CN107245103A - Antitumor recombinant protein IFTI and its encoding gene and application - Google Patents

Abstract

The invention discloses a kind of antitumor recombinant protein IFTI and its encoding gene and application, antitumor recombinant protein and its encoding gene are provided, and the method for the high efficient expression albumen, the protein that the recombinant protein IFTI partly or entirely blends for the Amino-terminal amino acid residue of cardiac muscle troponin I and the carboxy terminal amino acid residue sequence of the artificial small peptide with the amino acid residue sequence in SEQ ID No.2.The present invention can be used clinically for treating a variety of solid malignants as medicine, the features such as with efficient, wide spectrum, small toxic side effect, be expected to turn into brand-new antineoplastic.

Description

Antitumor recombinant protein IFTI and its encoding gene and application

Technical field

The present invention relates to bioengineering field.It is more particularly related to a kind of antitumor recombinant protein IFTI and Its encoding gene and application.

Background technology

Malignant tumour is a class disease of current harm human health most serious.The common method of current treating cancer has： Surgical operation therapy, chemical medicinal treatment, radiotherapy, biotherapy and Chinese medical and herbal therapy.Wherein with surgical operation therapy, Chemical medicinal treatment, radiotherapy are the most commonly used.Surgical operation therapy as most of oncotherapies first choice and major programme, Countless tumor patients is rehabilitated, extend the life-span or improve quality of life.But, in terms of oncotherapy, surgery hand Art treats the patient for being only applicable to certain condition and scope, and due to tumour can recur and shift, surgery excision is simultaneously The purpose of radical cure is not attained by.Also played very in terms of the treatment for being used in tumour of chemical medicinal treatment and radiotherapy Important and essential effect, and also constantly developing.But, the chemotherapeutics therapy and radiotherapy applied at present There are infringement, such as hemopoietic system, intestines and stomach to all active proliferative cells in body, so the patient of chemicotherapy often has blood As symptoms such as reduction, hemorrhage of gastrointestinal tract, Nausea and vomitings.It is additionally, since by patient's body and spirit aspect endurance and tumour There is the limitation of many-sided reason such as drug resistance in cell, and chemicotherapy can not kill all tumour cells, be the recurrence of tumour Hidden danger is left with transfer and the deterioration of disease.Biotherapy and Chinese medical and herbal therapy are also antineoplastic rational method, but Require further study to improve curative effect.Therefore, exploitation has obvious therapeutic effect, the antineoplastic of safety to have become the world of medicine The extremely urgent task faced.

Research shows, the not medium well of the unrestricted invasive growth and its transfer of malignant entity tumor dependent on new blood vessel Into so the growth of tumour can significantly be suppressed by suppressing angiogenesis.Suppress angiogenesis, it is different to block knurl body blood supply The new strategy treated in conventional anticancer, also referred to as " tumour hunger cure ", has become the focus of antineoplaston research.Just Under normal physiological status, except female reproductive system is in ovulation, corpus luteum is generated, the process such as pregnancy, there is of short duration new vessels generation Outside process, the vascular system hyperplasia of adult is in relative static conditions.But in tumor tissues, to adapt to the fast of tumor tissue Fast-growing is long, and angiogenesis is very active, with time for tumor tissue growth supply blood.Microvascular origin in tumor tissues has two Kind：A kind of is the VEGF of the generations such as tumour cell, induction knurl body generation capilary；Another is to remain in knurl The host blood vessel of body gradually becomes tumor vessel, and both host blood vessel was anti-tumor.It is different from normal blood vessels, tumor vascular blood vessel Endothelial cell is imperfect, and basilar memebrane is sparse and is easy to leakage.After Tumor Angiongesis further differentiation reconstruct into artery and Vein, no smooth muscle and nerve endings, belongs to passive blood vessel, and its blood flow places one's entire reliance upon body circulation.Newborn capilary is swollen The first stop that knurl infiltrates and shifted, tumour cell is distally shifted by tumor-microvessel wall into blood circulation.

