CN1837374A - Method for detecting anti-liver cancer efficacy of tyroserleutide, kit and gene chip used thereby - Google Patents

Abstract

The invention discloses a detecting method and its agent box and gene chip of tyr-ser-leu protein anti-hepatocarcinoma effect, which comprises the following steps: detecting the changing condidtions of gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21and P27 expression level in the biological material after acted by tyr-ser-leu protein; displaying downward for gene NDUFA2, NDUFA10,NDUFS1,SUCLG1,Akt1,Akt2 and upward for the expression level of gene Calreticulin,PTEN, P21,P27; possessing anti-hepatocarcinoma effect. The invention provides a rapid detecting path of tyr-ser-leu protein anti-hepatocarcinoma effect on the molecular level.

Description

Detect method and the used kit and the gene chip of anti-liver cancer efficacy of tyroserleutide

Technical field

The present invention relates to a kind of method and preparation of detection of drugs anticancer effect, particularly relate to method and used kit and gene chip that a kind of molecular level detects anti-liver cancer efficacy of tyroserleutide.

Background technology

Primary hepatocarcinoma is one of human modal malignant tumor of digestive tract, and its sickness rate occupies the 8th of the main cancer in the world, and the mortality ratio height is only second to cancer of the stomach, esophagus cancer and occupies the 3rd in mortality of malignant tumors.China is the country occurred frequently of primary hepatocarcinoma, and first of the M ﹠ M Jun Ju world, mortality ratio accounts for second in various cancers, have every year 110000 people to die from liver cancer approximately, accounts for about 45% of whole world PLC mortality number.The treatment of liver cancer at present still with operation, radiotherapy and chemotherapy as main means, but early hepatocarcinoma is found difficulty, the excision rate is low, thereby to seek effective medicine be the key that improves the liver cancer survival rate.

Tyroserleutide (Tyrosyl-seryl-leucine YSL), is a kind of tripeptide compound, and its chemical structure consists of L-tyrosyl-L-seryl-L-leucine, and molecular formula is C18H27N306, and molecular weight is 381.42, and molecular structural formula is as follows:

The experimentation on animals result shows that tyroserleutide demonstrates the effect of certain inhibition growth of tumour cell.When this medicine is 40～80 μ g/kg at dosage, can make ascitic type liver cancer H22 mouse increase in life span reach 60%～90%.Stable through four repeated experiments results.Tyroserleutide demonstrates the effect of human hepatocellular carcinoma BEL-7402 cell's inhibition of proliferation in experiment in vitro, and best tumour inhibiting rate can reach 36.29%.In the transplanted tumor in nude mice experiment, when dosage was 160～320 μ g/kg, tyroserleutide can significantly suppress the growth of people's liver cancer BEL-7402 transplanted tumor in nude mice, through five repeated experiments, inhibitory rate 40～50%.

Tyroserleutide also demonstrates good drug effect in clinical experiment, can obviously prolong the survival time of hepatocarcinoma patient.But find also that in clinical study the antihepatocarcinoma effect of tyroserleutide there are differences between different patients' individuality, promptly different individualities there are differences the susceptibility of tyroserleutide.Some patient is after giving the tyroserleutide treatment, and obvious inhibition has taken place tumor growth; And some patient tumor growth behind the injection tyroserleutide is not subjected to obvious influence.

Anticancer therapy is a secular process, needs the result of treatment of regular evaluation medicine to patient in therapeutic process.If certain medicine really can onset to this patient in therapeutic process, then prompting can continue to use this medicine and treats.If this patient is insensitive to this kind medicine, then should stop the predetermined treatment scheme as early as possible, reduce poisonous side effect of medicine, and use other schemes instead and continue treatment.Present stage, the judge whether a kind of cancer therapy drug can play a role to patient is often by observing the metamorphosis of patient's tumour behind the drug administration.But changing to the tumour form from drug administration often needs long period of time, and during this period of time, whether this medicine can play curative effect to this patient then can't be predicted.Tyroserleutide equally also faces such problem as a kind of new drug of anti-liver cancer.

Plastosome is the synthetic place of intracellular energy, on the mitochondrial inner membrane, mainly comprises the respiratory chain of being made up of the redox combined enzyme agent, ATP synthetic enzyme and adenosine transport body.Respiratory chain is made up of 70 multiple polypeptides, presses the height of redox-potential, can be divided into four complex bodys.Be respectively complex body I (nadh dehydrogenase), II (succinodehydrogenase), III (cytochrome reductase), IV (Terminal oxidase).Electronics finally transmits oxygen supply through respiratory chain, and membranous sub-gradient is striden in formation in the process of transmitting, and as the power that high-energy phosphate compound forms, ADP generates ATP through phosphorylation under the effect of ATP synthetic enzyme, supplies with the required energy of cellular metabolism.NDUFA2, NDUFA10 and NDUFS1 are the integral parts of complex body I (nadh dehydrogenase), SUCLG1 is the moiety of complex body II (succinodehydrogenase), and these expression of gene level declines can cause the impaired of cellular respiration chain function, thereby influences the function of cell mitochondrial.

Calreticulin is a kind of calcium binding protein in the endoplasmic reticulum, plays an important role for the adjusting of intracellular Ca2+ stable state and the transmission of calcium signal.Calreticulin crosses expression can increase Ca2+ stream by endoplasmic reticulum, reduces the potential energy of plastosome Ca2+ and film, makes the increase that Ca2+ upgrades between two kinds of organoids.

The PI3K approach is important signal transduction pathway in the cell, plays an important role in cell activities such as apoptosis, aging, propagation.PTEN and AKT are two containing factors of counterproductive on this path.The enhancing of PTEN function can suppress the cell proliferation signal transmission of PI3K path, and this will make the AKT in its downstream not be activated.

PTEN is the tumor suppressor gene that is positioned on the human 10q23 karyomit(e), after the activation of PTEN dephosphorylation, has the dual function of albumen and lipid Phosphoric acid esterase.The phosphoprotein phosphatase interaction energy of PTEN removes the phosphoric acid on the tyrosine, this effect and many cancer protein functions reversed, and may therefore act on the common substrate with many cancer proteins, performance suppresses the effect of tumor development.AKT then plays a significant role in the various kinds of cell activity, and the AKT kinases can be regulated many target spots by phosphorylation after being activated by phosphorylation, various kinases, transcription factor and other adjusting molecule.Promote apoptosis target gene (as Bad etc.) phosphorylation comprising making, thereby directly prolong the cells survival time.AKT also can influence some factor of cell cycle and promote the progress of cell cycle.

It is active and when causing AKT and not being activated, the p21 of passage downstream, p27 will be activated the inhibitory cell cycle, inhibition of cell proliferation to suppress PI3K at PTEN.P21 and p27 belong to CYC-CDK (cyclin-Cyclin Dependent Kinase) mixture are played inhibiting Cip/Kip albumen (p21, p27, p57) family.P27 is that PTEN activates the target site that the back suppresses cell cycle evolution.The tyrosine phosphorylation supressor can be cancelled its effect and promote the progress of cell cycle.P21 almost can combine with each CYC-CDK mixture, makes Rb albumen and the p53 albumen can not phosphorylation and suppress cell and enter the S phase.P21 can also suppress combining of PCNA and DNA polymerase, thereby suppresses the extension process of dna replication dna, causes the cell cycle to prevent, and makes tumour cell move towards apoptosis.

Summary of the invention

Purpose of the present invention is exactly in order to overcome the above problems, and a kind of method at molecular level rapid detection anti-liver cancer efficacy of tyroserleutide is provided, and used reagent or gene chip.

For achieving the above object, the present invention has adopted following technical scheme:

The invention discloses a kind of method that detects anti-liver cancer efficacy of tyroserleutide, described method comprises step:

In the biomaterial after the tyroserleutide effect, detect gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21 or P27 changes of expression level situation;

When detected result is: after the tyroserleutide effect, the expression level of gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Akt1, Akt2 descends, the expression level of gene C alreticulin, PTEN, P21, P27 rises, and shows that then tyroserleutide has the effect of anti-liver cancer to this biomaterial.

Concrete, in the described biomaterial after the tyroserleutide effect, detect gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21 or P27 changes of expression level situation, be meant, adopt the method for reverse transcription PCR to detect the expression changing conditions of the mRNA of these ten genes in the described biomaterial in tyroserleutide effect front and back, when after the tyroserleutide effect, gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Akt1, the mRNA expression level of Akt2 descends, gene C alreticulin, PTEN, P21, the mRNA expression level of P27 rises, and shows that then tyroserleutide has the effect of anti-liver cancer to this biomaterial.

Perhaps concrete, in the described biomaterial after the tyroserleutide effect, detect gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21 or P27 changes of expression level situation, be meant, adopt the method for gene chip, detect the expression changing conditions of cDNA before and after the tyroserleutide effect of these ten genes in the described biomaterial, when after the tyroserleutide effect, gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Akt1, the cDNA expression level of Akt2 descends, gene C alreticulin, PTEN, P21, the cDNA expression level of P27 rises, and shows that then tyroserleutide has the effect of anti-liver cancer to this biomaterial.

The cDNA sequence of described gene NDUFA2 comprises the sequence shown in the 74th～373 bit base among the sequence table SEQ ID No.23, and the 74th～373 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.23.

The cDNA sequence of described gene NDUFA10 comprises the sequence shown in the 81st～1148 bit base among the sequence table SEQ ID No.24; The 81st～1148 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.24.

The cDNA sequence of described gene NDUFS1 comprises the sequence shown in the 128th～2311 bit base among the sequence table SEQ ID No.25; The 128th～2311 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.25.

The cDNA sequence of described gene SUCLG1 comprises the sequence shown in the 32nd～1033 bit base among the sequence table SEQ ID No.26; The 32nd～1033 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.26.

The cDNA sequence of described gene C alreticulin comprises the sequence shown in the 69th～1322 bit base among the sequence table SEQ ID No.27; The 69th～1322 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.27.

The cDNA sequence of described gene PTEN comprises the sequence shown in the 1032nd～2243 bit base among the sequence table SEQ ID No.28; The 1032nd～2243 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.28.

The cDNA sequence of described gene A kt1 comprises the sequence shown in the 555th～1997 bit base among the sequence table SEQ ID No.29; The 555th～1997 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.29.

