CN1292002C - Arginine deacylase fusion protein, method for preparation and application - Google Patents

Abstract

The present invention relates to fusion protein (HSA-ADI) for human serum albumin (HSA) and mycoplasma arginine deacylase, a method for preparing the protein and application to preparing medicine for treating tumors, such as hepatoma, etc. The present invention adopts a eukaryotic expression philosophy and technology to clone the gene of the human serum albumin by a PCR method and chemically synthesize the gene of the mycoplasma arginine deacylase. A eukaryotic expression vector is used for constructing recombinant plasmids and converting eukaryotic cells to obtain a strain for stably expressing HSA-ADI. After the strain is fermented, fermentation liquid supernatant is purified by the methods of dialysis, ion exchange, a molecular sieve, etc. New molecules obtained by bioactivity examining verification have ADI activity. A protective agent is added to the purified product to be prepared into a freeze-dried pharmaceutical preparation. As proved by pharmacodynamics research results, the preparation can be used for the treatment of liver cancer, mammary cancer, melanoma, leukemia, lymphoma, glossopharyngeum tumor, etc.

Description

A kind of arginine deacylase fusion rotein, its manufacture method and application

The invention belongs to biological technical field, it is human serum albumin-arginine deiminase (HSA-ADI) that a kind of new fusion rotein is provided, and invention also relates to the manufacture method of this fusion rotein and this fusion rotein and is used for the treatment of application in the medicine of tumour in preparation.

Tumours such as liver cancer, mammary cancer, melanoma are the human body kinds of tumor.Studies show that mostly these tumours are arginine auxotroph, the cell of these tumours often can not synthesize arginine, must rely on the growth and breeding that extraneous arginine is kept cell, and this provides an important clue for tumor treatment.And the human normal cell can self synthesize arginic.Therefore, the arginine concentration in the blood circulation is reduced, will cause tumour cell nutritive deficiency, reach the effect that suppresses tumor growth.

Mycoplasma M.arginini is the pollutent microorganism of many cell cultures, a kind of enzyme arginine deacylase (arginine deiminase of its coding, ADI) have the arginic effect of single-minded degraded, in the presence of water, can be decomposed into citrulline and ammonia to arginine.ADI is by the arcA genes encoding, and Tot Prot accounts for 10% of M.arginini soluble protein.Except mycoplasma M.arginini, the ADI that encode such as other many microorganisms such as Mycoplasma hominis, Mycoplasma arthritidis, Mycobacterium tuberculosis, Lymedisease spirochetes and Streptococcus, the metabolism of latter's catalysis arginine provides energy for organism.The biomacromolecule of the ball-type that the ADI of M.arginini coding is made up of 410 amino acid, molecular weight is 46000, iso-electric point is 4.7, does not have intramolecular disulfide bond, occurs with dimeric forms.

Recent years, ADI has caused the great interest of people.It is reported that ADI concentration can suppress the growth of clones such as melanoma, leukemia, prostate cancer under the situation of ng/ml.Grow the necessary arginine except reducing specific tumor cells, research in recent years thinks that also this kind of enzyme participates in apoptosis, and have inhibition nitrogen protoxide (NO) synthetic, in and effect such as tumour necrosis factor.Infusively be, recently studies show that not only directly anticancer growth of ADI, and can suppress the generation of tumour neovascularity.And the formation of tumour neovascularity is considered to the important step of tumor growth.

U.S. Phoenix Pharmacologics is carrying out the III phase clinical study of ADI modifier treatment hepatocellular carcinoma now.Japan Nippon Mining company proves by experimentation on animals, and ADI can be in vivo, the growth of 6 kinds of tumor cell lines of vitro inhibition, prolongs the survival time of animal.R﹠D institution and the hospital's Combined Trials of Germany Essen have proved that ADI inhibition leukemia cell's growth fraction asparaginase is more effective, also can be suppressed to neurocytoma.The ADI that Seoul, South Korea is also expressed at research E.coli is suppressing the regulating and controlling effect of nitrogen protoxide aspect synthetic.