Entity tumor growth needs substantial amounts of blood supply.Many experimental studies confirm to suppress new blood vessel life in animal body Into, the blood supply that nutrient is provided for tumour growth can be effectively cut off, make the atrophy of knurl body so that disappearance.Anti- neovascularization medicaments TNP-470 and anti-bFGF, VEGF monoclonal antibody application, saltant type vegf receptor FlK-1 competitiveness blocking VEGF signals Conduction, the overexpression inducing endothelial cell apoptosis of α V β 3 and Angiostatin specifically suppress tumor angiogenesis etc., Tumour growth can be suppressed, be a kind of new, promising antineoplastic, Present clinical oncotherapy means can be made up Limitation and replace part treatment means.Because this kind of medicine is that suppression tumour life is reached by suppressing the formation of new vessels Long, and generation of the growth of the normal tissue cell of people independent of new vessels, so normal tissue is almost without poison Side effect.This kind of medicine is not directly placed on tumour cell, therefore suppressing tumour has broad spectrum activity without developing immunity to drugs, and is current Effective, safe, the promising antineoplastic generally acknowledged in the world.It is mainly used in situ and transfer solid tumor treatment and auxiliary The operative treatment of tumour is helped, may also be used for preventing the recurrence and transfer of tumour.Therefore, exploitation has blood vessel hyperplasia inhibitory action Antineoplastic, in terms of the preventing and treating of malignant tumour, with important clinical meaning.

Cardiac muscle troponin I is the subunit of cardiac troponin, and the mark of diagnosis of myocardial infarction, the heart are often used as at present Flesh Troponin I is the SEQ ID No.1 in sequence table, is made up of 210 amino acid residues, by experimental study (in State's patent：ZL03109861.4) confirmed myocardial Troponin I has obvious suppression vascular endothelial cell growth and antitumor work With.

Escherichia expression system is one of most successful expression system for expressing foreign protein, is developed in recent years non- It is often rapid, hundreds of kinds of albumen are had at present and are expressed in the expression system, and it grows rapid, and condition of culture is simple, can be with High density is continuously cultivated, and technology is quite ripe, is the high expression strain commonly used in industrial production.

The content of the invention

It is an object of the invention to provide a kind of antitumor recombinant protein IFTI and its encoding gene, there is provided antitumor with application Recombinant protein and its encoding gene, and the high efficient expression albumen method, the recombinant protein IFTI is cardiac muscle troponin I Amino-terminal amino acid residue and the artificial small peptide with the amino acid residue sequence in SEQ ID No.2 carboxy terminal amino acid The protein that residue sequence is partly or entirely blended, recombinant protein IFTI of the invention can be used clinically for controlling as medicine A variety of solid malignants are treated, the features such as with efficient, wide spectrum, small toxic side effect, are expected to turn into brand-new antineoplastic.

In order to realize according to object of the present invention and further advantage there is provided a kind of antitumor recombinant protein IFTI, The recombinant protein IFTI is for the Amino-terminal amino acid residue of cardiac muscle troponin I and with the amino acid in SEQ ID No.2 The protein that the carboxy terminal amino acid residue sequence of the artificial small peptide of residue sequence is partly or entirely blended.

Preferably, the recombinant protein IFTI is the amino of first amino acid of cardiac muscle troponin I with having SEQ The protein that the carboxyl of the end amino acid of the artificial small peptide of amino acid residue sequence in ID No.2 is combined to form.

Preferably, in described antitumor recombinant protein IFTI, the cardiac muscle troponin I is in SEQ ID No.1 Amino acid residue sequence or amino acid residue sequence in SEQ ID No.1 being taken by one or several amino acid residues Generation, missing and/or addition and with identical with the amino acid residue sequence in SEQ ID No.1 active by SEQ ID No.4 In amino acid residue sequence derived from protein.

Preferably, in described antitumor recombinant protein IFTI, the artificial small peptide is the amino in SEQ ID No.2 Sour residue sequence or by the amino acid residue sequence in SEQ ID No.2 by the replacing of one or several amino acid residues, lack Lose and/or add and with the active small peptide as derived from SEQ ID No.2 identical with SEQ ID No.2.