The cDNA sequence of described gene A kt2 comprises the sequence shown in the 204th～1649 bit base among the sequence table SEQ ID No.30; The 204th～1649 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.30.

The cDNA sequence of described gene P21 comprises the sequence shown in the 95th～589 bit base among the sequence table SEQ ID No.31; The 95th～589 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.31.

The cDNA sequence of described gene P27 comprises the sequence shown in the 466th～1062 bit base among the sequence table SEQ ID No.32.The 466th～1062 the interior base of scope is open reading frame (ORF) region of this gene among this SEQ ID No.32.

The invention also discloses a kind of test kit that is used to detect the tyroserleutide action effect, contain the primer sequence of the mRNA expression level that in the reverse transcription PCR method, detects gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21, P27 respectively in the described test kit.

Preferably, the primer sequence of described detection gene NDUFA2 is shown in SEQ ID No.1 and 2 in the sequence table;

The primer sequence of described detection gene NDUFA10 is shown in SEQ ID No.3 and 4 in the sequence table;

The primer sequence of described detection gene NDUFS1 is shown in SEQ ID No.5 and 6 in the sequence table;

The primer sequence of described detection gene SUCLG1 is shown in SEQ ID No.7 and 8 in the sequence table;

The primer sequence of described detection gene C alreticulin is shown in SEQ ID No.9 and 10 in the sequence table;

The primer sequence of described detection gene PTEN is shown in SEQ ID No.11 and 12 in the sequence table;

The primer sequence of described detection gene A kt1 is shown in SEQ ID No.13 and 14 in the sequence table;

The primer sequence of described detection gene A kt2 is shown in SEQ ID No.15 and 16 in the sequence table;

The primer sequence of described detection gene P21 is shown in SEQ ID No.17 and 18 in the sequence table;

The primer sequence of described detection gene P27 is shown in SEQ ID No.19 and 20 in the sequence table.

Further contain the primer sequence of the reverse transcription PCR of reference gene GAPDH in the described test kit, this primer sequence is shown in SEQ ID No.21 and 22 in the sequence table.

The invention also discloses a kind of gene chip that is used to detect the tyroserleutide action effect, being fixed with on the solid phase carrier of described gene chip can be respectively and the nucleotide sequence of the cDNA sequence hybridization of gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21, P27.

Because adopted above scheme, the beneficial effect that the present invention is had is:

Method of the present invention provides a kind of approach at molecular level rapid detection anti-liver cancer efficacy of tyroserleutide, using the tyroserleutide front and back by detecting, ten genes (NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21, P27) changes of expression level situation, thereby can comparatively fast understand tyroserleutide to this individual action effect, this time that detects the result from molecular level is shorter than the time that shows result of treatment from individual phenotype greatly.Thereby this method can for hepatocarcinoma patient in the process of using the tyroserleutide related drugs, the reference of definite medication effect is provided, make and take the invalid patient of this medicine and can cut out this medicine early, saved expense and will significantly reduce the toxic side effect of taking medicine.

Test kit of the present invention and gene chip can be applicable in the method for the present invention, from the anti-liver cancer efficacy of molecular level rapid detection tyroserleutide.

Description of drawings

Fig. 1 is that the RT-PCR method detects tyroserleutide to mitochondrial respiratory chain genes involved NDUFA2 in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte, the electrophoresis result figure of the influence that NDUFA10, NDUFS1 and SUCLG1 mRNA express.

The PT-PCR method that shows Fig. 2 detects tyroserleutide to mitochondrial respiratory chain genes involved NDUFA2 in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte, the statistical graph of the influence that NDUFA10, NDUFS1 and SUCLG1mRNA express: relatively with the physiological saline group, the tyroserleutide group is breathed all decline in various degree of mitochondrial respiratory chain mRNA expression of gene associated for above-mentioned 4 kinds, has statistical significance (P＜0.05).

Fig. 3 is that the RT-PCR method detects the electrophoresis result figure of tyroserleutide to the influence of the steady peptide regulatory gene of calcium Calreticulin mRNA expression in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte.

Fig. 4 shows the statistical graph of tyroserleutide to the influence of the steady peptide regulatory gene of calcium Calreticulin mRNA expression in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte: with the physiological saline group relatively, tyroserleutide group Calreticulin gene mRNA expression obviously raises, and has statistical significance (P＜0.05).

Fig. 5 is that the RT-PCR method detects the electrophoresis result figure of tyroserleutide to the influence of PTENmRNA expression in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte.

Fig. 6 is that the RT-PCR method detects the electrophoresis result figure of tyroserleutide to the influence of AKT1, AKT2mRNA expression in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte.

Fig. 7 is that the PT-PCR method detects the electrophoresis result figure of tyroserleutide to the influence of P21, P27mRNA expression in people's liver cancer BEL-7402 transplanted tumor in nude mice histocyte.

Fig. 8 shows the statistical graph of 160 μ g/kg/d tyroserleutides to the influence of people's liver cancer BEL-7402 transplanted tumor in nude mice tumor proliferation genes involved PTEN, AKT1, AKT2, P21, P27 mRNA expression: the mRNA that tyroserleutide can increase cancer suppressor gene PTEN, P21, P27 in the transplanted tumor in nude mice cell expresses, suppress the expression of oncogene AKT1, AKT2, the corresponding separately together physiological saline group data of tyroserleutide group said gene expression of results data relatively have statistical significance (P＜0.05).

Fig. 9 is YSL anti-human liver cancer BEL-7402 transplanted tumor in nude mice experiment Electronic Speculum result, wherein

A is the physiological saline group: well-grown liver cancer cell (* 7500);

B is a YSL group (160 μ g/kg/d): apoptotic cell (* 7500);

C is YSL group (160 μ g/kg/d): liver cancer cell mitochondrial swelling, reticulum dilatation are remarkable, visible calcium overload (* 12450) in plastosome and the endoplasmic reticulum cisterna;

D is a .YSL group (160 μ g/kg/d): plastosome endoplasmic reticulum vacuolation (* 12450).

Embodiment

Also the present invention is described in further detail in conjunction with the accompanying drawings below by specific embodiment.

People's liver cancer BEL7402 cell is waited by Chinese Chen Rui inscription and builds on 1975, and various features are similar to clinical human liver cell liver cancer, kept the malignant characteristics of tumour, and nude mice people liver cancer BEL7402 transplanted tumor has been preserved people's cancer original structure, function, and growth is stable.

The experimental technique of unreceipted actual conditions in the following example or the experimental example, the method that relates to conventional molecular biotechnology, these technology are extensively known in affiliated field, usually according to normal condition such as Molecular Cloning:ALaboratory Manual, the 3rd edition, the 1-3 volume, people such as Sambrook edit, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., the condition described in 2001, or the condition of advising according to manufacturer.

Experimental example 1

The experiment of YSL anti-human liver cancer BEL-7402 transplanted tumor in nude mice

The preparation of nude mice people liver cancer model, can referring to document (Han Rui. cancer therapy drug research and experiment technology [M]. first version. Beijing: the .1997 of combined publication society of China Concord Medical Science University of Beijing Medical University, 4:299).

Lotus people liver cancer BEL-7402 nude mice is available from Tumour Inst., Chinese Medical Academy thing center.Raise in the specified-pathogens free laminar flow cabinet of independent ventilation, feeds utilized, bedding and padding, tap water are aseptically process, in strict accordance with (the safety operation number: A3873-1) carry out of NIH standard.

In the present invention, choose diameter of tumor greater than 1cm, the lotus people liver cancer BEL-7402 nude mice that growth conditions is good, aseptic condition is cut into 2～4mm with fresh tumor tissue down ³Fritter, place the RPMI1640 nutrient solution, at the subcutaneous osculum of cutting in the nude mice belly outside, the knurl piece is transplanted in subcutaneous with Wugou ophthalmology tweezers.With the nude mice of inoculated tumour, (0.2ml only to be divided into the physiological saline group at random ^-1d ^-1), YSL various dose group (320 μ gkg ^-1d ^-1, 160 μ gkg ^-1d ^-1, 80 μ gkg ^-1d ^-1), each is organized medicine and is dissolved in respectively in the 0.2ml physiological saline, next day behind tumor inoculation, and through abdominal injection, once a day, successive administration 60 days.

Embodiment 1

Reverse transcription PCR (RT-PCR) method detects YSL to mitochondrial respiratory chain genes involved NDUFA2, NDUFA10, NDUFS1, SUCLG1 in the BEL-7402 transplanted tumor in nude mice cell, calcium homeostasis is regulated protein gene Calreticulin, and the influence expressed of the mRNA of gene PTEN, Akt1/2, p27, p21

Concrete steps are as follows:

(1) sample preparation

In the experiment of YSL anti-human liver cancer BEL-7402 transplanted tumor in nude mice, from physiological saline group and tyroserleutide 160 μ g/kg/d dosage groups, randomly draw 5 tumor bearing nude mices for every group, after the cervical vertebra dislocation is put to death, peel off tumor tissues with the eye scissors of no RNA enzyme, it is all right to choose tumor growth, there are not obviously necrosis, ulceration, the harder position of quality, be cut into the fritter of about 50mg, after tinfoil was wrapped, it was frozen standby to put into liquid nitrogen immediately.

(2) total RNA extracts

1) tissue homogenate: take out frozen tumor tissues and take by weighing 50～100mg on ice from liquid nitrogen, every part of tissue adds 1mlTRIZOL, and (15～30 ℃ leave standstill 5min for Invitrogen, Inc.USA.) homogenate on ice.

2) RNA separates: the ratio that adds 0.2ml with every 1mlTRIZOL adds chloroform, concuss 15s, and 15～30 ℃ leave standstill 2～3min, and 4 ℃ down 12, the centrifugal 15min of 000g draws the upper strata water to new pipe.

3) RNA precipitation: the RNA aqueous phase, the ratio that adds 0.5ml with every 1mlTRIZOL adds Virahol, and 15～30 ℃ leave standstill 10min, and 4 ℃ are descended 12, and the centrifugal 10min of 000g abandons supernatant.

4) RNA washing: precipitation adds 75% ethanol in the ratio that every 1mlTRIZOL adds 1ml, concuss, and 4 ℃ are descended 7, and the centrifugal 5min of 500g abandons supernatant.

