CN1228995A - Medicine for treating senils dementia, parkinsonism and cardio-cerebral angiopathy - Google Patents

Abstract

A medicine in the form of powdered or liquid injection, capsule, tablet and dropping pill for treating senile dementia, Parkinson's disease, cerebrovascular disease, coronary heart disease, hypertension and atheroscierosis is prepared from nerve growth factor (NGF), brain nerve nutrition factor (BDNF), nerve nutrition factors (NT-3, NT-4 and NT-5), ciliary nerve nutrition factor (CNTF), fibroblast growth factor (FGF), epidermal growth factor (EGF) ganlioside, acetylcholine (Ach), histamine (HA), calcitonin gene related peptide (CGRP), nerve peptide Y (NPY) and cyclonucleotide (cAMP). After controlled enzymolysis of said medicine, a small peptide chain is obtained, which can act on brain via blood-brain barrier.

Description

A kind of medicine for the treatment of senile dementia, Parkinson's disease, cardiovascular and cerebrovascular disease

The present invention relates to a kind of with neurotrophic factor, nerve growth factor, ganglioside, epidermal growth factor, bioactive substances such as fibroblast growth factor are treatment senile dementia, Parkinson's disease, cerebrovascular, coronary heart disease, hypertension, the atherosclerotic medicine that raw material is made.

Cardiovascular and cerebrovascular disease is the first disease of serious harm human life health, and senile dementia and Parkinson's disease also are old people's frequently-occurring diseases.According to World Health Organization (WHO) and each disease foundation statistics of the world, the expense that the world is used for smelting treatment cardiovascular diseases every year reaches 1,280 hundred million dollars, and senile dementia reaches 1,000 hundred million dollars.

The medicine of existing treatment cardiovascular and cerebrovascular disease, senile dementia, Parkinson's disease is as nitroglycerin (nitroglycerin), sorbide nitrate (isosorbide dinitrate, sorbitrate), calcium antagonist, B-adrenergic receptor blocker, a ₁Receptor blocking agent, diuretic, colestyramine (cholestyramine), examine for sending out (cholestipol), nicotinic acid, chlorine tool butyl ester, gemfibrozil (gemfibrozil) Lovastatin (loyastatin), acetylcholinesteraseinhibitors inhibitors (AchE), duxil (Duxil), flunarizine (Sibeline), trihexyphenidyl, kemadrin (Kemadrin), scopolamine, diphenhydramine (benadryl), breath crust (disipal), amitriptyline (amitryptyline), imipramine (imipramine), all can not cure above-mentioned incurable disease, can only the effect of " ineffectual remedy " on control and mitigate the disease, and above-mentioned chemicals all has toxic and side effects in various degree.

The purpose of this invention is to provide a kind of medicine for the treatment of senile dementia, Parkinson's disease, cardiovascular and cerebrovascular disease.This medicine can be regulated and repair because of aging and morbific brain and cardiovascular system, have the permanent curing action of " removing burning wood away under the boiler ", and this medicine is without any side effects to human body.

Senile dementia, Parkinson's disease, coronary heart disease among cerebrovascular and the cardiovascular diseases, hypertension, atherosclerosis all causes because of organism aging process, if with the patient because of aging and impaired brain and cardiovascular system reparation, regulate normal, disease controlling well just, even cure this disease, treatment principle of the present invention is: repair impaired brain by allowing the patient absorb, various growths and trophic factors that cardiovascular system is required, promote neurocyte, the growth of myocardial cell and propagation, promote the functional rehabilitation of interior cutaneous vessel cell and smooth muscle cell, and prolong the time-to-live, make patient's brain and cardiovascular system recover normal gradually.The various effective ingredient of this medicine have detailed description in " positive effect " described later.

The present invention is with nerve growth factor (nerve growth factor, NGF), brain derived neurotrophic factor (brain-derived neurotrophic facfor, BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-5 (NT-5), ciliary neurotrophic factor (ciliary neurotrophic fa-ctor.CNTF), fibroblast growth factor (fibroblast growth fa-ctor, FGF), epidermal growth factor (epidermal growth factor, EGF), ganglioside (ganglioside), acetylcholine (acetylcholine, Ach), histamine (his-tamine, HA), calcitonin-gene-related peptide (calcitonin gene-related pep-tide, CGRP), neuropeptide tyrosine (neuropeptide Y, NPY), cyclic nucleotide (cAMP), atrial natriuretic peptide (atrial natriuretic peptide, ANP), brain natriuretic peptide (brain natriureticpeptide, BNP), C type natriuretic peptide (c-typeNatriuretic peptide, CNP) prostacyclin (PGI ₂), endothelium derived relaxing factor (EDRF) is raw material, above-mentioned raw materials can separately or be mixed and made into the treatment senile dementia, Parkinson's disease, cerebrovascular, coronary heart disease, hypertension, atherosclerotic medicament.

The present invention treats the curative effect of senile dementia, Parkinson's disease, cardiovascular and cerebrovascular disease from the contained various effective ingredient of its raw material, these effective ingredient are confirmed by the most advanced and sophisticated medical science in west, physiological experimentation, are elaborated with regard to the various effective ingredient in this medicine below.