Preferably, in described antitumor recombinant protein IFTI, the cardiac muscle troponin I is that Amino-terminal amino acid is residual The amino acid residue of any part in sequence 1-152 of the initiation site of base in SEQ ID No.1.

Preferably, in described antitumor recombinant protein IFTI, the artificial small peptide is Amino-terminal amino acid residue The amino acid residue of any part in sequence 1-20 of the initiation site in SEQ ID No.2.

Preferably, in described antitumor recombinant protein IFTI, the cardiac muscle troponin I and the artificial small peptide are logical The dipeptides connection of glycine and serine formation is crossed, the gene coded sequence of the dipeptides is GGATCC.

A kind of antitumor recombinant protein IFTI encoding gene, is one of following nucleotide sequences：

The polynucleotides of SEQ ID No.1 protein sequences or the DNA sequence dna limited with it have in a, polynucleotide More than 90% homology, and coding identical function protein DNA sequence；

The polynucleotides of SEQ ID No.2 protein sequences or the DNA sequence dna limited with it have in b, polynucleotide More than 90% homology, and coding identical function protein DNA sequence；

C, the expression vector containing the gene in a and the b and cell line.

A kind of high-efficiency expression methods of antitumor recombinant protein IFTI in Escherichia coli, comprises the following steps：

Step 1: from the preferred codon of Escherichia coli, designing and synthesizing SEQ ID No.3 and SEQ ID No.4 portion Point or full sequence antigen-4 fusion protein gene sequence；

Step 2: the genetic fragment synthesized in step one is connected with carrier pET21b respectively, expression plasmid is obtained, is converted Expression engineering bacteria is obtained after Escherichia coli；

Step 3: the engineering bacteria obtained in incubation step two, expression product, obtain target protein.

Preferably, it is described big in high-efficiency expression methods of the described antitumor recombinant protein IFTI in Escherichia coli Enterobacteria is preferably BL21-DE3 Escherichia coli.

The present invention at least includes following beneficial effect：

The recombinant protein IFTI of present invention toxic side effect is small, the novel concept of tumour hunger cure is introduced, by special Property suppress the hyperplasia of vascular endothelial cell, the generation of new blood vessel is blocked, so that oncocyte lacks nutrition, it is impossible to growth and then dead Die, normal tissue is without influence；Simultaneously because it is protein medicaments, toxic side effect is small, hence it is evident that be different from current Clinical practice Antineoplastic.

The recombinant protein IFTI of present invention antitumor activity is high, and tumour inhibiting rate is up to 86%, and causes part knurl body disappearance.

The recombinant protein IFTI of present invention antitumor spectra is wide：The present invention can be used for all entity tumors, inorganization selection Property, therefore its scope of application is very wide, including but not limited to fibrosarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteosarcoma, Chordoma, angiosarcoma, lymphangioendothelial sarcoma, synovialoma, celiothelioma, according to Wen Shi knurls, leiomyosarcoma, eye socket rhabdomyosarcoma, Colon cancer, cancer of pancreas, breast cancer, oophoroma, prostate cancer, unicorn columnar epithelium cell cancer, stroma cell cancer, gland cancer, syringocarcinoma, skin Adipose gland cancer, mastoid process cancer, mastoid process gland cancer, cystadenocarcinoma, cephaloma, bronchiolar carcinoma, clear-cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, Sperm mother cell knurl, embryo's epithelioma, the nephroblastoma, cervix cancer, carcinoma of testis, lung cancer, ED-SCLC, carcinoma of urinary bladder, epithelium Cell cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, Kaposi sarcoma, pineal body Knurl, hemangioblastoma, auditory nerve knurl, oligodendroglioma, melanoma, neuroblastoma and retinoblastoma cell Knurl.

The antitumor recombinant protein that the expression that the present invention is provided is obtained accounts for the 30-70% of nutrient solution total protein, table Simple up to process operation, product cost is relatively low, available for large-scale production, has important meaning for the antineoplastic for developing new Justice.

Further advantage, target and the feature of the present invention embodies part by following explanation, and part will also be by this The research and practice of invention and be understood by the person skilled in the art.

