CN106890192A

CN106890192A - A kind of mixture from blood plasma for strengthening memory and its preparation method and application

Info

Publication number: CN106890192A
Application number: CN201710092755.2A
Authority: CN
Inventors: 崔文宏; 张丽丽; 孔毅荣; 郝敬雨; 石浩威; 任园园
Original assignee: Individual
Current assignee: Beijing Haosi Mass Spectrometry Technology Co ltd; Hunan Haosi Biotechnology Co ltd
Priority date: 2016-12-29
Filing date: 2017-02-21
Publication date: 2017-06-27
Anticipated expiration: 2037-02-21
Also published as: CN106890192B

Abstract

The invention belongs to biology field, and in particular to a kind of mixture from blood plasma of enhancing memory and its preparation method and application.The mixture includes multiple proteins and various small molecules, and the SDS PAGE denaturation gel electrophoresis of the mixture have the high-visible band of 7 naked eyes.The preparation method includes successively：Plasma collection, filter at low temperature, low temperature ultrafiltration, cold sulphur ammonium precipitation, SD inactivations centrifugation, anion exchange, for the first time concentration, molecular sieve, dialysis, second concentration step；Between the plasma collection, cold sulphur ammonium settling step, filter at low temperature, low temperature ultrafiltration step can also be included.The curative effect of mixture prepared by the present invention is mainly lifting old dementia patients human-subject test and enhancing memory；Each step of above-mentioned preparation method, parameter setting rationally, between act synergistically, the quality of final products is improve jointly；The method may be directly applied to amplify metaplasia product.

Description

A kind of mixture from blood plasma for strengthening memory and its preparation method and application

Technical field

The invention belongs to biology field, it is related to a kind of mixture from blood plasma, in particular it relates to one kind is from blood Mixture in slurry separate, for strengthening memory and preparation method thereof, the mixture can be used to preventing, improve and treating old Dementia disease, and with the effect of enhancing memory.

Background technology

Alzheimer disease (Alzhermer ' s disease, AD) is a kind of with acquired hypomnesia, cognitive function barrier The central nervous system degenerative disease for principal character such as hinder.As China human mortality aging process accelerates, the illness rate of AD In substantially increase trend.Alzheimer disease has become the No.1 chronic killer of the Health and Living for threatening the elderly.

The cause of disease of Alzheimer's disease is complicated, current to have researched and proposed various possible factors and hypothesis, popular The gene theory that has (research thinks that 4 kinds of genes are relevant with AD, and app gene is relevant with early onset familial AD, apo E (ApoE) gene and tardy AD and distribute that AD is relevant, presenilin-1 (PS-) and presenilin -2 (PS-2) gene are relevant with AD.About The cause of disease of 50%AD patient is relevant with said gene.), cholinergic theory (AD brain in patients region of interest cholinergic neurons Serious loss, denaturation and functional defect result in the generation of AD.), (the low of serum calcium level causes metabolic calcium disorder theory Nerve cell is denatured, promote AD develop), (big increasing for intracerebral aluminium content causes memory function to poisoning by aluminum theory The deposition gone down with big intracerebral part particular matter) etc..

The medicine of AD has two classes：One class is the medicine of the spiritual pathological symptom that control occurs together, such as anxiolytic, anti-suppression Strongly fragrant medicine and antipsychotic drug etc.；Another kind of is the medicine of nootropic or improvement cognitive function, is mainly used in improving cognitive function, is delayed Progression of disease, acts on the medicine and brain metabolic activation medicine of neurotransmitter.In a word, treatment there is no specific short at present, with right Based on disease treatment, the medicine being currently known all is accompanied by stronger toxic and side effect.Accordingly, it would be desirable to research and develop new more effective, safety Therapeutic AD medicines.

Research on animal model recently shows that the blood for being transfused young mice can improve the latter to AD model mices AD symptoms, (Middeldorp J, Lehallier B, Villeda SA, Miedema SS, Evans E, Czirr E, Zhang H,Luo J,Stan T,Mosher KI,Masliah E,Wyss-Coray T.Preclinical Assessment ofYoung Blood Plasma for Alzheimer Disease.JAMA Neurol.2016 Nov1；73(11):1325- 1333.).Stanford Univ USA carries out the clinical trial of the haemodialysis AD patient using the young man of less than 30 years old.

The content of the invention

For the defect of prior art, an object of the present invention is to provide a kind of mixing from blood plasma for strengthening memory Compound.

The second object of the present invention is the preparation method for providing said mixture.

The third object of the present invention is to provide the application of said mixture.

To achieve these goals, present invention employs following technical scheme：

A kind of mixture from blood plasma for strengthening memory, the SDS-PAGE denaturation gel electrophoresis of the mixture at least include 7 band, molecular size range is respectively：25kD、48kD、67kD、71kD、115kD、155kD、200kD.

In said mixture, used as a kind of preferred embodiment, the blood plasma derives from mammal；It is highly preferred that The blood plasma derives from the mankind.

In said mixture, as a kind of preferred embodiment, analyzed by protein spectrum, in the mixture at least Contain 67 kinds of albumen；Preferably, the mixture also includes and the protein bound small molecule；It is highly preferred that described 67 kinds Albumen is as shown in Table 1 below.

The preparation method of said mixture, comprises the following steps successively：Plasma collection, cold sulphur ammonium precipitation, SD inactivations, centrifugation Separation, anion exchange, for the first time concentration, molecular sieve, dialysis, second concentration step；Preferably, the plasma collection procedure Afterwards, before the cold sulphur ammonium settling step, also including filter at low temperature step and low temperature ultrafiltration step.

In the preferred embodiment of preparation method, in the plasma collection procedure, blood is collected using anticoagulant tube, then By the way that supernatant is collected by centrifugation, blood plasma is obtained；Preferably, in the centrifugation, centrifugal force is 500-1300g, and the time is 10-40 points Clock, centrifuging temperature is 0-5 DEG C.

In the preferred embodiment of preparation method, in the filter at low temperature step, under stress by the blood Slurry is filtered, and obtains filtrate；Preferably, the aperture of filter membrane is that, not less than 0.22 micron, the aperture of more preferably described filter membrane is 0.22 micron and 0.45 micron；Preferably, the temperature of the filtering is 0-15 DEG C, and pressure is 1-20MPa.

In the preferred embodiment of preparation method, in the low temperature ultrafiltration step, the filtrate is pressed by applying Power carries out film bag ultrafiltration, obtains low temperature ultrafiltration product；Preferably, the film bag molecular cut off is 3kD-10kD；Preferably, institute The temperature of ultrafiltration is stated for 0-15 DEG C, pressure is 1-20MPa；Preferably, the buffer solution that the ultrafiltration is used be phosphate buffer, One kind in Tris- hydrochloride buffers, HBS buffer solutions；It is highly preferred that the concentration of the Tris- hydrochloride buffers is 0.2- 300mM, pH value are 7.0-9.2, and the concentration of the phosphate buffer is 1.0-500mM, pH value is 6.0-9.0, the HBS bufferings The concentration of liquid is 0.5-250mM, pH value is 6.4-8.4；It is further preferred that it is that 250mM, pH value are that the buffer solution is concentration 8.0 Tris- hydrochloride buffers.

In the preferred embodiment of preparation method, in the cold sulphur ammonium settling step, by the low temperature ultrafiltration product Or blood plasma mix with the ammonium sulfate solution of saturation after stand, collect supernatant；The supernatant is mixed with the ammonium sulfate solution of saturation again Stood after conjunction, retain precipitation, obtain cold sulphur ammonium precipitated product；Preferably, the temperature of the ammonium sulfate solution of the saturation is 0-4 DEG C； Preferably, the volume ratio of the ammonium sulfate solution of the saturation and the low temperature ultrafiltration product or blood plasma is 1：(9-10)；Preferably, institute The volume ratio of the ammonium sulfate solution and the supernatant of stating the saturation is 1：(1-5)；Preferably, the dwell temperature is 0-10 DEG C, time of repose is 8-16 hours.

In the preferred embodiment of preparation method, in the SD inactivation steps, molecular weight retention product is used SD inactivators stand after suspending again, obtain inactivating product；Preferably, the dwell temperature is 4~37 DEG C, and the time is 6~20 Hour；More preferably described dwell temperature is 20 DEG C, and the time is 10 hours；Preferably, the SD inactivators contain 0.3~2% N- butyl triphosphate, 0.5~2% tween.

In the preferred embodiment of preparation method, in the step with centrifugal separation, after the inactivation product centrifugation Retain supernatant, obtain centrifugation product；Preferably, in the centrifugation, centrifugal force is 2000-10000g, and the time is 10-30 Minute, temperature is 0-10 DEG C.