5) RNA redissolves: precipitation dry air 5～10min is dissolved in 30 μ l and removes RNA enzyme water, 55～60 ℃ of water-bath 10min.Remove the sample that is used for RNA concentration and purity evaluation, all the other are all frozen in-80 ℃ of refrigerators.

6) RNA quantitative analysis: UV spectrophotometer measuring A325, A280, A260, A230 are to determine purity and the content of RNA, and the A260/A280 value is between 1.8～2.0.Formula: RNA measures (μ g/ml)=A260/0.025 * extension rate.

(3)RT-PCR

In the 50 μ l reaction volumes 5 */AMVTf1 damping fluid 10 μ l, AMV reversed transcriptive enzyme 1 μ l, Tf1 archaeal dna polymerase 1 μ l, dNTP1 μ l, Mg ²⁺2 μ l, each 50pmol of upstream and downstream primer, total RNA 1 μ g.The reverse transcription condition is 48 ℃ of reverse transcription 45min, 94 ℃ of AMV reversed transcriptive enzyme deactivation/RNA/cDNA primer sex change 2min, and PCR primer and amplification condition see the following form 1.And total RNA of every nude mice carries out these ten Gene RT-PCR respectively and detects, and the result of gained is carried out statistical procedures.

Table 1

The gene title	Primer sequence	Amplification condition
			NDUFA2	Sense 5’-TCATTGAGAAACGCTACGTGG-3’(SEQ ID No.1) antisense 5’-AAGGGACATTCGTCTCTTGGC-3’(SEQ ID No.2)	94℃ 1min， 57℃ 1min， 72℃ 1min， 35cycles

NDUFA10	Sense 5’-GAGGTCCAGAGGCGGATTC-3’(SEQ ID No.3) antisense 5’-CCTCGGTGTTGTACCCAGG-3’(SEQ ID No.4)	94℃ 1min， 56℃ 1min， 72℃ 1min， 35cycles
			NDUFS1	Sense 5’-TGCAGATCCCTCGATTCTGTT-3’(SEQ ID No.5) antisense 5’-GCACAAGCAGCTACAACCTTA-3’(SEQ ID No.6)	94℃ 1min， 55℃ 1min， 72℃ 1min， 35cycles
SUCLG1	Sense 5’-GGCACCAAACTCGTTGGAG-3’(SEQ ID No.7) antisense 5’-TGCTTGACTCGTAGCATGTCC-3’(SEQ ID No.8)	94℃ 1min， 55℃ 1min， 72℃ 1min， 35cycles
			Calreticulin	sense 5’-AGTTCCGGCAAGTTCTACGG-3’(SEQ ID No.9) antisense 5’-CTGAAAGGCTCGAAACTGGC-3’(SEQ ID No.10)	94℃ 1min， 57℃ 1min， 72℃ 1min， 35cycles
PTEN	sense：5’-GGACGAACTGGTGTAATGATATG-3’(SEQ ID No.11) antisense：5’-TCTACTGTTTTTGTGAAGTACAGC-3’(SEQ ID No.12)	94℃ 1min， 55℃ 1min， 72℃ 1min， 30cycles
			AKT1	sense：5’-ATGAGCGACGTGGCTATTGTGAAT-3’(SEQ ID No.13) antisense：5’-GAGGCCGTCAGCCACAGTCTGGATG-3’(SEQ ID No.14)	94℃ 1min， 55℃ 1min， 72℃ 1min， 30cycles
AKT2	sense：5’-ATGAATGAGGTGTCTGTCATCAAAGAAGGC-3’ (SEQ ID No.15) antisense：5’-TGCTTGAGGCTGTTGGCGACC-3’(SEQ ID No.16)	94℃ 1min， 55℃ 1min， 72℃ 1min， 30cycles
			P21	sense：5’-AAGACCATGTGGACCTGTCA-3’(SEQ ID No.17) antisense：5’-GGCTTCCTCTTGGAGAAGAT-3’(SEQ ID No.18)	95℃ 30sec， 55℃ 60sec， 72℃ 90sec， 30cycles
P27	sense：5’-ATGTCAAACGTGCGAGTGTCT-3’(SEQ ID No.19) antisense：5’-TTACGTTTGACGTCTTCTGA-3’(SEQ ID No.20)	94℃ 30sec， 57℃ 1min， 72℃ 1.5min， 30cycles
			GAPDH (confidential reference items)	sense：5’-CTCAGACACCATGGGGAAGGTGA-3’(SEQ ID No.21) antisense：5’-ATGATCTTGAGGCTGTTGTCATA-3’(SEQ ID No.22)

(4) evaluation of PCR product

1.5% agarose gel electrophoresis PCR product is a molecular weight marker with 100bp DNAMarker, and 80V constant voltage electrophoresis 40min observes under ultraviolet lamp and takes a picture.

(5) data processing

Adopt Scion Image software to read optical density value and gray-scale value.As internal reference,, adopt the analysis of variance statistics respectively to organize data with GAPDH with relative expression's abundance of target gene gray scale/GAPDH gray level ratio as mRNA.

(6) experimental result

1) RT-PCR result shows, compares with the physiological saline group, and tyroserleutide group (160 μ g/kg/d) transplanted tumor in nude mice cell NDUFA2, NDUFA10, NDUFS1, the mRNA expression level of SUCLG1 gene all descend in various degree.(Fig. 1, Fig. 2)

2) with GAPDH as internal reference, with the relative expression abundance of target gene/GAPDH gray level ratio as mRNA.The result shows that the mRNA that tyroserleutide can increase Calreticulin in people's liver cancer BEL-7402 transplanted tumor in nude mice cell expresses.(see Fig. 3, Fig. 4)

3) tyroserleutide energy structure promotes the expression of the mRNA of cancer suppressor gene PTEN, P21, P27 in people's liver cancer BEL-7402 transplanted tumor in nude mice tissue, suppresses the expression of the mRNA of oncogene AKT.(Fig. 5 to Fig. 8)

Embodiment 2

Be used to detect the test kit of tyroserleutide action effect

This test kit has a box body, comprises the sealed vessel of the solid-state dry powder primer that is loaded with SEQ ID No.1～22 respectively in the box body.

Can also contain the sealed vessel that is loaded with following substances respectively in this test kit:

Sterilization distilled water, 2.5mmol/L dNTP, 15mmol/L magnesium chloride, 10 * PCR damping fluid, AMV ThermoScript II, RNA enzyme inhibitors, Taq archaeal dna polymerase that DEPC handles.

Wherein, the preparation of 10 * PCR damping fluid (100ml volume) is as follows:

The sterilization distilled water 70mL that DEPC handles

Tutofusin tris (Tris) 0.158g

Repone K 0.373g

Triton x-100 0.1mL

Hydrochloric acid is transferred PH to 9.0

The sterilization distilled water that DEPC handles adds to 100mL

This test kit in use, can earlier solid-state dry powder primer be added the sterilization distilled water be 10pmol/ μ L wiring solution-forming by concentration, be made into reaction system (25 μ L system) according to following ratio then:

The sterilization distilled water 13 μ L that DEPC handles

2.5mmol/L dNTP 2.5μL

10pmol/ μ L RT-PCR primer 1.5 μ L

15mmol/L magnesium chloride 2 μ L

10XPCR damping fluid 2.5 μ L

AMV ThermoScript II 0.2 μ L

RNA enzyme inhibitors 0.3 μ L,

Taq archaeal dna polymerase 1 μ L

The sample RNA 2 μ L that extract

This test kit can be used for the experimentation among the embodiment 1.

Embodiment 3

Method with gene chip detects YSL to mitochondrial respiratory chain genes involved NDUFA2, NDUFA10, NDUFS1, SUCLG1 in the BEL-7402 transplanted tumor in nude mice cell, calcium homeostasis is regulated protein gene Calreticulin, and the influence expressed of the mRNA of gene PTEN, Akt1/2, p27, p21

Total RNA of the physiological saline group tumor bearing nude mice that extracting among the embodiment 1 is obtained, utilize primer and the reaction conditions of NDUFA2 in the table 1, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1/2, p27, p21 to carry out RT-PCR respectively, obtain the cDNA sequence of these ten genes.CDNA sequence point sample method routinely point sample on the slide carrier of these ten genes of obtaining is prepared gene chip.In addition, the cDNA sequence that GAPDH is also arranged on this gene chip is as positive reference gene, and blank spot is as negative reference point.

The gene chip for preparing is used for following experiment.

1 sample

In the experiment of YSL anti-human liver cancer BEL-7402 transplanted tumor in nude mice, randomly draw 1 part of physiological saline group people liver cancer BEL-7402 transplanted tumor in nude mice sample, 2 parts of tyroserleutide group (160 μ g/kg/d) people's liver cancer BEL-7402 transplanted tumor in nude mice samples.

The extracting of 2 total RNA and double-stranded cDNA's is synthetic

Total RNA in every part of tumor tissues sample of TRIZOL method extracting, with RNeasy mini kit purifying, spectrophotometer detects purity and the concentration of RNA.With the total RNA behind the purifying is template, synthesizes first chain and second chain of cDNA respectively with SupercriptII RT, E.coliDNA ligase and E.coli archaeal dna polymerase reverse transcription.

Synthetic, the mark of 3 cRNA and fragmentation

With cDNA is template, and the cRNA with T7 RNA polymerase in-vitro transcription synthesizing biotinylated mark uses RNeasy minikit purifying again, and spectrophotometer detects purity and the concentration of cRNA.The cRNA sample that obtains is split into the fragment of long 50～100 Nucleotide in the fragmentation damping fluid.

The test of 4 Test chip hybridizations

Be the quality that detects each sample cRNA and the stability of crossing system, before the formal chip hybridization, hybridize test experiments with the Test chip earlier.The cRNA of fragmentation adds hybridization solution, and mixing and Test gene chip after 16 hours, are selected corresponding wash-out and dyeing procedure according to chip type 45 ℃ of hybridization.Scan with gene chip scanning instrument at last.The display background signal meets requirement of experiment as a result, and standard control genetic expression meets the requirements, and chip quality and hybridization system are qualified, and sample quality qualified (3 '/5 '＜3) is so can continue experiment.

5 chip hybridizations

The cRNA of fragmentation adds hybridization solution, and mixing and gene chip after 16 hours, carry out wash-out and dyeing procedure 45 ℃ of hybridization.Scan with gene chip scanning instrument at last.