1 nerve growth factor (nerve growth factor, NGF) physiology and health-care effect: growth, the atomization of neurocyte have three phases, by specific neuroblast division growth, divided a word with a hyphen at the end of a line, stretched to particular portion then before this, formed the network that interknits at last.NGF induces axon growth promoting the neurocyte division growth, forms in this three phases of synapse network and is all bringing into play important effect.NGF is indispensable to the existence and the growth of sympathetic nerve and sensory neuron.Inject anti-NGF serum for the period of development animal, remove NGF natural in the body after, just aplasia of the sympathetic chain of animal.Experiment showed, that NGF can promote polyamines synzyme and ODC Ornithine decarboxylase (ODC) increased activity, sympathetic ganglionic cell is very strong to this zymoid dependency in the emergence period, and cubhood is strong more to the facilitation of enzyme.Take in the synthetic of the interior NGF energy accelerator nerve mediator of neurocyte.In sympathetoblast, can increase tyrosine hydroxylase activity, make the synthetic quickening of catecholamines mediator.This process is not to work by second message,second messenger cAMP, increases but pheron is synthetic.NGF also can promote the synthetic of sensory neuron P material.

(brain-derived neurotrophic factor BDNF) can separate from mammal brains such as pig 2 brain derived neurotrophic factors, is a kind of basic protein, isoelectric point, IP 10, molecular weight 12.3kD.Mainly be distributed in the Hippocampus position in the brain, every gram Medulla sus domestica can extract BDNF5ng.The physiology of BDNF and health-care effect; Can promote the growth and the survival of embryo's retinal ganglial cells and substantia nigra of midbrain dopaminergic neuron,, promote its axon growth, prolong the time-to-live of In vitro culture spinal cord Dorsal root ganglion cell and the also nutritious effect of cranial nerve sensory neuron.

(neurotrophin-3 NT-3) is the 3rd member in the NGF family to 3 neurotrophic factor-3s, claims NGF-2 or the Hippocampus NTF that derives again.Ripe NT-3 is the polypeptide that 119 aminoacid are formed, isoelectric point, IP 9.5.NT-3 can promote the axon growth of dorsal root ganglion sensory neuron and prolong the time-to-live, can quicken the growth of knot shape joint and sympathetic ganglionic cell aixs cylinder.

4 fibroblastic growths are at son (fibroblast growth factor, FGF) .FGF is a kind of albumen mitogen, in this family, successively find to have the somatomedin of 7 kinds of structural similarities, wherein the receptor of these two kinds of factors of FGF-1 and FGF-2 (or claiming aFGF and bFGF) is in close relations with the nervous system growth promoter.FGF can stimulate most of division growths that derive from mesoderm and neuroderm cell, as vascular endothelial cell, smooth muscle cell, osteoblast, chondrocyte, one-tenth Skeletal Muscle Cell, lens epithelial cell, iris cells etc.In early days embryonic development period, FGF can induce mesoblastic formation, and some embryonic cells are broken up towards the mesoblastema pattern.Discover that FGF is the cell growth factor with obvious neurotrophic activity.FGF-2 just can make the hippocampal neurons prolonged survival period in the cultivation in a small amount, and projection increases.FGF-2 has this similar effect to many axoneurons.FGF-1 to the existence of keeping the retinal ganglion cell apparently higher than FGF-2.FGF-2 can increase in the cholinergic every neuron survival, saves the neuron that sustains damage.FGF-2 also has nutritional activities to astrocyte; Can promote granulation tissue (mainly being little blood vessel and fibrocyte) new life, quicken the healing of wound.

(epidermal growth factor EGF) is the single chain polypeptide that is made of 53 aminoacid to 5 epithelium growth factors.Molecular weight 6045, isoelectric point, IP 4.6, heat-resisting, 30 minutes non-inactivations still in 100 ℃ of water.People EGF (hEGF) is very similar to mice EGF (mEGF) structure, in 53 aminoacid 37 identical, the position of cystine linkage is also identical.The effect of EGF mainly is chafe and mesoblastema growth.Galactophore epithelial cell, hide fiber cell, respiratory tract and gastrointestinal tract epithelial cell, smooth muscle cell, hepatocyte, vascular endothelial cell and gonad granulocyte etc. all there are growth promotion and proliferation activity.EGF can increase the neuron survival time, promotes axon growth, quickens the propagation of glial cell.Under the effect of EGF, K ⁺Enter with small-molecule substance such as glucose and to speed up in the cell, and quicken the decomposition of sugar, lactic acid is increased.EGF can also promote RNA, DNA and proteinic synthetic.

6, acetylcholine (acetylcholine, Ach).Ach is quite extensive to the biological effect of nervous tissue, comprises nerve---and joint, autonomic ganglion, cholinergic neuron and the neuron paths (periphery and maincenter) of muscle all have biological effect widely.Because cholinergic neuron distributes very wide at maincenter, what confirmation was arranged is up reticular system, limbic system, motor system, sensory system etc., the Ach effect has plenty of irritability, has plenty of inhibition, in general based on irritability, in addition Ach to ingest, adjusting maincenters such as drinking-water, body temperature, blood pressure all have specific effect, concrete biological effect has following several respects: 1. promote the synthetic of phosphate acid ester and phosphatidyl-4 alcohols.2. promote the release of biogenic amine; Ach promotes pheochromocyte to discharge catecholamine, impels mastocyte to discharge histamine.3. promote nervous tissue to discharge thiamine: to find in recent years, in the test of Ach perfusion nervous tissue, find to promote the release of thiamine class material.Confirmed that clinically Ach is effective in cure to the treatment senile dementia, can improve patient's memory and cognitive competence.

7, histamine (histamine, HA), its curative effect increases the capillary permeability except having blood vessel dilating, also have the 1. function of limbic system: histaminergic nerve unit is projected to structures such as many limbic system structures such as Hippocampus, corpus amygdaloideum, nucleus accumbens septi, thereby think the control of itself and emotion, memory function is relevant with the adjusting of vegetalitas function.2. to neural effect: can cause the vasodilation and the axon reflex of reverse property, transmit relevant with the initial sum of peripheral nerve sensation.