In the preferred embodiment of preparation method, in the anion exchange step, by the centrifugation product Step level wash-out is carried out in addition anion-exchange column, the eluent for obtaining every time is collected, anion exchanged product is obtained after mixing； Preferably, in the step level wash-out, first eluted with first time elution buffer, obtain first time eluent；Again with washing for the second time De- liquid wash-out, obtains waste liquid；Third time elution is used again, obtains third time eluent；Merge the first time eluent With third time eluent, anion exchanged product is obtained；It is highly preferred that containing 50- in the first time eluent buffer solution The sodium chloride of 80mM, second eluent buffer solution contains the sodium chloride of 250-300mM, the third time eluent buffering Liquid contains the sodium chloride of 450-500mM；It is highly preferred that the first time buffer elution liquid, second buffer elution liquid, third time Buffer elution liquid is concentration for 0.2-200mM, pH value are the Tris- hydrochloride buffers of 7.0-9.2.

In the preferred embodiment of preparation method, in the first time concentration step, by the anion exchanged product Concentrated, obtained first time enriched product；Preferably, the volume of the dialysis product is the enriched product not less than 50 Times, more preferably 50-500 times；It is highly preferred that the volume of the first time enriched product is 500 microlitres -5 milliliters；Preferably, The concentration is centrifuged using concentration tube, and the concentration tube allows the material of 1.0-2.5KD to pass through, in the centrifugation, centrifugal force It is 500-3000g, temperature is 2-8 DEG C；Preferably, the concentration is that, using concentration cup pressurization, the concentration cup allows 1.0- The material of 2.5KD passes through, and the pressure of the pressurization is 1-15MPa.

In the preferred embodiment of preparation method, in the molecular sieve step, the first time enriched product is being divided Eluted in son sieve, obtain molecular sieve product；Preferably, the sodium chloride containing 150-500mM in the elution buffer of the wash-out； Preferably, the elution buffer is Tris- hydrochloride buffers, and its concentration is 0.2-100mM, and its pH value is 7.0-9.2；It is more excellent Selection of land, the elution buffer is Tris- hydrochloride buffers, and its concentration is 20mM, and its pH value is 8.0；Preferably, start to collect Elution volume during eluent is 40-50ml, and elution volume when stopping collecting eluent is 70-80ml.

In the preferred embodiment of preparation method, in the dialysis step, the molecular sieve product is dialysed, Collect the product in Dialysis container and obtain product of dialysing；Preferably, the elution buffer for using of dialysing is for Tris- hydrochloric acid is slow One kind in fliud flushing, phosphate buffer, HBS buffer solutions；It is highly preferred that the concentration of the Tris- hydrochloride buffers is 0.2- 300mM, pH value are 7.0-9.2, and the concentration of the phosphate buffer is 1.0-500mM, pH value is 6.0-9.0, the HBS bufferings The concentration of liquid is 0.5-250mM, pH value is 6.4-8.4；It is further preferred that it is 8 that the elution buffer is 1mM, pH value Tris hydrochloride buffers；Preferably, the time of the dialysis is 20-72h；Preferably, the dialysis volume ratio of the dialysis is 1: (100-10000)。

In the preferred embodiment of preparation method, in the concentration step, the dialysis product is carried out second Concentration, obtains second enriched product, as described mixture；Preferably, the volume of the dialysis product is that the concentration is produced Thing not less than 50 times, more preferably 50-200 times；Preferably, the concentration is centrifuged using concentration tube, and the concentration tube is permitted Perhaps the material of 1.0-3.5KD passes through, and in the centrifugation, centrifugal force is 1000-3000g；Preferably, the concentration is using concentration Cup pressurization, the concentration cup allows the material of 1.0-3.5KD to pass through, and the pressure of the pressurization is 1-20MPa.

A kind of pharmaceutical composition, comprising said mixture and pharmaceutically acceptable carrier.

In a preferred embodiment, the pharmaceutically acceptable carrier is：Pharmaceutically acceptable buffer solution, albumen One or more in matter, gelatin, monose, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surfactant.

In a preferred embodiment, described pharmaceutical composition includes following component：1 times of said mixture of volume, 9 times The 8.5wt%NaCl of the volume or PBS of 1.5M, pH7.0；Preferably, albumin, glucose are also included in described pharmaceutical composition And glutamine；It is highly preferred that quality percent by volume of the albumin in described pharmaceutical composition is 2%, it is described Quality percent by volume of the glucose in described pharmaceutical composition is 1%, and the glutamine is in described pharmaceutical composition Quality percent by volume be 3%.

A kind of sustained release preparation, comprising said mixture or aforementioned pharmaceutical compositions and pharmaceutically acceptable biology Compatible substances；Preferably, the formulation of the sustained release preparation is liposome, microballoon, hydrogel, Osmotic minipumps or microcapsules.

A kind of kit, comprising said mixture, preceding claim pharmaceutical composition, or above-mentioned sustained release preparation.

Said mixture, aforementioned pharmaceutical compositions, above-mentioned sustained release preparation, mentioned reagent box, are preventing, improving or/and are controlling Treat the application in senile dementia.

Said mixture, aforementioned pharmaceutical compositions, above-mentioned sustained release preparation, mentioned reagent box, in memory medicine is strengthened Application.

Compared to prior art, the present invention has the advantages that：

A) curative effect of mixture prepared by the present invention is mainly lifting old dementia patients human-subject test and enhancing memory The two aspects；The mixture can be used for preventing, improve and treating senile dementia, and it may also operate as enhancing memory in addition The effect of power；B) each step of preparation method of the invention, parameter setting rationally, between act synergistically, improve jointly most The quality of finished product.C) preparation method of the invention may be directly applied to amplify metaplasia product, directly batch production can prepare greatly The mixture of amount.D) mixture that the present invention is provided can more effectively lift patients of senile dementia than blood plasma and current medicine Human-subject test.At the same time, this mixture of long-term taking can substantially reduce side effect that long-term taking Western medicine brings and Pharmacological dependence.

Brief description of the drawings

Fig. 1：In test case 1, the HC4201616 mixtures SDS- denaturation gel electrophoresis qualification result electrophoretograms of embodiment 1.Swimming Road M：Protein Marker；Swimming lane 1-4：HC4201616 mixtures.

Fig. 2：In test case 3, the microphoto of the primary hippocampal cells after different disposal.There is primary sea toward different cultures HC4201616 mixtures, and untreated human plasma are separately added into the DMEM culture mediums of horse cell, as experimental group；Control Physiological saline is added in group hippocampal cell.Hippocampal cell is imaged under the microscope after one day；(a), (b), (c) in figure It is respectively the primary hippocampal cells after HC4201616 mixtures, untreated human plasma, physiological saline (i.e. control group) treatment.

Fig. 3：In test case 3, the HC4201616 mixtures of embodiment 1 suppress the active of primary hippocampal cells apoptosis and dissipate Point diagram.HC4201616 mixtures are separately added into the DMEM culture mediums for there are primary hippocampal cells toward different cultures, and it is untreated Human plasma, as experimental group；Only add physiological saline in control group hippocampal cell；Hippocampal cell is entered under the microscope after one day Row counting, experiment with computing group cell number and cellular control unit number.

Fig. 4：In test case 3, Synaptic formation between the HC4201616 mixtures promotion primary hippocampal cells of embodiment 1 The block diagram of activity；HC4201616 mixtures are separately added into the DMEM culture mediums for there are primary hippocampal cells toward different cultures, With untreated human plasma, as experimental group；Only add physiological saline in control group hippocampal cell；Under the microscope to prominent after one day Tactile number is counted, experiment with computing group cynapse number and control group cynapse number.

Fig. 5：In test case 3, HC4201616 mixtures lifting alzheimer's disease (senile dementia) of embodiment 1 are small The block diagram of the memory animal model data of mouse.HC4201616 mixture systems are expelled to alzheimer's disease mouse In model；By water maze laboratory, castering action of the administration to alzheimer's disease mouse memory power is evaluated.

Specific embodiment

In order to preferably be illustrated to technical characteristic of the invention and effect, below using specific embodiment to this hair It is bright to be described in detail, but the present invention is not limited thereto.

Present invention firstly provides a kind of mixture from blood plasma for strengthening memory, i.e., the mixture isolated from blood plasma HC4201616, the mixture includes multiple proteins and various small molecules, the SDS-PAGE denaturation gel electrophoresis of the mixture With the high-visible band of 7 naked eyes, the molecular weight of the band is：25kD、48kD、67kD、71kD、115kD、155kD、 200kD。

Analyzed by protein spectrum, the mixture at least contains 67 kinds of albumen, as shown in table 1；Also at least contain with it is upper State 67 kinds of protein bound small molecules.

Table 1

Specifically, analyzed and identified by protein spectrum (such as MS/MS), albumen contained by the mixture is as follows：

The blood plasma derives from mammal, such as mankind, muroid etc. are preferably derived from the mankind.

