CN103820479B - The preparation of targeting antineoplastic amalgamation protein matter LPO and application - Google Patents

Abstract

The invention discloses targeting antineoplastic amalgamation protein matter LPO, it is by the genes of interest fragment of synthetic<i>lHRH-PEA</i>transferring film peptide-<i>oNC</i>(referred to as LPO), insert in PET28a plasmid, convert to e. coli bl21 (DE3), the fusion protein expressed, the experiment proved that fusion protein LPO is to there being LHRH receptor, including colon cancer LOVO cell, lung cancer A549 cell, oral cancer KB cell, prostate carcinoma PC-3 M cells etc., tumor cell line all has obvious inhibitory action, to without the lymphoma Hut102 of LHRH receptor and leukemia HL60 then unrestraint effect, there is targeting specific, safe and reliable.

Description

The preparation of targeting antineoplastic amalgamation protein matter LPO and application

Technical field

The invention belongs to biological technical field, specifically targeting antineoplastic amalgamation protein matter LPO and its preparation method and application.

Background technology

Along with development and the scientific and technological progress of society, the mankind have broken away from the puzzlement of common infectious disease gradually, and malignant tumor (cancer) had become the maximum killer of human life already.World Health Organization's current research data show, will increase by 50% to the year two thousand twenty whole world cancer morbidity, namely every year will newly-increased 15,000,000 cancer patients.Moreover, the death toll of cancer also rapidly rises in the whole world.Within 2007, the whole world has 7,600,000 people to die from cancer, it is contemplated that the year two thousand thirty, this numeral will increase to 13,200,000, and the authority's report defending planning commission's issue according to country claims China's new cancer and because cancer mortality case load accounts for the 20% and 24% of the whole world respectively.So, the treatment of malignant tumor has become the vital task of current medical science.

Ribonuclease (RNase) is prevalent in natural environment and human body, and they can kill cell by the ribonucleic acid in degradation of cell.Plant greatly Cobra venom endonuclease and excision enzymes in liver cell containing about more than 20, they participate in the biological actions such as RNA metabolism, cell maturation and apoptosis, the formation of promotion new vessels, Initiative Defense RNA viruses.Although ribonuclease exists in a large number, but due to the inhibitory action of ribonuclease inhibitor (RI), histiocyte is not damaged.

RNaseA superfamily Major Members have RNaseA, people's pancreas ribonuclease, eosinophil derived neurotoxin, addicted to eosin cationic protein, angiogenesis factor, cattle seminal fluid ribonuclease and amphibian ribonuclease (such as ranpirnase and bull frog agglutinin) etc..RNaseA superfamily is played a role by degradation of rna, its family member has similar aminoacid sequence and tertiary structure, typically contain 4 pairs of disulfide bond (angiogenesis factor contains 3 pairs of disulfide bond), and all contain identical catalytic triads, including 2 histidine and 1 lysine.Owing to many members of this family derive from people or mammal, their immunogenicity is than deriving from the low of plant or microorganism.

Ranpirnase, is originally called P-30 albumen, and post-registration Onconase(by name is called for short ONC), when the 19th-century seventies, Shogen and Yoon separates in the embryo of the arctic leopard frog at first and proposes some extracts with active anticancer.In 1987, AlfacellM separated and is purified into its main active.Those active substances are the basic proteins of a kind of little (about 12kDa) maternal origin (being present in a large number at present in unfertilized oocyte).Its complete structure is determined in the laboratory of ShailendraK.Saxena et al..It is made up of 104 amino acid residues, relative molecular mass is 11835Da, isoelectric point, IP is 9.7, its sequence and pancreas ribonuclease A (RNaseA, EC3.1.27.5) with other members of ribonuclease super A family very similar, with the base sequence homology that RNaseA has 30%, and tertiary structure is similar, is single domain albumen minimum in known pancreatic ribonuclease superfamily member.Single for ribonuclease activity, RNaseA is higher than ranpirnase and cattle seminal fluid ribonuclease (BS-RNase), but owing to being subject to the suppression of RI, tumor cell is not had fragmentation effect by RNaseA；RNaseA in amphibian animal body is owing to can avoid RI, therefore has killing tumor cell effect.Especially ranpirnase, is the ribonucleic acid enzyme medicine of first entrance III phase clinical research, is mainly used in treatment malignant mesothe, and the research treating nonsmall-cell lung cancer at present has also come into clinical II phase experimental stage.