8, (calcitonin gene-related peptide-, CGRP), the CGRP precursor is made up of 128 amino acid residues, molecular weight is 16KD to calcitonin-gene-related peptide, contains 76 amino acid whose N terminal sequences identical with the CT precursor.CGRP has powerful vasodilative effect, and cerebral blood flow is obviously increased.

9, neuropeptide tyrosine (neuropeptide Y, NPY).NPY can increase the sensitivity of the smooth muscle of sympathetic innervation to norepinephrine, can bring high blood pressure down, decreased heart rate.

11 cyclic nucleotides.It is the important interior courier of cell; In many cell systems, cell is by 3 ＇ to replying of various adjusting signals, 5 ＇, one cyclic adenosine monophosphates (cAMP) mediation.In nervous system, neurotransmitter, hormone and neuroregulator pair cell cAMP content or (with) 3 ＇, 5 ＇, one cyclic guanylic acids (cGMP) have important regulatory role, cAMP and cGMP are some neururgic cell important regulatory role of mediation, and cAMP and cGMP are couriers in some neururgic cells of mediation.G-protein (G albumen) plays an important role striding in the film transmission of receptor signal.In nervous system, many effect protein matter are subjected to the proteic adjusting of G as the activity of adenyl cyclase, phospholipase C, phospholipase A2 and phosphodiesterase etc.Striding in the film transmission of information, G albumen is " relay station " of contact receptor and effect protein matter.

12, ganglioside (ganglioside): in nervous system, ganglioside has many important function.It is the membrane-associated activity material, concentrates especially in neuronic synapse position.In each stage of nervous system development, along with cell differentiation, synapse foundation and myelin form, and special variation often takes place for the content of ganglioside and composition.Ganglioside participates in the interaction and the information exchanging process of nervous system and environment, and is also relevant with senior activities such as the plasticity of nerve and learning and memories.In addition, ganglioside also has the regeneration that promotes behind the neuronal damage.

13, sharp sodium peptide family comprises atrial natriuretic peptide, brain natriuretic peptide, C type natriuretic peptide, the effect that have sharp sodium, diuresis, vasodilator, brings high blood pressure down.

Aforementioned polypeptides class neurotrophic factor in preparation through controlled enzymolysis, protease digestion NGF as after handling with CNBr in 11 polypeptide that obtain, has 100 times NGF activity by bonded 10-25 peptide of cystine linkage and 75-88 peptide, these little peptide chains can pass through blood brain barrier, act on brain.Perhaps use pepsin and thermolysin (thermoly-sin) digestion to obtain three little peptide chains.More than be injectable lyophilised powder and injection, as for oral formulations such as capsule, tablet, drop pill etc. need not through the controlled enzymolysis of engineered protein enzyme, but (medicine) being taken before meal is used, drink simultaneously into 300-500 milliliter boiled water, making medicine shorter in the time of gastric enzymolysis, is a kind of controlled naturally enzymolysis, makes polypeptide constituents in the medicine that enters small intestinal, resolve into activated little peptide chain, these little peptide chains can pass through blood brain barrier.

Select 89 routine senile dementia patients in the test, inject this medicine 5 μ g every day, matched group 55 examples are taken placebo every day.Through test in 4 months, this medicine effective percentage 81% as a result.The test group conditions of patients takes a turn for the better, and shows as: hypermnesia, and disorientation alleviates, and the CT examination cerebral cortex stops atrophy.

Select 50 routine Parkinsonians in the test, inject this medicine 5 μ g every day; Matched group 50 example is taken placebo every days.Through test in 4 months, this medicine effective percentage 74% as a result, and test group patient's the state of an illness alleviates, as the minimizing of trembling, or gradually had language, an ability of thinking from the packinson dementia.

This medicine is used for the prevention and the treatment of cerebrovascular.

Select 100 people in the test in the cerebrovascular high-risk group, wherein 50 people classify test group as, inject this medicine 3 μ g every day; Matched group 50 people take placebo every day, and through test in 4 months, test group did not have 1 example hemorrhagic or ischemic cerebrovascular take place, and 2 example morbidities are arranged in the matched group; Test in back 1 year, test group does not have 1 example morbidity, and matched group has 7 example morbidities.

Select 64 routine patients with cerebrovascular disease in the test, wherein test I group 30 examples and be Patients with Hemorrhagic Cerebrovascular Disease, test II group 34 examples are the ischemic cerebrovascular patient, and I group patient injects this medicine 5 μ g every day and takes with hyperosmotic dehydration agent (20% mannitol, glycerol saline) or diuretic.II group patient injects this medicine 4 μ g every day and takes the choke piperazine of requiring mental skill.Matched group 50 routine patients with cerebrovascular disease, wherein contrast I group 25 examples and be Patients with Hemorrhagic Cerebrovascular Disease, take placebo every day and take and use hyperosmotic dehydration agent and diuretic, contrast II group 25 examples are the ischemic cerebrovascular patient, take placebo and brain choke piperazine every day.Through test in 6 months, the result showed that this medicine is 68% to the hemorrhagic apoplexy effective percentage, is 76% to the ischemic cerebrovascular effective percentage.

Select 100 routine patients with coronary heart disease in the test, inject this medicine 5 μ g every day and take and use nitroglycerin; Matched group 80 examples are taken placebo every day and are taken and use nitroglycerin; Through test in 5 months, test group patient's angina pectoris or myocardial infarction frequency reduced 85% than test is preceding, and matched group is not seen minimizing.