6 chip results data analyses

With Microarray Suite 5.0 softwares data are carried out analysis-by-synthesis relatively, the genetic expression of tyroserleutide group (160 μ g/kg/d) people's liver cancer BEL-7402 transplanted tumor in nude mice and physiological saline group are relatively, difference has or not significance one-sideWilcoxon ' s signed rank test to test, and raise for genetic expression p＜0.0025 significance; There is significance p＞0.9975 for down regulation of gene expression.

The result is as follows, and shown in the result consistent with the result who obtains with the RT-PCR method:

Gene	Multiple	The P value
			SUCLG1	2.32	0.9998
NDUFA10	2.41	0.9996
			NDUFA2	2.32	0.9991
NDUFS1	2.41	0.9979
			AKT1	2.08	0.9995
AKT2	1.16	0.9982
			PTEN	2.56	0.0009
CALR	2.79	0.0016
			p21	2.13	0.0021
p27	2.24	0.0018

Experimental example 2

In YSL anti-human liver cancer BEL-7402 transplanted tumor in nude mice is tested, get tumour by only cuing open next day after administration finishes, weighing, record knurl weigh, and check that tumour has or not situations such as necrosis infection.With 3 mutually perpendicular diameters of vernier caliper measurement tumour, the substitution following formula calculates: V=(1/6) π ABC.V is a gross tumor volume, and A, B, C are 3 diameters of knurl body.Calculate tumor control rate (%)=(the average knurl of the average knurl weight-experimental group of control group is heavy)/average knurl of control group heavy * 100%.

The result is as follows, and when the YSL dosage was 80 μ g/kg/d～320 μ g/kg/d, tumor control rate was respectively 21.66%, 41.34% and 34.78%, with the physiological saline group significant difference (P＜0.05) (table 2) is arranged more all.

Table 2 YSL is to the restraining effect of nude mice people liver cancer BEL-7402 transplanted tumor

Group	Drug dose (kg/d)	Number of animals		Gross tumor volume (cm 3)	Knurl heavy (g)	Inhibiting rate (%)
							Beginning	At last
		YSL YSL YSL physiological saline	320μg 160μg 80μg -						10 10 10 10	10 10 10 10	0.6276±0.1482 * 0.6050±0.2528 * 0.9523±0.3684 0.9630±0.3624	0.5744±0.1685 * 0.5167±0.2131 * 0.6900±0.2607 0.8808±0.3978	34.78 41.34 21.66 -

*: compare P＜0.05 with physiological saline

Simultaneously, it is all right to choose tumor growth, does not have obviously necrosis, ulceration, quality than position, huttriall clip 1-2mm ³The knurl piece be soaked in immediately and fill in 2.5% glutaraldehyde, JEOL-100CX type transmission electron microscope is observed the tumour cell ultrastructural change down.

The result is, YSL can cause the chromatolysis of tumour cell, and necrosis and apoptosis appear in cell.Mitochondrial swelling, dissolving, reticulum dilatation, see in the endoplasmic reticulum of expansion has the calcium particle that is dispersed in more, and prompting has calcium overload phenomenon to take place.(see figure 9)

Sequence table

＜110〉Yitai Medicine Research (Shenzhen) Co., Ltd.

＜120〉method and the used kit and the gene chip of detection anti-liver cancer efficacy of tyroserleutide

<130>CY0510517

<160>32

<170>PatentIn version 3.3

<210>1

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>1

tcattgagaa acgctacgtg g 21

<210>2

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>2

aagggacatt cgtctcttgg c 21

<210>3

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>3

gaggtccaga ggcggattc 19

<210>4

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>4

cctcggtgtt gtacccagg 19

<210>5

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>5

tgcagatccc tcgattctgt t 21

<210>6

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>6

gcacaagcag ctacaacctt a 21

<210>7

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>7

ggcaccaaac tcgttggag 19

<210>8

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>8

tgcttgactc gtaccatgtc c 21

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>9

agttccggca agttctacgg 20

<210>10

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>10

ctgaaaggct cgaaactggc 20

<210>11

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>11

ggacgaactg gtgtaatgat atg 23

<210>12

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>12

tctactgttt ttgtgaagta cagc 24

<210>13

<211>24

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>13

atgagcgacg tggctattgt gaat 24

<210>14

<211>25

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>14

gaggccgtca gccacagtct ggatg 25

<210>15

<211>30

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>15

atgaatgagg tgtctgtcat caaagaaggc 30

<210>16

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>16

tgcttgaggc tgttggcgac c 21

<210>17

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>17

aagaccatgt ggacctgtca 20

<210>18

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>18

ggcttcctct tggagaagat 20

<210>19

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>19

atgtcaaacg tgcgagtgtc t 21

<210>20

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>20

ttacgtttga cgtcttctga 20

<210>21

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>21

ctcagacacc atggggaagg tga 23

<210>22

<211>23

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

＜223〉primer

<400>22

atgatcttga ggctgttgtc ata 23

<210>23

<211>600

<212>DNA

<213>Homo sapiens

<400>23

gagggtccgc ggttggtcag accggggcac ttggcctgaa gacctggaat tggcgacttc 60

gatattaaca aggatggcgg cggccgcagc aagtcgagga gtcggggcaa agctgggcct 120

gcgtgagatt cgcatccact tatgtcagcg ctcgcccggc agccagggcg tcagggactt 180

cattgagaaa cgctacgtgg agctgaagaa ggcgaatccc gacctaccca tcctaatccg 240

cgaatgctcc gatgtgcagc ccaagctctg ggcccgctac gcatttggcc aagagacgaa 300

tgtccctttg aacaacttca gtgctgatca ggtaaccaga gccctggaga acgttctaag 360

tggtaaagcc tgaagcctcc actgaggatt aagagcaaca gccccagagc ctgggctctg 420

ctggacttag tataatgtga aaaaaatgtg ttctcctatt cctcataaag cttgtgctgt 480

aaaatacttt ctcagggtgt tcttgtcctc atctaccctc taccccttac tgtgcaacca 540

ctgaggcaaa gtagcttaat ataaaaataa aactttattc tgtctcaaaa aaaaaaaaaa 600

<210>24

<211>1557

<212>DNA

<213>Homo sapiens

<400>24

ggcagcgcgc cggccgcgag agagggcccc gtcgcgaccg cgtccccttg ggtccttgat 60

cctgagctga ccgggtagcc atggccttgc ggctcctgaa gctggcagcg acgtccgcgt 120

ccgcccgggt cgtggcggcg ggcgcccagc gcgtgagagg aattcatagc agtgtgcagt 180

gcaaactgcg ctatggaatg tggcatttcc tacttgggga taaagcaagc aaaagactga 240

cagaacgcag cagagtgata actgtagatg gcaatatatg tactggaaaa ggcaaacttg 300

caaaagaaat agcagagaaa ctaggcttca agcactttcc tgaagcgggg attcattatc 360

cagacagtac cacaggagat gggaagcccc tcgccaccga ctataatggc aactgtagtt 420

tggagaaatt ttacgatgat ccgagaagca atgatggcaa cagttaccgc ctgcagtcct 480

ggttgtacag cagtcgcctg ctgcagtact cagatgcctt ggagcacttg ctgaccacag 540

gacaaggtgt tgtgttggag cgctccatct tcagtgactt tgtgttcctg gaggcgatgt 600

acaaccaggg attcatccga aagcagtgtg tggaccacta caacgaggtg aagagcgtca 660

ccatctgcga ttacctgccc ccccacctgg tgatttacat cgatgtgccc gttccagagg 720

tccagaggcg gattcagaag aaaggagatc cacatgaaat gaagatcacc tctgcctatc 780

tacaggacat tgagaatgcc tataagaaaa cctttctccc tgagatgagt gaaaaatgtg 840

aggttttaca atattctgca agggaagctc aagattcaaa aaaggtggta gaggacattg 900

aatacctgaa gttcgataaa gggccgtggc tcaagcagga caatcgcact ttataccacc 960

tgcgattact ggttcaggat aagtttgagg tgctgaatta cacaagcatt cctatctttc 1020

tcccggaagt caccattgga gctcatcaga ctgaccgtgt cttacatcag ttcagagagc 1080

tgccgggccg caagtacagc cctgggtaca acaccgaggt gggagacaag tggatctggc 1140

tgaagtgaac gggccgcctt ctgctccagc tgcatcacag tgatggccaa gctgcatcag 1200

ccgcactctc ctggacgcca tatagcttta agatcggggg agggtaaata atgcaaaaat 1260

tgcacagtgg aagaaggggt ctcacaaaaa gcaatccatc ctgtagtata ggtaatggag 1320

ttgggggaag cagcttccat tctggatgtt tggaaccctt tagctttgtt ttggaatggc 1380

ccaccattct cactggaaaa cagtggtctg ctgtgaaagg ccagctctcg gcagcccctg 1440

tggtttcagc gctgccgctc tgtgtcattc aggttgtgca cattgttttt cttctgactt 1500

ccagaaataa aagtgtttcc atgggaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1557