ONC is similar to known RNaseA structure, 2 antiparallel β lamellas and 3 αhelix the kidney forming core structure constituted, its enzyme active center is a conservative catalytic triads.RobertF.Gahl et al. in experiments it is found that in ONC oxidative folding process can form stable intermediate, so can keep its effective active form.DanielE.Holloway et al. first reports in 100K and complicated sulfate ion, ONC crystal structure under atom definition, the electron density map that quality is sufficiently large significantly shows several nonplanar peptide bond, it is possible to be very easy to determine most of amino acid residues in its active center.

In order to determine the amino acid residue being responsible for toxicity in onc protein, You-DiLiao et al. clones the rpr gene of the leopard frog from chromosome set, and has carried out the expression of various ways in e. coli bl21 (DE3).Expression one: methionine is arranged in the N-end of ONC recombinant, found that this recombiant protein heat stability reduces and catalysis activity reduces with antigenicity.They utilize dependence endogenous Escherichia coli Methionine Aminopeptidase and apelB signal peptide cutting two ways to produce the ONC not having methionine, it has been found that recombiant protein is consistent substantially with the physics of the ONC of natural origin, chemistry, biological nature.Proving that the N-end at ONC has the pyroglutamic acid of uridine-guanines substrate preferences, the cytotoxicity of ranpirnase is had bigger help by N-end pyroglutamic acid that is complete and that expose simultaneously.

Compared with RNaseA, the distinctive architectural feature of ONC is the disulfide bond of the lactams of its N end and C end.Ranpirnase N end Gln is folded inverted against N end spiral, forms hydrogen bond network with the carbonyl group of Val96 on the nitrilo of side chain Lys9 and C end β lamella.After Met is added to ranpirnase N end by the proof such as Didnato, the mutant catalytic action to all RNA and the toxicity to tumor cell all significantly reduce, and illustrate that this cyclized structure is most important to ribonuclease activity and the cytotoxicity of ranpirnase.Whole protein molecular contains 4 disulfide bond, and the C end disulfide bond that wherein Cys87-Cys104 is formed exists only in amphibian RNaseA, is the important feature territory stablizing its enzymatic activity.ONC is the albumen that a kind of structural stability is very strong, can reach 90 DEG C in pH6.0 time variation temperature, and medicine Half-life in vivo is also 3h.Except N end lactams and C end disulfide bond, the intramolecular hydrophobic environment of enzyme has also functioned to the effect of Stable conformation, and this is likely to be the major reason of RNase activity and the nephrotoxicity causing it.Mutant (M23L)-ONC has been fully retained anti-tumor activity, it is possible to reduces nephrotoxicity but stability and decreases.

YouNengWu et al. I¹²⁵Labelling ONC, and make ONC be combined in the special combination position of 9L neuroglial cytoma of artificial culture.Through Scatchard methods analyst labelling result, it was demonstrated that the binding site of ONC and 9L neuroglial cytoma has two, its dissociation constant respectively Kd=6.2 10^-8With Kd=2.5 10^-7.Every cell can in conjunction with 3 10⁵Individual ONC molecule, the dissociation constant of low level binding site is similar to IC50, and ONC is combined on cell amplification culture, and temperature is increased to 37 DEG C from 4 DEG C, it is possible to increase the cell sensitivity to ONC toxicity.Metabolic poison, sodium azide and deoxyglucose can suppress the cytotoxicity of ONC, and monensin can make cytotoxicity increasing ten times greater.Because for ONC toxicity, ONC molecular alkyl is the essential condition of ribonuclease activity, it is possible to strengthens and suppresses intracellular protein synthesis capability.In 9L cell, ONC suppresses protein synthesis to be by chance consistent with intracellular degraded, and action target is 28s and 18sRNA subunit.Compared with ribonuclease A, ONC can resist the degraded of two kinds of RNase, and one is placental ribonuclease, and another kind is ACETM inhibitor.

It is a kind of special RNase that RobertF.Gahl et al. demonstrates ONC, and it has selecting cell toxicity.The cytotoxicity of ONC depends on AP-2/ clathrin-mediated endocytosis effect, enters cell by the internalization of endocytotic vesicles, and the RNA of degradation of cell is thus killing cell.IntaeLee etc. find that when studying the antitumaous effect of ribonuclease ONC with the nude mice that A549 people's pulmonary carcinoma is transplanted ONC stops protein synthesis mode to kill tumor cell by decomposing t-RNA, and this perhaps can as a kind of new way substituting Cisplatin Treating Lung Cancer.

ONC can suppress the growth of A549 tumor cell significantly, and has significant dose dependent.In zoopery, the ONC repeatedly giving low dose is more effective than disposable heavy dose, and few side effects.The minimizing growth of tumour cell that ONC is more significantly with the mode of cisplatin combined medication energy, but for relatively large tumor body, ONC can suppress the growth of tumor cell, but cisplatin then can not.