Secondly the present invention provides the preparation method of said mixture HC4201616, and the mixture is extracted from blood plasma and prepared Form, the preparation method is comprised the following steps successively：Plasma collection, filter at low temperature, low temperature ultrafiltration, cold sulphur ammonium precipitation, SD go out Work, centrifugation, anion exchange, for the first time concentration, molecular sieve, dialysis, second concentration step；As preferred embodiment party Formula, between the plasma collection, cold sulphur ammonium settling step, also including filter at low temperature, low temperature ultrafiltration step, to better control over The quality of final products.Each step is described in detail below.

(I) plasma collection：

Blood is collected using anticoagulant tube, then by the way that supernatant is collected by centrifugation, obtains blood plasma；Preferably, the centrifugal force is 500-1300g, centrifugation time is 10-40min, and centrifuging temperature is 0-5 DEG C.For example：Above-mentioned centrifugal force can for 500g, 750g, The middle arbitrary value such as 800g, 1000g, 1300g or any number range between the two, above-mentioned centrifugation time can for 10min, The middle arbitrary value such as 15min, 20min, 25min, 30min, 40min or any number range between the two, above-mentioned centrifuging temperature Can be 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, the middle arbitrary values such as 5 DEG C or any number range between the two.

In a preferred embodiment, also need to carry out filter at low temperature and low temperature ultrafiltration step after this step, then carry out The steps such as cold sulphur ammonium precipitation afterwards.

(II) filter at low temperature：The purpose of this step is to remove the haemocyte that may be remained in blood plasma.

The blood plasma that the plasma collection procedure is obtained is added in filter, and applying pressure using peristaltic pump is filtered (pressure Power scope is 1-20MPa), obtain filtrate；The filter sizes of the filter are not less than 0.22 micron, and preferably 0.22 or 0.45 is micro- Rice, the temperature control of whole filter at 0-15 DEG C, preferably 0-4 DEG C.For example：Above-mentioned pressure can for 1MPa, 5MPa, The middle arbitrary value such as 10MPa, 15MPa, 20MPa or any number range between the two, said temperature can for 0 DEG C, 5 DEG C, 8 DEG C, 10 DEG C, the middle arbitrary values such as 15 DEG C or any number range between the two.

Above-mentioned filter sizes can not be less than 0.22 micron, and not so haemocyte can not be removed by filtration；The temperature for filtering simultaneously Degree not above 15 DEG C, preferably 0-4 DEG C, not so the albumen inside final product can variability so as to lose activity.

(III) low temperature ultrafiltration：The purpose of this step is to replace in the buffer solution determined in pH the material inside blood plasma, So as to the pH of convenient control solution system.

The filtrate that the filter at low temperature is obtained carries out film bag ultrafiltration, using peristaltic pump apply pressure (scope be 1~ 20MPa), low temperature ultrafiltration product is obtained；In film bag ultra-filtration process, active ingredient can enter in buffer solution；The film bag retention Molecular weight be 3kD-10kD, the buffer solution that ultrafiltration is used be phosphate buffer, Tris- hydrochloride buffers and HBS buffer solutions Etc. conventional buffer solution, the temperature control of whole ultrafiltration apparatus is at 0-15 DEG C, it is preferable that the concentration of the Tris- hydrochloride buffers For 0.2-300mM, pH value are 7.0-9.2, the concentration of the phosphate buffer is 1-500mM, pH value is 6.0-9.0, the HBS The concentration of buffer solution is 0.5-250mM, pH value is 6.4-8.4；It is highly preferred that it is that 250mM, pH value are that the buffer solution is concentration 8.0 Tris- hydrochloride buffers.For example, above-mentioned pressure can be the middle arbitrary values such as 1MPa, 5MPa, 10MPa, 15MPa, 20MPa Or any number range between the two；Above-mentioned film bag molecular cut off can for the middle arbitrary value such as 3kD, 5kD, 7kD, 10kD or Any number range between the two；The concentration of above-mentioned Tris- hydrochloride buffers can for 0.2mM, 1mM, 5mM, 10mM, The middle arbitrary value such as 50mM, 100,125mM, 150mM, 200mM, 250mM, 300mM or any number range between the two, pH value Can be the middle arbitrary values such as 7.0,8.0,8.5,8.8,9.2 or any number range between the two；Above-mentioned phosphate buffer Concentration can be the middle arbitrary values or any two such as 1mM, 10mM, 50mM, 100mM, 200mM, 350mM, 400mM, 450mM, 500mM Number range between person, pH value can be the middle arbitrary values such as 6.0,7.0,8.0,9.0 or any number range between the two； The concentration of above-mentioned HBS buffer solutions can be the middle arbitrary values such as 0.5mM, 1mM, 10mM, 50mM, 100mM, 200mM, 250mM or appoint Meaning number range between the two, pH value can be the middle arbitrary values such as 6.4,7.0,7.5,8.0,8.4 or it is any between the two Number range；Said temperature can be 0 DEG C, 5 DEG C, 8 DEG C, 10 DEG C, the middle arbitrary values such as 15 DEG C or any numerical value model between the two Enclose.

Above-mentioned film bag aperture can not be more than 10kD, and not so some effective albumen are lost from ultra-filtration process；On Stating the temperature of ultrafiltration should control at 0-15 DEG C, not so the albumen inside final product can variability so as to lose activity.

(IV) cold sulphur ammonium (i.e. ammonium sulfate) precipitation：The purpose of this step is to be settled out powerful protein component in the present invention And these protein bound small molecules.

The low temperature ultrafiltration product (as omitted step 2, three, then by blood plasma) is carried out into cold sulphur ammonium precipitation twice, obtains cold Sulphur ammonium precipitated product；Wherein, for the first time precipitate in, the ammonium sulfate solution of saturation after 0-4 DEG C of precooling, and low temperature ultrafiltration product (or Blood plasma) with 1：(9-10) volume ratio is mixed, mixing after 0-10 DEG C of placement, until precipitation automatic sedimentation to become it is real (herein " becoming real " refers to：Supernatant precipitation has obvious line of demarcation, and supernatant becomes bright, and precipitation quantity no longer increases), obtain herein Be precipitated as discarded object, retain supernatant；During second precipitates, then take the supernatant for precipitating obtain for the first time, and saturation ammonium sulfate solution With volume ratio (5-1)：1 mixing is placed after 0-10 DEG C up to precipitation automatic sedimentation is to becoming real, retains precipitation, is sunk as cold sulphur ammonium Shallow lake product；In precipitating twice, the time of placement is both preferably 8-16 hours, more preferably 12 hours.For example：The temperature of above-mentioned precooling Degree can be 0 DEG C, 1 DEG C, 3 DEG C, 3 DEG C, the middle arbitrary values such as 4 DEG C or any number range between the two, after above-mentioned mixing twice The temperature of placement can be 0 DEG C, 1 DEG C, 3 DEG C, 5 DEG C, 8 DEG C, the middle arbitrary values such as 10 DEG C or any number range between the two, the The ammonium sulfate solution of above-mentioned saturation and the volume ratio of low temperature ultrafiltration product (or blood plasma) are 1 in primary sedimentation：9、1：9.5、1：9.8、 1：Arbitrary value or any number range between the two in 10 grades, the ammonium sulfate solution of supernatant saturation is with volume in second precipitation Than being 5:1、4:1、2.5:1、2:1、1：Arbitrary value or any number range between the two in 1 grade.

Different volumes than above-mentioned saturation ammonium sulfate solution and the albumen that is precipitated out of low temperature ultrafiltration product (or blood plasma) be Different；Place temperature after above-mentioned mixing to preferably must be held in the range of 0-10 DEG C, the albumen being not so settled out may be denatured.

(V) SD inactivations：The potential virus that the purpose of this step is so that in initial feed human blood lose activity, so as to improve The security of final products.

The cold sulphur ammonium precipitated product is suspended again with SD inactivators, 4~37 DEG C is put in and is stood 6~20h, preferably 20 DEG C 10h is stood, obtains inactivating product；For example, the time of above-mentioned standing can be in 6h, 8h, 10h, 12h, 15h, 18h, 20h etc. Meaning value or any number range between the two, temperature can be 4 DEG C, 10 DEG C, 15 DEG C, 20 DEG C, 25 DEG C, 28 DEG C, 30 DEG C, 37 DEG C Arbitrary value or any number range between the two in.

SD inactivators contain percent by volume for 0.3~2%TnBP (N- butyl triphosphate) and 0.5~2% Tween80, preferably contains 1%TnBP and 1%Tween80.For example：The percent concentration of above-mentioned TnBP can for 0.3%, 0.5%th, the middle arbitrary value such as 1%, 1.5%, 2% or any number range between the two, the percent concentration of above-mentioned Tween80 Can be the middle arbitrary value such as 0.5%, 1%, 1.5%, 2% or any number range between the two.The preparation of SD inactivators is such as Under：By taking the SD inactivators for preparing 100mL as an example, 0.3-2mlTnBP is dissolved in suitable quantity of water, it is subsequently adding 0.5~ 2mLTween80, is mended to 100mL after being well mixed with water.