Brief description of the drawings

Fig. 1 is the first pure products IFTI electrophoretograms of BL21-DE3 E. coli broths after IPTG of the present invention is induced Spectrum.

Embodiment

A kind of antitumor recombinant protein IFTI, the recombinant protein IFTI are the Amino-terminal amino acid of cardiac muscle troponin I The carboxy terminal amino acid residue sequence part of residue and the artificial small peptide with the amino acid residue sequence in SEQ ID No.2 or The protein all blended.

The recombinant protein IFTI is for the amino of first amino acid of cardiac muscle troponin I and with SEQ ID No.2 In amino acid residue sequence artificial small peptide end amino acid the protein that combines to form of carboxyl.

In described antitumor recombinant protein IFTI, the cardiac muscle troponin I is that the amino acid in SEQ ID No.1 is residual Basic sequence or by the amino acid residue sequence in SEQ ID No.1 by one or several amino acid residues substitution, missing and/ Or add and there is the amino acid in the active No.4 by SEQ ID identical with the amino acid residue sequence in SEQ ID No.1 Protein derived from residue sequence.

In described antitumor recombinant protein IFTI, the artificial small peptide is the amino acid residue sequence in SEQ ID No.2 Amino acid residue sequence in SEQ ID No.2 is passed through the substitution of one or several amino acid residues, lacks and/or add by row Plus and with the active small peptide as derived from SEQ ID No.2 identical with SEQ ID No.2.

In described antitumor recombinant protein IFTI, the cardiac muscle troponin I is the starting of Amino-terminal amino acid residue The amino acid residue of any part of the site in SEQ ID No.1 in sequence 1-152；It is for example following to combine：Myocardium myo calcium egg The initiation site of white I Amino-terminal amino acid residue sequence 1-210 in SEQ ID No.1, it by SEQ ID No.2 with being derived Small peptide carboxy terminal amino acid residue and SEQ ID No.1 in the Amino-terminal amino acid residue of sequence 1 blend the people to be formed Work small peptide merges to form fusion protein；The initiation site of the Amino-terminal amino acid residue of cardiac muscle troponin I is in SEQ ID No.1 Middle sequence 101-210, its with the carboxy terminal amino acid residue of small peptide and SEQ ID No.101 as derived from SEQ ID No.2 The Amino-terminal amino acid residue of sequence 1 blends the artificial small peptide to be formed and merges to form fusion protein；The cardiac muscle troponin I Amino-terminal amino acid residue initiation site in SEQ ID No.1 sequence 153-210, its with by SEQ ID No.2 derive Small peptide carboxy terminal amino acid residue and SEQ ID No.1 in the Amino-terminal amino acid residue of sequence 153 blend what is formed Artificial small peptide merges to form fusion protein.

In described antitumor recombinant protein IFTI, in described antitumor recombinant protein IFTI, the artificial small peptide is The amino acid residue of any part in sequence 1-20 of the initiation site of Amino-terminal amino acid residue in SEQ ID No.2； It is for example following to combine：The c-terminus ammonia of Amino-terminal amino acid residue and sequence 1-23 in SEQ ID No.2 in SEQ ID No.1 Base acid residue blends to form cardiac muscle troponin I, and it merges to be formed with the amino acid residue of sequence 1-23 in SEQ ID No.2 Fusion protein；The carboxyl Amino End Group of Amino-terminal amino acid residue and sequence 16-23 in SEQ ID No.2 in SEQ ID No.1 Sour residue blends to form cardiac muscle troponin I, and it merges to be formed with the amino acid residue of sequence 16-23 in SEQ ID No.2 Fusion protein；The carboxyl Amino End Group of Amino-terminal amino acid residue and sequence 20-23 in SEQ ID No.2 in SEQ ID No.1 Sour residue blends to form cardiac muscle troponin I, and it merges to be formed with the amino acid residue of sequence 20-23 in SEQ ID No.2 Fusion protein；

In described antitumor recombinant protein IFTI, the cardiac muscle troponin I and the artificial small peptide pass through glycine With the dipeptides connection of serine formation, the gene coded sequence of the dipeptides is GGATCC.

