CN103805621A - Novel preparation technology of targeting antitumor fusion protein LPO (lipid peroxidation) - Google Patents

Abstract

The invention discloses a novel preparation technology of targeting antitumor fusion protein (lipid peroxidation). The novel preparation method comprises the steps of building a fusion expression protein which takes the glutathione S-transferase A (TrxA)-His-tag (His6-Tag)-SUMO protease recognition substrate as a protein soluble expression auxiliary fragment and takes LHRH (luteinizing hormone releasing hormone)-PEA (phenylethylamine) trans-cell penetrating peptides-ONC (LPO for short) as a target segment, connecting the two proteins with each other by a correct read frame and carrier positive sequence and converting to enter into expression bacteria, finally building fusion protein which is connected with six expression substances in series, crudely extracting, carrying out metal chelating medium purification in the presence of imidazole, and carrying out SUMO protease digestion. Compared with the LPO prepared by a conventional method, the LPO prepared by the novel preparation technology disclosed by the invention can obviously improve the inhibiting effect on the tumor cell lines such as colon cancer HT-29 cells, ovarian cancer OVCAR3 cells, cervical adenocarcinoma HeLa cells and liver cancer HepG-2 cells.

Description

The novel preparation process of targeting antineoplastic amalgamation protein matter LPO

Technical field

The present invention relates to genetically engineered field, specifically the novel preparation process of targeting antineoplastic amalgamation protein matter LPO.

Background technology

Malignant tumour (cancer) had become human life's maximum killer already.World Health Organization's current research data presentation, will increase by 50% to the year two thousand twenty whole world cancer morbidity, will increase every year 1500 ten thousand cancer patientss newly.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, estimate the year two thousand thirty this numeral will be increased to 1,320 ten thousand, according to health ministry issue authority report claim the new cancer of China and because of cancer mortality case load account for respectively the whole world 20% and 24%.So the treatment of malignant tumour becomes one of vital task of current medical science.

Rnase (RNase) is prevalent in physical environment and human body, they can be by the Yeast Nucleic Acid in degradation of cell cell killing.In liver cell, approximately plant endonuclease and excision enzyme containing more than 20, they participate in the biological actions such as RNA metabolism, cell maturation and apoptosis, the formation of promotion new vessel, Initiative Defense RNA viruses.Although rnase exists in a large number, due to the restraining effect of ribonuclease inhibitor (RI), the RNase in body does not damage histocyte.

RNase A superfamily Major Members has RNase A, people's pancreas rnase, eosinophil derived neurotoxin, has a liking for eosin cationic protein, angiogenesis factor, ox seminal fluid rnase and batrachians rnase (as ranpirnase and bullfrog lectin) etc.RNase A superfamily plays a role by degradation of rna, its family member has similar aminoacid sequence and tertiary structure, generally contain 4 pairs of disulfide linkage (angiogenesis factor contains 3 pairs of disulfide linkage), and all contain identical catalysis triplet, comprise 2 Histidines and 1 Methionin.Because many members of this family derive from people or Mammals, their immunogenicity is lower than the toxin complex that derives from plant or microorganism.

In the time of the 19th-century seventies, Shogen separates at first and proposes some extracts with antitumour activity with Yoon in the embryo of the arctic leopard frog.In 1987, the main active ingredient of extract separated by Alfacell M and purifying out.Those active substances are basic proteins of a kind of little (approximately 12kDa) maternal instinct origin (being present in a large number at present in unfertilized ovocyte).Be called as ranpirnase (ranpimase), definite designation is afterwards that Onconase(is called for short ONC), it is made up of 104 amino-acid residues, relative molecular mass is 11835Da, iso-electric point is 9.7, its sequence and pancreas ribonuclease A (RNase A, EC 3.1.27.5) with very similar of other members of the super A of rnase family, there is 30% base sequence homology with RNase A, and tertiary structure is similar, be single domain albumen minimum in known pancreatic ribonuclease superfamily member.Single with regard to ribonuclease activity, RNase A will be higher than ranpirnase and ox seminal fluid rnase (BS-RNase), but owing to being subject to the inhibition of RI, RNase A does not have fragmentation effect to tumour cell; RNase A in amphibian animal body is owing to can avoiding RI, therefore there is killing tumor cell effect.Especially ranpirnase, is the first Yeast Nucleic Acid enzyme medicine that enters the clinical study of III phase, is mainly used in treating malignant mesothe, and the research for the treatment of at present nonsmall-cell lung cancer has also entered clinical II phase experimental stage.

ONC and known RNase A structural similitude, the kidney forming core structure having been formed by 2 antiparallel β lamellas and 3 αhelix, its enzyme active center is a conservative catalysis triplet.The people such as Robert F. Gahl find can form stable intermediate in ONC oxidative folding process in test, can keep like this its effective active form.Daniel E. Holloway etc. has reported in 100K electric field and complicated sulfate ion, the crystalline structure of ONC, enough in large electron density map, significantly demonstrate several nonplanar peptide bonds a quality, can be very easy to determine most of residues in its active centre.

In order to determine the amino-acid residue of being responsible for toxicity in onc protein, the people such as You-Di Liao clone the rpr gene of the leopard frog from genome, and in e. coli bl21 (DE3), have carried out the expression of various ways.Expression method one: methionine(Met) is arranged in to the N-end of ONC recombinant chou, found that this recombinant protein thermostability reduces and catalytic activity and antigenicity reduction.They utilize dependence endogenous intestinal bacteria methionine(Met) aminopeptidase and two kinds of modes of apelB signal peptide cutting to produce the ONC that there is no methionine(Met), find that recombinant protein conforms to substantially with physics, chemistry, the biological nature of the ONC of natural origin.The N-end simultaneously proving at ONC has the Pyrrolidonecarboxylic acid of uridine-guanine substrate preferences, and N-end Pyrrolidonecarboxylic acid complete and that expose has larger help to the cytotoxicity of ranpirnase.

