope after the nail varnish mounting, and the result sees Fig. 6.

Conclusion: visible by the fluorescence in situ hybridization result; After being positioned nuclear Afu albumen and combining to form complex body with endonuclear tRNA; TRNA is stranded in the nucleus, limited tRNA from nucleus to cytoplasmic transhipment, caused the minimizing of ripe tRNA in the tenuigenin.Embodiment 7: through the regulation and control of BrdU marker detection Afu albumen to the cell cycle of tumour cell

1. the deckglass of bacterium of will going out is put into the culture hole of 6 orifice plates, in normal ratio with passage to deckglass, place 37 ℃, cultivate about 24h in the 5%CO2 incubator and reach about 90% to cell degree of converging, carry out transfection;

2. the HeLa cell culture medium with 24h after the transfection changes the DMEM substratum that contains 12mM BrdU into, and 37 ℃ are continued to cultivate 21h;

3. cell is with the fixing 1h of 4% Paraformaldehyde 96 room temperature;

Under the room temperature with PBS rinsing 15min;

5. the H2O2 that adds an amount of 3% concentration, 37 ℃ of effect 10min;

Under the room temperature with PBS rinsing 3 times, altogether 15min adds an amount of 2N HCl, 37 ℃ of effect 30min;

7. clean with the thorough rinsing of PBS under the room temperature, add confining liquid (1% BSA, 0.5% Tween, 20,0.1% sodiumazide, 5% FBS), 37 ℃ of effect 15min;

8. change the BrdU monoclonal antibody into, 37 ℃ of continuation effect 2h reclaim antibody;

Under the room temperature with PBS rinsing 3 times, 15min altogether, the sheep anti mouse fluorescence two that adds the rhodamine mark then is anti-, 37 ℃ of effect 2h reclaim two anti-;

Under the room temperature with PBS rinsing 3 times, altogether 15min adds DAPI transfect cell nuclear, room temperature effect 15min;

11. use the PBS rinsing under the room temperature, on slide glass, drip anti-fluorescent quenching mounting liquid, cover deckglass with cell, under fluorescent microscope, observe after the nail varnish mounting, the result sees Fig. 7.

Conclusion: visible by Fig. 7, have in the proteic cell of Afu in expression, almost detect signal less than BrdU, show that these cells do not have to pass through the dna replication dna stage of cell cycle, promptly cell does not have and can pass through the S phase.The result shows that the proteic expression of Afu makes the CDC of tumour cell receive regulation and control, and cell is stuck in the G1/S phase.

Embodiment 8: detect the regulation and control of Afu albumen cell cycle through flow cytometry

1. get the HeLa cell of 18h and 24h after the transfection, after the pancreatin room temperature treatment with 0.25% digests, the increase serum termination reaction.The cell of collecting cleans 3-5 time with PBS solution, piping and druming gently, microscopy cell and counting;

2. the gained single cell suspension is with 4 ℃ of fixed overnight of 70% ethanol;

3. the cell after centrifugal collection is fixing cleans 2 times with PBS solution;

4. add 37 ℃ of reactions of RNase A (100ug/ml) 30min;

5. add 4 ℃ of lucifuge reactions of PI dyestuff (50ug/ml) 30min;

6.1h interior upflowing cell instrument detects, excitation wavelength is 488nm, uses Cell QuestTM software version 3.1 softwares to carry out cell cycle analysis.The sample in each period repeats to prepare three times, and the result sees Fig. 8.

Conclusion: visible by Fig. 8; The Afu-Hela and the GFP cell that are in the G1/G0 phase have reduced 10.34% (50.83%-40.49%) and 18.73% (68.20%-49.47%) respectively, explain that Afu albumen makes the Hela cell tend to be stuck in the G1/G0 phase and do not change to the S phase.Form with it obviously correlated be the cell of G2/M phase in the Afu-Hela cell at 18h after the transfection to almost not changing between the 24h; And keep an extremely low ratio (2.9%-3.77%), and the cell of G2/M phase has increased by 15.71% (2.92%-18.63%) in the control cells under initial ratio situation much at one.Explanation is in expressing the proteic tumour cell of Afu, and the cell cycle has received retardance through the S phase to G2/M phase transforming process.

Embodiment 9:Afu albumen is to the regulation and control of HeLa cell proliferation

Get the HeLa cell of 24h after the transfection, after the pancreatin room temperature treatment with 0.25% digests, the increase serum termination reaction.Count behind the cell dilution of collecting, get 4000 cells and be resuspended in the DMEM substratum that contains 0.35% soft agar, continue to cultivate and contain on the 35mm culture plate of 0.5% soft agar at bottom.It forms the situation of population of cells the back observation of 4 weeks, and the result sees Fig. 9.Conclusion: visible by Fig. 9, after the growth through 4 weeks, expressing has the proteic tumour cell of Afu almost to divide formation population of cells, and the division of control cells is normal.