<210>25

<211>3417

<212>DNA

<213>Homo sapiens

<400>25

ttctccaggc ccggctgaca gagttagccg aggccgccat attgaataag cgacccggcc 60

tcctaggggg tcgtcgtggt ccagacagtt tagcagaaca gcctccgcgg ctccggggag 120

aagcaatatg ttaaggatac ctgtaagaaa ggccttagta ggcctttcta agtctcctaa 180

aggatgtgtt cgaacaactg ccacagcagc aagcaacttg attgaagtat ttgttgatgg 240

tcagtctgtc atggtggaac cgggaacgac cgtcctccaa gcttgtgaga aggttggcat 300

gcagatccct cgattctgtt atcatgaaag gttgtctgtt gctggaaact gcaggatgtg 360

ccttgttgaa attgagaaag cccctaaggt tgtagctgct tgtgccatgc cagtaatgaa 420

gggttggaat atcctaacaa actcagaaaa atccaaaaaa gccagggaag gtgtgatgga 480

gttcttatta gcaaatcacc cattggactg tcctatttgt gaccagggag gtgaatgtga 540

tctgcaggac cagtccatga tgtttggaaa tgataggagc cgatttttag aggggaagcg 600

tgctgtggaa gacaagaaca ttgggccatt ggtaaagacc atcatgacaa gatgtataca 660

gtgtactcgc tgcatcaggt ttgcaagtga gattgcagga gtagatgatt tgggaacaac 720

aggcagagga aatgatatgc aagttggcac atacattgaa aagatgttca tgtctgaact 780

gtctgggaat atcattgata tctgccctgt aggtgcccta acctctaagc cctatgcctt 840

tactgcccgg ccttgggaaa caagaaagac agaatccatt gatgtaatgg atgcggttgg 900

aagtaatatt gtggttagca caagaactgg agaagtgatg aggattttgc cacgtatgca 960

tgaggacatc aatgaagagt ggatctctga taaaaccaga tttgcctatg atgggctaaa 1020

acgtcaaaga cttaccgagc caatggtcag aaatgaaaaa gggcttttaa cctatacttc 1080

ttgggaggat gcgctctctc gcgtagctgg aatgttgcag agttttcaag gcaaagatgt 1140

ggcagcaatt gcaggtggct tggtggatgc tgaagccctg gtagctctca aagatttgct 1200

taatagagtg gactctgaca ccttatgcac tgaagaggtc ttccccactg caggagctgg 1260

cacagatttg cgttccaatt atcttcttaa tactacaatt gctggtgtgg aagaggcaga 1320

tgttgttctt ctggttggta caaacccacg ttttgaggca ccactgttta atgctagaat 1380

tcgaaagagc tggctgcata atgacttaaa agtggccctt ataggcagtc cagtggacct 1440

cacttacaca tatgaccacc tgggagactc ccccaaaatt cttcaagaca ttgcttcggg 1500

aagccatcca tttagccagg tcctaaagga agctaaaaaa ccaatggtgg ttttaggcag 1560

ttctgcactc caaagaaatg atggagcagc aattcttgca gctgtttcta gcattgcaca 1620

aaagattcgg atgactagtg gtgttactgg tgattggaaa gttatgaata tccttcatag 1680

gattgcaagt caagtagctg ctttggacct tggctataag cctggggtgg aagcaattcg 1740

gaagaaccct cccaaggtgc tgtttctcct gggagcagat ggaggttgta tcacacgaca 1800

ggatttgcca aaggattgtt tcattattta tcaaggacat catggtgatg ttggggctcc 1860

catagctgat gttattctcc caggagctgc ttacacagag aagtctgcta catatgtcaa 1920

cactgagggt agagctcagc agactaaggt agcagtgaca cctcctggct tggcaagaga 1980

agactggaaa attataagag cactctctga gattgctgga atgactcttc catatgatac 2040

tctggatcaa gtaaggaaca gattggaaga agtctctcct aatcttgttc gatatgatga 2100

tattgaaggg gctaattact tccagcaagc aaatgagctc tcaaagctag tgaaccagca 2160

gcttcttgct gacccacttg ttccacctca gctaactata aaagacttct acatgacaga 2220

ttcaattagc agagcctcac agacaatggc caaatgtgtc aaagctgtca cagagggtgc 2280

ccaggcagta gaggaaccat ccatatgctg aagcttctac taggatccca gttttgccgc 2340

agataattaa tggacaactg tagtgcagtg atcctttaca ggtttatttc tttgtaaaaa 2400

aaaataataa taatttgaat catgtaatat ttaaggttat actatgccta tttgaaaatg 2460

atattagtta tcaactttgc agtttgaaaa acatgtattg tgtgtaaagg ttaaataaca 2520

aaactatgca gatgctctta aaagcattga taacctttgt gacgaacata aagagatcct 2580

taaattatga gttgttggct tatcttcata aataatttgt ctgtaaaatg gatgaaatga 2640

aaagaggttc aattaaaacc tacttttttc tagtgctaaa gaaaagattt aagcaccttg 2700

tcaagctggg taaataggaa aaatacataa tcatgctcag atatgtatct aggataatta 2760

taattaataa taatcatagt aacaatggct aatgataatt tagctttatt atatgtgcta 2820

agcactctgg ttttacatgc attatcttct tttgttcttg ctacaaccct gtgagatagt 2880

agtattatct cattttactg atgaagactg aagcctaggt atattaaata gcttgcccaa 2940

ggccacacag caaaagtcac caactcaaac ctacttctta tttactccaa agcctgttgt 3000

tcttaactgc aacatattat ttcgtctcat taatgttgat tctataggtt gttacttcta 3060

aaaattagta ttgagtttaa tggtgaacac atttttctat tttctcttga atctgcttct 3120

ataatgtcat ggtgatttat gtggcttttt tttttttata agttatacat gtatgcatgt 3180

atacttatga gacctccctt ggaatgaggg aggtctcaag agatataatt tagattctca 3240

ttgatgttct gtattcatta tcctaacacc atctgtagtg ttaaatcaac taaattattt 3300

cagcaatagg agacaaaaca accagctttc ataattttta attgtcaaaa ccaaaaggaa 3360

tcagaataag gatcactgag aatttaaaaa aataaaaaag gaagtaaaaa ttttaca 3417

<210>26

<211>1227

<212>DNA

<213>Homo sapiens

<400>26

aacccttgcc gctgccgctg acatcgctac catggtctcc ggcagcagcg gcctcgccgc 60

cgcccgtctc ctgtcgcgca gcttcctcct gccgcagaat ggaattcggc attgttccta 120

cacagcttct cggcaacatc tctatgttga taaaaataca aagattattt gccagggttt 180

cactggcaaa cagggcacct ttcacagcca gcaggcattg gaatatggca ccaaactcgt 240

tggaggaaca ctcccaggga aaggaggcca gacacatctg ggcttacctg tctttaatac 300

tgtgaaggag gccaaagaac agacaggagc aacggcttct gtcatttatg ttcctccgcc 360

ttttgctgct gctgccatta atgaagctat tgaggcagaa attcccttgg ttgtgtgtat 420

cactgaagga attccccagc aggacatggt acgagtcaag cacaaactgc tgcgccagga 480

aaagacaagg ctaattgggc ccaactgccc tggagtcatc aatcctggag aatgtaaaat 540

tgggatcatg cctggccata ttcacaaaaa aggaaggatt ggcattgtgt ccagatctgg 600

caccctgact tatgaagcag ttcaccaaac aacgcaagtt ggattggggc agtctttgtg 660

cgttggcatt ggaggtgatc cttttaatgg aacagatttt attgactgcc tcgaaatctt 720

tttgaacgat tctgccacag aaggcatcat attgattggt gaaattggtg gtaatgcaga 780

agagaatgct gcagaatttt tgaagcaaca taattcaggt ccaaattcca agcctgtagt 840

gtccttcatt gctggtttaa ctgctcctcc tgggagaaga atgggtcatg ccggggcaat 900

tattgctgga ggaaaaggtg gagctaaaga gaagatctct gcccttcaga gtgcaggagt 960

tgtggtcagt atgtctcctg cacagctggg aaccacgatc tacaaggaat ttgaaaagag 1020

gaagatgcta tgaaagaaaa aaaaaattcc taaaactgtg gaatggatca cgtagacatg 1080

taacccagca gcagtttgct tctgttgtcc actgattaat cagcctatgt gcctgacact 1140

ggtcttgcag tacaactgga agccaaaaca aggtggaaga tgtcctgaat taagacgttt 1200

tcaccacatt gtattacaga gacagcc 1227

<210>27

<211>1899

<212>DNA

<213>Homo sapiens

<400>27

gtccgtactg cagagccgct gccggagggt cgttttaaag ggccgcgttg ccgccccctc 60

ggcccgccat gctgctatcc gtgccgctgc tgctcggcct cctcggcctg gccgtcgccg 120

agcccgccgt ctacttcaag gagcagtttc tggacggaga cgggtggact tcccgctgga 180

tcgaatccaa acacaagtca gattttggca aattcgttct cagttccggc aagttctacg 240

gtgacgagga gaaagataaa ggtttgcaga caagccagga tgcacgcttt tatgctctgt 300

cggccagttt cgagcctttc agcaacaaag gccagacgct ggtggtgcag ttcacggtga 360

aacatgagca gaacatcgac tgtgggggcg gctatgtgaa gctgtttcct aatagtttgg 420

accagacaga catgcacgga gactcagaat acaacatcat gtttggtccc gacatctgtg 480

gccctggcac caagaaggtt catgtcatct tcaactacaa gggcaagaac gtgctgatca 540

acaaggacat ccgttgcaag gatgatgagt ttacacacct gtacacactg attgtgcggc 600

cagacaacac ctatgaggtg aagattgaca acagccaggt ggagtccggc tccttggaag 660

acgattggga cttcctgcca cccaagaaga taaaggatcc tgatgcttca aaaccggaag 720

actgggatga gcgggccaag atcgatgatc ccacagactc caagcctgag gactgggaca 780

agcccgagca tatccctgac cctgatgcta agaagcccga ggactgggat gaagagatgg 840

acggagagtg ggaaccccca gtgattcaga accctgagta caagggtgag tggaagcccc 900

ggcagatcga caacccagat tacaagggca cttggatcca cccagaaatt gacaaccccg 960

agtattctcc cgatcccagt atctatgcct atgataactt tggcgtgctg ggcctggacc 1020

tctggcaggt caagtctggc accatctttg acaacttcct catcaccaac gatgaggcat 1080

acgctgagga gtttggcaac gagacgtggg gcgtaacaaa ggcagcagag aaacaaatga 1140

aggacaaaca ggacgaggag cagaggctta aggaggagga agaagacaag aaacgcaaag 1200

aggaggagga ggcagaggac aaggaggatg atgaggacaa agatgaggat gaggaggatg 1260

aggaggacaa ggaggaagat gaggaggaag atgtccccgg ccaggccaag gacgagctgt 1320

agagaggcct gcctccaggg ctggactgag gcctgagcgc tcctgccgca gagcttgccg 1380

cgccaaataa tgtctctgtg agactcgaga actttcattt ttttccaggc tggttcggat 1440

ttggggtgga ttttggtttt gttcccctcc tccactctcc cccaccccct ccccgccctt 1500

tttttttttt tttttaaact ggtattttat cctttgattc tccttcagcc ctcacccctg 1560

gttctcatct ttcttgatca acatcttttc ttgcctctgt gccccttctc tcatctctta 1620

gctcccctcc aacctggggg gcagtggtgt ggagaagcca caggcctgag atttcatctg 1680