The research such as ShailendraK.Saxena shows that the cytotoxicity of ONC is primarily directed to the degraded of tRNA.ONC is joined and can produce such as cell inhibiting, cytotoxicity and antiviral activity effect in the mammalian cell culture grown by they, and ONC enters can selectively and not detectable decomposition tRNA in the mammalian cell lived.Additionally, ONC can narrow spectrum reticulocyte lysate liquid and the pure RNA substrate of utilizing in vitro, and demonstrate the surprising specificity to tRNA degraded in vivo, and rRNA and mRNA stand intact.

MihailS.Iordanov etc. are when studying the molecular determinants of ONC inducing cell apoptosis, it was shown that the toxic mechanism of cell is different from protein synthesis inhibitory action by it.There is apoptosis in the cell of ONC effect, simultaneously with cell shrinkage, karyopyknosis or even break (karyorhexis), nuclear dna cracks, activate half Guang-Aspartase simultaneously, existing evidence proves that ONC exists the mechanism of a kind of inducing cell apoptosis, and this Apoptosis mechanism can independently suppress albumen to synthesize.Use half Guang-aspartic acid enzyme inhibitor benzyloxycarbonyl group-Val-Ala-Asp (OMe, omeprazole) fluorine ketone (zVADfmk), the ONC apoptosis caused can be stoped when certain concentration completely.As can be seen here, as long as the toxic action of cell is showed both ways by ONC: the tRNA that primary effect is degradation of cell suppresses cell protein synthesis, causes cell death, the Apoptosis mechanism of secondary role active cell, causes apoptosis.Therefore, ONC can as strong cancer therapy drug candidate albumen.Owing to ONC enters cell by endocytosis, the selectivity of cell is poor, in druggability research, have to take into account that the killing of human normal cell is caused bigger side effect by it.

For these reasons, we have carried out bigger improvement in drug design, for making ONC play maximum tumor cytotoxicity effect in vivo, reduce side effect, it is necessary to make ONC molecule effectively select tumor cell, increase ONC simultaneously and enter the efficiency of cell.

Based on the specific binding theory of receptor-ligand, use the aglucon LHRH(of LHRH receptor to be also known as GnRH) analog carry out the guiding component of anti-tumor drugs targeting and be used and become the important directions that current antineoplastic medicine designs.

The agonist and the inhibitor that use LHRH carry out gonad dependency prostate cancer therapy and have had the history in more than 30 years, illustrate that the LHRH agonist analog treatment carcinoma of prostate of application synthesis obtains significant achievement as nineteen eighty-two Schally etc. writes articles, alleviate the mental burden of patient physical's castration on the one hand, and obtain satisfied tumor suppression and therapeutical effect.

LHRH receptor at cancerous tissue surface high-affinity is to be developed by the reason of some the unknowns and come.This hypothesis is tentatively being confirmed with Mus, normal hamster pancreatic cell film does not detect LHRH receptor by RLA method, produce after pancreas tumor through induction, corresponding cell membrane has measured the LHRH receptor of high-affinity, and has become the tumor initial stage namely to can detect that.The new target drone that LHRH receptor is cancerous tissue, cell is demonstrated by multiple method detections such as RT-PCR method, immunohistochemical location method and radioligand assay methods (RLA).Kakar(1995) in breast carcinoma and prostate cancer tissue and carcinoma of endometrium and ovarian cancer tissue with cell strain, be found that the high-affinity LHRH receptor similar to hypophysis, and propose the carcinoma of prostate of 86%, the breast carcinoma of 50% and 80% ovarian cancer and carcinoma of endometrium have high-affinity LHRH receptor to be distributed.

By the work of long-term new drug research and external numerous documents, we prove that luteinising hormone-releasing hormo receptor (LHRHR is also known as gonadotropin-releasing hormone receptor, GnRHR) is distribution of specific on tumor tissue cell surface, have confirmed that the new target drone into tumor.

It is based on the design of the LHRH antitumor drug being targeting that ligand-receptor is specific binding and receptor is established by the fact that tumor cell surface overexpression.

The single chain protein matter that people LHRHR is made up of 328 aminoacid, it forms outer 7 the cross-film districts being connected with born of the same parents' inner annular structure of born of the same parents, has the construction features of g protein coupled receptor.LHRH receptor is found in hypophysis the earliest, accepts the luteinising hormone-releasing hormo (LHRH) that hypothalamus produces, promotes pituitary promoting sexual gland hormone LH and FSH, and rear both act on gonad, thus playing the adjustment effect to gonad function.Recently as going deep into that LHRH and receptor thereof are studied, it is widely distributed that increasing clinical and experimental evidence shows that LHRH receptor presents specificity in cancerous tissue.