Embodiment 10: through the regulation and control of BrdU marker detection Afu albumen to the cell cycle of Xenopus laevis and zebrafish embryo cell

1. the zygote with Xenopus laevis of choosing and zebra fish moves on in the petridish, before a cell stage, uses UltraMicroPump II equipped with the Micro4 ^TMMicroSyringe Pump Controller microinjection instrument injection foreign DNA, concentration is 0.15 μ g/ μ L, the about 2.3nL of DNA volume.After the injection of the animal pole of zygote finishes, slowly extract entry needle out, zygote putting into is equipped with the petridish of 1 * Half ' s nutrient solution, make it recover to grow.

2. will pass through the young (19 phases of Xenopus laevis, zebra fish 14h) after the microinjection is positioned over and cultivates 2-3d (40 phases of Xenopus laevis, zebra fish 72h) in the nutrient solution that contains 3mM BrdU.

3. take out the individual body and function 4% Paraformaldehyde 96 fixed overnight after handling

4. with the PBS solution rinsing 2 times that contains 0.5% NP40, each 10min;

5. use 25%, 50%, 75%, 100% ethanol (being diluted among the PBS) dehydration successively, each gradient treatment time is 15min, on ice operation;

With 5% H2O2 at 4 ℃ of bleaching 1-2h, room temperature is shaken;

7. use 25%, 50%, 75%, 100% PBS (being diluted in the ethanol) dehydration behind the ethanol rinsing 10min successively, each gradient treatment time is 15min, on ice operation;

8. with PBSST (adding 5% sera, 0.1% Triton among the PBS) rinsing, 4 ℃ of 1h;

9. add and contain the PBSST that BrdU one resists, 4 ℃ are spent the night, and control group does not add one and resists;

10. with PBSST rinsing 2 times, each 15min, room temperature is shaken; Continue rinsing 4 times, each 15min, 4 ℃ are shaken;

11. add the PBSST that contains two anti-(AP-conjugtaed gota anti-mouse IgG), 4 ℃ are spent the night;

12. with PBSST rinsing 2 times, each 15min, room temperature is shaken, and continues rinsing 4 times at 4 ℃ then;

13. with adding the NBT/BCIP colour developing behind the TM damping fluid rinsing 15min, the result sees Figure 10.

Conclusion: can detect mixing of BrdU in contrast individual tail end mitotic division vigorous zone, in expressing the proteic individuality of Afu, then detect less than, explain that these cells are not accomplished dna replication dna and through the S phase.This result proves, no matter in tumour cell still was normal cell, tRBP albumen can both cause the disorder of cell cycle.

Embodiment 11: analyze the signal path that the regulation and control of Afu albumen cell cycle relate to through Western blotting

Detect the halogen worm individual (a) of different development stage and express Afu protein 24 h, the expression and the phosphorylation situation (Figure 11) of following cell cycle GAP-associated protein GAP are sought the related signal path of this regulation process: cyclinA, cyclin D1, D2 in the HeLa cell (b) behind 36h and the 48h; D3, cyclinE2, CDK4, CDK6, chk2; 4EBP1, E2F1, E2F2, E2F3, p15; C-Jun and phosphor-specific antibodies against Rb (T356, S807/811and T780), Akt (T308), ERK1/2 (T202/Y204), FOXO3a (T32; S318/321, and S253), RSK (S221 and S380), MEK (T286, T292 and S298); P38 (T180/Y182), JNK (T183/Y185), Fos (S32), FOXO1 (S256) and RNA POL II (S2).Participate in the signal path letter of regulation and control shows like Figure 12 about Afu albumen.

Conclusion: Afu albumen is accumulated in nucleus, the nucleo-cytoplasmic transport of regulation and control tRNA and thus the stagnation of cell cycle in the G1/S phase of modulate tumor be through MAPK, signal paths such as PI3K-AKT and FKHR/FoxO to cyclin D/CDK6/Rb are accomplished.

Embodiment 12:Afu albumen is to the growth-inhibiting and the cell cycle regulating of nude mice Subcutaneous tumor

1. use Adenovirus Dual Expression Kit that the Afu gene is connected in the Ad5 virus vector, packing is purified to 10 ⁹Pfu;

2. collect HeLa and HT1080 cell, it is subcutaneous to be expelled to the BALB/c nude mice, and becoming after the knurl is cut into small pieces tumour expands each 30 mouse to;

3. select lotus that 100mm is arranged ³The mouse of tumour is divided into experimental group and control group, injects adenovirus that contains the Afu gene and the contrast virus of 108pfu respectively, 2 times weekly.Mensuration tumour size;

4. before the last injecting virus, to mouse peritoneal injection BrdU (50mg/kg), sampling after 10 hours, the fixing back of Paraformaldehyde 96 specimens paraffin embedding slices detects mixing of BrdU;

5. get the Western blotting detection that the tumour of handling without BrdU is carried out signaling pathway protein.