Compared with RNase A, the distinctive constitutional features of ONC is the lactan of its N end and the disulfide linkage of C end.Ranpirnase N end Gln is folded inverted near N end spiral, holds the carbonyl group of Val96 on β lamella to form hydrogen bond network with nitrogen base and the C of side chain Lys9.The proofs such as Didnato are added to Met after ranpirnase N end, and the katalysis of mutant to all RNA and the toxicity of tumour cell is all significantly reduced illustrates that ribonuclease activity and the cytotoxicity of this cyclized structure to ranpirnase is most important.Whole protein molecular contains 4 disulfide linkage, and the C end disulfide linkage that wherein Cys87-Cys104 forms is only present in amphibian RNase A.ONC is the albumen that a kind of structural stability is very strong, can reach 90 ℃ in pH6.0 time variation temperature, and medicine Half-life in vivo is also 3h.Except N end lactan and C end disulfide linkage, the intramolecular hydrophobic environment of enzyme has also played the effect of Stable conformation, and this is probably to cause its low RNA enzymic activity and the major reason of renal toxicity.Mutant (M23L)-ONC has retained anti-tumor activity completely, and stability decreases, and likely reduces renal toxicity.

The people I such as YouNeng Wu ¹²⁵mark ONC, and make ONC be combined in the special combination position of the 9L neuroglial cytoma of artificial culture.Through Scatchard methods analyst mark result, prove that the binding site of ONC and 9L neuroglial cytoma has two, its dissociation constant is respectively Kd=6.2 ╳ 10 ^-8with Kd=2.5 ╳ 10 ^-7.Every cell can be in conjunction with 3 ╳ 10 ⁵individual ONC molecule, the dissociation constant of low level binding site is similar to IC50, and ONC is combined in enlarged culturing on cell, and temperature is elevated to 37 ℃ from 4 ℃, can increase the susceptibility of cell to ONC toxicity.Metabolic poison, sodium azide and deoxyglucose can suppress the cytotoxicity of ONC, and monensin can make cytotoxicity increase ten times.Because for ONC toxicity, ONC molecular alkyl is the prerequisite of ribonuclease activity, can strengthen and suppress intracellular protein synthesis capability, and at least strengthen 100 times.In 9L cell, ONC arrestin matter is synthetic is 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 RNA enzymes, and one is placenta rnase, and another kind is ACETM inhibitor.

The people such as Robert F. Gahl have proved that ONC is a kind of special RNA enzyme, and it has selecting cell toxicity.The cytotoxicity of ONC depends on the endocytosis of AP-2/ clathrin mediation, enters cell by the internalization of endocytosis vesicle, thus the RNA cell killing of degradation of cell.Intae Lee etc. find that in the time that the nude mice of A549 people's lung cancer transplanting is studied the antitumous effect of rnase ONC with it ONC stops protein synthesis mode to kill tumour cell by decomposing t-RNA, and perhaps this can be used as a kind of novel mode of alternative Cisplatin Treating Lung Cancer.

ONC can suppress the growth of A549 tumour cell significantly, and has significant dose-dependently.In experimentation on animals, repeatedly give low dose of ONC more effective than disposable heavy dose, and few side effects.The mode of ONC and cisplatin combined medication can reduce growth of tumour cell more significantly, but for relatively large tumour body, the growth that ONC can inhibition tumor cell, but cis-platinum can not.

The research of Shailendra K. Saxena etc. shows that the cytotoxicity of ONC is mainly the degraded for tRNA.They join ONC in the mammalian cell culture of growing and can produce such as cell inhibition, cytotoxicity and antiviral activity effect, and ONC enters in mammalian cell alive can be selectively and the decomposition tRNA not discovered.In addition, ONC can narrow spectrumly in vitro utilize reticulocyte lysate and pure RNA substrate, and demonstrates in vivo the surprising specificity to tRNA degraded, and rRNA and mRNA are excellent.

Mihail S. Iordanov etc., in the time of the molecule determinant of research ONC cell death inducing, show that its toxic mechanism to cell is different from protein synthesis restraining effect.There is apoptosis in the cell of ONC effect, follow cell shrinkage simultaneously, karyopyknosis and even break (karyorhexis), nuclear dna generation cracking, activate half Guang-L-Aspartase simultaneously, existing evidence proves that ONC exists a kind of mechanism of cell death inducing, this apoptosis mechanism can be synthesized by independent arrestin, use half Guang-aspartic acid enzyme inhibitors carbobenzoxy-(Cbz)-Val-Ala-Asp (OMe, omeprazole) fluorine ketone (zVADfmk), the apoptosis that can stop ONC to cause completely in the time of certain concentration.As can be seen here, ONC needs only performance both ways to the toxic action of cell: the tRNA that primary effect is degradation of cell suppresses cell protein and synthesizes, and causes necrocytosis, and the apoptosis mechanism of secondary role active cell, causes apoptosis.Therefore, ONC can be used as strong cancer therapy drug candidate albumen.Because ONC enters cell by endocytosis, poor to the selectivity of cell, aspect patent medicine Journal of Sex Research, having to consider the larger side effect that it causes killing and wounding of human normal cell.

For these reasons, we have carried out larger improvement aspect medicinal design, for making ONC bring into play in vivo maximum tumor cytotoxicity effect, reduce side effect, must make ONC molecule effectively select tumour cell, the efficiency that increases ONC simultaneously and enter cell.