Select 90 routine hyperpietics in the test, inject this medicine 4 μ g every day and take verapamil (verapamil verapamil), test group patient mean blood pressure before the test: SBP179mmHg and DBP109mmHg; Matched group 90 examples are taken placebo and verapamil every day.Through test in 6 months, the result showed this medicine effective percentage 92%, test group patient's mean blood pressure: SBP136mmHg, DBP93mmHg.The matched group mean blood pressure only reduces 3-5mmHg before testing.

Select 70 routine atherosclerotics in the test, inject this medicine 3 μ g every day, matched group 70 examples are taken placebo every day, and through test in 6 months, the result showed this medicine effective percentage 87%.

The invention will be further described below in conjunction with embodiment.

The present invention is with nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-5 (NT-5), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), epidermal growth factor (EGF), ganglioside (ganglioside), acetylcholine (Ach), histamine (HA), calcitonin-gene-related peptide (CGRP), neuropeptide tyrosine (NPY), cyclic nucleotide (cAMP), atrial natriuretic peptide (atrialnatriuretic peptide.ANP), brain natriuretic peptide (brain natriuretic peptide-.BNP), C type natriuretic peptide (c-type Natriuetic peptide.CNP), prostacyclin (PGI ₂), endothelium derived relaxing factor (EDRF) is a raw material, above-mentioned raw materials can separately or be mixed and made into treatment senile dementia, Parkinson's disease, cerebrovascular, coronary heart disease, hypertension, atherosclerotic medicament.Above-mentioned raw materials all can extract from the animal organ, or uses the technique for gene engineering mass production, and producing of raw material is prior art, below is several examples of producing raw material with technique for gene engineering:

A, epidermal growth factor (EGF).

One, structure and character

The gene mapping of hEGF is in No. 4 chromosomal q24 of people～q26 band.HEGF is the single chain polypeptide that 53 aminoacid are formed, molecular weight about 6200; Isoelectric point, IP is 4.7; Intramolecularly is not with glycosyl, and the N end is agedoite, and the C end is arginine; Three intrachain disulfide bonds are arranged, be distributed in Cys ⁶-Cys ²⁰, Cys ¹⁴-Cys ³¹And Cys ³³-Cys ¹²Between, in the secondary structure of molecule, form three disulphide rings, very important to keeping the required stable space configuration of its biological activity.

It is latent qualitative that hEGF has heat; Put protease hydrolysis easily; Its three disulfide bond are difficult to correct pairing, after the mispairing, cause the protein molecule refolding and biological activity is reduced or disappear.Therefore, utilize engineering bacterium expression hEGF, keep the correctness of these three disulfide bond most important.Usually adopt secretion type expression to help forming correct space structure.

Two, production technology

The mature peptide of hEGF does not contain glycosylation site, and is active irrelevant with glycosyl, so be suitable for expressing in E.coli.Existing both at home and abroad how tame laboratory and company in E.coli successful expression hEGF, the promoter that is adopted comprises Ecoli alkaline phosphatase promoter (phoA), outer membrane protein promoter (omp), Trp promoter and pk promoter etc.Because hEGF is micromolecule polypeptide, in bacterial cell, is difficult for forming inclusion body, and causes protease in the cohesion rally of born of the same parents and attack and degrade.Therefore, the optimal strategy of expression is to adopt secretion type expression.At present the most successful is the secretion type expression that adopts E.coli alkaline phosphatase gene (phoA) promoter and signal peptide.E.coli phoA promoter is subjected to phoA, and synthetic a large amount of alkaline phosphatases (APase) are induced in the adjusting of phoM and phoR gene outcome under the hypophosphate condition.In bacterial cell, Apase is synthetic to have the signal propeptide, mediates sophisticated APase by signal peptide then and passes cell membrane and enter pericentral siphon.If the eukaryotic protein gene is connected with the phoA signal peptide gene, do not keep any unnecessary aminoacid between the two, the phoA signal peptide will obtain processing, and with the eucaryon mature transporter peptide outside bacterial membrane in the pericentral siphon.The foreign protein of expressing can need not the antibacterial of breaking with the method purification of osmotic shock.

Made up the excretion vector of coding phoA promoter and signal peptide first.One of them is pTA1529.This carrier has single Hind III limiting enzyme point at 3 ＇ of phoA signal peptide end, can easily insert foreign structural gene in this position, and no any base is inserted or disappearance between phoA signal peptide and the insertion body.Cut pTA1529 DNA with the Hind III, repair sticky end, and, reclaim Sal I-big fragment of Hind III with the digestion of Sal I restriction endonuclease with the T4DNA polymerase.From cloned plasmids carrier pTA1002, cut out the hEGF structural gene of synthetic with the same manner, make it to be connected, be built into hEGF secreted expression carrier pTA1522 with Sal I-Hind III fragment of pTA1529 with the T4DNA ligase.With pTA1522 Transformed E .coli APase deletion mutation strain YK537, transform bacterial strain under the hypophosphate condition, 30 ℃ of inducing culture 9h collect thalline, and osmotic shock is handled, lyophilizing.Product is collected the hEGF eluting peak through SephadexG-50 post gel filtration, lyophilizing, and dry is soluble in water, again through μ bond pakc18 column chromatography, can get highly purified reorganization hEGF, and output is 2413 μ g/L cultures, or each cell 1.8 * 10 ⁵Molecule.

Because identical signal peptide is used in the secretion of the hEGF of Apase and expression,, suppress the secretion of hEGF so the endogenous APase of E.coli may compete with the secretion of hEGF.Use Apase defective strain YK537 can avoid the appearance of this problem, the yield that makes reorganization hEGF is than 5 times of raisings in non-APase defective strain (as C600 etc.).Result of the test shows in addition, induces 9h than inducing 6h output to improve 2 times at 37 ℃ at 30 ℃.