The result of numerous bases and clinical study report has confirmed the pharmacological action of ADI, can expect optimistically that in view of the above ADI becomes a strong anti-tumor medicine.But relative human body, this kind of enzyme belongs to heterologous protein, cause allergic reaction easily, simultaneously this protein in human body easily by enzyme liberating.

Human serum albumin is the important component of blood plasma, also is the carrier of many castle's intrinsic factors and external source medicine.585 amino acid of human serum albumin total length, molecular weight about 66500.Human serum albumin is not easy to see through renal glomerulus under the normal circumstances, and plasma half-life is more than two weeks.These characteristics all help it to become a drug modification molecule.

The purpose of this invention is to provide a kind of new fusion rotein is HSA-ADI, and this albumen has following characteristics: 1, specificity degraded arginine.2, immunogenicity is lower than ADI.3, be longer than ADI plasma half-life.

This new fusion rotein HSA-ADI is used for the treatment of tumours such as liver cancer, melanoma and has remarkable advantages: 1, reduce anaphylaxis, be convenient to many courses of treatment or SM.2, increase plasma half-life, reduce frequency injection, prolong dosing interval, reduce the patient suffering, improve patient's compliance.

The present invention adopts following technical proposals:

(1) in order to obtain human serum albumin gene and mycoplasma ADI gene, can adopt two genes of method amplification of PCR or RT-PCR, equally also can take complete synthesis method.Preferable methods is complete synthesis ADI gene, and the codon TGA of mycoplasma coding colors propylhomoserin is adapted into TGG, so that express in yeast or mammalian cell.Preferred method that obtains the HSA gene is that the method with RT-PCR obtains from people's fetal liver total tissue RNA.

(2) with HSA gene and the series connection of mycoplasma ADI gene.Preferred series connection method is the upstream that the HSA gene is connected on the ADI gene, can improve expression amount.Preferred scheme is the gene order that adds one section coding connection peptides in the middle of two genes.Described connection peptides mainly is made up of glycine, Serine or proline(Pro).The length of connection peptides can be 1-100 amino acid, and preferred connection peptides length is 10-30 amino acid.Preferred connection peptides sequence is (Gly-Gly-Gly-glycine-Serine) n, and n is less than 10.The adding of connection peptides will help improving the activity of fusion rotein.

Suitable carrier for expression of eukaryon is arrived in the gene clone that connects, and mammalian cell expression can be selected for use but be not limited only to the pcDNA serial carrier, and preferred mammalian cell expression vector is the pcDNA series plasmid that carries dihydrofolate reductase gene.Mammalian host cell can be selected but be not limited only to Chinese hamster ovary celI series, preferably Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary celI.

Yeast expression can be selected for use but be not limited only to the pPIC serial carrier, and preferred yeast vector is pPIC9.Can select tandem gene is placed the alpha factor downstream of pPIC9 carrier.Host's yeast can be selected but be not limited only to pichia spp, most preferably pichia spp GS115.

(3) fermentation engineering bacterial classification.The fermentation of mammalian cell can be selected for use but is not limited only to DMEM, 1640, MEM or the special-purpose serial substratum of CHO.When adopting pPIC9 to transform the barms fermentation of GS115 method acquisition, can adopt conventional substratum,, add a certain proportion of phosphoric acid salt, vitriol, vitamin H or the like composition in addition with the glycerine carbon source.Carry out 0.5-2 hour hunger after glycerine runs out, add methyl alcohol then, induced 48-96 hour.

(4) tunning is carried out purification process, comprising dialysis, positive anion-exchange chromatography and gel exclusion chromatography etc.The character of target product can have slight variation because of the size of connection peptides.In preferred yeast secreted expression, because the target product molecular weight is bigger, and yeast culture supernatant composition is fairly simple, can adopt but is not limited only to means such as salt precipitation, dialysis, ion-exchange, gel exclusion.Preferred purifying process process is dialysis-anionresin-S200.This technology can be that the purpose product purity reaches more than 98%, and can remove intracellular toxin, obtains meeting the stoste of producing the preparation requirement.