Specific binding theory based on acceptor-aglucon, is used the aglucon LHRH(of LHRH acceptor to be also called GnRH) or its analogue guiding component of carrying out anti-tumor drugs targeting be used and become the hot topic of current antitumor drug design.

The agonist of use LHRH and inhibitor carry out sexual gland dependency prostate cancer the history of more than 30 years, as writing articles the synthetic LHRH agonist analogue treatment prostate cancer of application, nineteen eighty-two Schally etc. obtains significant achievement, alleviate the mental burden of patient's physics castration on the one hand, and obtained satisfied tumor suppression and therapeutic action.

The LHRH acceptor that has high-affinity on cancerous tissue surface is to develop by some unknown reasons.This hypothesis is tentatively confirmed with it mouse, on normal hamster pancreatic cell film, do not detect LHRH acceptor by RLA method, after induction produces pancreatic neoplasm, on corresponding cytolemma, measure the LHRH acceptor of high-affinity, and become the knurl initial stage to detect.Detect and proved that LHRH acceptor is the new target drone of cancerous tissue, cell by several different methods such as RT-PCR method, immunohistochemical methods localization method and radioligand assay methods (RLA).Kakar(1995) mammary cancer and prostate cancer tissue and carcinoma of endometrium and ovarian cancer tissue with in cell strain, found the high-affinity LHRH acceptor similar to hypophysis, and propose 86% prostate cancer, 50% mammary cancer and 80% ovarian cancer and carcinoma of endometrium and have high-affinity LHRH acceptor to distribute.

We prove luteinising hormone-releasing hormo acceptor (LHRHR by long-term new drug research work and external numerous document, also be called gonadotropin-releasing hormone receptor, GnRHR) be distribution of specific on tumor tissue cell surface, confirmed as the new target drone of tumour.

The design of the antitumor drug take LHRH as target is that the distribution based on its acceptor determines.

The single chain protein matter that people LHRHR is made up of 328 amino acid, it forms outer 7 the cross-film districts that are connected with ring structure in born of the same parents of born of the same parents, has the constructional feature of g protein coupled receptor.LHRH acceptor is found in hypophysis the earliest, accepts the luteinising hormone-releasing hormo (LHRH) that hypothalamus produces, and impels pituitary gonad-stimulating hormone LH and FSH, and both act on sexual gland afterwards, thus the regulating effect of performance to gonad function.In recent years along with to the going deep into of LHRH and acceptor thereof research, increasing clinical and experimental evidence shows that LHRH acceptor presents specificity and extensively distributes in cancerous tissue.

Bono etc. (2002) have studied the distribution and characteristics of LHRHR on prostate gland canceration and optimum sample, measure simultaneously and have compared the expression of LHRH receptor mrna in Various Tissues, and result shows that 86% canceration sample membranin is to [DTrp ⁶] LHRH performance specificity high-affinity, there is 86% prostate cancer cell surface expression LHRH acceptor simultaneously, also LHRH Receptor Gene Expression as seen in benign prostate tissue, but its content and avidity are significantly lower than canceration group.

Gene content and the expression of S.Dharap etc. (2005) had detected and analyzed coding LHRH acceptor cDNA with quantitative RT-PCR in various kinds of cell, tissue homogenate.Measure the expression that human ovarian cancer, mammary cancer and prostate cancer and multiple normal organ tissue comprise the LHRH acceptor that (comprises same patient's ovarian cancer tissue and healthy tissues) in the heart, liver, spleen, lung, kidney, brain, thymus gland and skeletal muscle.Result shows that the gene of LHRH acceptor in tumour cell is overexpression, but LHRH expression of receptor but do not detected in the normal tissue cells such as the heart, liver, spleen, lung, kidney, brain, thymus gland and skeletal muscle.

Grundker(2002), Schally(2005) utilize synthetic p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 [DLys ⁶] as fairlead, take Zorubicin or derivatives thereof as drug component, coupling becomes 3 kinds of LHRH-Zorubicins (DOX) or derivatives thereof AN152, AN201, AN207, these medicines can pass through LHRH specific binding tumour cell, and by Zorubicin or Zorubicin derivative performance cytotoxic effect, specific killing tumour cell, its tumor killing effect is far superior to simple p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2 and Zorubicin or derivatives thereof, and has greatly extended survival time and the amount of survival of tumor bearing nude mice.And compared with using with simple Zorubicin, dosage can be brought up to 5 times above (molar dose ratios) of Zorubicin.This project has completed clinical trial together as Germany, United States Government's Funded Projects, and odd-numbered day dosage reaches 267mg/m2, is obtaining consistent approval (referring to United States Patent (USP) 5843903) aspect mammary cancer, prostate cancer therapy.Simultaneously they have also carried out the distribution that specificity RT-PCR and RLA detect LHRHR and have shown: LHRH acceptor is mainly distributed in kinds of tumors tissue, cell surface, the acceptor containing utmost point trace only in the reproductive system organs such as normal ovary, uterine endometrium, uterine tube, other healthy tissues is if heart, liver, lungs, muscle and bone etc. are all without the distribution of LHRH acceptor.

The experiment of target validation that we carry out in the R&D process of PTS LHRH-PE40, has determined that the kinds of tumor cells surfaces such as oral carcinoma, nasopharyngeal carcinoma, the esophageal carcinoma, lung cancer, intestinal cancer, cancer of the stomach, mammary cancer, carcinoma of the pancreas, liver cancer, prostate cancer, Humanmachine tumour, uterus carcinoma, ovarian cancer, epithelial cancer contain high-affinity LHRH acceptor (referring to Chinese patent ZL 99122205.9).