Bono etc. (2002) have studied distribution and the feature of LHRHR in prostate canceration and optimum specimen, measures simultaneously and compare the expression of LHRH receptor mrna in Various Tissues, and result shows that the canceration specimen memebrane protein of 86% is to [DTrp⁶] LHRH shows specificity high-affinity, has the prostate gland cancer cell surface expression LHRH receptor of 86% simultaneously, also visible LHRH Receptor Gene Expression in benign prostate tissue, but its content and affinity are substantially less than canceration group.

S.Dharap etc. (2005) detect and analyze the cDNA of coding LHRH receptor gene content in various kinds of cell, tissue homogenate and receptor protein expression with quantitative RT-PCR.Determine human ovarian cancer, breast carcinoma and carcinoma of prostate and the content of the multiple normal organ tissue LHRH receptor that includes (including ovarian cancer tissue and the normal structure of same patient) in the heart, liver, spleen, lung, kidney, brain, thymus and skeletal muscle.Result shows that in tumor cell the gene of LHRH receptor is abundant and receptor protein is overexpression, but in the normal tissue cells such as the heart, liver, spleen, lung, kidney, brain, thymus and skeletal muscle, but it is not detected by LHRH acceptor gene, also it is not detected by receptor protein simultaneously and expresses.

Grundker(2002), Schally(2005) utilize the p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 [DLys synthesized⁶] as fairlead, with amycin or derivatives thereof for drug component, coupling becomes 3 kinds of LHRH-amycin (DOX) or derivatives thereofs: AN152, AN201, AN207, these medicines can pass through the specific binding tumor cell of LHRH, and play cytotoxic effect by amycin or doxorubicin derivative, specific killing tumor cell, its tumor killing effect is far superior to simple p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 and amycin or derivatives thereof, and substantially prolongs life span and the amount of survival of tumor bearing nude mice.And compared with using with simple amycin, dosage can bring up to more than 5 times (molar dose ratios) of amycin.This project has been completed a clinical trial phase as Germany, U.S. Government support project, and odd-numbered day dosage reaches 267mg/m², in breast carcinoma, prostate cancer therapy, obtain consistent accreditation (referring to United States Patent (USP) 5843903).Simultaneously they have also carried out specificity RT-PCR and RLA and detect the distribution of LHRHR and show: LHRH receptor is mainly distributed on kinds of tumors tissue, cell surface, only containing the receptor of pole trace in the reproductive system organs such as normal ovary, endometrium, fallopian tube, other normal structure such as heart, liver, lungs, muscle and skeleton etc. are all without the distribution of LHRH receptor.

The target validation experiment that we carry out in the R&D process of PTS LHRH-PE40, it is determined that high-affinity LHRH receptor (referring to Chinese patent ZL99122205.9) is contained on the kinds of tumor cells surfaces such as oral cancer, nasopharyngeal carcinoma, the esophageal carcinoma, pulmonary carcinoma, intestinal cancer, gastric cancer, breast carcinoma, cancer of pancreas, hepatocarcinoma, carcinoma of prostate, Humanmachine tumour, uterus carcinoma, ovarian cancer, epithelial cancer.

The LHRH-PE40 pharmacodynamic study that we carry out, find that tumor bearing nude mice utilizes LHRH-PE40 tail intravenously administrable to treat, inhibitory rate is to more than 60%, p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2+LHRH-PE40 the mixture of synthesis gives tumor bearing nude mice, tumour inhibiting rate reduces to 40%, give merely p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2, the tumour inhibiting rate of tumor bearing nude mice is 23%, this description of test LHRH-PE40 is by the LHRH LHRHR in conjunction with tumor cell surface, drug specificity is acted on tumor tissues, reach oncotherapy effect, p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2+LHRH-PE40 mixture treatment tumor bearing nude mice, tumour inhibiting rate reduces explanation p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 can be emulative in conjunction with LHRH receptor, stop the tumor-inhibiting action of LHRH-PE40.Simple p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 also has certain tumor-inhibiting action, and only tumor suppression is relatively inefficient.These experiments have absolutely proved the LHRH practicality meaning as neoplasm targeted therapy.

Summary of the invention

It is an object of the invention to provide targeting antineoplastic amalgamation protein matter LPO.