(5) pharmaceutical excipient of pure product adding can be but be not limited only to stablizer and solubility promoters such as medicinal N.F,USP MANNITOL and phosphate buffered saline buffer, and through filtration sterilization, lyophilize becomes freeze-dried products.Use water for injection dissolving back, can be used for tumor treatment such as liver cancer, melanoma, mammary cancer.

(6) biological activity of the new fused protein of the external arginine degraded experimental examination of employing.

(7) carry out pharmacodynamic experiment with planting the nude mice that is implanted with tumours such as liver cancer, check the inhibition effect of new fusion rotein tumor growth.

The technical program provides a kind of new protein, and this protein is produced by recombinant DNA technology, is easy to amplify, and can realize industrialization.This fused protein has the ADI activity, has application prospect in the treatment of liver cancer mammary cancer, melanoma, leukemia, lymphoma, glossopharyngeum tumour etc.

Description of drawings:

Human serum albumin gene nucleotide sequence and encoded protein matter sequence thereof that accompanying drawing 1.RT-PCR method obtains.

Accompanying drawing 2. has the arginine deacylase gene nucleotide series and the encoded protein matter sequence thereof of connection peptides encoding sequence.

Accompanying drawing 3.HSA-ADI fusion gene nucleotide sequence and encoded protein matter sequence thereof.

Accompanying drawing 4. plasmid construction synoptic diagram.HSA and ADI gene successively are inserted in the carrier.

The present invention describes with following example:

The acquisition of embodiment 1:HSA gene

Design two primers according to the design of primers principle, and add EcoRI restriction enzyme site and BamHI restriction enzyme site respectively at upstream and downstream primer 5 ' end.With the total RNA of people's embryonic liver tissue is that template is carried out RT-PCR, obtains the full-length gene of human serum albumin maturation protein, this gene 3 ' end disappearance stop code.Referring to accompanying drawing 1.Connect into the pBV220 carrier after order-checking is correct, be built into pBV220-HSA.

HSA1：5’CggAATTCgATgCACACAAgA 3’

HSA2：5’CgggATCCTAAgCCTAAggCA 3’

Embodiment 2: mycoplasma ADI full length gene is segmental synthetic

According to biochemical theory, positive and negative two chains to full gene carry out salvage respectively, anneal then, connect.TGG be should be to the codon TGA of mycoplasma coding colors propylhomoserin in synthetic, and BamHI restriction enzyme site and SalI restriction enzyme site added respectively at genes encoding chain 5 ' end.This genes encoding chain 5 ' end has also added (Gly-Gly-Gly-Gly-Ser) except adding restriction enzyme site ₂Encoding sequence, see accompanying drawing 2.Connect into the pBV220-HSA carrier after order-checking is correct, be built into the pBV220-HSA-ADI carrier.

The structure of embodiment 3:HSA-ADI fusion protein expression vector

Design two primer HSA-ADI1 and HSA-ADI2 according to the design of primers principle, and add XhoI restriction enzyme site and EcoRI restriction enzyme site respectively at primer 5 ' end.With the pBV220-HSA-ADI carrier is that template is carried out PCR, obtains the HSA-ADI gene.Be connected to carrier for expression of eukaryon behind this gene double digestion, preferred pPCI9 is built into pPCI9-HSA-ADI.Connection peptides between this fusion rotein first district and second district is that 12 amino acid are Gly-Ser-(Gly-Gly-Gly-Gly-Ser) ₂The encoding sequence of fusion rotein is seen accompanying drawing 3.The plasmid construction process of embodiment 1,2,3 is referring to accompanying drawing 4.