A kind of antitumor recombinant protein IFTI encoding gene, is one of following nucleotide sequences：

The polynucleotides of SEQ ID No.1 protein sequences or the DNA sequence dna limited with it have in a, polynucleotide More than 90% homology, and coding identical function protein DNA sequence；

The polynucleotides of SEQ ID No.2 protein sequences or the DNA sequence dna limited with it have in b, polynucleotide More than 90% homology, and coding identical function protein DNA sequence；

C, the expression vector containing the gene in a and the b and cell line.

A kind of high-efficiency expression methods of antitumor recombinant protein IFTI in Escherichia coli, comprises the following steps：

Step 1: from the preferred codon of Escherichia coli, designing and synthesizing SEQ ID No.3 and SEQ ID No.4 portion Point or full sequence antigen-4 fusion protein gene sequence；

Step 2: the genetic fragment synthesized in step one is connected with carrier pET21b respectively, expression plasmid is obtained, is converted Expression engineering bacteria is obtained after Escherichia coli；

Step 3: the engineering bacteria obtained in incubation step two, expression product, obtain target protein.

In high-efficiency expression methods of the described antitumor recombinant protein IFTI in Escherichia coli, the Escherichia coli are preferred For BL21-DE3 Escherichia coli.

The technical program is merged the part or all of molecular structure and artificial small peptide of cardiac muscle troponin I, synthesis The new warm body of the protein with antitumor activity.The fusion has the effect of obvious anti-mouse entity tumor, is disliking Property tumour treatment in terms of, it is significant.

The recombinant protein IFTI that the technical program is obtained can be used for treating a variety of solid malignants, include but is not limited to fibre Tie up sarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, lymphangioendothelial sarcoma, synovialoma, Rind gall, according to Wen Shi knurls, leiomyosarcoma, eye socket rhabdomyosarcoma, colon cancer, cancer of pancreas, breast cancer, oophoroma, prostate cancer, Unicorn columnar epithelium cell cancer, stroma cell cancer, gland cancer, syringocarcinoma, carcinoma of sebaceous glands, mastoid process cancer, mastoid process gland cancer, cystadenocarcinoma, cephaloma, Bronchiolar carcinoma, clear-cell carcinoma, liver cancer, cholangiocarcinoma, choriocarcinoma, sperm mother cell knurl, embryo's epithelioma, the nephroblastoma, uterus Neck cancer, carcinoma of testis, lung cancer, ED-SCLC, carcinoma of urinary bladder, cell carcinoma, glioma, astrocytoma, pith mother cells Knurl, craniopharyngioma, ependymoma, Kaposi sarcoma, pinealoma, hemangioblastoma, auditory nerve knurl, oligodendroglia Knurl, melanoma, neuroblastoma and retinoblastoma.

In the technical program, it is necessary to when, in using above-mentioned antitumor recombinant protein as the medicine of active component, may be used also To add one or more pharmaceutically acceptable carriers, including it is the conventional diluent of pharmaceutical field, excipient, filler, viscous Mixture, wetting agent, disintegrant, sorbefacient, surfactant, absorption carrier and lubricant etc., while note can also be made The diversified forms such as liquid, freeze drying powder injection are penetrated, the medicine of above-mentioned various formulations can be prepared according to the conventional method of pharmaceutical field.

<Embodiment 1>

The Escherichia coli vivoexpression of antitumor recombinant protein：

1st, the synthesis of artificial gene：SEQ ID No.3 (encoding gene of cardiac muscle troponin I) in artificial synthesized sequence table Part or full length sequence, and its 5 ' end plus BamH I, 3 ' end add NotI restriction enzyme sites, obtain SEQ ID in sequence table No.3 cloned sequence.Design and synthesize the part of the artificial small peptide amino acid residue encoding genes of SEQ ID No.2 in sequence table Or whole nucleotide sequences, and BamH I restriction enzyme sites are added plus NdeI, 3 ' ends at its 5 ' end, obtain SEQ ID in sequence table No.4 cloned sequence.The nucleotide sequence and the heart for the artificial small peptide amino acid residue encoding gene that digestion is synthesized are distinguished with BamH I The nucleotide sequence of flesh Troponin I amino acid residue encoding gene, purifying, connection, synthesizes the nucleotide sequence of fusion protein.