ctctccttcc tggagcccag aggagggcag cagaaggggg tggtgtctcc aaccccccag 1740

cactgaggaa gaacggggct cttctcattt cacccctccc tttctcccct gcccccagga 1800

ctgggccact tctgggtggg gcagtgggtc ccagattggc tcacactgag aatgtaagaa 1860

ctacaaacaa aatttctatt aaattaaatt ttgtgtctc 1899

<210>28

<211>3416

<212>DNA

<213>Homo sapiens

<400>28

cctcccctcg cccggcgcgg tcccgtccgc ctctcgctcg cctcccgcct cccctcggtc 60

ttccgaggcg cccgggctcc cggcgcggcg gcggaggggg cgggcaggcc ggcgggcggt 120

gatgtggcgg gactctttat gcgctgcggc aggatacgcg ctcggcgctg ggacgcgact 180

gcgctcagtt ctctcctctc ggaagctgca gccatgatgg aagtttgaga gttgagccgc 240

tgtgaggcga ggccgggctc aggcgaggga gatgagagac ggcggcggcc gcggcccgga 300

gcccctctca gcgcctgtga gcagccgcgg gggcagcgcc ctcggggagc cggccggcct 360

gcggcggcgg cagcggcggc gtttctcgcc tcctcttcgt cttttctaac cgtgcagcct 420

cttcctcggc ttctcctgaa agggaaggtg gaagccgtgg gctcgggcgg gagccggctg 480

aggcgcggcg gcggcggcgg cacctcccgc tcctggagcg ggggggagaa gcggcggcgg 540

cggcggccgc ggcggctgca gctccaggga gggggtctga gtcgcctgtc accatttcca 600

gggctgggaa cgccggagag ttggtctctc cccttctact gcctccaaca cggcggcggc 660

ggcggcggca catccaggga cccgggccgg ttttaaacct cccgtccgcc gccgccgcac 720

cccccgtggc ccgggctccg gaggccgccg gcggaggcag ccgttcggag gattattcgt 780

cttctcccca ttccgctgcc gccgctgcca ggcctctggc tgctgaggag aagcaggccc 840

agtcgctgca accatccagc agccgccgca gcagccatta cccggctgcg gtccagagcc 900

aagcggcggc agagcgaggg gcatcagcta ccgccaagtc cagagccatt tccatcctgc 960

agaagaagcc ccgccaccag cagcttctgc catctctctc ctcctttttc ttcagccaca 1020

ggctcccaga catgacagcc atcatcaaag agatcgttag cagaaacaaa aggagatatc 1080

aagaggatgg attcgactta gacttgacct atatttatcc aaacattatt gctatgggat 1140

ttcctgcaga aagacttgaa ggcgtataca ggaacaatat tgatgatgta gtaaggtttt 1200

tggattcaaa gcataaaaac cattacaaga tatacaatct ttgtgctgaa agacattatg 1260

acaccgccaa atttaattgc agagttgcac aatatccttt tgaagaccat aacccaccac 1320

agctagaact tatcaaaccc ttttgtgaag atcttgacca atggctaagt gaagatgaca 1380

atcatgttgc agcaattcac tgtaaagctg gaaagggacg aactggtgta atgatatgtg 1440

catatttatt acatcggggc aaatttttaa aggcacaaga ggccctagat ttctatgggg 1500

aagtaaggac cagagacaaa aagggagtaa ctattcccag tcagaggcgc tatgtgtatt 1560

attatagcta cctgttaaag aatcatctgg attatagacc agtggcactg ttgtttcaca 1620

agatgatgtt tgaaactatt ccaatgttca gtggcggaac ttgcaatcct cagtttgtgg 1680

tctgccagct aaaggtgaag atatattcct ccaattcagg acccacacga cgggaagaca 1740

agttcatgta ctttgagttc cctcagccgt tacctgtgtg tggtgatatc aaagtagagt 1800

tcttccacaa acagaacaag atgctaaaaa aggacaaaat gtttcacttt tgggtaaata 1860

cattcttcat accaggacca gaggaaacct cagaaaaagt agaaaatgga agtctatgtg 1920

atcaagaaat cgatagcatt tgcagtatag agcgtgcaga taatgacaag gaatatctag 1980

tacttacttt aacaaaaaat gatcttgaca aagcaaataa agacaaagcc aaccgatact 2040

tttctccaaa ttttaaggtg aagctgtact tcacaaaaac agtagaggag ccgtcaaatc 2100

cagaggctag cagttcaact tctgtaacac cagatgttag tgacaatgaa cctgatcatt 2160

atagatattc tgacaccact gactctgatc cagagaatga accttttgat gaagatcagc 2220

atacacaaat tacaaaagtc tgaatttttt tttatcaaga gggataaaac accatgaaaa 2280

taaacttgaa taaactgaaa atggaccttt ttttttttaa tggcaatagg acattgtgtc 2340

agattaccag ttataggaac aattctcttt tcctgaccaa tcttgtttta ccctatacat 2400

ccacagggtt ttgacacttg ttgtccagtt gaaaaaaggt tgtgtagctg tgtcatgtat 2460

ataccttttt gtgtcaaaag gacatttaaa attcaattag gattaataaa gatggcactt 2520

tcccgtttta ttccagtttt ataaaaagtg gagacagact gatgtgtata cgtaggaatt 2580

ttttcctttt gtgttctgtc accaactgaa gtggctaaag agctttgtga tatactggtt 2640

cacatcctac ccctttgcac ttgtggcaac agataagttt gcagttggct aagagaggtt 2700

tccgaagggt tttgctacat tctaatgcat gtattcgggt taggggaatg gagggaatgc 2760

tcagaaagga aataatttta tgctggactc tggaccatat accatctcca gctatttaca 2820

cacacctttc tttagcatgc tacagttatt aatctggaca ttcgaggaat tggccgctgt 2880

cactgcttgt tgtttgcgca ttttttttta aagcatattg gtgctagaaa aggcagctaa 2940

aggaagtgaa tctgtattgg ggtacaggaa tgaaccttct gcaacatctt aagatccaca 3000

aatgaaggga tataaaaata atgtcatagg taagaaacac agcaacaatg acttaaccat 3060

ataaatgtgg aggctatcaa caaagaatgg gcttgaaaca ttataaaaat tgacaatgat 3120

ttattaaata tgttttctca attgtaacga cttctccatc tcctgtgtaa tcaaggccag 3180

tgctaaaatt cagatgctgt tagtacctac atcagtcaac aacttacact tattttacta 3240

gttttcaatc ataatacctg ctgtggatgc ttcatgtgct gcctgcaagc ttcttttttc 3300

tcattaaata taaaatattt tgtaatgctg cacagaaatt ttcaatttga gattctacag 3360

taagcgtttt ttttctttga agatttatga tgcacttatt caatagctgt cagccg 3416

<210>29

<211>3008

<212>DNA

<213>Homo sapiens

<400>29

taattatggg tctgtaacca ccctggactg ggtgctcctc actgacggac ttgtctgaac 60

ctctctttgt ctccagcgcc cagcactggg cctggcaaaa cctgagacgc ccggtacatg 120

ttggccaaat gaatgaacca gattcagacc ggcaggggcg ctgtggttta ggaggggcct 180

ggggtttctc ccaggaggtt tttgggcttg cgctggaggg ctctggactc ccgtttgcgc 240

cagtggcctg catcctggtc ctgtcttcct catgtttgaa tttctttgct ttcctagtct 300

ggggagcagg gaggagccct gtgccctgtc ccaggatcca tgggtaggaa caccatggac 360

agggagagca aacggggcca tctgtcacca ggggcttagg gaaggccgag ccagcctggg 420

tcaaagaagt caaaggggct gcctggagga ggcagcctgt cagctggtgc atcagaggct 480

gtggccaggc cagctgggct cggggagcgc cagcctgaga ggagcgcgtg agcgtcgcgg 540

gagcctcggg caccatgagc gacgtggcta ttgtgaagga gggttggctg cacaaacgag 600

gggagtacat caagacctgg cggccacgct acttcctcct caagaatgat ggcaccttca 660

ttggctacaa ggagcggccg caggatgtgg accaacgtga ggctcccctc aacaacttct 720

ctgtggcgca gtgccagctg atgaagacgg agcggccccg gcccaacacc ttcatcatcc 780

gctgcctgca gtggaccact gtcatcgaac gcaccttcca tgtggagact cctgaggagc 840

gggaggagtg gacaaccgcc atccagactg tggctgacgg cctcaagaag caggaggagg 900

aggagatgga cttccggtcg ggctcaccca gtgacaactc aggggctgaa gagatggagg 960

tgtccctggc caagcccaag caccgcgtga ccatgaacga gtttgagtac ctgaagctgc 1020

tgggcaaggg cactttcggc aaggtgatcc tggtgaagga gaaggccaca ggccgctact 1080

acgccatgaa gatcctcaag aaggaagtca tcgtggccaa ggacgaggtg gcccacacac 1140

tcaccgagaa ccgcgtcctg cagaactcca ggcacccctt cctcacagcc ctgaagtact 1200

ctttccagac ccacgaccgc ctctgctttg tcatggagta cgccaacggg ggcgagctgt 1260

tcttccacct gtcccgggag cgtgtgttct ccgaggaccg ggcccgcttc tatggcgctg 1320

agattgtgtc agccctggac tacctgcact cggagaagaa cgtggtgtac cgggacctca 1380

agctggagaa cctcatgctg gacaaggacg ggcacattaa gatcacagac ttcgggctgt 1440

gcaaggaggg gatcaaggac ggtgccacca tgaagacctt ttgcggcaca cctgagtacc 1500

tggcccccga ggtgctggag gacaatgact acggccgtgc agtggactgg tgggggctgg 1560

gcgtggtcat gtacgagatg atgtgcggtc gcctgccctt ctacaaccag gaccatgaga 1620

agctttttga gctcatcctc atggaggaga tccgcttccc gcgcacgctt ggtcccgagg 1680

ccaagtcctt gctttcaggg ctgctcaaga aggaccccaa gcagaggctt ggcgggggct 1740

ccgaggacgc caaggagatc atgcagcatc gcttctttgc cggtatcgtg tggcagcacg 1800

tgtacgagaa gaagctcagc ccacccttca agccccaggt cacgtcggag actgacacca 1860

ggtattttga tgaggagttc acggcccaga tgatcaccat cacaccacct gaccaagatg 1920

acagcatgga gtgtgtggac agcgagcgca ggccccactt cccccagttc tcctactcgg 1980

ccagcggcac ggcctgaggc ggcggtggac tgcgctggac gatagcttgg agggatggag 2040

aggcggcctc gtgccatgat ctgtatttaa tggtttttat ttctcgggtg catttgagag 2100

aagccacgct gtcctctcga gcccagatgg aaagacgttt ttgtgctgtg ggcagcaccc 2160

tcccccgcag cggggtaggg aagaaaacta tcctgcgggt tttaatttat ttcatccagt 2220

ttgttctccg ggtgtggcct cagccctcag aacaatccga ttcacgtagg gaaatgttaa 2280

ggacttctgc agctatgcgc aatgtggcat tggggggccg ggcaggtcct gcccatgtgt 2340

cccctcactc tgtcagccag ccgccctggg ctgtctgtca ccagctatct gtcatctctc 2400

tggggccctg ggcctcagtt caacctggtg gcaccagatg caacctcact atggtatgct 2460

ggccagcacc ctctcctggg ggtggcaggc acacagcagc cccccagcac taaggccgtg 2520

tctctgagga cgtcatcgga ggctgggccc ctgggatggg accagggatg ggggatgggc 2580

cagggtttac ccagtgggac agaggagcaa ggtttaaatt tgttattgtg tattatgttg 2640

ttcaaatgca ttttgggggt ttttaatctt tgtgacagga aagccctccc ccttcccctt 2700

ctgtgtcaca gttcttggtg actgtcccac cgggagcctc cccctcagat gatctctcca 2760