The temperature of above-mentioned standing cannot be below 4 DEG C, and the time can not be shorter than 6 hours, and not so the effect of inactivation of virus can compare Typically, the inactivation time of different virus is also different.

(VI) centrifugation：The purpose of this step is to remove some to be denatured aggregate and precipitate because of SD viral inaction steps Foreign protein, not so foreign proteins of these denaturation coagulations can disturb the anion exchange step in downstream.

The inactivation product is centrifuged, retains supernatant, as centrifugation product；Preferably, it is described from Mental and physical efforts are 2000-10000g, and centrifugation time is 10-30min, and centrifuging temperature is 0-10 DEG C.For example, above-mentioned centrifugal force can be The middle arbitrary value such as 2000g, 2500g, 5000g, 7500g, 8000g, 10000g or any number range between the two, the time can Think the middle arbitrary value such as 10min, 12min, 15min, 20min, 25min, 30min or any number range between the two, temperature Degree can be 0 DEG C, 1 DEG C, 2 DEG C, 3 DEG C, 4 DEG C, 6 DEG C, 8 DEG C, the middle arbitrary values such as 10 DEG C or any number range between the two.

Above-mentioned centrifugal force can not be less than 2000g, and centrifugation time can not be less than 10min, not so be denatured the miscellaneous egg of aggregate and precipitate The white situation for occurring that sedimentation is unreal.

(VII) anion exchange：The purpose of this step prepares the albumen containing active ingredient and protein bound with these Small molecule.

Step level wash-out in centrifugation product addition anion-exchange column (such as SourceQ), will be carried out, collects every The secondary eluent for obtaining, obtains anion exchanged product after mixing；

Wherein, in the step level wash-out, first eluted with first time elution buffer, obtain first time eluent；Again with Secondary elution buffer wash-out, obtains waste liquid；Eluted with third time elution buffer again, obtain third time eluent；Merge institute First time eluent and third time eluent are stated, anion exchanged product is obtained；

The purpose that selection wash-out is 3 times is to remove the waste liquid for affording for the second time；Because second liquid of wash-out, to god It is toxic through first cell, if not removing, it will substantially reduce the curative effect of final whole mixture；In above-mentioned each wash-out, purple is waited External detector shows and start when there is albumen collection, stops collecting when albumen has gone out, i.e., the wash-out for being eluted each time Liquid；

Sodium chloride containing 50-80mM in the first time elution buffer, second elution buffer contains The sodium chloride of 250-300mM, the third time elution buffer contains the sodium chloride of 450-500mM；Three times the wash-out of wash-out delays Fliud flushing is concentration for 0.2-200mM, pH value are the Tris- hydrochloride buffers of 7.0-9.2；Each elution speed is controlled in instrument In the range of device is allowed, such as 2-5ml/min.For example, in above-mentioned first time elution buffer sodium chloride concentration can for 50mM, The middle arbitrary value such as 60mM, 70mM, 75mM, 80mM or any number range between the two, in above-mentioned second elution buffer The concentration of sodium chloride can for the middle arbitrary value such as 250mM, 260mM, 270mM, 280mM, 290mM, 300mM or it is any between the two Number range, in above-mentioned third time elution buffer the concentration of sodium chloride can for 450mM, 460mM, 470mM, 480mM, The middle arbitrary value such as 490mM, 500mM or any number range between the two, the concentration of above-mentioned Tris- hydrochloride buffers can be The middle arbitrary value such as 0.2mM, 0.5mM, 1mM, 10mM, 50mM, 100mM, 150mM, 200mM or any numerical value model between the two Enclose, pH value can be the middle arbitrary values such as 7.0,8.0,8.5,9.2 or any number range between the two.

This step can greatly improve the curative effect of HC4201616.

(VIII) concentrate for the first time：The purpose of this step is to reduce the volume of sample, the molecular sieve loading behaviour after convenience Make.

The anion exchanged product is carried out into first time concentration, first time enriched product is obtained；Wherein, condensate precursor product Be concentration after volume not less than 50 times, preferably 50-500 times；Preferably, the volume after concentration is 500 μ L-5mL；It is preferred that Ground, the concentration is carried out by the way of concentration tube centrifugation, and the concentration tube allows the material of 1.0-2.5KD to pass through, institute Centrifugal force is stated for 1000-3000g, temperature is 2-8 DEG C, cycles of concentration stops concentration after reaching requirement；Preferably, it is described dense Contracting is carried out by the way of concentration cup pressurization, and the concentration cup allows the material of 1.0-2.5KD to pass through, the pressure of the pressurization Power scope is 1~15MPa.For example, above-mentioned multiple can be the middle arbitrary values such as 50,100,200,300,400,500 times or any Number range between the two, the concentration tube, concentration cup allow the size of the material for passing through be 1.0KD, 1.25KD, The middle arbitrary value such as 1.5KD, 1.75KD, 2KD, 2.25KD, 2.5KD or any number range between the two, the centrifugal force is The middle arbitrary value such as 1000g, 1500g, 2000g, 2500g, 3000g or any number range between the two, the temperature can be with Be 2 DEG C, 4 DEG C, 5 DEG C, the middle arbitrary values such as 8 DEG C or any number range between the two, the pressure be 1MPa, 5MPa, The middle arbitrary value such as 7.5MPa, 10MPa, 15MPa or any number range between the two.

Some when concentration tube used by this step or concentration cup size are more than 2.5kD, inside the final mixture for preparing Active ingredient can be lost in, but when concentration tube or concentration cup size are less than 1.0kD, concentration speed also can be very slow, time-consuming to compare It is long；When the pressure that concentration is used is less than 1MPa or centrifugal force less than 1000g, concentration speed can be very slow, takes long；It is dense When contracting temperature is higher than 8 DEG C, some active ingredients may be denatured so as to lose activity because of temperature drift in concentration process, dense When contracting temperature is less than 2 DEG C, the price of temperature control system again can be of a relatively high, while there is enriched product because the too low hair of temperature The risk of raw aggregate and precipitate.

(IX) molecular sieve：The purpose of this step is to prepare the albumen containing active ingredient and protein bound small point with these Son.

By in first time enriched product addition molecular sieve, molecular sieve product is obtained after wash-out；Wherein, elution buffer In contain 150-500mM sodium chloride, only wash-out once；Preferably, start to collect eluent when elution volume is 40-50ml, Stop collecting eluent when elution volume is 70-80ml, the eluent that collection is obtained is HC4201616 stostes, as molecule Sieve product；Preferably, described buffer solution is Tris- hydrochloride buffers, and concentration is 0.2-100mM, pH value is 7.0-9.2；More Preferably, described buffer solution is Tris- hydrochloride buffers, and concentration is 20mM, pH value is 8.For example, in above-mentioned elution buffer The concentration of sodium chloride can be the middle arbitrary values or any two such as 150mM, 175mM, 200mM, 250mM, 300mM, 400mM, 500mM Number range between person, it is above-mentioned start collect eluent when elution volume can be 40ml, 42ml, 45ml, 48ml, 50ml Arbitrary value or any number range between the two in, elution volume when eluent is collected in above-mentioned stopping can for 70ml, The middle arbitrary value such as 72ml, 75ml, 78ml, 80ml or any number range between the two, above-mentioned Tris- hydrochloride buffers it is dense Degree can be the middle arbitrary values such as 0.2mM, 0.5mM, 1mM, 10mM, 50mM, 75mM, 100mM or any numerical value model between the two Enclose, pH value can be the middle arbitrary values such as 7.0,8.0,8.5,9.2 or any number range between the two.

When sodium chloride concentration is less than 20mM in elution buffer, it is heavy that Partial Protein can occur aggregation because solubility is low Form sediment, cause pillar to block the loss with active ingredient；When sodium chloride concentration inside elution buffer is higher than 500mM, this step Rapid product can produce very big osmotic pressure because sodium chloride concentration is too high, so that following dialysis step needs more Time long and the more dialyzates of consuming；Within the above-mentioned time period for starting and collecting and stop collection eluent, it is collected into Eluent is only active ingredient, otherwise the composition being not so collected into is containing many invalid or even poisonous compositions, otherwise it is exactly one A little active ingredients generation losses are reduced so as to cause curative effect.

(X) dialyse：The purpose of this step be removal before impurity in step, such as inactivator, to prevent the medium under Trip product is prepared and produces harmful effect.