The result sees Figure 13.

Conclusion: Afu albumen all has the obvious suppression effect to the growth of two kinds of tumour cells, detects through BrdU to show, expressing has the proteic tumour cell of Afu all not have dna replication dna, promptly is stuck in the G1/S phase.The detection of Western blotting shows that Afu albumen is through MAPK to the cell cycle regulating and the growth-inhibiting of tumour, and signal paths such as PI3K-AKT and FKHR/FoxO to cyclin D/CDK6/Rb are accomplished.

SEQUENCE LISTING

< 110>Zhejiang University

< 120>participate in that the tRNA of cell cycle regulating is conjugated protein, encoding sox and application

<130>

<160> 18

<170> PatentIn version 3.4

<210> 1

<211> 404

<212> PRT

<213> Unknown

<220>

< 223>artificial sequence

<400> 1

Met Ala Glu Asn Ile Gln His Asp Glu Arg Ile Leu Arg Lys Val Arg

1 5 10 15

Lys Gln Ile Glu Phe Tyr Leu Ser Asp Ala Asn Leu Ser Lys Asp Arg

20 25 30

Val Thr Cys Cys Thr Tyr Ser Glu Ala Ile Ser Ser Gly Gly Ile Pro

35 40 45

Ala Asp Phe Phe Leu Lys Cys Asn Arg Val Lys Glu Arg Ile Thr Thr

50 55 60

Val Ala Glu Ile Glu Glu Ala Leu Lys Thr Ser Lys Tyr Leu Gln Phe

65 70 75 80

Ala Asp Gly Lys Val Ser Arg Lys Phe Pro Phe Gln Pro Arg Thr Asn

85 90 95

Gln Asp Arg Cys Thr Ile Tyr Val Glu Asn Ile Pro Thr Tyr Ala Thr

100 105 110

Gln Glu Lys Val Arg Lys Trp Phe Arg Pro Tyr Gly Lys Val Val Tyr

115 120 125

Val Ser Leu Pro Lys Ser Lys Val Gly Thr Ile Lys Gly Tyr Ala Phe

130 135 140

Val Glu Phe Asn Thr Glu Glu Glu Ala Glu Ile Cys Met Leu Ser Tyr

145 150 155 160

Gln Glu Ser Gly Arg Tyr Ile Glu Asn Arg Asp Pro Ala Glu Leu Leu

165 170 175

Ser Val Lys Thr Phe Glu Gly Phe Glu Asp Ala Glu Val Glu Thr Glu

180 185 190

Thr Ala Glu Pro Glu Tyr Gln Pro Phe Glu Ser Ala Glu Glu Ala His

195 200 205

Pro Gln Glu Val Thr Lys Glu Asp Val Thr Thr Gln Phe Arg Ile Leu

210 215 220

Ser Arg Asn Asp Trp Lys Lys Glu Arg Asn Lys Tyr Leu Asn Asn Trp

225 230 235 240

Arg Lys Glu Gly Lys Ala Arg Arg Gln Glu Ile Trp Arg Gln Gln Met

245 250 255

Lys Ile Asn Arg Arg Arg Lys Ile Ala Glu Glu Glu Lys Ala Leu Gln

260 265 270

Pro Lys Thr Glu Leu Asn Phe Gln Ser Gly Leu Ile Val Lys Ile Ser

275 280 285

Leu Lys Glu Glu Ile His Asp Pro Lys Ser Ile Lys Glu Gln Leu Lys

290 295 300

Ala Ile Gly Gly Ile Lys Tyr Val Glu Ala Met Gln Gly Ala Val Glu

305 310 315 320

Ala Ile Val Arg Thr Glu Ser Pro Asp Val Ala Ser Ser Leu Ile Lys

325 330 335

Asn Pro Leu Gly Lys Gly Glu Val Leu Thr Gly Ala Glu Glu Leu Glu

340 345 350

Tyr Trp Phe Lys Ile Leu Ser Gly Lys Glu Ala Lys Leu Ser Ala Ser

355 360 365

Lys Glu Val Asn Lys Val Lys Val Lys Lys Lys Lys Lys Lys Ala Gln

370 375 380

His Ile Arg Phe Asp Asp Asp Glu Asn Thr Lys Ser Glu Ile Glu Thr

385 390 395 400

Thr Asp Val Ile

<210> 2

<211> 1425

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 2

ggcacgaggg caaatgcgag tttccttaca agaagaaaaa gaaatggcag aaaatatcca 60

acatgatgaa cgcatacttc gaaaagttag gaagcaaatt gaattttact taagtgatgc 120

aaacttgtct aaagaccggg taacttgctg tacttactct gaggccatct caagtggagg 180

tattcccgca gatttcttct tgaaatgcaa tagagtaaaa gaacgcatta caacagttgc 240

tgaaattgaa gaagcgctga agacatctaa atatttgcaa tttgcagatg gaaaagtctc 300

cagaaaattt ccttttcaac ctcgaacaaa ccaggataga tgcaccattt atgtggaaaa 360

tattcccacg tatgcaacgc aagagaaagt tcgtaaatgg ttccgacctt acggaaaggt 420

agtttatgtc tcccttccta aatcaaaagt agggacaatt aaaggatatg cttttgttga 480