2nd, antitumor recombinant protein vivoexpression purifying

(1) above-mentioned artificial synthesized complete sequence is respectively charged into pGEM-T-EASY plasmids, builds pGEM-IFTIx clones Carrier.

(2) structure of expression plasmid

Using the efficient expression plasmid pET21b purchased from Novagen companies of the U.S., digestion is distinguished with SnabI and NotI Target fragment is collected in pGEM-IFTI plasmids and pET21b plasmids, gel electrophoresis, and with T4DNA ligases, 16 DEG C connect overnight.Often The conversion of rule method, chooses bacterium, and amplification obtains expression plasmid pET-IFTIx.

(3), through culture, selected in the inverted importing BL21-DE3 Escherichia coli of plasmid pET-IFTI respectively, screening etc. Process, obtains monoclonal bacterium.

(4) the monoclonal bacterium selected, is inoculated in 5ml culture mediums, and 37 DEG C overnight, are then transferred in 1000ml culture mediums, Continue to cultivate to OD600=0.8, add IPTG to final concentration of 1%, 37 DEG C of induced expressions 4 hours.

(5) the bacterium solution 8000g of culture is centrifuged 20 minutes, collects thalline, the thalline that every liter of bacterium solution is collected adds 50ml and split Solve buffer solution (wherein：Tris-HCl concentration is 50mmol/L, and EDTA concentration is 1mmol/L, and the concentration of 2 mercapto ethanol is 20mmol/L, the concentration of sodium chloride is 100mmol/L), room temperature is placed 30 minutes, then adds 10%Triton-X-100 2.5ml, adds 0.1M PMSF0.25ml, and 37 DEG C are incubated 15 minutes, carry out ultrasonication, and 20000g is centrifuged 20 minutes afterwards, is abandoned Supernatant, it is 8mol/L Urea Lysis Buffers that precipitation, which adds 20ml concentration, is stirred 30 minutes, and supernatant is collected in centrifugation.It will contain CTnI supernatant in solution A (wherein：TEA/HCl Ph7.5 concentration is 25mmol/L, and the concentration of urea is 8mol/L, EDTA concentration is 2mmol/L, and DTT concentration is 1mmol/L) in dialyse at room temperature 2-3 hour, 4 DEG C of 30 points of centrifugations of 12000g Clock.DEAE Sepharose posts are balanced with solution A, then by centrifuged supernatant loading, to adsorb foreign protein, efflux are collected. CM Sepharose posts are balanced with solution A again, with collected efflux loading, now destination protein will be attracted to filler On, then destination protein is washed out with solution B (sodium chloride being added in solution A, the wherein concentration of sodium chloride is 1mol/L).With point Light photometry determines protein content.Protein solution 5ug after purification is taken, 12%SDS-PAGE electrophoresis is used, and through coomassie Bright blue dyeing.IFTI1 (fusion protein of small peptide 20-23, GS dipeptides and cardiac muscle troponin I complete sequence) result such as Fig. 1 institutes Show, the purity of the destination protein of gained is 96%.

<Embodiment 2>

Influence of the antitumor recombinant protein of the present invention to culture vascular endothelial cell value-added functionality：

Human umbilical vein endothelial cells EVC304 cell lines are purchased from Military Medical Science Institute of PLA；RPMI-1640 culture mediums, Hyclone, trypsase are purchased from Hyclone companies, and other reagents are purchased from Sigma companies, antitumor recombinant protein (IFTI1) it is expression and purification product that purity is 96%.