Similar process route is also adopted in the production of domestic reorganization hEGF.The hEGF structural gene of synthetic is made up of 6 synthetic fragments, and totally 159 base pairs add EcoR I point of contact at gene 5 ＇ end, and 3 ＇ hold interpolation.Hind III point of contact.Synthetic hEGF gene and multicopy plasmid pI218R are used EcoR I-Hind III double digestion equally, and the reorganization connection, with connecting product Transformed E .coli JM103 bacterial strain, Analysis and Screening goes out recombiant plasmid pEG14.Utilize synthetic E.coli phoA gene promoter, signal coding sequence and above-mentioned hEGF gene constructed expression plasmid p-AE-8.Transform YK537 with this plasmid, filter out hEGF and express the highest bacterial strain EE-86 as engineered strain.With 37 ℃ of fermentation culture of engineered strain, make bacterial growth to more than the A660nm=1, in containing low phosphorus hydrochlorate culture medium, begin to induce it to efficiently express.After inducing 10h, be secreted in the culture medium more than the hEGF90% of expression, reach as high as the 30mg/L culture medium.Get fermented liquid supernatant, through ammonium sulfate precipitation, dialysis, gel permeation chromatography, ion-exchange chromatography and reversed-phase HPLC, purity can reach more than 98%.

Utilize fed-batch culture (Fed-Batch culture), in E.coli mass production hEGF.The expression plasmid carrier that they make up is pTRLBT1, and this plasmid contains trp promoter, terminal 6 the amino acid whose coded sequences of trpL gene N-and fuses synthetic hEGF structural gene in its downstream.Change than E.coliHB101 with this plasmid, filter out engineered strain.Because hEGF produces the acetate assimilation of bacteria growing inhibiting and antibacterial,, cell culture is divided into two steps so engineering bacterium fermentation adopts the fed-batch culture mode.The first step adds tryptophan and suppresses trp promoter expression hEGF gene, makes the cell growth; Second step, from culture medium, remove tryptophan, induce trp promoter expression hEGF gene, produce hEGF.Engineering bacteria is cultivated in 5 liters of fermentation tanks, and initial working volume is 2L, comprises 0.2L spend the night strain, 1g/L glucose and 0.2ml foam reducing composition.PH keeps 7,37 ℃ of temperature, gas flow rate 2L/min, mixing speed 400～1000r/min.Transfer pH with ammonia (6mol/L).By changing mixing speed dissolved oxygen levels is controlled at 1～3ppm.When mixing speed reaches the highest 1000r/min, replenish 50% oxygen to replace air to fermentation tank.By detecting acetate concentration control charging rate, so that acetate concentration is maintained below the 33mmol/L.Raise culture medium (feeding medium) by the 200g/L glucose, the 1000g/L casein hydrolysate, the 60g/L yeast extract, 0.8g/L tryptophan and 0.1g/L ampicillin are formed, pH7.0.When inducing hEGF gene expression, from raise culture medium, get rid of yeast extract and tryptophan.Antibacterial culturing 8h changes culture medium.Culture medium one changes hEGF and begins at once to produce, and cultivates when finishing, and output reaches 60mg/L.Reorganization hEGF produces with the fusion rotein form that the N-end fuses proteic 6 amino acid residues of TrpL, and is present in the cell with the form of inclusion body.

Three, the method for inspection

1. quality testing reorganization hEGF product is transparent supernatant liquid; SDS-PAGE determining molecular weight 6045; It is 4.55～5.20 that isoelectric focusing electrophoresis is measured isoelectric point, IP; It is consistent with standard substance that reversed-phase HPLC is analyzed peptide figure; It is consistent with document that the amino acid sequence analysis instrument is measured the n terminal amino acid sequence; HPLC measures purity＞95%.

2. biological activity determination is a labelled amount 80%～120% with MTT A431 cell inhibition method mensuration biological value.

B, fibroblast growth factor (FGF).

One, the gene of bFGF

People bFGF gene is positioned at chromosome No. 4, is single copy gene, is made up of 3 exons and 2 introns, and length is greater than 38kb.First intron is between the 60th and 61 bit codons, and the 2nd intron is between the 94th and 95 bit codons.BFGF mRNA has two kinds of forms, is respectively 4.6kb and 2.2kb.CDNA sequence analysis by the mRNA reverse transcription shows in the readable framework of bFGF cDNA, a common AUG start codon and UGA termination codon are arranged.This frame amino acid sequence coded and the bFGF consensus amino acid sequence of from tissue, purifying.In addition, three uncommon start codon CUG are arranged on three diverse locations in common start codon front.Confirmed that these CUG codons start the synthetic of the bFGF of three kinds of terminal prolongation forms (molecular weight be respectively 24,23.1 and 22kD) respectively.

Two, the structure of bFGF

BFGF is a single chain polypeptide.By 155 precursor molecules that aminoacid is formed of primary transcript coding that cDNA infers, molecular weight is about 18kD, does not wherein contain signal peptide.The bFGF peptide chain of purifying from different tissues is different in size.Separate the bFGF that purifies with hypophysis from Medulla Bovis seu Bubali the earliest and form, and corpus luteum bFGF only is made up of 130 aminoacid by 146 aminoacid.Being less than 155 amino acid whose bFGF is the amino terminal clipped form.Confirmed that the shortening of bFGF amino terminal is less than 25 aminoacid and does not influence its activity, illustrated that n terminal amino acid does not participate in combining of biological activity or bFGF receptor with the cell table.