gttcaataca gaagaagaag cagaaatctg catgttatcc tatcaagaat ctgggagata 540

tattgaaaac cgtgacccag cggagctgtt atcagttaaa acgtttgaag gatttgaaga 600

tgctgaggtt gaaactgaaa cagcggaacc tgaatatcag ccctttgaga gcgctgaaga 660

ggcccatcct caggaagtca caaaagagga tgtcaccact cagtttagga ttcttagtag 720

gaacgattgg aaaaaggaga gaaacaagta cctgaataac tggaggaaag aaggaaaggc 780

acgtaggcaa gaaatttgga ggcagcagat gaaaattaac cgaaggagga aaatagccga 840

ggaggaaaaa gctttacagc ctaaaacgga actaaacttc caaagcggct tgattgtcaa 900

gatttctttg aaggaagaaa ttcatgatcc aaaatctata aaggaacaat taaaagctat 960

tggcggcata aaatatgttg aagccatgca aggtgcagtg gaagcaattg tccgtacaga 1020

aagcccagat gtggccagct cacttatcaa aaatccatta ggaaagggtg aagtccttac 1080

tggtgctgag gaactagaat attggtttaa aatactttca ggcaaggaag cgaagctttc 1140

ggcatcaaaa gaagtcaata aagtaaaggt gaagaagaag aagaaaaagg ctcagcatat 1200

tcggtttgat gatgatgaaa atacaaaatc ggaaatagaa acaacagacg tgatttgata 1260

agctgttttc cgaaaagcgt agtttattga cgtgcattat ttttttatgt attttacttt 1320

tttacttttt acaatattta ttttttactt tcttgtaaga ttacatttat acgtcaatac 1380

attacatgat taatagaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 1425

<210> 3

<211> 22

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 3

tagtctccct tccaaataca aa 22

<210> 4

<211> 22

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 4

tacctaaact gagtgcggac at 22

<210> 5

<211> 22

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 5

acgagggaca tataaaggta at 22

<210> 6

<211> 22

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 6

cggacatcct cttttgtgac tt 22

<210> 7

<211> 30

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 7

ccgggatcct agcgagaaat ataccaacta 30

<210> 8

<211> 33

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 8

ccgctcgagt tatcaataca cgtctgttgt ttc 33

<210> 9

<211> 30

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 9

cgtctagaac ctcgaacaaa ccaggtaaga 30

<210> 10

<211> 29

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 10

ggatatctat ttttacgccg cataacgtt 29

<210> 11

<211> 35

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 11

tatagaagtc agacgcgttc gctataccgc acgcg 35

<210> 12

<211> 35

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 12

ttacgacggt cgcttaacca actggcgcaa gagac 35

<210> 13

<211> 35

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 13

ctaagagtat cgaactcttc gtacttacga tacct 35

<210> 14

<211> 35

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 14

aaacgcgaac cgtctaccaa ctgacgtaac aagga 35

<210> 15

<211> 35

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 15

cagtcttcgc gcttaaacca acttgcgtac cgaga 35

<210> 16

<211> 35

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 16

tcgagtctct cgccttaacc actcgcgcta agtcg 35

<210> 17

<211> 32

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 17

ccgctcgaga aatggcagaa ataatccaac ta 32

<210> 18

<211> 33

<212> DNA

<213> Unknown

<220>

< 223>artificial sequence

<400> 18

ccgggatcct tatcaataca cgtctgttgt ttc 33

Claims (4)

1. a tRNA who participates in cell cycle regulating is conjugated protein, and its aminoacid sequence is shown in SEQ ID No.1.

2. protein-bonded encoding sox of the said tRNA of claim 1.

3. encoding sox as claimed in claim 2 is characterized in that said gene order is shown in SEQ ID No.2.

4. the conjugated protein application in the medicine in preparation modulate tumor cell cycle of tRNA as claimed in claim 1.

Images