Dialysed during the molecular sieve product (HC4201616 stostes) is put into bag filter or pipe, collected saturating after dialysis Product in analysis bag or pipe, as dialysis product；Wherein, the elution buffer for being used during the dialysis is Tris- hydrochloride buffers Liquid, phosphate buffer or HBS buffer solutions；Preferably, the concentration of the Tris- hydrochloride buffers is that 0.2-300mM, pH value are 7.0-9.2, the concentration of the phosphate buffer is 1-500mM, pH value is 6.0-9.2, and the concentration of the HBS buffer solutions is 0.5- 250mM, pH value are 6.4-8.4；It is highly preferred that the elution buffer is 1mM, the Tris hydrochloride buffers that pH value is 8；Dialysis Time is 20-72h, and dialysis volume ratio is 1:(100-10000)；During the dialysis volume ratio is dialysis system, dialysis tubing or dialysis The volume ratio of the liquid outside liquid and the dialysis tubing or bag filter in bag is (such as：Dialysis system is that bag filter is placed in beaker, Then the dialysis volume ratio is referred to：Liquid volume in bag filter:The volume of liquid (in beaker in the volume-bag filter of liquid)). For example, the concentration of above-mentioned Tris- hydrochloride buffers can for 0.2mM, 1mM, 5mM, 10mM, 50mM, 100,125mM, 150mM, The middle arbitrary value such as 200mM, 250mM, 300mM or any number range between the two, pH value can for 7.0,8.0,8.5, 8.8th, the middle arbitrary values such as 9.2 or any number range between the two；The concentration of above-mentioned phosphate buffer can for 1mM, 10mM, The middle arbitrary value such as 50mM, 100mM, 200mM, 350mM, 400mM, 450mM, 500mM or any number range between the two, pH Value can be the middle arbitrary values such as 6.0,7.0,8.0,9.0 or any number range between the two；The concentration of above-mentioned HBS buffer solutions Can be the middle arbitrary values such as 0.5mM, 1mM, 10mM, 50mM, 100mM, 200mM, 250mM or any numerical value model between the two Enclose, pH value can be the middle arbitrary values such as 6.4,7.0,7.5,8.0,8.4 or any number range between the two；Above-mentioned dialysis body Product ratio can be 1:100、1:500、1:1000、1:2500、1:5000、1:In 10000 grades arbitrary value or it is any between the two Number range.

Dialysis time is defined in this step；When dialysis time is less than 20 hours, it may occur that magazine removes incomplete feelings Condition；When dialysis time was more than 72 hours, the time cost of production can be dramatically increased again.

(XI) concentrate for second：The first purpose of this step is to improve active principle in the mixture HC4201616 Concentration, so as to improve the curative effect of unit composition；The second purpose is the osmotic pressure that solution is adjusted by concentration.

Preparation method of the invention need to concentrate twice because：Concentration is, in order to reduce volume, to facilitate following for the first time Molecular sieve step implementation；Second concentration is to meet to efficiently control the osmotic pressure of final product HC4201616 to face The bed requirement of physiologic infusion agent.

The dialysis product is carried out into second concentration, second enriched product, as mixture HC4201616 is obtained； Wherein, condensate precursor product be concentration after volume not less than 50 times, preferably 50-200 times；Preferably, the concentration is to use What the mode of concentration tube centrifugation was carried out, the concentration tube allows the material of 1.0-3.5KD to pass through, and the centrifugal force is 500- 3000g, temperature is 2-8 DEG C, and cycles of concentration stops concentration after reaching requirement；Preferably, the concentration is using concentration cup What the mode of pressurization was carried out, the concentration cup allows the material of 1.0-3.5KD to pass through, the pressure limit of the pressurization for 1~ 20MPa.For example, above-mentioned multiple can for 50 times, 80 times, 100 times, 120 times, 150 times, 180 times, the middle arbitrary values such as 200 times or Any number range between the two, the concentration tube, concentration cup allow the size of the material for passing through be 1.0KD, 1.25KD, The middle arbitrary value such as 1.5KD, 1.75KD, 2KD, 2.25KD, 2.5KD, 2.75KD, 3KD, 3.25KD, 3.5KD or it is any between the two Number range, the centrifugal force be the middle arbitrary value such as 500g, 1000g, 1500g, 2000g, 2500g, 3000g or it is any both Between number range, the temperature can be 2 DEG C, 4 DEG C, 5 DEG C, the middle arbitrary values such as 8 DEG C or any numerical value model between the two Enclose, the pressure be the middle arbitrary value such as 1MPa, 5MPa, 7.5MPa, 10MPa, 15MPa, 17.5MPa, 20MPa or it is any both it Between number range.

Define that concentration tube or concentration cup allow the magnitude range of the molecular weight of the material for passing through, use concentration in this step Centrifugal force scope during pipe, using concentration cup when pressure limit, concentration when temperature range；Concentration tube or concentration cup used When size is more than 3.5kD, some active ingredients inside the final mixture for preparing can be lost in, but when concentration tube or concentration cup are big During less than 1.0kD, concentration speed also can be very slow, takes long；The pressure that concentration is used is less than 1MPa or centrifugal force During less than 1000g, concentration speed can be very slow, takes long；When thickening temperature is higher than 8 DEG C, some are effective in concentration process Composition may be denatured so as to lose activity because of temperature drift, when thickening temperature is less than 2 DEG C, the price of temperature control system Again can be of a relatively high, while there is enriched product because the too low risk that aggregate and precipitate occurs of temperature.

The various common agents used in above-mentioned preparation method are prepared using conventional method.

Furthermore the present invention provides a kind of pharmaceutical composition, comprising the mixture HC4201616 and pharmaceutically acceptable Carrier.The pharmaceutically acceptable carrier includes：Pharmaceutically acceptable buffer solution, protein, gelatin, monose, polysaccharide, ammonia One or more in base acid, chelating agent, sugar alcohol, polyethylene glycol and surfactant.

Used as preferred embodiment, described pharmaceutical composition includes following component：1 times of mixture of volume HC4201616, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0；Preferably, also include in described pharmaceutical composition Albumin, glucose and glutamine, wherein, quality percent by volume of the albumin in described pharmaceutical composition is 2%, quality percent by volume of the glucose in described pharmaceutical composition is 1%, and the glutamine is in the medicine Quality percent by volume in composition is 3%.

Furthermore the present invention provides a kind of sustained release preparation, comprising the mixture HC4201616 and pharmaceutically acceptable Biocompatible substance (is also called pharmaceutically acceptable carrier)；Preferably, the formulation of the sustained release preparation is liposome, micro- Ball, hydrogel, Osmotic minipumps or microcapsules；Preferably, the pharmaceutically acceptable biocompatible substance includes：For aqueous PH buffer solutions, including the buffer solution of phosphate, citrate or other organic acids, ascorbic acid or other antioxidants are low Molecular weight (being no more than 10 residues) polypeptide, seralbumin, gelatin, immunoglobulin or other protein, polyvinyl pyrrole Alkanone or other hydrophilic polymers, glycine, glutamine, asparagine, arginine, lysine or other amino acid, list Sugar, disaccharides, glucose, mannose, dextrin or other carbohydrates, EDTA or other chelating agents, mannitol, sorbierite or other sugar Alcohol, sodium ion or other into salt counter ion,Polyethylene glycol (PEG),Or other nonionics Surfactant.

Furthermore the present invention provides a kind of kit, includes：The mixture HC4201616 or/and comprising the mixing The aforementioned pharmaceutical compositions of thing HC4201616 or/and the above-mentioned sustained release agent comprising the mixture HC4201616.

Furthermore the present invention provides the mixture HC4201616 or/and comprising the above-mentioned of the mixture HC4201616 Pharmaceutical composition or/and the above-mentioned sustained release agent comprising the mixture HC4201616 or/and comprising the mixture The mentioned reagent box of HC4201616, the application in prevention, the medicine for improving or/and treating senile dementia.

The present invention finally provides the mixture HC4201616 or/and comprising the above-mentioned of the mixture HC4201616 Pharmaceutical composition or/and the above-mentioned sustained release agent comprising the mixture HC4201616 or/and comprising the mixture The mentioned reagent box of HC4201616, the application in the medicine of enhancing memory.

Preparation, identification and application below by embodiment to inventive mixture are illustrated.Related in following examples And for example unreceipted specific experimental condition of molecular biology manipulations and method, refer to SambrookJ etc. chief editor, scientific publication Society, 2002, the specification of Molecular Cloning:A Laboratory guide (third edition) or corresponding product.

Preparation, identification and application below by embodiment to inventive mixture are illustrated.Make in following examples Primary hippocampal cells are according to following bibliography culture：Guo,W.,Y.Ji,et al.(2014)."Neuronal activity alters BDNF-TrkB signaling kinetics and downstream functions."J Cell Sci 127(Pt 10):2249-2260。

Embodiment 1

The present embodiment is the preparation method of mixture HC4201616, and the method is comprised the following steps：

(I) blood plasma is collected

Obtained by hospital donates blood, heparin tube is anticoagulant tube to the blood of Healthy People, rocks blood sampling in blood collection procedure in bloodletting Pipe, prevents blood clotting；Heparin tube containing blood is put into centrifuge, centrifugal force as 1000g is set, is centrifuged 20 minutes, from Heart temperature is 4 DEG C；Then supernatant is carefully drawn with pipettor, the human plasma being as collected into.