Genetic fragment LPO, its base sequence is such as shown in sequence table SEQ IDNO:1；

Fused protein LPO, its aminoacid sequence is such as shown in sequence table SEQ IDNO:2；

The preparation method of fused protein LPO, it includes:

1) the synthetic such as gene shown in sequence table SEQ IDNO:1；

2) utilize the gene shown in NcoI, EcoRI double digestion PET28a plasmid and sequence table SEQ IDNO:1, connect, build plasmid PET28a-LPO and turn the amplification of JM109 engineering bacteria；

3) in positive plasmid transformed competence colibacillus e. coli bl21 (DE3), abduction delivering；

4) purification.

Another object of the present invention is fused protein LPO application in preparing antitumor；

Described tumor is the tumor having LHRH receptor；

Described tumor is colon cancer LOVO, pulmonary carcinoma A549, oral cancer KB or carcinoma of prostate PC-3M.

Detailed Description Of The Invention, comprehensively our basic research for many years and LHRH-PE40 new drug research and research experience of abroad going together, it is considered that LHRH can be used as the targeted molecular of antitumor drug, when intellectual property is not conflicted, we utilize the more rational p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2-antineoplastic pharmaceutical compositions of design, develop anti-cancer agent.

First in the selection of p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2, We conducted substantial amounts of screening operation, based on the active polypeptide chain that natural LHRH is decapeptide, PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, owing to the first aminoacid is pyroglutamic acid, gene engineering expression synthesis acquires a certain degree of difficulty, it is mostly default or it is sported glutamic acid that generation deposits analog, but through our experience of gene expression LHRH-toxin fusion protein and follow-up pharmacological effect activity test for many years, prove that the first amino acid mutation is that glutamine more can play its Receptor recognition, binding ability, so, we design and have synthesized the codon that the first amino acid mutation is glutamine.Again owing to natural LHRH the 6th amino acids is glycine, its receptor affinity is relatively low, Half-life in vivo is only 5min, so its 6th glycine has been also carried out artificial mutation by us, tryptophan (Trp) is utilized to substitute glycine, its receptor binding capacity improves 7-10 times, half-life reaches 2 hours, be conducive to medicine integrity in vivo and long-lasting, with reference to Pseudomonas aeruginosa Exotoxin A and diphtheria toxin, diphtherotoxin construction features with enter born of the same parents' mechanism, C-terminal at LHRH adds a generally acknowledged transferring film peptide, so as to afterbody forms Furin enzyme action recognition site, once pharmaceutical composition is attached to tumor cell surface, when forming endocytotic vesicles under receptor acting, Furin enzyme in endocytotic vesicles plays enzyme action effect when pH5.2, by in antitumor drug active transport to tumor cell.

Term used herein " LPO ", refer to through detailed design, utilizing gene synthesis technology to obtain gene order and fragment, enable to obtain high efficient expression in escherichia coli through genetic engineering operation, the protein of purification can have the protein of targeting anti-tumor effect in vivo and in vitro.

Amino acid symbols used in literary composition is trigram dummy suffix notation generally in the art, wherein with the addition of NcoI restriction enzyme enzyme recognition site when gene chemical synthesis in fusion gene 5` end in order to gene is easy to connect, and utilize the ATG in NcoI restriction enzyme enzyme recognition site as the first setting up password, accurate translation fusion protein first place aminoacid Met.Add the recognition sequence GAATTC of restricted enzyme EcoRI at the 3` end of the protein gene merged, in order to make genetic fragment and carrier be respectively formed sticky end when connecting, be beneficial to gene and connect simultaneously.For making expression product reach high efficient expression, escherichia coli bias codon is incorporated into wherein, so that expression product is adapted at expression in escherichia coli.

Term used herein " IC50 " refers to the half cell-lethal concentration utilizing the biological product described by Products in China code that activity of tumor cells standard verification method is measured, namely the human tumor cells utilizing In vitro culture reaches concentration (the reference Chinese Pharmacopoeia annex of medicine when half suppresses, antitumor drug Bioactivity Assay, its concentration represents with μ g/ml).

The invention further relates to multiple types protein or the method for polypeptide fused in tandem expression destination protein matter of producing with recombinant DNA technology, the method includes: (1) provides and carries T7 strong promoter and the expression vector containing multiple clone site；(2) the coding gene sequence that synthetic gene is connected in series, is cloned in the expression vector of linearizing step (1) with correct reading frame；(3) convert suitable host cell with the expression vector of step (2)；(4) the described host cell being converted is cultivated when being suitable to expressed fusion protein matter；(5) from cell culture, utilize suitable purifying process recovery the fused protein described in purification.