HSA-ADI1：TCTCTCgAgAAAAgAgAggCTATggATgCACACAAgA

HSA-ADI2：5’CggAATTCTTACCACTTAACATCTTTAC

Embodiment 4: transform and screening engineering bacteria bacterial classification and fermentation

Select for use suitable endoenzyme such as SacI, SalI to cut the pPCI9-HSA-ADI plasmid, transform pichia spp GS115, according to the rules way screening Mut ⁺, His ⁺The clone shakes and carries out methanol induction after cultivating in the bottle, select expression amount high as engineering bacteria.Being that carbon source was cultivated about 24 hours with glycerine in fermentor tank, is that carbon source was induced 48-96 hour then with methyl alcohol.Fermention medium adopts conventional substratum.Gather in the crops culture supernatant after the fermentation ends.

Embodiment 5: the separation of target product, purifying

Fusion rotein HSA-ADI mainly exists in the culture supernatant with soluble form.The supernatant of results is at first dialysed with phosphoric acid buffer, and culture medium solution is fallen in displacement, adopts means such as anionresin and molecular sieve to carry out separation and purification then.

Embodiment 6: the evaluation of target product

SDS-PAGE measures the fusion protein molecule amount and is about 114KD, and purity is all more than 98%.

Adopt external arginine degradation experiment to check the biological activity of fusion rotein.Ultimate principle is: ADI catalysis arginine is transformed into citrulline, and the latter combines with diacetyl mono-oxime, at the 460nm place strong absorption is arranged, and in certain linearity range, the variable quantity of reaction solution 460nm place absorbancy is directly proportional with the amount of amino acid of reaction.Operation steps is: hatched enzyme and substrate mixture 1 hour for (1) 37 ℃, mixture is formed (50mM arginine 150ul, 50mM acetate buffer (PH5.5) 2.3ml, enzyme are an amount of, the sodium azide 3.6ul of concentration 0.05%).

(2) the hydrochloric acid 0.5ml termination reaction of interpolation 2M.(3) add H in the supernatant liquor that 1ml stopped reacting ₃PO ₄-H ₂SO ₄(3/1, V/V) 10% methanol solution of 1.5ml and 250ul diacetyl mono-oxime (1.5%), 95 ℃ were reacted 30 minutes in the dark, cooled off 10 minutes.

(4) 460nm photometry absorption value, this value has been reacted the growing amount of citrulline.

The unit of activity definition: the enzyme amount that interior catalysis 1 micromole's arginine of 1 minute reaction times changes 1 micromole's citrulline into is defined as 1 unit of activity (IU).

30L fermentor tank one time fermentation can obtain the above HSA-ADI of 100,000 IU.

Embodiment 7: sterile filtration, packing, freeze-drying

Pure product can add auxiliary materials such as N.F,USP MANNITOL and make small-volume injection or freeze-dried preparation.Can adopt (but being not limited only to) buffer system is 10mM PB, pH6.8,5% N.F,USP MANNITOL.Reach at environment cleanliness under hundred grades the condition, with 0.22 μ M filtering with microporous membrane, carry out packing, freeze-drying then, seal, labeling, vanning.Be stored in 2～8 ℃ the cold storage environment.

Embodiment 8:HSA-ADI anti-tumor in vivo cell proliferation test

Select 30 of kind of the nude mices that is implanted with hepatocellular carcinoma cells for use, diameter of tumor is 1-3 centimetre.Be divided into low dosage administration group (10), high dosage administration group (10) and control group (10).Adopt the tail vein injection medication, the administration capacity is every mouse 1ml.Low, high dosage administration group is injected the HSA-ADI fusion rotein and be respectively 100IU or 400IU every day, and control group is given the physiological saline with same capability.2 weeks of successive administration, 7 days and the variation of observing and measuring diameter of tumor in 14 days respectively after the administration, the record diameter of tumor increases numerical value.Observe measuring result twice and show that administration treated animal tumor tissue growth is slow, compares significant difference with control group.Last observations is carried out statistical analysis show, two administration treated animal diameter of tumor are compared all with control group and are obviously dwindled, and the P value is all less than 0.01.Illustrate that the HSA-ADI fusion rotein has the obvious suppression effect to liver cancer cell growth.The last record data are as follows:

Table .HSA-ADI anti-tumor in vivo cell proliferation test data sheet (diameter of tumor changes, centimetre).