(II) filter at low temperature：

During the blood plasma that will be collected into adds filter sizes to be 0.22 micron of filter, peristaltic pump is used to apply pressure

10MPa is filtered, and temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

(III) low temperature ultrafiltration：

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 8kD, pressure 10MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 250mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

(IV) cold sulphur ammonium precipitation：

First carry out first time precipitation：By the 0-4 DEG C of ammonium sulfate solution of the saturation of precooling and the low temperature ultrafiltration product according to volume Than 1:9.5 mixing, then at 0 DEG C of placement 12h, become real to precipitation automatic sedimentation；Second precipitation is carried out again：Take and precipitate for the first time The supernatant for arriving, and saturation ammonium sulfate solution with volume ratio 3：1 mixing is placed 12 hours after 0 DEG C, until precipitation automatic sedimentation is extremely Become real, obtain cold sulphur ammonium precipitated product.

(V) SD inactivations：

The cold sulphur ammonium precipitated product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 20 DEG C are put in 10 hours are stood, the product after standing is inactivation product.

(VI) centrifugation：

The inactivation product is centrifuged 15 minutes in 6000g, 0-4 DEG C, retains supernatant, as centrifugation product.

(VII) anion exchange：

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 30 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 65mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 275mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 475mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Each elution speed is 4ml/min, and loading speed is 3ml/ml.

In above-mentioned each wash-out, start to collect when waiting UV-detector to show and albumen occur, albumen goes out to be over, and stops Collect, i.e., the eluent for being eluted each time.

(VIII) concentrate for the first time：

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 2.5kD that concentration tube allows size Material pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 1500g, design temperature is 4 DEG C, starts centrifuge, Start concentration, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, with Volume is reached 2mL, be centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, Plasma component after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

(IX) molecular sieve：

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 150mM sodium chloride Be 20mM, pH value for 8) elute, when elution volume be 45ml when start collect eluent, when elution volume be 75ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201616 stostes, as molecular sieve product.

(X) dialyse：

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 1mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:5000；Dialysis temperature is 4 DEG C, and dialysis time is 24 hours, collects the product in bag filter as dialysis product.

(XI) concentrate for second：

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, it is the material of 2.5kD that concentration tube allows size Pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 2000g, design temperature is 4 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201616.

Embodiment 2

In the present embodiment, in addition to step (VIII) is different from embodiment 1, other preparation processes are same as Example 1, In the present embodiment, step (VIII) is specific as follows：

Anion exchanged product prepared by the step of embodiment 1 (VII) adds the concentration cup for allowing 1.5kD materials to pass through In, concentration cup lid is covered, the chromatography cabinet that cup is put into will be concentrated, concentration cup is then connected into liquid nitrogen bottle, liquid nitrogen bottle air valve is opened, Set pressure as 10MPa；Under this air pressure, concentration cup starts product after concentration dialysis, observes the volume of product after concentrating and is Before concentration 1/50 when, stop concentration, obtain first time enriched product.

Embodiment 3

In the present embodiment, in addition to step (XI) is different from embodiment 1, other preparation processes are same as Example 1, In the present embodiment, step (XI) is specific as follows：

Dialysis product prepared by the step of embodiment 1 (X) is added in the concentration cup for allowing 1.5kD materials to pass through, and is covered dense Contracting cup lid, will concentrate the chromatography cabinet that cup is put into, and concentration cup then is connected into liquid nitrogen bottle, open liquid nitrogen bottle air valve, set pressure It is 10MPa；Under this air pressure, concentration cup starts product after concentration dialysis, and the volume for observing product after concentrating is before concentrating When 1/50, stop concentration, obtain second enriched product, as mixture HC4201616.

Embodiment 4

(I) blood plasma is collected：Operating method is identical in the step of with embodiment 1 (I).

(II) filter at low temperature：

During the blood plasma that will be collected into adds filter sizes to be 0.45 micron of filter, peristaltic pump is used to apply pressure 15MPa Filtered, temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

(III) low temperature ultrafiltration：

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 7kD, pressure 20MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 250mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

(IV) cold sulphur ammonium precipitation：

First carry out first time precipitation：By the 0-4 DEG C of ammonium sulfate solution of the saturation of precooling and the low temperature ultrafiltration product according to volume Than 1：9 mixing, then at 0 DEG C of placement 16h, become real to precipitation automatic sedimentation；Second precipitation is carried out again：Precipitation for the first time is taken to obtain Supernatant, and saturation ammonium sulfate solution with volume ratio 5：1 mixing is placed 16 hours after 10 DEG C, until precipitation automatic sedimentation extremely becomes It is real, obtain cold sulphur ammonium precipitated product.

(V) SD inactivations：

The cold sulphur ammonium precipitated product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 4 DEG C are put in 20 hours are stood, the product after standing is inactivation product.

(VI) centrifugation：

The inactivation product is centrifuged 30 minutes in 2000g, 0-4 DEG C, retains supernatant, as centrifugation product.

(VII) anion exchange：

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 30 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 65mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 275mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 475mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product.

Every time in wash-out, start to collect when waiting UV-detector to show and albumen occur, albumen goes out to be over, stop receiving Collection, i.e., the eluent for being eluted each time.

(VIII) concentrate for the first time：

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 1.5kD that concentration tube allows size Material pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 1000g, design temperature is 2 DEG C, starts centrifuge, Start concentration, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, with Volume is reached 2mL, be centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, Plasma component after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

(IX) molecular sieve：

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 500mM sodium chloride Be 1mM, pH value for 8) elute, when elution volume be 40ml when start collect eluent, when elution volume be 70ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201616 stostes, as molecular sieve product.

(X) dialyse：

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 1mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:100；Dialysis temperature is 4 DEG C, and dialysis time is 50 hours, obtains product of dialysing.

(XI) concentrate for second：

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, it is the material of 3.5kD that concentration tube allows size Pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 1500g, design temperature is 6 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201616.

Embodiment 5

(II) filter at low temperature：

During the blood plasma that will be collected into adds filter sizes to be 0.45 micron of filter, peristaltic pump is used to apply pressure 20MPa Filtered, temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

(III) low temperature ultrafiltration：

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 10kD, pressure 1MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 250mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

(IV) cold sulphur ammonium precipitation：

First carry out first time precipitation：By the 0-4 DEG C of ammonium sulfate solution of the saturation of precooling and the low temperature ultrafiltration product according to volume Than 1：10 mixing, then at 4 DEG C of placement 8h, become real to precipitation automatic sedimentation；Second precipitation is carried out again：Precipitation for the first time is taken to obtain Supernatant, and saturation ammonium sulfate solution with volume ratio 1：1 mixing is placed 8 hours after 4 DEG C, until precipitation automatic sedimentation extremely becomes It is real, obtain cold sulphur ammonium precipitated product.

(V) SD inactivations：

The cold sulphur ammonium precipitated product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 37 DEG C are put in 6 hours are stood, the product after standing is inactivation product.

(VI) centrifugation：

The inactivation product is centrifuged 10 minutes in 10000g, 4 DEG C, retains supernatant, as centrifugation product.

(VII) anion exchange：

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 30 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 50mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 250mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 450mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Wherein, each elution speed is 4ml/min, and loading speed is 3ml/ml.

(VIII) concentrate for the first time：

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 2.5kD that concentration tube allows size Material pass through；Concentration tube is put into centrifuge again, sets centrifugal force as 2000g, design temperature is 8 DEG C, starts centrifuge, Start concentration, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, with Volume is reached 2mL, be centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, Plasma component after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

(IX) molecular sieve：

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 400mM sodium chloride Be 20mM, pH value for 8) elute, when elution volume be 50ml when start collect eluent, when elution volume be 80ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201616 stostes, as molecular sieve product.

(X) dialyse：

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 1mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:10000；Dialysis temperature is 4 DEG C, and dialysis time is 20 hours, obtains product of dialysing.

(XI) concentrate for second：

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size to lead to for the material of 3kD Cross；Concentration tube is put into centrifuge again, sets centrifugal force as 3000g, design temperature is 2 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201616.

Embodiment 6

(II) filter at low temperature：

During the blood plasma that will be collected into adds filter sizes to be 0.22 micron of filter, apply pressure 1MPa using peristaltic pump and enter Row filtering, temperature control of whole filter obtains filtrate at 0-4 DEG C during it.

(III) low temperature ultrafiltration：

By the film bag ultrafiltration that the molecular weight that the filtrate retains is 3kD, pressure 15MPa, its process are applied using peristaltic pump The middle buffer solution for using is the Tris hydrochloride buffers of concentration 250mM, pH8.0, and the temperature control of whole ultrafiltration apparatus is in 0-4 DEG C, collect the buffer solution after ultrafiltration and obtain low temperature ultrafiltration product.