Next we have also carried out the pharmacodynamic study of targeting antitumor new drug LHRH-PE40, and more than finding that tumor bearing nude mice utilizes LHRH-PE40 tail intravenously administrable treatment inhibitory rate to 60%, synthetic p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2+LHRH-PE40 mixture treatment tumor bearing nude mice, tumour inhibiting rate drops to 40%, simple giving and p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2, tumor bearing nude mice, tumour inhibiting rate 23%, this description of test LHRH-PE40 is the LHRHR in conjunction with tumor cell surface by LHRH, drug specificity is acted on to 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 emulative combination LHRH acceptor, 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 efficiency is relatively low.These experiments have absolutely proved the practicality meaning of LHRH as neoplasm targeted therapy.

By us fundamental research for many years and LHRH-PE40 new drug research and the research experience of abroad going together, we think that the targeted molecular that LHRH can be used as antitumor drug is used, in the situation that intellecture 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, exploitation anti-cancer agent.

First in the selection of p-GLU-HIS-TRP-SER-TYR-D-TRP-LEU-ARG-PRO-GLY-NH2, we have carried out a large amount of screening operations, the active polypeptide chain that is decapeptide based on natural LHRH, PGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2, because the first amino acid is Pyrrolidonecarboxylic acid, gene engineering expression is synthetic to acquire a certain degree of difficulty, it is mostly default or it is sported to L-glutamic acid that generation is deposited analogue, but through our experience of genetic expression LHRH-toxin fusion rotein and follow-up pharmacological effect activity test for many years, prove that the first amino acid mutation is that glutamine more can be brought into play its Receptor recognition, binding ability, so, we design, and to have synthesized the first amino acid mutation be glutamine.Again because natural LHRH the 6th amino acids is glycine, its acceptor avidity is lower, Half-life in vivo is only 5min, so the 6th glycine also carried out artificial mutation by we, utilize tryptophane (Trp) to substitute glycine, its receptor binding capacity improves 7-10 doubly, transformation period reaches 2 hours, be conducive to integrity in vivo of medicine and long-lasting, with reference to Pseudomonas Exotoxin A and diphtheria toxin constructional feature with enter born of the same parents' mechanism, add a generally acknowledged transferring film peptide at the C-terminal of LHRH, make it afterbody formation Furin enzyme and cut recognition site, once pharmaceutical composition is attached to tumor cell surface, form endocytosis vesicle under receptor acting time, Furin enzyme in endocytosis vesicle is brought into play enzyme and is cut effect in the time of PH5.2, by antitumor drug active transport to tumour cell.

In the Chinese patent of declaring " preparation and the application of targeting antineoplastic amalgamation protein matter LPO " our successful construction expression take LHRH as guiding, prescind peptide as transferring film effect take Furin enzyme in Pseudomonas Exotoxin A, targeting anti-tumor fused protein take ranpirnase as cytotoxic agent, but this target protein is with inclusion body form expression and purification, total institute is known, the protein expression product of inclusion body form must pass through sex change, renaturation can be carried out purifying and be recovered its biological activity, the purification step more complicated that this technique relates to, simultaneously due to the lower cause of renaturation yield, its product yield is low, be that production cost will increase.For the purifying of this target protein of convenient carrying out, reduce production costs, we have carried out gene reconstitution and have optimized its phraseology on the basis of the preparation of original LPO, make target protein with soluble form amalgamation and expression, and have simplified its purifying process.

Summary of the invention

Object of the present invention is in order to solve targeting antineoplastic amalgamation protein matter LPO with inclusion body form expression and purification, the problem that renaturation yield is lower, and the novel preparation process of a kind of targeting antineoplastic amalgamation protein matter LPO is provided.

A kind of fusion gene fragment tHS-LPO, its large base order is as shown in sequence table SEQ ID NO:3.

A kind of expression vector, it is the expression vector with T7 promotor, and after T7 promotor, has inserted the gene as shown in sequence table SEQ ID NO:3;

Described expression vector is PET28a, that is: PET28a- tHS-LPO.

The novel preparation method of targeting antineoplastic amalgamation protein matter LPO, it comprises:

A. prosthesis synthetic gene as shown in sequence table SEQ ID NO:3 outward;

B. use NdeI, EcoRI double digestion, insert in PET28a, obtain PET28a- tHS-LPO;

C. by PET28a- tHS-LPOin transformed competence colibacillus intestinal bacteria, express; Extract purified fusion protein;

D. under imidazoles exists, carry out metal-chelating medium purification, with SUMO proteolytic enzyme, purified fused protein is carried out to enzyme and cut, purifying.

A kind of targeting antineoplastic amalgamation protein matter LPO, it is prepared with aforesaid method.

Term used herein " LPO ", refer to the gene engineering product that can bring into play in vivo and in vitro anti-tumor activity function, according to the preferred embodiments of the invention, for faithful to natural LPO aminoacid sequence, after specially designing when synthetic gene the codon CAG of the first amino acid Gln of natural LPO being placed in to SUMO proteolytic enzyme identification substrate aminoacid sequence Gly-Gly, said LPO is with the genetically engineered restructuring LPO containing bit codon headed by Met not.

Term as used herein " LPO " refers to the gene engineering product that can bring into play in vivo and in vitro antitumor action, the product that its biological activity should be equal to or prepare higher than former technique due to the transformation of expression-form.

Wherein in the time that gene is synthetic, add NdeI restriction enzyme enzyme recognition site in the 5` of antigen-4 fusion protein gene end for gene is easy to connect, and utilize ATG in NdeI restriction enzyme enzyme recognition site as the first setting up password, the first amino acid Met of accurate translation fusion rotein.Simultaneously add the recognition sequence GAATTC of restriction enzyme EcoRI at the 3` of the protein gene merging end, to all form sticky end when gene fragment is connected with carrier, be beneficial to gene connection.For making expression product reach high efficient expression, intestinal bacteria bias codon is incorporated into wherein, so that expression product is adapted at expression in escherichia coli.