cggtagcact tgaccttttc gacgcttaac ctttccgctg tcgccccagg ccctccctga 2820

ctccctgtgg gggtggccat ccctgggccc ctccacgcct cctggccaga cgctgccgct 2880

gccgctgcac cacggcgttt ttttacaaca ttcaacttta gtatttttac tattataata 2940

taatatggaa ccttccctcc aaattcttca ataaaagttg cttttcaaaa aaaaaaaaaa 3000

aaaaaaaa 3008

<210>30

<211>1715

<212>DNA

<213>Homo sapiens

<400>30

gaattccagc ggcggcgccg ttgccgctgc cgggaaacac aaggaaaggg aaccagcgca 60

gcgtggcgat gggcgggggt agagccccgc cggagaggct gggcggctgc cggtgacaga 120

ctgtgccctg tccacggtgc ctcctgcatg tcctgctgcc ctgagctgtc ccgagctagg 180

tgacagcgta ccacgctgcc accatgaatg aggtgtctgt catcaaagaa ggctggctcc 240

acaagcgtgg tgaatacatc aagacctgga ggccacggta cttcctgctg aagagcgacg 300

gctccttcat tgggtacaag gagaggcccg aggcccctga tcagactcta ccccccttaa 360

acaacttctc cgtagcagaa tgccagctga tgaagaccga gaggccgcga cccaacacct 420

ttgtcatacg ctgcctgcag tggaccacag tcatcgagag gaccttccac gtggattctc 480

cagacgagag ggaggagtgg atgcgggcca tccagatggt cgccaacagc ctcaagcagc 540

gggccccagg cgaggacccc atggactaca agtgtggctc ccccagtgac tcctccacga 600

ctgaggagat ggaagtggcg gtcagcaagg cacgggctaa agtgaccatg aatgacttcg 660

actatctcaa actccttggc aagggaacct ttggcaaagt catcctggtg cgggagaagg 720

ccactggccg ctactacgcc atgaagatcc tgcgaaagga agtcatcatt gccaaggatg 780

aagtcgctca cacagtcacc gagagccggg tcctccagaa caccaggcac ccgttcctca 840

ctgcgctgaa gtatgccttc cagacccacg accgcctgtg ctttgtgatg gagtatgcca 900

acgggggtga gctgttcttc cacctgtccc gggagcgtgt cttcacagag gagcgggccc 960

ggttttatgg tgcagagatt gtctcggctc ttgagtactt gcactcgcgg gacgtggtat 1020

accgcgacat caagctggaa aacctcatgc tggacaaaga tggccacatc aagatcactg 1080

actttggcct ctgcaaagag ggcatcagtg acggggccac catgaaaacc ttctgtggga 1140

ccccggagta cctggcgcct gaggtgctgg aggacaatga ctatggccgg gccgtggact 1200

ggtgggggct gggtgtggtc atgtacgaga tgatgtgcgg ccgcctgccc ttctacaacc 1260

aggaccacga gcgcctcttc gagctcatcc tcatggaaga gatccgcttc ccgcgcacgc 1320

tcagccccga ggccaagtcc ctgcttgctg ggctgcttaa gaaggacccc aagcagaggc 1380

ttggtggggg gcccagcgat gccaaggagg tcatggagca caggttcttc ctcagcatca 1440

actggcagga cgtggtccag aagaagctcc tgccaccctt caaacctcag gtcacgtccg 1500

aggtcgacac aaggtacttc gatgatgaat ttaccgccca gtccatcaca atcacacccc 1560

ctgaccgcta tgacagcctg ggcttactgg agctggacca gcggacccac ttcccccagt 1620

tctcctactc ggccagcatc cgcgagtgag cagtctgccc acgcagagga cgcacgctcg 1680

ctgccatcac cgctgggtgg ttttttaccc ctgcc 1715

<210>31

<211>2140

<212>DNA

<213>Homo sapiens

<400>31

agctgaggtg tgagcagctg ccgaagtcag ttccttgtgg agccggagct gggcgcggat 60

tcgccgaggc accgaggcac tcagaggagg cgccatgtca gaaccggctg gggatgtccg 120

tcagaaccca tgcggcagca aggcctgccg ccgcctcttc ggcccagtgg acagcgagca 180

gctgagccgc gactgtgatg cgctaatggc gggctgcatc caggaggccc gtgagcgatg 240

gaacttcgac tttgtcaccg agacaccact ggagggtgac ttcgcctggg agcgtgtgcg 300

gggccttggc ctgcccaagc tctaccttcc cacggggccc cggcgaggcc gggatgagtt 360

gggaggaggc aggcggcctg gcacctcacc tgctctgctg caggggacag cagaggaaga 420

ccatgtggac ctgtcactgt cttgtaccct tgtgcctcgc tcaggggagc aggctgaagg 480

gtccccaggt ggacctggag actctcaggg tcgaaaacgg cggcagacca gcatgacaga 540

tttctaccac tccaaacgcc ggctgatctt ctccaagagg aagccctaat ccgcccacag 600

gaagcctgca gtcctggaag cgcgagggcc tcaaaggccc gctctacatc ttctgcctta 660

gtctcagttt gtgtgtctta attattattt gtgttttaat ttaaacacct cctcatgtac 720

ataccctggc cgccccctgc cccccagcct ctggcattag aattatttaa acaaaaacta 780

ggcggttgaa tgagaggttc ctaagagtgc tgggcatttt tattttatga aatactattt 840

aaagcctcct catcccgtgt tctccttttc ctctctcccg gaggttgggt gggccggctt 900

catgccagct acttcctcct ccccacttgt ccgctgggtg gtaccctctg gaggggtgtg 960

gctccttccc atcgctgtca caggcggtta tgaaattcac cccctttcct ggacactcag 1020

acctgaattc tttttcattt gagaagtaaa cagatggcac tttgaagggg cctcaccgag 1080

tgggggcatc atcaaaaact ttggagtccc ctcacctcct ctaaggttgg gcagggtgac 1140

cctgaagtga gcacagccta gggctgagct ggggacctgg taccctcctg gctcttgata 1200

cccccctctg tcttgtgaag gcagggggaa ggtggggtac tggagcagac caccccgcct 1260

gccctcatgg cccctctgac ctgcactggg gagcccgtct cagtgttgag ccttttccct 1320

ctttggctcc cctgtacctt ttgaggagcc ccagcttacc cttcttctcc agctgggctc 1380

tgcaattccc ctctgctgct gtccctcccc cttgtctttc ccttcagtac cctctcatgc 1440

tccaggtggc tctgaggtgc ctgtcccacc cccaccccca gctcaatgga ctggaagggg 1500

aagggacaca caagaagaag ggcaccctag ttctacctca ggcagctcaa gcagcgaccg 1560

ccccctcctc tagctgtggg ggtgagggtc ccatgtggtg gcacaggccc ccttgagtgg 1620

ggttatctct gtgttagggg tatatgatgg gggagtagat ctttctagga gggagacact 1680

ggcccctcaa atcgtccagc gaccttcctc atccacccca tccctcccca gttcattgca 1740

ctttgattag cagcggaaca aggagtcaga cattttaaga tggtggcagt agaggctatg 1800

gacagggcat gccacgtggg ctcatatggg gctgggagta gttgtctttc ctggcactaa 1860

cgttgagccc ctggaggcac tgaagtgctt agtgtacttg gagtattggg gtctgacccc 1920

aaacaccttc cagctcctgt aacatactgg cctggactgt tttctctcgg ctccccatgt 1980

gtcctggttc ccgtttctcc acctagactg taaacctctc gagggcaggg accacaccct 2040

gtactgttct gtgtctttca cagctcctcc cacaatgctg aatatacagc aggtgctcaa 2100

taaatgattc ttagtgactt taaaaaaaaa aaaaaaaaaa 2140

<210>32

<211>2422

<212>DNA

<213>Homo sapiens

<400>32

gtcagcctcc cttccaccgc catattgggc cactaaaaaa agggggctcg tcttttcggg 60

gtgtttttct ccccctcccc tgtccccgct tgctcacggc tctgcgactc cgacgccggc 120

aaggtttgga gagcggctgg gttcgcggga cccgcgggct tgcacccgcc cagactcgga 180

cgggctttgc caccctctcc gcttgcctgg tcccctctcc tctccgccct cccgctcgcc 240

agtccatttg atcagcggag actcggcggc cgggccgggg cttccccgca gcccctgcgc 300

gctcctagag ctcgggccgt ggctcgtcgg ggtctgtgtc ttttggctcc gagggcagtc 360

gctgggcttc cgagaggggt tcgggccgcg taggggcgct ttgttttgtt cggttttgtt 420

tttttgagag tgcgagagag gcggtcgtgc agacccggga gaaagatgtc aaacgtgcga 480

gtgtctaacg ggagccctag cctggagcgg atggacgcca ggcaggcgga gcaccccaag 540

ccctcggcct gcaggaacct cttcggcccg gtggaccacg aagagttaac ccgggacttg 600

gagaagcact gcagagacat ggaagaggcg agccagcgca agtggaattt cgattttcag 660

aatcacaaac ccctagaggg caagtacgag tggcaagagg tggagaaggg cagcttgccc 720

gagttctact acagaccccc gcggcccccc aaaggtgcct gcaaggtgcc ggcgcaggag 780

agccaggatg tcagcgggag ccgcccggcg gcgcctttaa ttggggctcc ggctaactct 840

gaggacacgc atttggtgga cccaaagact gatccgtcgg acagccagac ggggttagcg 900

gagcaatgcg caggaataag gaagcgacct gcaaccgacg attcttctac tcaaaacaaa 960

agagccaaca gaacagaaga aaatgtttca gacggttccc caaatgccgg ttctgtggag 1020

cagacgccca agaagcctgg cctcagaaga cgtcaaacgt aaacagctcg aattaagaat 1080

atgtttcctt gtttatcaga tacatcactg cttgatgaag caaggaagat atacatgaaa 1140

attttaaaaa tacatatcgc tgacttcatg gaatggacat cctgtataag cactgaaaaa 1200

caacaacaca ataacactaa aattttaggc actcttaaat gatctgcctc taaaagcgtt 1260

ggatgtagca ttatgcaatt aggtttttcc ttatttgctt cattgtacta cctgtgtata 1320

tagtttttac cttttatgta gcacataaac tttggggaag ggagggcagg gtggggctga 1380

ggaactgacg tggagcgggg tatgaagagc ttgctttgat ttacagcaag tagataaata 1440

tttgacttgc atgaagagaa gcaattttgg ggaagggttt gaattgtttt ctttaaagat 1500

gtaatgtccc tttcagagac agctgatact tcatttaaaa aaatcacaaa aatttgaaca 1560

ctggctaaag ataattgcta tttattttta caagaagttt attctcattt gggagatctg 1620

gtgatctccc aagctatcta aagtttgtta gatagctgca tgtggctttt ttaaaaaagc 1680

aacagaaacc tatcctcact gccctcccca gtctctctta aagttggaat ttaccagtta 1740

attactcagc agaatggtga tcactccagg tagtttgggg caaaaatccg aggtgcttgg 1800

gagttttgaa tgttaagaat tgaccatctg cttttattaa atttgttgac aaaattttct 1860

cattttcttt tcacttcggg ctgtgtaaac acagtcaaaa taattctaaa tccctcgata 1920

tttttaaaga tctgtaagta acttcacatt aaaaaatgaa atatttttta atttaaagct 1980

tactctgtcc atttatccac aggaaagtgt tatttttaaa ggaaggttca tgtagagaaa 2040

agcacacttg taggataagt gaaatggata ctacatcttt aaacagtatt tcattgcctg 2100

tgtatggaaa aaccatttga agtgtacctg tgtacataac tctgtaaaaa cactgaaaaa 2160

ttatactaac ttatttatgt taaaagattt tttttaatct agacaatata caagccaaag 2220

tggcatgttt tgtgcatttg taaatgctgt gttgggtaga ataggttttc ccctcttttg 2280