ECV304 cells are cultivated by endothelial cell conventional method, and the normal ECV304 cells of the growth of culture are pressed per hole 5*104 cells are inoculated in 96 orifice plates respectively, and 37 DEG C of cultures, 12 hours Hou Change contain the antitumor of sequence IFTI 1 expression purification The μ g/ml of recombinant protein 5 fresh medium, blank Dui Zhao Change contain the fresh medium of isometric physiological saline, 37 DEG C of continuation Culture 36 hours, proliferative activity is determined with mtt assay.96 well culture plates are taken out, MTT solution (5mg/ml) 20 μ is added per hole L, 37 DEG C are continued to terminate culture after cultivating 4 hours, and careful inhale abandons culture supernatant in hole, and 150 μ l DMSO, vibration are added per hole 10 minutes, crystal is fully dissolved, select 490nm wavelength, the light absorption value in each hole is determined on ELIASA.As a result with blank pair Compared according to group, the μ g/ml of antitumor recombinant protein 5 of IFTI expression purification cell proliferation inhibition rate is respectively 86 ± 7%.

<Embodiment 3>

The mouse tumor inhibition of the antitumor recombinant protein of the present invention：24 kunming mices are taken, are divided into phosphate-buffered Liquid and expression purification liquid group, respectively neck subcutaneous vaccination S180 ascites cells knurl strain, are subcutaneously injected phosphate and delay respectively after three days Fliud flushing and expression purification liquid 1.0mg albumen/kg, every injection in 12 hours once, continuous injection 7 days.Observe and cut tumor tissue, Weigh, as a result phosphate buffer and expression purification liquid sequence IFTI group knurl body weight be respectively 1.09 ± 0.31 grams and 0.15 ± 0.05 gram, tumour inhibiting rate is 86.2%.

Although embodiment of the present invention is disclosed as above, it is not restricted in specification and embodiment listed With it can be applied to various suitable the field of the invention completely, can be easily for those skilled in the art Other modification is realized, therefore under the universal limited without departing substantially from claim and equivalency range, the present invention is not limited In specific details and shown here as the embodiment with description.

<110>Medicinal Plants of Guangxi garden

<120>Antitumor recombinant protein IFTI and its encoding gene and application

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Met Ala Asp Gly Ser Ser Asp Ala Ala Arg Glu Pro Arg Pro Ala

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Pro Ala Pro Ile Arg Arg Arg Ser Ser Asn Tyr Arg Ala Tyr Ala

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Thr Glu Pro His Ala Lys Lys Lys Ser Lys Ile Ser Ala Ser Arg

35 40 45

Lys Leu Gln Leu Lys Thr Leu Leu Leu Gln Ile Ala Lys Gln Glu

50 55 60

Leu Glu Arg Glu Ala Glu Glu Arg Arg Gly Glu Lys Gly Arg Ala

65 70 75

Leu Ser Thr Arg Cys Gln Pro Leu Glu Leu Ala Gly Leu Gly Phe

80 85 90

Ala Glu Leu Gln Asp Leu Cys Arg Gln Leu His Ala Arg Val Asp

95 100 105

Lys Val Asp Glu Glu Arg Tyr Asp Ile Glu Ala Lys Val Thr Lys

110 115 120

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

125 130 135

Arg Gly Lys Phe Lys Arg Pro Thr Leu Arg Arg Val Arg Ile Ser

140 145 150

Ala Asp Ala Met Met Gln Ala Leu Leu Gly Ala Arg Ala Lys Glu

155 160 165

Ser Leu Asp Leu Arg Ala His Leu Lys Gln Val Lys Lys Glu Asp

170 175 180

Thr Glu Lys Glu Asn Arg Glu Val Gly Asp Trp Arg Lys Asn Ile

185 190 195

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

200 205 210

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EDKSPVPNEQCEKENSANEMASM

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atgtcccgcc ctctttcaga ccaggataag agaaagcaaa tcagcgttcg tggcttagcc 60

ggagtggaaa atgtgtcggg atccatggcg gatgggagca gcgatgcggc tagggaacct 120

cgccctgcac cagccccaat cagacgccgc tcctccaact accgcgctta tgccacggag 180

ccgcacgcca agaaaaaatc taagatctcc gcctcgagaa aattgcagct gaagactctg 240

ctgctgcaga ttgcaaagca agagctggag cgagaggcgg aggagcggcg cggagagaag 300

gggcgcgctc tgagcacccg ctgccagccg ctggagttgg ccgggctggg cttcgcggag 360

ctgcaggact tgtgccgaca gctccacgcc cgtgtggaca aggtggatga agagagatac 420

gacatagagg caaaagtcac caagaacatc acggagattg cagatctgac tcagaagatc 480

tttgaccttc gaggcaagtt taagcggccc accctgcgga gagtgaggat ctctgcagat 540

gccatgatgc aggcgctgct gggggcccgg gctaaggagt ccctggacct gcgggcccac 600

ctcaagcagg tgaagaagga ggacaccgag aaggaaaacc gggaggtggg agactggcgc 660

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Claims (10)