Utilize LPO and former process products prepared by the inventive method to carry out active contrast, found that the standby sample activity of patent system of the present invention is significantly better than former handicraft product.

Term used herein " IC50 " refers to the half cell-lethal concentration of utilizing the described biological products of Products in China rules to measure activity of tumor cells standard verification method, while utilizing the human tumor cells of vitro culture to reach half to suppress, the concentration of medicine is (with reference to Chinese Pharmacopoeia appendix, antitumor drug biological activity determination method, its concentration represents with μ g/ml).

The invention further relates to the method for producing multiple types protein or polypeptide fused in tandem expression target protein matter with recombinant DNA technology, the method comprises: (1) the expression vector that carries T7 strong promoter and contain multiple clone site is provided; (2) the coding gene sequence that synthetic gene is connected in series, is cloned into correct reading frame in the expression vector of linearizing step (1); (3) use the expression vector of step (2) to transform suitable host cell; (4) under the condition that is suitable for expressed fusion protein matter, cultivate the said host cell being converted; (5) from cell culture, utilize suitable purifying process to reclaim and the said fused protein of purifying.

Take N end Met as initial protein molecule for building the THS-LPO molecule of fused protein of the present invention, add successively the gene order, the gene order of HIS6 of being convenient to purifying and the SUMO enzyme that are beneficial to the transsulfurase A that protein disulfide forms and cut identification substrate gene order, it is the gene order of LHRH thereafter, in order to increase the binding ability of LHRH and its acceptor and to extend its transformation period, the 6th amino acids in LHRH sequence is replaced with to Trp by Gly.After complete LHRH decapeptide sequence, and then connect PEA transferring film decapeptide sequence brachymemma and that can be identified by furin enzyme, finally connect 104 amino acid of ONC, and finish with terminator codon TAA.

Because temporary transient shortage merged the ready-made sequence of LPO molecule as the present invention, so use in the present invention the nucleotide sequence of general DNA synthesizer these fusion roteins of composite coding on 1000A solid phase carrier.For the ease of being connected with specific recombinant vectors, can introduce suitable endonuclease (as NdeI and EcoRI) restriction enzyme site at 5 ' of synthetic antigen-4 fusion protein gene sequence and/or 3 ' end, and cause the sticky end that is suitable for connection.Can be according to recombinant technology well known by persons skilled in the art, the gene (or with its cDNA or genomic dna form) that these synthetic reading frames of clones coding are unobstructed, in DNA reorganization operation, generally use the gene clone of standard and Subcloned technology to carry out transfer and the connection of gene fragment.Use Restriction Enzyme cutting method to identify the exactness of sequence closure and possible sudden change, finally carry out sequence confirmation with DNA sequencing.

Analyze with garden two chromatograms and detect and prove that LPO protein molecules are to fold with correct disulfide linkage form by technician computer.

Recombinant protein plasmagene will be operably connected on suitable expression control sequenc, the T that is suitable for using in intestinal bacteria as being connected to ₇, on trp or λ promotor, ribosome bind site and transcription termination signal.Can use known method for transformation, as the electroporation that is suitable for the calcium chloride facture of prokaryotic cell prokaryocyte or is suitable for mammalian cell by recombinant plasmid transformed of the present invention in the host cell of selecting.Can select the positive cell being converted by the antibiotics resistance based on contained antibiotics resistance gene is given on plasmid.Once express required fused protein, can separate and this fused protein of purifying according to methods known in the art.For example, can be from fermenting culture centrifugal collection somatic cells and with N,O-Diacetylmuramidase and ultrasonic treatment it, then ultracentrifugation add saturated ammonium sulphate to carry out fractional precipitation in low phosphorus hydrochlorate (about 20mM) solution.Successively through ion exchange chromatography (IEC) and the required LPO recombinant protein of IMAC chromatography purification.

In general, use polyacrylamide gel to analyze each column chromatography wash-out part with SDS-PAGE electrophoretic method, and monitor it with immunoblotting.Recombinant fusion protein purified product is carried out to inhibiting tumour cells test to detect the biologic activity (Products in China rules 2010 editions are shown in concrete operations) of recombinant protein.

Can be using LPO protein of the present invention as primary activity composition, and add one or more pharmaceutically acceptable carrier or vehicle, make the pharmaceutical composition that is suitable for clinical application.Said carrier or vehicle comprise but be not only limited to phosphate buffered saline (PBS), physiological saline, etc. ooze glucose solution, dextran, dextran etc.According to the difference of treated disease, can in pharmaceutical composition of the present invention, add one or more and protein of the present invention to have auxiliary or synergistic other natural, active compounds that synthesize or restructuring.In addition, can in pharmaceutical composition of the present invention, add low molecular weight peptide, glycine or Methionin and metallic cation (as Zn ²⁺, Mn ²⁺, Mg ²⁺and Ca ²⁺) protein protectant, and the stablizer of polyoxyethylene glycol, carboxymethyl cellulose, polyglycine, gsh.

Can be by the outer approach of conventional route of administration, the particularly gi tract pharmaceutical composition of the present invention that comes into operation, for example, by administration in intravenously, intraperitoneal, intramuscular, intracutaneous, subcutaneous or mucous membrane.The effective dosage ranges of pharmaceutical composition of the present invention can be from several nanogram(ng)s to tens mg/kg body weight/day, but for the concrete dosage of each given patient by according to the character of disease to be treated or pathological state and severity, patient's age, body weight, the factor such as response capacity and administering mode of medicine is determined.