The bFGF intramolecule has two potential heparin lands, and one is positioned at N-terminal the 18th～22 residue, and another is positioned at the 107th～110 residue of c-terminus, therefore heparin is had very high affinity.Secondary structure analysis shows, the 37th～40 and the 78th～81 residue is putting upside down of the fine essence-Gan-Radix Asparagi-X sequence that connects plain (fibronectin) just, may be present in proteinic outside, and these residues can make bFGF be attached to the recognition site of cell surface.

BFGF has four cysteine, forms two disulfide bond.BFGF is extremely conservative during evolution, as the bFGF of people and Niu, has only two differences in 146 aminoacid, and homology reaches 98.7%, and bird bFGF forms identical with the acid of cattle bFGF nitrilo.

The 26S Proteasome Structure and Function of bFGF is similar to aFGF, but the specific activity aFGF of bFGF is big 10 times～and 100 times, and the cell distribution of aFGF is more limited, mainly in cerebral tissue, retina, kidney and osseous tissue, bFGF then is distributed in brain, hypophysis, retina, kidney, adrenal gland, Placenta Hominis, prostate, thymus, bone, immune system and the various tumor.Basically all cells all have specificity, the high-affinity receptor of bFGF.Therefore, bFGF is a kind of basic regulatory factor, works by autocrine and two kinds of mechanism of paracrine.

Three, the physicochemical property of bFGF

The isoelectric point, IP of bFGF is 9.6: to thermally labile, easily by proteasome degradation; Heparin is had very strong affinity, and after heparin combined, biological activity was constant, but the stability of temperature and acid is strengthened, and was difficult for by proteasome degradation.

Four, recombinant human bfgf's production technology

Utilize the T7 promoter expression system in E.coli, to express people bFGF, and utilize heparin-Sepharose affinity column chromatography to realize the single step purification of recombinant bfgf.

1. the basis of the structure of expression plasmid pJU1005 structure bFGF expression vector is plasmid pET-3b.This plasmid is the derivant of pBR322, contains φ 10 gene promoters, translation initiation position and preceding 11 codons of T7 phage and the transcription stop signals of T φ-t7 rna polymerase identification.These sequences are inserted in the BamH of pBR322 I site, make to transcribe counterclockwise to carry out.With the two enzymic digestions of EcoR I-EcoRv, remove a 185bp fragment that contains endogenous EcoR I, Cla I and Hind III point of contact among the pET-3b.Insert a synthetic poly joint (Polylinker) at BamH I point of contact, wherein, near the T7 promoter be BamH I point of contact, distal-most end be Xho II point of contact.With the Bgl II-Sall fragment among a 650bp EcoR I-Sall fragment (having eliminated inner BamH I and Hind III point of contact with bisulfites mutation) the displacement pET-3b of homologous tet gene, recovered the tet gene of this plasmid, made it have amicillin resistance and tetracyclin resistance simultaneously.This plasmid called after pJU1003.Be respectively the joint at BamH I and Nco I point of contact with synthetic two ends, bFGF cDNA sequence directly is connected with T7 phage gene 10 among the pJU1003, obtain expression plasmid pJU1005

2.bFGF expression and purification with plasmid pJU1005 Transformed E .coli BL21 (DE3) bacterial strain, filter out positive transformed bacteria.Transformed bacteria is cultured to the logarithm later stage in 30 ℃ of culture medium that containing tetracycline, adds 0.1mmol/L IPTG and induce bFGF to express.Gather in the crops thalline behind the 2h, ice bath cooling fast.In 4 ℃ centrifugal, every gram cell precipitation is resuspended in the 4ml buffer A (50mmol/L Tris-HCl pH7.5), uses the French cell press smudge cells.Cell pyrolysis liquid is transferred to the 0.5mol/L sodium chloride concentration, in 4 ℃ centrifugal, remove cell debris.The suspension composition that contains recombinant bfgf further is diluted to the volume corresponding to the 5ml/g wet cell weight with the buffer A that contains 0.5mol/L sodium chloride, directly is added to and uses chromatography on the equilibrated heparin of same buffer-Sepharose affinity column in advance.Wash post with the level pad that diploid is long-pending, then, reuse contains the buffer A of 1mol/L sodium chloride to be washed, and reaches baseline value until the A280nm of effluent.With the buffer A that contains 2mol/L sodium chloride eluting bFGF from the post, obtain highly purified bFGF.

The recombinant bfgf that this expression system produces accounts for more than 10% of bacterial protein, and output reaches 40mg/L culture/A260nm unit.

Five, the determination of activity of recombinant bfgf

By ³H-TdR mixes method, measures its mitogen activity with mice 3T3 fibroblast.

C, nerve growth factor (NGF).

NGF is extracted by the snake venom gland, is purified by the separation of mice lower jaw gland again afterwards.The NGF that purifies from mice lower jaw gland is that a kind of sedimentation coefficient is the complex of 7S, contains three kinds of protein subunits of α, β and γ, consists of α 2 β γ 2, molecular weight 130kD.The γ subunit is the specific ester peptidase of a kind of arginine, participates in the processing of β-NGF precursor.α subunit and γ subunit have the homology more than 80%, but do not have the ester peptidase activity.Have only the β subunit to have the NGF activity.