1. a kind of antitumor recombinant protein IFTI, it is characterised in that the recombinant protein IFTI is the amino of cardiac muscle troponin I Terminal amino acid residue sequence and the carboxy terminal amino acid of the artificial small peptide with the amino acid residue sequence in SEQ ID No.2 are residual The protein that basic sequence is partly or entirely blended.

2. antitumor recombinant protein IFTI as claimed in claim 1, it is characterised in that the recombinant protein IFTI is myocardium myo The end of the amino of first amino acid of calcium protein I and the artificial small peptide with the amino acid residue sequence in SEQ ID No.2 The protein that the carboxyl of terminal amino acid is combined to form.

3. antitumor recombinant protein IFTI as claimed in claim 1, it is characterised in that the cardiac muscle troponin I is SEQ Amino acid residue sequence in SEQ ID No.1 is passed through one or several amino by amino acid residue sequence in ID No.1 The substitution of sour residue, missing and/or addition and with it is identical with the amino acid residue sequence in SEQ ID No.1 it is active by Protein derived from amino acid residue sequence in SEQ ID No.4.

4. antitumor recombinant protein IFTI as claimed in claim 1, it is characterised in that the artificial small peptide is SEQ ID The amino acid residue sequence or amino acid residue sequence in SEQ ID No.2 is residual by one or several amino acid in No.2 The substitution of base, missing and/or addition and with the active small peptide as derived from SEQ ID No.2 identical with SEQ ID No.2.

5. antitumor recombinant protein IFTI as claimed in claim 3, it is characterised in that the cardiac muscle troponin I is amino The amino acid residue of any part in sequence 1-152 of the initiation site of terminal amino acid residue in SEQ ID No.1.

6. antitumor recombinant protein IFTI as claimed in claim 4, it is characterised in that the artificial small peptide is amino Amino End Group The amino acid residue of any part in sequence 1-20 of the initiation site of sour residue in SEQ ID No.2.

7. antitumor recombinant protein IFTI as claimed in claim 1, it is characterised in that the cardiac muscle troponin I and described Artificial small peptide is connected by the dipeptides of glycine and serine formation, and the gene coded sequence of the dipeptides is GGATCC.

8. the encoding gene of the antitumor recombinant protein IFTI as described in any in a kind of 1-7 such as claim, it is characterised in that be One of following nucleotide sequences：

The polynucleotides of SEQ ID No.1 protein sequences or the DNA sequence dna limited with it have 90% in a, polynucleotide Above homology, and coding identical function protein DNA sequence；

The polynucleotides of SEQ ID No.2 protein sequences or the DNA sequence dna limited with it have 90% in b, polynucleotide Above homology, and coding identical function protein DNA sequence；

C, the expression vector containing the gene in a and the b and cell line.

9. high efficient expression sides of the antitumor recombinant protein IFTI in Escherichia coli as described in any in a kind of 1-7 such as claim Method, it is characterised in that comprise the following steps：

Step 1: from the preferred codon of Escherichia coli, design and synthesize SEQ ID No.3 and SEQ ID No.4 part or The antigen-4 fusion protein gene sequence of full sequence；

Step 2: the genetic fragment synthesized in step one is connected with carrier pET21b respectively, expression plasmid is obtained, large intestine is converted Expression engineering bacteria is obtained after bacillus；

Step 3: the engineering bacteria obtained in incubation step two, expression product, obtain target protein.

10. high-efficiency expression methods of the antitumor recombinant protein IFTI in Escherichia coli as claimed in claim 9, its feature exists In the Escherichia coli are preferably BL21-DE3 Escherichia coli.