Our laboratory has proved that the LPO albumen that LPO protein prepared by the inventive method is prepared than former technique is all significantly improved to the restraining effect of the tumor cell lines such as colon cancer cell line HT-29, ovarian cancer OVCAR3 cell, adenocarcinoma of cervix HeLa cell and HepG-2 cell line.

Accompanying drawing explanation

Fig. 1 shows the LPO construction of recombinant plasmid figure for expressing LPO fusion rotein;

Fig. 2 shows the THS-LPO construction of recombinant plasmid figure for expressing LPO fusion rotein.

Embodiment

Further illustrate the present invention by embodiment below, but it will be appreciated by those of skill in the art that these embodiment do not form the await the reply restriction of claim scope to the present invention.

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

The Construction and identification of recombinant expression plasmid

A. gene is synthetic: design the prosthesis synthetic following sequence outward of in the present invention, carrying out restriction enzyme site and LPO complete genome sequence according to previously described design concept:

NcoI restriction endonuclease recognition sequence (CCATGG)+GC+LHRH polypeptide gene+PEA transferring film peptide+ONC sequence+terminator codon (TAA)+EcoRI restriction endonuclease recognition sequence (GAATTC), gene order is shown in SEQ ID NO:1, this composition sequence Direct Cloning enters in the T carrier that Dalian Takara company provides, and transform e. coli jm109 bacterium, identify and determine positive clone strain through PCR, positive clone strain enlarged culturing, conventional molecule clone technology extracts plasmid DNA, utilize NcoI, EcoRI this plasmid of double digestion and PET28a plasmid, reclaim object fragment and PET28a plasmid vector sticky end fragment, with correct reading frame at T ₄under ligase enzyme (Promega) exists, object fragment is inserted in the PET28a plasmid vector that same enzyme cuts, the double-stranded expressing gene of construction of fusion protein also transforms JM109 engineering bacteria.

B. identify and determine positive clone strain through PCR, positive clone strain enlarged culturing, conventional molecule clone technology extracts plasmid DNA, utilize NcoI, EcoRI double digestion PET28a-LPO plasmid to identify, correct person carries out DNA sequencing, then positive plasmid transformed competence colibacillus e. coli bl21 (DE3), and by the microbial culture being converted in containing in the LB substratum of kantlex (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, identify with endonuclease cutting and sepharose (2%) electrophoretic method, then positive recombinant plasmid carries out DNA sequence analysis.

Fig. 1: the structure that has shown recombinant plasmid pET28a-LPO.

embodiment 2:the expression of LPO fused protein and the purifying of product:

The e. coli bl21 (DE3) that carries the conversion of recombination plasmid (is contained to T ₇rna polymerase gene) cultivate on the LB agar plate that contains kantlex (50 μ g/ml).After cultivation, select kalamycin resistance bacterium colony, in containing 37 ℃ of cultivations in the LB substratum of kantlex (50 μ g/ml), work as A ₆₀₀reach at approximately 0.4 ~ 0.6 o'clock and add 1mM isopropylthio-β-D-galactoside (IPTG) (final concentration 1mM), 37 ℃ are continued to cultivate 3-4 hour, to induce the expression of object product.Then centrifugal separating cell and substratum, and damping fluid composition will be added in the thalline that contain target protein matter, final concentration reaches 50mM Tris-HCl, pH8.0,1mM EDTA, ultrasonication, 4 ℃ centrifugal (20,000g, 30 minutes), get precipitation (insolubility part) and be fusion rotein crude extract.

Crude extract is through washing, denaturation renaturation processing, the recombinant protein matter obtaining is carried out ion exchange chromatography purifying: the DEAE-Sepharose Fast Flow post (Pharmacia) of damping fluid balance, with TE damping fluid (the 20mM Tris-HCl that contains 0 ~ 0.5M NaCl, pH8.0,1mM EDTA) continuous gradient wash-out, and collect the each component peaks part of protein.Object component peaks part is changed after liquid salt adding through 30 minutes ultrafiltration and concentration of small hollow fibrous ultrafilter (Milipore) effect, make enriched material contain final concentration 1.15M NaCl, use 20mM Tris-HCl, pH8.0,1.15M NaCl damping fluid balance XK 1.6 × 20cm Phenyl-Sepharose Fast Flow post (Pharmacia), then loading, damping fluid scrub stream Chuan Feng, finally utilize the damping fluid (20mM TrisHCl, pH8.0) that contains 1.15M-0.05M NaCl to carry out 100%-0% gradient elution.Collect target protein peak part, utilize the concentrated target protein of small hollow fibrous ultrafilter (Milipore) to 2mg/ml.Utilize 20mM PB, pH7.0,0.15M NaCl damping fluid balance XK 1.6 × 100cm Superdex75 gel-filtration column (Pharmacia), utilizes loading ring to inject sample 5ml, and flow velocity 1ml/min carries out gel-filtration separation and purification, collects protein peak value (A ₂₈₀) partly at-20 ℃, store for future use.So lipidated protein >97% of purifying.

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

Cytopathic effect inhibition test:

By the human tumor cells monolayer cell of cultivating, through tryptic digestion, piping and druming collecting cell suspension, utilizes after cell counting count board numeration, and adjusting cell quantity is 60000/ml, joins (every hole 5000 cells) in 96 well culture plates, 5%CO according to 80 μ l/ holes ₂, under 37 ℃ of conditions, cultivate 4h.The LPO protein example concentration of adjusting preparation is 1mg/ml, and the degerming after filtration of quantitative sample, by waiting times dilution method that the sample of difference amount is added in each cell hole, is then supplied substratum, and making it cumulative volume is 100 μ 1,5%CO ₂, under 37 ℃ of conditions, cultivate 24h.Then discard supernatant liquor in cell plate, every hole adds 50 μ 1 staining fluids, room temperature is placed after 30min, washes away staining fluid with flowing water, and blots residual moisture, every hole adds destainer 100 μ 1, room temperature place 5min, after mixing, by microplate reader take 630nm as reference wavelength, go out to measure absorbancy at wavelength 570nm, record measurement result.