(IV) cold sulphur ammonium precipitation：

First carry out first time precipitation：By the 0-4 DEG C of ammonium sulfate solution of the saturation of precooling and the low temperature ultrafiltration product according to volume Than 1：9.5 mixing, then at 8 DEG C of placement 10h, become real to precipitation automatic sedimentation；Second precipitation is carried out again：Take and precipitate for the first time The supernatant for arriving, and saturation ammonium sulfate solution with volume ratio 4：1 mixing is placed 10 hours after 8 DEG C, until precipitation automatic sedimentation is extremely Become real, obtain cold sulphur ammonium precipitated product.

(V) SD inactivations：

The cold sulphur ammonium precipitated product is suspended again with SD inactivators (containing 1%TnBP, 1%Tween80), 10 DEG C are put in 15 hours are stood, the product after standing is inactivation product.

(VI) centrifugation：

The inactivation product is centrifuged 20 minutes in 4000g, 6 DEG C, retains supernatant, as centrifugation product.

(VII) anion exchange：

The centrifugation product is added into anion-exchange column (such as SourceQ, purchased from GE Healthcare, average grain It is 3 microns to spend, and anion exchange column volume is 25 milliliters) in, first with the Tris- hydrochloride buffers containing 80mM sodium chloride (100mM, pH8.0) carries out first time wash-out, retains eluent；Then with the Tris- hydrochloride buffers containing 300mM sodium chloride (100mM, pH8.0) carries out second wash-out, discards eluent；Finally with the Tris- hydrochloride buffers containing 500mM sodium chloride (100mM, pH8.0) carries out third time wash-out, retains eluent；Merge above-mentioned first time and third time afford eluent, As anion exchanged product；Wherein, each elution speed is 4ml/min, and loading speed is 3ml/ml.

(VIII) concentrate for the first time：

Take a small amount of Anion separation product to be added in the concentration tube that volume is 2mL, it is 2kD's that concentration tube allows size Material passes through；Concentration tube is put into centrifuge again, sets centrifugal force as 3000g, design temperature is 6 DEG C, starts centrifuge, is opened Begin to concentrate, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that Volume reaches 2mL, is centrifuged with identical parameter, is again concentrated to final volume for 500 microlitres；So circulation concentration, directly Plasma component to after by dialysis is finally concentrated to volume for 500 microlitres, used as first time enriched product.

(IX) molecular sieve：

By first time enriched product add molecular sieve in, with the Tris- hydrochloride buffer (concentration containing 200mM sodium chloride Be 20mM, pH value for 8) elute, when elution volume be 45ml when start collect eluent, when elution volume be 75ml when stop receive Collection eluent, the eluent that collection is obtained is HC4201616 stostes, as molecular sieve product.

(X) dialyse：

The molecular sieve product is added into aperture to be about in 0.25 nanometer of bag filter, then the bag filter is put into the burning of 1L In cup, then to 1mM Tris hydrochloride buffers (pH8.0) of 1L are added outside bag filter in the beaker, dialyse while stirring, dialyse Volume ratio is 1:2500；Dialysis temperature is 4 DEG C, and dialysis time is 72 hours, obtains product of dialysing.

(XI) concentrate for second：

Take a small amount of dialysis product to be added in the concentration tube that volume is 2mL, concentration tube allows size to lead to for the material of 1kD Cross；Concentration tube is put into centrifuge again, sets centrifugal force as 2000g, design temperature is 8 DEG C, starts centrifuge, is started dense Contracting, until final volume is 500 microlitres；Afterwards, by the addition again of the remaining dialysis product centrifuge tube, so that volume 2mL is reached, is centrifuged with identical parameter, be again concentrated to final volume for 500 microlitres；So circulation concentration, until inciting somebody to action Plasma component after dialysis is finally concentrated to volume for 500 microlitres, used as enriched product, as said mixture HC4201616.

Test case 1

Mixture HC4201616 prepared by embodiment 1-6 is carried out into SDS-PAGE denaturation gel electrophoresis identifications, the authentication method Comprise the following steps：

Step one, takes out 2 microlitres, under ultraviolet 280nm in the mixture HC4201616 for being prepared from each embodiment respectively Its absorbance is determined, so as to calculate the protein concentration of mixture HC4201616.

Step 2, takes the mixture HC4201616 of certain volume, and (Beijing is purchased from 1 microlitre of 5 × albumen sample-loading buffer Lan Bolide Bioisystech Co., Ltd, article No. D621) mixing, as need to carry out the sample of electrophoresis, sample the inside has 10 The protein of microgram.

Step 3,100 DEG C are warming up to by electrophoresis Sample, and heating makes protein denaturation in 20 minutes, immediately after puts sample Arrive on ice, start to run SDS-PAGE denaturation after waiting 5 minutes and go back virgin rubber (compound method that virgin rubber is gone back in SDS denaturation is as follows：30 (w/v) the acrylamide Acr-Bis (being purchased from GE Healthcare) of % takes 1.3ml, 1.5M Tris- hydrochloride buffers (pH8.8, purchased from GE Healthcare) takes 1.3ml, the SDS of 10 (w/v) % and takes the 0.05ml, (purchase of 10% (w/v) ammonium persulfate From GE Healthcare) take that 0.05ml, TEMED (purchased from GE Healthcare) take 0.003ml and water takes 2.3ml, altogether 5ml, plastic can be solidified after mixing in room temperature).When running glue, the albumen applied sample amount of each swimming lane is 10 micrograms, and glue electricity is run in setting It is 100V to press, and starts to run glue, runs the glue time for 1 hour.

Step 4, after running through glue, with coomassie brilliant blue staining liquid, (preparation method of the dyeing liquor is：By 2.5g coomassies Light blue R-250 is dissolved in the ethanol solutions of 500ml 95%, adds the acetic acid solution of 100ml 85%, then, is supplemented to distilled water 1000ml, this dye liquor puts 4 DEG C and keeps stabilization at least 6 months) glue is dyeed.

Embodiment 1 prepare mixture HC4201616 testing result referring to Fig. 1, wherein：Swimming lane M：Molecular weight of albumen mark It is accurate；Swimming lane 1-4：HC4201616 mixtures；At least contain 11 visible polypeptides of naked eyes, its molecule in mixture HC4201616 Amount be respectively be successively from small to large 25kD, 37kD, 41kD, 51kD, 66kD, 69kD, 88kD, 105kD, 145kD, 170kD, 200kD.The result of mixture HC4201616 prepared by embodiment 2-6 is same as Example 1.

Test case 2

Proteomic image identification is carried out to mixture HC4201616 prepared by embodiment 1-6, the authentication method includes as follows Step：

, be transferred to mixture HC4201616 prepared by embodiment 1-6 in FALCON pipes respectively by step one, adds twice (formula of buffer solution is the sample buffer of volume：7.5M urea UREA, 1.5M thiocarbamide THIOUREA, 4 (w/v) %3- [3- (courage amido propyl) dimethylamino] propane sulfonic acid inner salt CHAPS, 0.05 bis- sulphur Soviet Union of (w/v) % lauryl sodium sulfate SDS, 100mM Sugar alcohol DDT, the shown concentration before each component is concentration of its respective components in buffer solution), and by 3kDa molecular Weight cut-off spin columns (Pall GmbH, Austria) centrifuge tube centrifugal concentrating, obtains concentrate.

Step 2, concentrate carries out reduction reaction with dithiothreitol (DTT), obtains reduzate；Wherein, concentrate and two sulphur The volume ratio of threitol is 1：3, the reaction time is 15 minutes, and temperature is room temperature.

Step 3, then the reduzate and iodoacetamide are reacted, obtain alkylate；Wherein, this is also originated in The volume ratio 1 of thing and iodoacetamide：1, the reaction time is 15 minutes, and temperature is room temperature；

Step 4, then carries out digestion reaction overnight with trypsase at 37 DEG C, wherein alkylate and trypsase Volume ratio 1:1, obtain postdigestive peptide fragment.

Step 5, Trypsin Induced gained peptide fragment obtains sample by C18 chromatographies.

Step 6, gained sample traditional vacuum is dried and is then stored in -20 DEG C of refrigerators and analyzed for MS/MS.MS/MS Analysis is specific as follows：HPLC's is the systems of Ultimate 3000, wherein being equipped with PepMap100 C-18 trap column (300 μ m 5mm) and two pillars of PepMap100 C-18 analytical column (75 μ m 250mm).Mass spectrograph is adopted It is 400-1400m/z with Amazon speed ETD, MS data acquisition range, the peptide fragment process range of MS/MS is 100- 2800m/z.Then, each MS data can automatically search for the top-quality CID MS/MS peaks spectrum of matched three.Jet hole Voltage is set to 1400 volts.The temperature of nitrogen protection is 150 DEG C, and flow velocity is 3 liters/min.The protein identification of MS and without mark Quantitative (LFQ) data analysis of note uses open-source software MaxQuant 1.3.0.5.By searching for SwissProt databases (version 1,0/2,003 20354) is identified protein, mixture HC4201616 qualification results ginseng prepared by embodiment 1 It is shown in Table 1.The result of mixture HC4201616 prepared by embodiment 2-6 is same as Example 1.