Group	1	2	3	4	5	6	7	8	9	10
											High dosage low dosage control group	0.1 0.2 0.6	-0.2 0.3 0.7	0.3 0.4 0.7	-0.1 -0.1 0.8	-0.2 -0.2 0.9	0.0 0.1 1.0	0.1 0.1 0.8	0.1 -0.2 1.3	-0.3 0.0 0.6	-0.4 -0.1 0.4

Sequence table

＜110〉Beijing Shuanglu Pharmaceutical Co., Ltd.

＜120〉a kind of arginine deacylase fusion rotein, its manufacture method and application

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gaatgcttct tgcaacacaa agatgacaac ccaaacctcc cccgattggt gagaccagag 360

gttgatgtga tgtgcactgc ttttcatgac aatgaagaga catttttgaa aaaatactta 420

tatgaaattg ccagaagaca tccttacttt tatgccccgg aactcctttt ctttgctaaa 480

aggtataaag ctgcttttac agaatgttgc caagctgctg ataaagctgc ctgcctgttg 540

ccaaagctcg atgaacttcg ggatgaaggg aaggcttcgt ctgccaaaca gagactcaag 600

tgtgccagtc tccaaaaatt tggagaaaga gctttcaaag catgggcagt agctcgcctg 660

agccagagat ttcccaaagc tgagtttgca gaagtttcca agttagtgac agatcttacc 720

aaagtccaca cggaatgctg ccatggagat ctgcttgaat gtgctgatga cagggcggac 780

cttgccaagt atatctgtga aaatcaagat tcgatctcca gtaaactgaa ggaatgctgt 840

gaaaaacctc tgttggaaaa atcccactgc attgccgaag tggaaaatga tgagatgcct 900

gctgacttgc cttcattagc tgctgatttt gttgaaagta aggatgtttg caaaaactat 960

gctgaggcaa aggatgtctt cttgggcatg tttttgtatg aatatgcaag aaggcatcct 1020

gattactctg tcgtgctgct gctgagactt gccaagacat atgaaaccac tctagagaag 1080

tgctgtgccg ctgcagatcc tcatgaatgc tatgccaaag tgttcgatga atttaaacct 1140

cttgtggaag agcctcagaa tttaatcaaa caaaattgtg agctttttga gcagcttgga 1200

gagtacaaat tccagaatgc gctgttagtt cgttacacca agaaagtacc cgaagtgtca 1260

actccaactc ttgtagaggt ctcaagaaac ctaggaaaag tgggcagcaa atgttgtaaa 1320

catcctgaag caaaaagaat gccctgtgca gaagactatc tatccgtggt cctgaaccag 1380

ttatgtgtgt tgcatgagaa aacgccagta agtgacagag tcaccaaatg ctgcacagaa 1440

tccttggtga acaggcgacc atgcttttca gctctggaag tcgatgaaac atacgttccc 1500

aaagagttta atgctgaaac attcaccttc catgcagata tatgcacact ttctgagaag 1560

gagagacaaa tcaagaaaca aactgcactt gttgagctcg tgaaacacaa gcccaaggca 1620

acaaaagagc aactgaaagc tgttatggat gatttcgctg cttttgtaga gaagtgctgc 1680

aaggctgacg ataaggagac ctgctttgcc gaggagggta aaaaacttgt tgctgcaagt 1740

caagctgcct taggcttagg atccggtggt ggtggttctg gtggtggtgg ttctatgtct 1800

gtatttgaca gtaaatttaa aggtattcac gtttattcag aaattggtga attagaatca 1860

gttctagttc acgaaccagg acgcgaaatt gaetatatta caccagctag actagatgaa 1920

ttattattct cagctatctt agaaagccac gatgctagaa aagaacacaa acaattcgta 1980

gcagaattaa aagcaaacga catcaatgtt gttgaattaa ttgatttagt tgctgaaaca 2040

tatgatttag catcacaaga agctaaagac aaattaatcg aagaattttt agaagactca 2100

gaaccagttc tatcagaaga acacaaagta gttgtaagaa acttcttaaa agctaaaaaa 2160

acatcaagag aattagtaga aatcatgatg gcagggatca caaaatacga tttaggtatc 2220

gaagcagatc acgaattaat cgttgaccca atgccaaacc tatacttcac acgtgaccca 2280

tttgcatcag taggtaatgg tgtaacaatc cactacatgc gttacaaagt tagacaacgt 2340

gaaacattat tctcaagatt tgtattctca aatcacccta aactaattaa cactccatgg 2400

tactacgacc cttcactaaa attatcaatc gaaggtgggg acgtatttat ctacaacaat 2460

gacacattag tagttggtgt ttctgaaaga actgacttac aaacagttac tttattagct 2520

aaaaacattg ttgctaataa agaatgtgaa tttaaacgta ttgttgcaat taacgttcca 2580

aaatggacaa acttaatgca cttagacaca tggctaacaa tgttagacaa ggacaaattc 2640

ctatactcac caatcgctaa tgacgtattt aaattctggg attatgactt agtaaacggt 2700

ggagcagaac cacaaccagt tgaaaacgga ttacctctag aaggattatt acaatcaatc 2760

attaacaaaa aaccagtttt aattcctatc gcaggtgaag gtgcttcaca aatggaaatc 2820

gaaagagaaa cacacttcga tggtacaaac tacttagcaa ttagaccagg tgttgtaatt 2880

ggttactcac gtaacgaaaa aacaaacgct gctctagaag ctgcaggcat taaagttctt 2940

coattccacg gtaaccaatt atcattaggt atgggtaacg ctcgttgtat gtcaatgcct 3000

ttatcacgta aagatgttaa gtggtag 3027

<210>6

<211>1008

a fusion protein

<212>PRT

＜213〉artificial sequence (Artificial Sequence)

<220>

<221>CHAIN

<222>(1)...(1008)