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

embodiment 4:the preparation of THS-LPO expression vector and engineering bacteria

The Construction and identification of recombinant expression plasmid

A. gene is synthetic: design the prosthesis synthetic following sequence outward of in the present invention, carrying out restriction enzyme site and THS-LPO complete genome sequence according to previously described design concept:

NdeI restriction endonuclease recognition sequence (CATATG)+TrxA+His6+SUMO+LHRH polypeptide gene+PEA transferring film decapeptide+ONC sequence+terminator codon (TAA)+EcoRI restriction endonuclease recognition sequence (GAATTC), gene order is shown in SEQ ID NO:1, this composition sequence Direct Cloning enters in the T carrier that Dalian Takara company provides, and transform e. coli jm109 bacterium, identify and determine positive clone strain through PCR, positive clone strain enlarged culturing, conventional molecule clone technology extracts plasmid DNA, utilize NdeI, EcoRI this plasmid of double digestion and PET28a plasmid, reclaim object fragment and PET28a plasmid vector sticky end fragment, with correct reading frame at T ₄under ligase enzyme (Promega) exists, object fragment is inserted in the PET28a plasmid vector that same enzyme cuts, the double-stranded expressing gene of construction of fusion protein also transforms JM109 engineering bacteria.

B. identify and determine positive clone strain through PCR, positive clone strain enlarged culturing, conventional molecule clone technology extracts plasmid DNA, utilize NdeI, EcoRI double digestion PET28a-THS-LPO plasmid to identify, then positive plasmid transformed competence colibacillus e. coli bl21 (DE3), and by the microbial culture being converted in containing in the LB substratum of kantlex (50 μ g/ml), with amplified plasmid dna.After cultivation completes, smudge cells, centrifugal collection plasmid plasmid DNA purification order-checking, the coli strain that contains the plasmid that order-checking is correct is engineering strain, and SEQ ID NO:1 has shown the nucleotide sequence of measured fusion rotein recombination.Figure I has shown the structure of recombinant plasmid pET28a-THS-LPO.

embodiment 4:the purifying of the expression of fused protein and LPO product:

The e. coli bl21 (DE3) that carries the conversion of recombination plasmid (is contained to T ₇rna polymerase gene) cultivate on the LB agar plate that contains kantlex (50 μ g/ml).After cultivation, select kalamycin resistance bacterium colony, in containing 37 ℃ of cultivations in the LB substratum of kantlex (50 μ g/ml), work as A ₆₀₀reach at approximately 0.4 ~ 0.6 o'clock and add 1mM isopropylthio-β-D-galactoside (IPTG) (final concentration 1mM), 37 ℃ are continued to cultivate 3-4 hour, to induce the expression of object product.Then centrifugal separating cell and substratum, and will in the thalline that contain target protein matter, add damping fluid composition, final concentration reaches 50mM Tris-HCl, pH8.0,1mM EDTA, ultrasonication, 4 ℃ centrifugal (20,000g, 30 minutes), get soluble fractions and be fusion rotein crude extract.

Crude extract is through the DEAE-Sepharose Fast Flow post (Pharmacia) of damping fluid balance, with TE damping fluid (the 20mM Tris-HCl that contains 0 ~ 0.5M NaCl, pH8.0,1mM EDTA) continuous gradient wash-out, and collect the each component peaks part of protein.Object component peaks part is changed after liquid through 30 minutes ultrafiltration and concentration of small hollow fibrous ultrafilter (Milipore) effect, make enriched material add 20mM imidazoles, then by using 20mM Tris-HCl, pH8.0,0.15M NaCl, 1.6 × 20cm IMAC post (Pharmacia) that 20mM imidazole buffer balance is crossed, and with the damping fluid that contains 0.15M NaCl (20mM TrisHCl, pH8.0 200mM imidazoles) wash-out.Collect target protein peak part, utilize SUMO enzyme to cut target protein, 30 ℃, 4hr, and again cross IMAC liquid-phase chromatographic column, collect protein peak value (A ₂₈₀) part thoroughly dialysis in 30mM PBS, after dialysis, at-20 ℃, store for future use.So lipidated protein >95% of purifying.

embodiment 5: utilize the inhibiting tumour cells method of vitro culture to measure the biologic activity effect of fusion rotein, (concrete operations are with reference to 2010 editions the 3rd appendix XC of Chinese Pharmacopoeia).

Cytopathic effect inhibition test:

By the human tumor cells monolayer cell of cultivating, through tryptic digestion, piping and druming collecting cell suspension, utilizes after cell counting count board numeration, and adjusting cell quantity is 60000/ml, joins (every hole 5000 cells) in 96 well culture plates, 5%CO according to 80 μ l/ holes ₂, under 37 ℃ of conditions, cultivate 4h.LPO protein example concentration prepared by new, old two kinds of techniques of adjustment preparation is 1mg/ml, and the degerming after filtration of quantitative sample, by waiting times dilution method that the sample of difference amount is added in each cell hole, is then supplied substratum, and making it cumulative volume is 100 μ 1,5%CO ₂, under 37 ℃ of conditions, cultivate 24h.Then discard supernatant liquor in cell plate, every hole adds 50 μ 1 staining fluids, room temperature is placed after 30min, washes away staining fluid with flowing water, and blots residual moisture, every hole adds destainer 100 μ 1, room temperature place 5min, after mixing, by microplate reader take 630nm as reference wavelength, go out to measure absorbancy at wavelength 570nm, record measurement result.