It is with N end Met for initial protein molecule for building the LPO molecule of the fused protein of the present invention, after increasing restriction enzyme site, in order to adjust ORF, the codon GGC of Gly is added after ATG codon, it is followed by the sequence of LHRH, in order to increase the binding ability of LHRH and its receptor and extend its half-life, the 6th amino acids in LHRH sequence is replaced with Trp by Gly.And then connect the transferring film of PEA and the decapeptide sequence of furin enzyme identification after complete LHRH decapeptide sequence, finally connect 104 aminoacid of ONC, and terminate with termination codon TAA.

Owing to temporarily lacking the ready-made sequence merging LPO molecule as the present invention, so using general DNA synthesizer nucleotide sequence of these fusion protein of composite coding on 1000A solid phase carrier in the present invention.For the ease of being attached with specific recombinant vector, suitable Cobra venom endonuclease (such as NcoI and EcoRI) restriction enzyme site can be introduced at 5 ' and/or 3 ' ends of the antigen-4 fusion protein gene sequence of synthesis, and cause the sticky end being adapted to.Can according to recombinant technique well known by persons skilled in the art, the gene (or with its cDNA or genomic DNA form) that the reading frame of these synthesis of clones coding is unobstructed, in DNA reorganization operation, generally use the gene clone of standard and Subcloned technology carries out transfer and the connection of genetic fragment.Use Restriction Enzyme pressure method to identify the correctness of sequence closure and possible sudden change, finally carry out sequence confirmation with DNA sequencing.

Here should it is emphasized that, due to relative to LHRH molecule, transferring film peptide and the ONC moieties amount of amalgamation and expression are bigger than normal, so the fused protein produced by three will be likely formed new advanced space conformation, cause that the curling of leading part is wrapped, affect activity and the spatial conformation of ONC.But analyzed by technician computer and prove that LPO protein molecule is to carry out folding with correct disulfide patterns with C. D. spectrum detection.

Recombiant protein plasmagene will be operatively coupled on suitable expression control sequenc, as being connected to the T being suitable to use in escherichia coli₇, trp or λ promoter, on ribosome binding site and transcription stop signals.Known method for transformation can be used, as appropriate for the calcium chloride facture of prokaryotic cell or be suitable to the electroporation of mammalian cell by the recombinant plasmid transformed of the present invention to the host cell selected.The positive cell that is converted can be selected based on the antibiotic resistance that antibiotics resistance gene contained on plasmid gives.Once have expressed required fused protein, can separate and this fused protein of purification according to methods known in the art.Such as, can be centrifuged from fermentation culture medium collection somatic cells and with lysozyme and ultrasonic treatment it, then ultracentrifugation carry out inclusion body washing in low phosphorus hydrochlorate (20mM), urea liquid, then utilize 8M Urea Lysis inclusion body, it is thus achieved that protein by renaturation agent exist under recover natural structure.Then LPO recombinant protein needed for ion-exchange chromatography (IEC) and hydrophobic chromatography, gel filtration chromatography successively.

It is, in general, that use polyacrylamide gel to analyze each column chromatography elution fraction with SDS-PAGE electrophoresis method, and monitor it with immunoblotting.Recombinant fusion protein purified product is carried out the inhibiting tumour cells test biologic activity (Products in China code 2010 editions is shown in concrete operations) to detect recombinant protein.

Using the LPO protein of the present invention as primary activity composition, and one or more pharmaceutically acceptable carrier or excipient can be added, make the pharmaceutical composition being suitable to clinical practice.Described carrier or excipient include but are not limited solely to phosphate buffer, normal saline, isotonic glucose solution, glucosan, dextran etc..Difference according to the disease treated, can add one or more in the pharmaceutical composition of the present invention has auxiliary or other natural, synthesis or restructuring reactive compounds synergistic with the protein of the present invention.It addition, low molecular weight peptide, glycine or lysine and metal cation can be added in the pharmaceutical composition of the present invention (such as Zn²⁺、Mn²⁺、Mg²⁺And Ca²⁺) protein protectant, and the stabilizer of Polyethylene Glycol, carboxymethyl cellulose, polyglycine, glutathion.

Conventional administration route, particularly parenteral routes can be passed through come into operation the pharmaceutical composition of the present invention, for instance by administration in intravenous, intraperitoneal, intramuscular, Intradermal, subcutaneous or mucosa.The effective dosage ranges of pharmaceutical composition of the present invention can from several milligammas to tens mg kg of body weight/sky, but for the concrete dosage of each given patient by the character according to disease to be treated or pathological state and the order of severity, the age of patient, body weight, the factor such as respond and administering mode of medicine is determined.

Our laboratory has turned out the LPO protein of the present invention all has obvious inhibitory action to tumor cell lines such as including colon cancer LOVO cell, lung cancer A549 cell, oral cancer KB cell, prostate carcinoma PC-3 M cells, to without the lymphoma Hut102 of LHRH receptor and leukemia HL60 then unrestraint effect.