<400>6

Met Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly

1 5 10 15

Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu

20 25 30

Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr

35 40 45

Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp

50 55 60

Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr

65 70 75 80

Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu

85 90 95

Pro Gly Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn

100 105 110

Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe

115 120 125

His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala

130 135 140

Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys

145 150 155 160

Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala

165 170 175

Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala

180 185 190

Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly

195 200 205

Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe

210 215 220

Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr

225 230 235 240

Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp

245 250 255

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

260 265 270

Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser

275 280 285

His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro

290 295 300

Ser Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr

305 310 315 320

Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala

325 330 335

Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys

340 345 350

Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His

355 360 365

Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu

370 375 380

Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly

385 390 395 400

Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val

405 410 415

Pro Glu Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly

420 425 430

Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro

435 440 445

Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu

450 455 460

His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu

465 470 475 480

Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu

485 490 495

Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala

500 505 510

Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr

515 520 525

Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln

530 535 540

Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys

545 550 555 560

Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu

565 570 575

Val Ala Ala Ser Gln Ala Ala Leu Gly Leu Gly Ser Gly Gly Gly Gly

580 585 590

Ser Gly Gly Gly Gly Ser Met Ser Val Phe Asp Ser Lys Phe Lys Gly

595 600 605

Ile His Val Tyr Ser Glu Ile Gly Glu Leu Glu Ser Val Leu Val His

610 615 620

Glu Pro Gly Arg Glu Ile Asp Tyr Ile Thr Pro Ala Arg Leu Asp Glu

625 630 635 640

Leu Leu Phe Ser Ala Ile Leu Glu Ser His Asp Ala Arg Lys Glu His

645 650 655

Lys Gln Phe Val Ala Glu Leu Lys Ala Asn Asp Ile Asn Val Val Glu

660 665 670

Leu Ile Asp Leu Val Ala Glu Thr Tyr Asp Leu Ala Ser Gln Glu Ala

675 680 685

Lys Asp Lys Leu Ile Glu Glu Phe Leu Glu Asp Ser Glu Pro Val Leu

690 695 700

Ser Glu Glu His Lys Val Val Val Arg Asn Phe Leu Lys Ala Lys Lys