Experimental data adopts computer program to carry out parametric regression calculating: the data acquisitions such as the each experimental point OD value to each laboratory sample, pre-extension rate, sample gradient are processed with computer program.That calculates respectively each laboratory sample partly imitates extension rate, and calculates the concentration (in table 2) of 50% effect point.

<110> MILITARY VETERINARY INST ACADE

the novel preparation process of <120> targeting antineoplastic amalgamation protein matter LPO

<160> 3

<210> 1

<211> 380

<212> DNA

<213> is artificial

<400> 1

ccatgggcca gcactggtcc tacggcctgc gtccgggtac ccgccatcgt cagccgcgcc 60

aggattggct gaccttccag aagaaacata tcactaacac ccgcgatgta gactgcgaca 120

acatcatgtc taccaatctg ttccactgta aggataaaaa cacctttatc tacagtcgtc 180

cagaaccggt aaaggctatc tgtaaaggca ttatcgcgag taagaacgtg ctgactacct 240

ccgagttcta cctgtctgat tgcaacgtta cctcccgccc gtgcaaatat aaactgaaga 300

aaagcactaa caaattttgc gtaacctgcg aaaaccaggc tccggtacat ttcgttggag 360

tcggcagctg ctaagaattc 380

<210> 2

<211> 123

<212> PRT

<213> is artificial

<400> 2

Met Gly Gln His Trp Ser Tyr Gly Leu Arg Pro Gly Thr Arg His Arg

1 5 10 15

Gln Pro Arg Gln Asp Trp Leu Thr Phe Gln Lys Lys His Ile Thr Asn

20 25 30

Thr Arg Asp Val Asp Cys Asp Asn Ile Met Ser Thr Asn Leu Phe His

35 40 45

Cys Lys Asp Lys Asn Thr Phe Ile Tyr Ser Arg Pro Glu Pro Val Lys

50 55 60

Ala Ile Cys Lys Gly Ile Ile Ala Ser Lys Asn Val Leu Thr Thr Ser

65 70 75 80

Glu Phe Tyr Leu Ser Asp Cys Asn Val Thr Ser Arg Pro Cys Lys Tyr

85 90 95

Lys Leu Lys Lys Ser Thr Asn Lys Phe Cys Val Thr Cys Glu Asn Gln

100 105 110

Ala Pro Val His Phe Val Gly Val Gly Ser Cys

115 120

<210> 3

<211> 1080

<212> DNA

<213> is artificial

<400> 3

catatgagcg ataaaattat tcacctgact gacgacagtt ttgacacgga tgtactcaaa 60

gcggacgggg cgatcctcgt cgatttctgg gcagagtggt gcggtccgtg caaaatgatc 120

gccccgattc tggatgaaat cgctgacgaa tatcagggca aactgaccgt tgcaaaactg 180

aacatcgatc aaaaccctgg cactgcgccg aaatatggca tccgtggtat cccgactctg 240

ctgctgttca aaaacggtga agtggcggca accaaagtgg gtgcactgtc taaaggtcag 300

ttgaaagagt tcctcgacgc taacctggcc ggttctggtt ctggccatgg taccggcagc 360

agccatcatc atcatcatca tggcagcggt ctggtgccgc gtggcagcgc gagcatgagc 420

gatagcgagg tgaaccagga agcgaagccg gaggtcaagc cggaggtcaa gccggagacg 480

cacatcaacc tgaaggtcag cgatggcagc tctgagattt tcttcaagat caagaagacc 540

acgccgctgc gtcgtctgat ggaggcgttc gctaagcgtc aaggcaagga gatggacagc 600

cttcgcttcc tgtacgatgg catccgcatt caagctgatc agactccgga ggacctggat 660

atggaggaca acgacatcat cgaagctcat cgtgagcaga tcggaggcca gcactggtcc 720

tacggcctgc gtccgggtac ccgccatcgt cagccgcgcc aggattggct gaccttccag 780

aagaaacata tcactaacac ccgcgatgta gactgcgaca acatcatgtc taccaatctg 840

ttccactgta aggataaaaa cacctttatc tacagtcgtc cagaaccggt aaaggctatc 900

tgtaaaggca ttatcgcgag taagaacgtg ctgactacct ccgagttcta cctgtctgat 960

tgcaacgtta cctcccgccc gtgcaaatat aaactgaaga aaagcactaa caaattttgc 1020

gtaacctgcg aaaaccaggc tccggtacat ttcgttggag tcggcagctg ctaagaattc 1080

Claims (5)

1. a fusion gene fragment tHS-LPO, its large base order is as shown in sequence table SEQ ID NO:3.

2. an expression vector, it is the expression vector with T7 promotor, and after T7 promotor, has inserted the gene as shown in sequence table SEQ ID NO:3.

3. a kind of expression vector claimed in claim 2, described expression vector is PET28a, that is: PET28a- tHS-LPO.

4. the novel preparation method of targeting antineoplastic amalgamation protein matter LPO, it comprises:

A. prosthesis synthetic gene as shown in sequence table SEQ ID NO:3 outward;

B. use NdeI, EcoRI double digestion, insert in PET28a, obtain PET28a- tHS-LPO;

C. by PET28a- tHS-LPOin transformed competence colibacillus intestinal bacteria, express; Extract purified fusion protein;

D. under imidazoles exists, carry out metal-chelating medium purification, with SUMO proteolytic enzyme, purified fused protein is carried out to enzyme and cut, purifying.

5. a targeting antineoplastic amalgamation protein matter LPO, it is prepared by method claimed in claim 4.

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