Accompanying drawing explanation

Fig. 1 shows the construction of recombinant plasmid figure for expressed fusion protein.

Detailed description of the invention

It is further elucidated with the present invention, it will be recognized by those skilled in the art that these embodiments are not intended that the restriction to accompanying claims scope of the present invention below by embodiment.

The preparation of embodiment 1:LPO expression vector and engineering bacteria

The structure of recombinant expression plasmid and qualification

A. gene chemical synthesis: carry out according to previously described design concept in the present invention synthesizing following sequence outside the design of restriction enzyme site and LPO complete genome sequence prosthesis:

NcoI endonuclease recognition sequence (CCATGG)+GC+LHRH polypeptide gene+PEA transferring film peptide+ONC sequence+termination codon (TAA)+EcoRI endonuclease recognition sequence (GAATTC), gene order is shown in SEQIDNO:1, this composition sequence Direct Cloning enters in the carrier T that Dalian Takara company provides, and convert e. coli jm109 antibacterial, identify through PCR and determine positive clone strain, positive clone strain amplification culture, common molecular clone technology extracts plasmid DNA, utilize NcoI, EcoRI this plasmid of double digestion and PET28a plasmid, reclaim purpose fragment and PET28a plasmid vector sticky end fragment, with correct reading frame at T₄Ligase (Promega) exists and lower is inserted in the PET28a plasmid vector of same enzyme action by purpose fragment, and construction of fusion protein double-strand expressing gene also converts JM109 engineering bacteria.

B. identify through PCR and determine positive clone strain, positive clone strain amplification culture, common molecular clone technology extracts plasmid DNA, NcoI, EcoRI double digestion PET28a-LPO plasmid is utilized to identify, correct person carries out DNA sequencing, then positive plasmid transformed competence colibacillus e. coli bl21 (DE3), and by the antibacterial culturing that is converted in the LB culture medium containing kanamycin (50 μ g/ml), with amplified plasmid dna.After cultivation completes, smudge cells, centrifugal collection plasmid plasmid DNA purification order-checking, the correct plasmid that checks order will be converted coli strain, identifying with endonuclease cutting and agarose gel (2%) electrophoresis method, then positive recombiant plasmid carries out DNA sequence analysis.

Fig. 1: show the structure of recombiant plasmid pET28a-LPO.

The expression of embodiment 2:LPO fused protein and the purification of product:

The e. coli bl21 (DE3) of recombination Plastid transformation will be carried (containing T₇Rna polymerase gene) cultivate on the LB agar plate containing kanamycin (50 μ g/ml).Selecting kalamycin resistance bacterium colony after cultivation, in the LB culture medium containing kanamycin (50 μ g/ml), 37 DEG C of cultivations, work as A₆₀₀Adding 1mM isopropylthio-β-D-galactoside (IPTG) (final concentration 1mM) when reaching about 0.4 ~ 0.6,37 DEG C are continued to cultivate 3-4 hour, to induce the expression of purpose product.Being then centrifuged for separating cell and culture medium, and will add buffer components in the thalline containing destination protein matter, final concentration reaches 50mMTris-HCl, pH8.0,1mMEDTA, ultrasonication, 4 DEG C centrifugal (20,000g, 30 minutes), take precipitation (insolubility part) and be fusion protein crude extract.

Crude extract processes through washing, denaturation renaturation, the recombinant protein matter obtained carries out cation exchange chromatography: the DEAE-SepharoseFastFlow post (Pharmacia) of buffer balance, with the TE buffer (20mMTris-HCl containing 0 ~ 0.5MNaCl, pH8.0,1mMEDTA) continuous gradient eluting, and collect each component peaks part of protein.Purpose component peaks part through small hollow fibrous ultrafilter (Milipore) act on 30 minutes be concentrated by ultrafiltration change liquid salt adding after, make concentrate containing final concentration 1.15MNaCl, with 20mMTris-HCl, pH8.0,1.15MNaCl buffer balance XK1.6 × 20cmPhenyl-SepharoseFastFlow post (Pharmacia), then loading, buffer solution Liu Chuan peak, finally utilize and carry out 100%-0% gradient elution containing the buffer (20mMTris HCl, pH8.0) of 1.15M-0.05MNaCl.Collect destination protein peak part, utilize small hollow fibrous ultrafilter (Milipore) to concentrate destination protein to 2mg/ml.Utilize 20mMPB, pH7.0,0.15MNaCl buffer balance XK1.6 × 100cmSuperdex75 solvent resistant column (Pharmacia), utilize loading ring to inject sample 5ml, flow velocity 1ml/min and carry out gel filtration separation purification, collect protein peak value (A₂₈₀) partly store for future use at-20 DEG C.The lipidated protein of such purification > 97%.