705 710 715 720

Thr Ser Arg Glu Leu Val Glu Ile Met Met Ala Gly Ile Thr Lys Tyr

725 730 735

Asp Leu Gly Ile Glu Ala Asp His Glu Leu Ile Val Asp Pro Met Pro

740 745 750

Asn Leu Tyr Phe Thr Arg Asp Pro Phe Ala Ser Val Gly Asn Gly Val

755 760 765

Thr Ile His Tyr Met Arg Tyr Lys Val Arg Gln Arg Glu Thr Leu Phe

770 775 780

Ser Arg Phe Val Phe Ser Asn His Pro Lys Leu Ile Asn Thr Pro Trp

785 790 795 800

Tyr Tyr Asp Pro Ser Leu Lys Leu Ser Ile Glu Gly Gly Asp Val Phe

805 810 815

Ile Tyr Asn Asn Asp Thr Leu Val Val Gly Val Ser Glu Arg Thr Asp

820 825 830

Leu Gln Thr Val Thr Leu Leu Ala Lys Asn Ile Val Ala Asn Lys Glu

835 840 845

Cys Glu Phe Lys Arg Ile Val Ala Ile Asn Val Pro Lys Trp Thr Asn

850 855 860

Leu Met His Leu Asp Thr Trp Leu Thr Met Leu Asp Lys Asp Lys Phe

865 870 875 880

Leu Tyr Ser Pro Ile Ala Asn Asp Val Phe Lys Phe Trp Asp Tyr Asp

885 890 895

Leu Val Asn Gly Gly Ala Glu Pro Gln Pro Val Glu Asn Gly Leu Pro

900 905 910

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

915 920 925

Pro Ile Ala Gly Glu Gly Ala Ser Gln Met Glu Ile Glu Arg Glu Thr

930 935 940

His Phe Asp Gly Thr Asn Tyr Leu Ala Ile Arg Pro Gly Val Val Ile

945 950 955 960

Gly Tyr Ser Arg Asn Glu Lys Thr Asn Ala Ala Leu Glu Ala Ala Gly

965 970 975

Ile Lys Val Leu Pro Phe His Gly Asn Gln Leu Ser Leu Gly Met Gly

980 985 990

Asn Ala Arg Cys Met Ser Met Pro Leu Ser Arg Lys Asp Val Lys Trp

995 1000 1005 1008

Claims (5)

1, a kind of human serum albumin-arginine deiminase who is used for the treatment of malignant tumor of liver, this proteinic aminoacid sequence is shown in Seq ID No.6.

2, the human serum albumin-arginine deiminase of claim 1 is characterized in that: the N end of fusion rotein is a human serum albumin, and the C end is a mycoplasma arginine deacylase, is called first district and second district.

3, the human serum albumin-arginine deiminase of claim 2, it is characterized in that: can be provided with connection peptides between this fusion rotein first district and second district, described connection peptides length is 1-100 amino acid, mainly is made up of glycine, Serine or proline(Pro).

4, the manufacture method of the described fusion rotein of a kind of claim 3 comprises:

(1) Auele Specific Primer of the synthetic seralbumin gene that is used to clone people;

(2) synthetic ADI full-length gene;

(3) be that template is carried out RT-PCR amplification HSA gene with people's tire liver total rna;

(4) the gene series connection that (2) and (3) is obtained is inserted into suitable carriers, makes up the recombination high efficiency expression vector;

(5) use yeast, zooblast or vegetable cell as the host, express (4) resulting efficient expression vector, set up engineering bacteria.

(6) engineering bacteria that (5) are obtained ferments;

(7) handle the tunning that (6) obtain with dialysis, ion-exchange and molecular sieve method, obtain the pure product of HSA-ADI fusion rotein;

5, the described human serum albumin-arginine deiminase of claim 3 is used for the treatment of application in the medicine of malignant tumor of liver in preparation.

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