The α of 7SNGF, β and γ subunit be by weak non-covalent bond combination, and be only o'clock stable in pH5～8.When pH＞8 or pH＜5, perhaps simple rare falling.7SNGF can be dissociated into α, β and γ subunit.Under physiological condition, NGF exists with 7S complex form.Each subunit isoelectric point, IP difference of 7SNGF can separate (seeing Table fully by ion-exchange chromatography

Utilize the carboxymethyl cellulose column chromatography to obtain the β-NGF of active unit of NGF, it has whole physiological effecies of NGF.Sedimentation coefficient is 2.5, molecular weight 26kD, and isoelectric point, IP is 9.3, by two non-covalent dimers that connect into of identical strand, every chain is made up of 118 aminoacid.The N-end is that Ser.C-is terminal for Arg, contains three intramolecular disulfide bonds.

(2) people NGF (hNGF) and gene thereof

Because synthetic NGF amount is extremely low in the tissue, the research of hNGF is very difficult.

Utilize 665bpHga I-Hinc II fragment of mice β-NGF clone cDNA from people's gene group library, to filter out h β-ngf gene DNA sequence of 3.9kb as probe.This sequence contains two zones that are respectively 124bp and 738bp, with the propetide of mNGF former (prepropeptide) gene height homology, is two exons of hNGF gene, and wherein big exon of 738bp contains ripe h β-NGF coded sequence.The intervening sequence that a 6.6kb is arranged between these two exons is between-126 and-125.-187 ,-121 and-119 three ATG triplets are arranged, wherein-121 ATG may be the propetide protogene start codon of h β-NGF.-121 upstream, the little exon that comprises 124bp may be 5 ＇ non-translated sequences.The signal peptide of β-NGF (121-104) and ripe β-NGF high conservative between people and mice, homology is respectively 94.5% and 90%.The-terminal amino acid of ripe h β-NGF is Ser, and identical with mNGF, the C-end has more two aminoacid Arg and Ala than mNGF.Therefore, h β-NGF is 120 aminoacid, and the 45th Asn wherein may be the N-glycosylation site.

(3) character of hNGF

The propetide of hNGF is former to contain 241 aminoacid, and signal peptide is 18 aminoacid, and ripe β-NGF is 120 aminoacid.Molecular weight 13000 has a N-glycosylation site, and six Cys residues form three intrachain disulfide bonds.Isoelectric point, IP is 8.86.Exist with dimer in vivo.

(4) reorganization hNGF production technology

At present, reorganization hNGF has obtained to express in E.coli, yeast and zooblast.But, according to the test report of having announced, the expression of reorganization hNGF is generally all lower, and the NGF activity of E.coli and yeast expression only is equivalent to 1/200～1/1000 of mice lower jaw gland mNGF, and equates with mNGF is active with the NGF of animal cell expression.

Utilize the expression plasmid pTB1058 that makes up in Chinese hamster ovary celI, to express hNGF.

1. the structure of expression vector plasmid, filters out one and has the positive colony that 15kb inserts body as probe with the 0.38kb hNGF coded sequence of synthetic from people's leukocyte genome λ EBL3 library.This nucleotide sequence that inserts an open reading frame in the body is identical with the open reading frame of the coding prepro-NGF (by Met-121 to Ala120) of report.Insert Bcl I-Apa I fragment of isolating a 0.8kb the body DNA, the C-end parts of coded signal peptide from this.Peptide former (propeptide) and ripe NGF.The synthetic DNA of this fragment with coded signal peptide N-end parts is connected, obtain complete hNGF gene, be inserted into the downstream of murine leukemia poison (MuLV) long terminal repeat (LTR) among the plasmid pTB399 (having removed II-2cDNA wherein), obtain plasmid pTB1054.From pTB1054, isolate the 2kb Cla I fragment that contains MuLV LTR and hNGF gene, insert the plasmid pTB348 contain dihydrofolate reductase (DHFR) cDNA (Cla I point of contact obtains expression plasmid pTB1058

(5) check of reorganization hNGF

Reorganization hNGF molecular weight by the SDS-PAGE electrophoretic determination is 13000; Isoelectric point, IP is 9～10; The n terminal amino acid sequence with infer from the hNGF gene nucleotide series identical; The carboxyl terminal aminoacid of determining according to hydrazinolysis is Ala, and manying two aminoacid with the c-terminus of the ripe hNGF of deduction such as Ullrich than mNGF is Arg ¹¹⁹And Ala ¹²⁰Conclusion conform to; Use pepsin and thermolysin (thermolysin) digestion to obtain three fragments successively, each segmental amino acid sequence analysis determines that three disulfide bond are positioned at Cys ¹⁵-Cys ⁸⁰, Cys ⁵⁸-Cys ¹⁰⁸And Cys ⁶⁸-Cys ¹¹⁰, identical with mNGF.

According to stimulating the growth of PC12 cell axon and supporting special biologic activity and the 2.5SmNGF of the reorganization hNGF that the survival of chick embryonic dorsal root ganglion sensory neuron is measured to be complementary.D, brain derived neurotrophic factor (BDNF).

(1) structure and character

The protein that BDNF is made up of 119 aminoacid, molecular weight are 13500; Intramolecularly has three disulfide bond and a glycosylation site; Isoelectric point, IP is 9.99.Exist with dimer in vivo, mainly be distributed in the central nervous system, repair neuronic growth, differentiation and damage back that the cholinergic neuron of the dopamine neuron of the sensory neuron of multiple neuron such as motor neuron, neural ridge and substrate origin, black substance, basal forebrain and Hippocampus, cortex, optic segment etc. are located facilitation.Recent findings, BDNF also has the function that promotes learning and memory.

(2) production technology of reorganization BDNF

(also have NT3, gene structure NT4/5) is similar to NGF, and the coded sequence of maturation protein is positioned at single exon, does not have intron at interval for BDNF.Therefore, can utilize PCR method directly is that template amplification comes out with human gene group DNA.He Xiaolong etc. (1995) utilize this method to prepare the encoding gene of BDNF, and obtain than high expressed in E.coli.