ttaaataata tggctatgct taaaaggttg catactgagc caagtataat tttttgtaat 2340

gtgtgaaaaa gatgccaatt attgttacac attaagtaat caataaagaa aacttccata 2400

gctaaaaaaa aaaaaaaaaa aa 2422

Claims (9)

1, a kind of method that detects anti-liver cancer efficacy of tyroserleutide, described method comprises step:

In the biomaterial after the tyroserleutide effect, detect gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21 or P27 changes of expression level situation;

When detected result is: after the tyroserleutide effect, the expression level of gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Akt1, Akt2 descends, the expression level of gene C alreticulin, PTEN, P21, P27 rises, and shows that then tyroserleutide has the effect of anti-liver cancer to this biomaterial.

2, a kind of method that detects anti-liver cancer efficacy of tyroserleutide according to claim 1, it is characterized in that, in the described biomaterial after the tyroserleutide effect, detect gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21 or P27 changes of expression level situation, be meant, adopt the method for reverse transcription PCR to detect the expression changing conditions of the mRNA of these ten genes in the described biomaterial in tyroserleutide effect front and back, when after the tyroserleutide effect, gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Akt1, the mRNA expression level of Akt2 descends, gene C alreticulin, PTEN, P21, the mRNA expression level of P27 rises, and shows that then tyroserleutide has the effect of anti-liver cancer to this biomaterial.

3, a kind of method that detects anti-liver cancer efficacy of tyroserleutide according to claim 1, it is characterized in that, in the described biomaterial after the tyroserleutide effect, detect gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21 or P27 changes of expression level situation, be meant, adopt the method for gene chip, detect the expression changing conditions of cDNA before and after the tyroserleutide effect of these ten genes in the described biomaterial, when after the tyroserleutide effect, gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Akt1, the cDNA expression level of Akt2 descends, gene C alreticulin, PTEN, P21, the cDNA expression level of P27 rises, and shows that then tyroserleutide has the effect of anti-liver cancer to this biomaterial;

4, according to the arbitrary described a kind of method that detects anti-liver cancer efficacy of tyroserleutide of claim 1～3, it is characterized in that the cDNA sequence of described gene NDUFA2 comprises the sequence shown in the 74th～373 bit base among the sequence table SEQ ID No.23;

The cDNA sequence of described gene NDUFA10 comprises the sequence shown in the 81st～1148 bit base among the sequence table SEQ ID No.24;

The cDNA sequence of described gene NDUFS1 comprises the sequence shown in the 128th～2311 bit base among the sequence table SEQ ID No.25;

The cDNA sequence of described gene SUCLG1 comprises the sequence shown in the 32nd～1033 bit base among the sequence table SEQ ID No.26;

The cDNA sequence of described gene C alreticulin comprises the sequence shown in the 69th～1322 bit base among the sequence table SEQ ID No.27;

The cDNA sequence of described gene PTEN comprises the sequence shown in the 1032nd～2243 bit base among the sequence table SEQ ID No.28;

The cDNA sequence of described gene A kt1 comprises the sequence shown in the 555th～1997 bit base among the sequence table SEQ ID No.29;

The cDNA sequence of described gene A kt2 comprises the sequence shown in the 204th～1649 bit base among the sequence table SEQ ID No.30;

The cDNA sequence of described gene P21 comprises the sequence shown in the 95th～589 bit base among the sequence table SEQ ID No.31;

The cDNA sequence of described gene P27 comprises the sequence shown in the 466th～1062 bit base among the sequence table SEQ ID No.32.

5, a kind of test kit that is used to detect the tyroserleutide action effect is characterized in that: the primer sequence that contains the mRNA expression level that detects gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21, P27 in the reverse transcription PCR method respectively in the described test kit.

6, a kind of test kit that is used to detect the tyroserleutide action effect according to claim 5, it is characterized in that: the primer sequence of described detection gene NDUFA2 is shown in SEQ ID No.1 and 2 in the sequence table;

The primer sequence of described detection gene NDUFA10 is shown in SEQ ID No.3 and 4 in the sequence table;

The primer sequence of described detection gene NDUFS1 is shown in SEQ ID No.5 and 6 in the sequence table;

The primer sequence of described detection gene SUCLG1 is shown in SEQ ID No.7 and 8 in the sequence table;

The primer sequence of described detection gene C alreticulin is shown in SEQ ID No.9 and 10 in the sequence table;

The primer sequence of described detection gene PTEN is shown in SEQ ID No.11 and 12 in the sequence table;

The primer sequence of described detection gene A kt1 is shown in SEQ ID No.13 and 14 in the sequence table;

The primer sequence of described detection gene A kt2 is shown in SEQ ID No.15 and 16 in the sequence table;

The primer sequence of described detection gene P21 is shown in SEQ ID No.17 and 18 in the sequence table;

The primer sequence of described detection gene P27 is shown in SEQ ID No.19 and 20 in the sequence table.

7, according to claim 5 or 6 described a kind of test kits that are used to detect the tyroserleutide action effect, it is characterized in that: further contain the primer sequence of the reverse transcription PCR of reference gene GAPDH in the described test kit, this primer sequence is shown in SEQ ID No.21 and 22 in the sequence table.

8, a kind of gene chip that is used to detect the tyroserleutide action effect is characterized in that: being fixed with on the solid phase carrier of described gene chip can be respectively and the nucleotide sequence of the cDNA sequence hybridization of gene NDUFA2, NDUFA10, NDUFS1, SUCLG1, Calreticulin, PTEN, Akt1, Akt2, P21, P27.

9, a kind of gene chip that is used to detect the tyroserleutide action effect according to claim 8 is characterized in that: the cDNA sequence of described gene NDUFA2 comprises the sequence shown in the 74th～373 bit base among the sequence table SEQ ID No.23;

The cDNA sequence of described gene NDUFA10 comprises the sequence shown in the 81st～1148 bit base among the sequence table SEQ ID No.24;

The cDNA sequence of described gene NDUFS1 comprises the sequence shown in the 128th～2311 bit base among the sequence table SEQ ID No.25;

The cDNA sequence of described gene SUCLG1 comprises the sequence shown in the 32nd～1033 bit base among the sequence table SEQ ID No.26;

The cDNA sequence of described gene C alreticulin comprises the sequence shown in the 69th～1322 bit base among the sequence table SEQ ID No.27;

The cDNA sequence of described gene PTEN comprises the sequence shown in the 1032nd～2243 bit base among the sequence table SEQ ID No.28;

The cDNA sequence of described gene A kt1 comprises the sequence shown in the 555th～1997 bit base among the sequence table SEQ ID No.29;

The cDNA sequence of described gene A kt2 comprises the sequence shown in the 204th～1649 bit base among the sequence table SEQ ID No.30;

The cDNA sequence of described gene P21 comprises the sequence shown in the 95th～589 bit base among the sequence table SEQ ID No.31;

The cDNA sequence of described gene P27 comprises the sequence shown in the 466th～1062 bit base among the sequence table SEQ ID No.32.

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