Embodiment 3: utilize the inhibiting tumour cells method of In vitro culture to measure the biologic activity effect of fusion protein, (concrete operations are with reference to 2010 editions the 3rd annex XC of Chinese Pharmacopoeia).

Cell phenotype conversion:

By the human tumor cells cell monolayer of cultivation through trypsinization, cell suspension is collected in piping and druming, and after utilizing cell counting count board numeration, adjusting cell quantity is 60000/ml, joins (every hole 5000 cell), 5%CO in 96 well culture plates according to 80 μ l/ holes₂, under 37 DEG C of conditions, cultivate 4h.The LPO protein example concentration adjusting preparation is 1mg/ml, and different amounts of sample, through filtration sterilization, is added in each cell hole by equimultiple dilution method, then supplies culture medium, so as to cumulative volume is 100 μ 1,5%CO by quantitative sample₂, under 37 DEG C of conditions, cultivate 24h.Then supernatant in cell plates is discarded, every hole adds 50 μ 1 dyeing liquors, room temperature washes away dyeing liquor with flowing water, and blots residual moisture after placing 30min, every hole adds destaining solution 100 μ 1, room temperature places 5min, after mixing, by microplate reader with 630nm for reference wavelength, go out to measure absorbance at wavelength 570nm, record measurement result.

Experimental data adopts computer program to carry out parametric regression calculating: the data acquisition computer programs such as each experimental point OD value of each laboratory sample, pre-dilution multiple, sample gradient are processed.Calculate half effect extension rate of each laboratory sample respectively and calculate the concentration (result following table) of 50% effect point.

<110>MILITARY VETERINARY INST ACADE

<120>preparation of targeting antineoplastic amalgamation protein matter LPO and application

<160>2

<210>1

<211>380

<212>DNA

<213>artificial

<400>1

ccatgggccagcactggtcctacggcctgcgtccgggtacccgccatcgtcagccgcgcc60

aggattggctgaccttccagaagaaacatatcactaacacccgcgatgtagactgcgaca120

acatcatgtctaccaatctgttccactgtaaggataaaaacacctttatctacagtcgtc180

cagaaccggtaaaggctatctgtaaaggcattatcgcgagtaagaacgtgctgactacct240

ccgagttctacctgtctgattgcaacgttacctcccgcccgtgcaaatataaactgaaga300

aaagcactaacaaattttgcgtaacctgcgaaaaccaggctccggtacatttcgttggag360

tcggcagctgctaagaattc380

<210>2

<211>123

<212>PRT

<213>artificial

<400>2

MetGlyGlnHisTrpSerTyrGlyLeuArgProGlyThrArgHisArg

151015

GlnProArgGlnAspTrpLeuThrPheGlnLysLysHisIleThrAsn

202530

ThrArgAspValAspCysAspAsnIleMetSerThrAsnLeuPheHis

354045

CysLysAspLysAsnThrPheIleTyrSerArgProGluProValLys

505560

AlaIleCysLysGlyIleIleAlaSerLysAsnValLeuThrThrSer

65707580

GluPheTyrLeuSerAspCysAsnValThrSerArgProCysLysTyr

859095

LysLeuLysLysSerThrAsnLysPheCysValThrCysGluAsnGln

100105110

AlaProValHisPheValGlyValGlySerCys

115120

Claims (6)

1. genetic fragment LPO, its base sequence is such as shown in sequence table SEQ IDNO:1.

2. fused protein LPO, its aminoacid sequence is such as shown in sequence table SEQ IDNO:2.

3. the preparation method of fused protein LPO, it includes:

1) the synthetic such as gene shown in sequence table SEQ IDNO:1；

2) utilize the gene shown in NcoI, EcoRI double digestion PET28a plasmid and sequence table SEQ IDNO:1, connect, build plasmid PET28a-LPO and turn the amplification of JM109 engineering bacteria；

3) in positive plasmid transformed competence colibacillus e. coli bl21 (DE3), abduction delivering；

4) purification.

4. the application in preparing antitumor drug of the fused protein LPO described in claim 2.

5. the application piece described in Ju claim 4, it is characterised in that: described tumor is the tumor having LHRH receptor.

6. application according to claim 5, it is characterised in that: described tumor is colon cancer LOVO, pulmonary carcinoma A549, oral cancer KB or carcinoma of prostate PC-3M.