At first design two kinds of PCR primers: primer I contains N-end parts coded sequence and the Nde I restriction enzyme site of ripe BDNF, and primer 2 contains the complementary series of ripe BDNF C-end parts coded sequence, and adds BamH I restriction enzyme site after termination codon.The BDNF gene that pcr amplification is gone out inserts between the Nde I and BamH I site of plasmid pET-3a and pET-16a, and Transformed E .coli JM109 strain through Screening and Identification, obtains containing the recombiant plasmid pET-3B and the pET-16B of BDNF gene respectively.These two plasmids are all controlled the expression of BDNF by the T7 promoter.

With recombiant plasmid pET-3B and pET-16B Transformed E .coli BL21 (DE3) strain again, and usefulness IPTG abduction delivering (37 ℃, 3h).The SDS-PAGE electrophoresis of cellular lysate liquid shows, the pET-3B engineering bacterium expression protein of the about 15kD of molecular weight, be nonfused BDNF, account for 15.4% of bacterial protein.N-hold preceding 15 amino acid sequence analysis results except that initiating terminal many Met, in full accord with bibliographical information, but molecular weight and theoretical value that SDS-PAGE shows have certain difference, reason is unclear.The pET-16B engineering bacterium expression molecular weight be the protein of 16.5kD, N-end has merged the BDNF of 21 peptides, accounts for 47.2% of bacterial protein.21 peptides wherein contain 10 successive histidine and factor Xa specific recognition site.Therefore, can be with special histidine in conjunction with resin (Novage company product) purification, 21 peptides that merge with factor Xa excision N-end obtain ripe BDNF then.

E, ciliary neurotrophic factor (CNTF).

(1) structure and properties

CNTF is made up of 200 aminoacid, and molecular weight is 24kD, and N-holds not band signal peptide, is not typical excreted factor.Intramolecularly only has a Cys residue at the 17th, does not contain disulfide bond, does not also contain glycosylation site.Isoelectric point, IP is 5.78, is acidic protein.CNTF all has the survival of promotion and injury protection effect to sensory neuron, motor neuron, Hippocampus cholinergic neuron, nigral dopaminergic neuron unit etc.

(2) production technology

From people CNTF cDNA cloning vehicle pKSH CNTF plasmid, cut out the CNTF genetic fragment with the EooR I, orientation is inserted into the EcoR I point of contact of the carrier pBPL that contains the PL promoter, Transformed E .coliJM83, obtain expressing year this pBLHc through Screening and Identification, Transformed E .coliHB101 obtains the CNTF expression strain.This bacterial strain is through cultivating and thermal induction, and having expressed molecular weight is the protein of 24kD, accounts for 25% of bacterial protein.

Collect thalline, ultrasonic degradation, centrifuging and taking supernatant.The supernatant ammonium sulfate precipitation is collected the deposited components of 30%～60% saturation, and (50mmol/L Tris-HCl, 5mmol/LEDTA pH7.5) after the dissolving, dialyse to TE with the TE buffer.Go up DEAE-A50 ion exchange-cross-linked dextran gel column chromatography then.Add 0.5mol/L sodium chloride (pH7.5) with TE and TE and carry out linear gradient elution, collect the CNTF enriched composition,, go up Sephacryl S-200 post again, use the TE eluting the TE dialysis, must purification CNTF, purity is more than 95%, yield 25.4%.

The activity of measuring recombinant C NTF with the chick embryonic dorsal root ganglion culture method is 1.3TU/ng.ITU (active unit) is illustrated in the mass number that can make neurocyte reach the required sample of the several halfs of maximum keep alive in the 1ml culture fluid herein.

Polypeptide class neurotrophic factor in the raw material needs the protease digestion after CNBr handles, obtain the active small peptide chain after, make injectable lyophilised powder or injection.

Oral formulations need not through protease digestion, but after raw material made cold-dry powder, directly makes capsule, tablet, drop pill or other oral formulations.

Claims (3)

1, a kind of treatment senile dementia, Parkinson's disease, the medicine of cardiovascular and cerebrovascular disease, it is characterized in that it is with nerve growth factor (nerve growth factor, NGF), brain derived neurotrophic factor (brain-derived neurotrophic factor, BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-5 (NT-5), ciliary neurotrophic factor (ciliary neurotrophic fa-ctor, CNTF), fibroblast growth factor (fibroblast growth factor, FGF), epidermal growth factor (epidermal growth factor, EGF), ganglioside (ganglioside), acetylcholine (acetylcholine, Ach), histamine (his-tamine, HA), calcitonin-gene-related peptide (calcitonin gene-related pep-tide, CGRP), neuropeptide tyrosine (neuropeptide Y, NPY), cyclic nucleotide (cAMP), atrial natriuretic peptide (atrial natriuretic peptide, ANP), brain natriuretic peptide (brain natriuretic peptide, BNP), C type natriuretic peptide (C-typeNatriuretic peptide, CNP), prostacyclin (PGI ₂), endothelium derived relaxing factor (EDRF) is raw material, above-mentioned raw materials can separately or be mixed and made into treatment senile dementia, Parkinson's disease, cerebrovascular, coronary heart disease, hypertension, atherosclerotic medicament.

2, medicine according to claim 1 is characterized in that said medicament is a said dosage form on any pharmaceutics.

3, medicine according to claim 1 is used for the treatment of senile dementia, Parkinson's disease, cerebrovascular, coronary heart disease, hypertension, atherosclerosis.