Test case 3

The activity of mixture HC4201616 prepared by detection embodiment 1-6 with external suppression primary hippocampal cells apoptosis, The activity of Synaptic formation between promotion hippocampal cell.The detection method is comprised the following steps：

Step one, takes out 2 microlitres, ultraviolet in the mixture HC4201616 for being prepared respectively from embodiment 1-6 respectively Its absorbance is determined under 280nm, so as to calculate the protein concentration of said mixture HC4201616.

Step 2, with 24 orifice plate culture primary hippocampal cells, culture used medium is DMEM, and condition of culture is 37 DEG C, 5vol% carbon dioxide.

Step 3, when cell density reaches every hole 4 × 10⁵During individual cell, toward culture medium in add mixture HC4201616, the mixture HC4201616 micrograms containing protein 10 00 added in each hole, as experimental group 1- mixtures HC4201616.Also include in experimental group simultaneously：It is 1000 micrograms i.e. toward protein content is separately added into the culture medium in different holes Human plasma (the step of 1 preparation method of embodiment (1) prepares), is named as experimental group 2- human plasmas.Control group is set simultaneously, In control group, when cell density reaches every hole 4 × 10⁵During individual cell, 100 microlitres of physiology is added toward the culture medium in hole Salt solution.

Step 4, continues cultured cells 24 hours, then under an optical microscope to each experimental group under conditions of original With the counting that control group carries out cell quantity and cynapse quantity.

Result such as Fig. 2-4 of mixture HC4201616 prepared by embodiment 1, wherein：

Fig. 2 (a), (b), (c) is respectively according to after experimental group 1-HC4201614, experimental group 2- human plasmas, control group treatment The primary hippocampal cells for obtaining.Can observe the cynapse quantity that either hippocampal cell quantity is still formed, experimental group 1- HC4201616 mixtures are far longer than control group, more than experimental group 2- human plasmas.

In Fig. 3, the quantity of the primary hippocampal cells of experimental group 1 reaches about 800-900/square centimeter, more than experimental group 2 About 500-600/square centimeter, and control group about 500-550/square centimeter, it was demonstrated that mixture HC4201616 has Suppress the activity of primary hippocampal cells apoptosis.

In Fig. 4, the Synaptic formation quantity of experimental group 1 reaches about 80-100/square centimeter, more than the about 45- of experimental group 2 60/square centimeter, and control group about 35-60/square centimeter, it was demonstrated that mixture HC4201616 has and promotes hippocampus thin The activity of Synaptic formation between born of the same parents.

The result of mixture HC4201616 prepared by embodiment 2 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 800-900/square centimeter, the about 400-500 of/square centimeter individual more than the about 500-600 of experimental group 2, and control group Individual/square centimeter, it was demonstrated that mixture HC4201616 has the activity for suppressing primary hippocampal cells apoptosis.The cynapse shape of experimental group 1 Reach about 80-100/square centimeter into quantity, more than about 40-60/square centimeter of experimental group 2, and control group about 40- 50/square centimeter, it was demonstrated that mixture HC4201616 has the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201616 prepared by embodiment 3 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 700-850/square centimeter, the about 300-400 of/square centimeter individual more than the about 400-500 of experimental group 2, and control group Individual/square centimeter, it was demonstrated that mixture HC4201616 has the activity for suppressing primary hippocampal cells apoptosis.The cynapse shape of experimental group 1 Reach about 80-95/square centimeter into quantity, more than about 40-50/square centimeter of experimental group 2, and control group about 30- 40/square centimeter, it was demonstrated that mixture HC4201616 has the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201616 prepared by embodiment 4 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 900-1000/square centimeter, the about 300-400 of/square centimeter individual more than the about 400-500 of experimental group 2, and control group Individual/square centimeter, it was demonstrated that mixture HC4201616 has the activity for suppressing primary hippocampal cells apoptosis.The cynapse shape of experimental group 1 Reach about 90-100/square centimeter into quantity, more than about 40-50/square centimeter of experimental group 2, and control group about 30- 40/square centimeter, it was demonstrated that mixture HC4201616 has the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201616 prepared by embodiment 5 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 800-900/square centimeter, the about 300-400 of/square centimeter individual more than the about 400-500 of experimental group 2, and control group Individual/square centimeter, it was demonstrated that mixture HC4201616 has the activity for suppressing primary hippocampal cells apoptosis.The cynapse shape of experimental group 1 Reach about 80-90/square centimeter into quantity, more than about 40-50/square centimeter of experimental group 2, and control group about 30- 40/square centimeter, it was demonstrated that mixture HC4201616 has the activity of Synaptic formation between promotion hippocampal cell.

The result of mixture HC4201616 prepared by embodiment 6 is as follows：The quantity of the primary hippocampal cells of experimental group 1 reaches To about 750-900/square centimeter, the about 300-400 of/square centimeter individual more than the about 400-500 of experimental group 2, and control group Individual/square centimeter, it was demonstrated that mixture HC4201616 has the activity for suppressing primary hippocampal cells apoptosis.The cynapse shape of experimental group 1 Reach about 75-90/square centimeter into quantity, more than about 40-50/square centimeter of experimental group 2, and control group about 30- 40/square centimeter, it was demonstrated that mixture HC4201616 has the activity of Synaptic formation between promotion hippocampal cell.

Test case 4

Mixture HC4201616 prepared by detection embodiment 1-6 can effectively lift alzheimer's disease (senile dementia Disease) mouse memory.The detection method is comprised the following steps：

Step one, using 5XFAD Elderly dementia patients models, orders in U.S. jackson laboratory, and according to Zoopery standard is bred and is raised；Each of which experimental model mouse all carries out identified for genes by rat-tail, it is ensured that The stabilization mutation of app gene and PS1 genes.

Mouse is grouped into：Experimental group 1- mixtures HC4201616：Using 20 14 week old 5XFAD male mices, injection The mixture HC4201616 that embodiment 1-6 is prepared respectively；Experimental group 2- human plasmas：It is small using 20 14 week old 5XFAD males The blood plasma that the step of mouse, injection 1 preparation method of embodiment (1) prepares；Control group：Using 20 14 week old 5XFAD males Mouse, injecting normal saline.

Various administrations are entered in Mice Body by tail vein injection, experimental group ensure 30 micrograms of protein/time dosage, Behaviors survey (specially water maze laboratory, learning ability and memory for detecting mouse) in first 24 days every three days to Medicine 1 time, is administered 8 times altogether.

Step 2, water maze laboratory：Carried out between 8 points of every morning at 1 point in afternoon.Water maze spatial memory training period is 4 days, four times a day, training interval time was 10 minutes every time；In an experiment, every four mouse are randomly divided into a training group. For each training group, the position of platform of water maze is probabilistically assigned, and keeps constant in whole training.In training, mouse It is released into water maze from optional position, and allows it that hiding platform was searched in 120 seconds.If mouse is not at 120 seconds Platform is inside found, it will be directed into platform.The time used by platform is found in training every time with the distance passed through by intelligence Camera automatically records.

Water maze test is carried out after last time is trained 48 hours.Every time in test, every mouse is released into not to be had In the water maze of placement platform, and allow its freely activity 60 second.Its travelling route is automatically recorded by intelligent video camera head.Survey The examination phase is shorter than time training period one times, to avoid mouse from producing Depressive behavior.Time that mouse is spent in target quadrant and its The time that his three quadrants are spent is recorded, for the assessment to mouse memory power.

The test result of embodiment 1 such as Fig. 5, the target quadrant stop memory time of experimental group 1 is about 90 seconds, is longer than experiment About 55 seconds of group 2 and about 50 seconds of control group.

The result of mixture HC4201616 prepared by embodiment 2 is as follows：The target quadrant of experimental group 1 stops memory time About 100 seconds, considerably longer than about 10 seconds of about the 25 of experimental group 2 second and control group.

The result of mixture HC4201616 prepared by embodiment 3 is as follows：The target quadrant of experimental group 1 stops memory time About 85 seconds, it is longer than about 55 seconds of experimental group 2 and about 50 seconds of control group.

The result of mixture HC4201616 prepared by embodiment 4 is as follows：The target quadrant of experimental group 1 stops memory time About 100 seconds, it is longer than about 55 seconds of experimental group 2 and about 50 seconds of control group.

The result of mixture HC4201616 prepared by embodiment 5 is as follows：The target quadrant of experimental group 1 stops memory time About 95 seconds, it is longer than about 55 seconds of experimental group 2 and about 50 seconds of control group.

The result of mixture HC4201616 prepared by embodiment 6 is as follows：The target quadrant of experimental group 1 stops memory time About 90 seconds, it is longer than about 55 seconds of experimental group 2 and about 50 seconds of control group.

Comparative example 1

In addition to the molecular weight of the film bag retention used in step (III) low temperature ultrafiltration step is for 13kD, other operation steps It is rapid same as Example 1.

The final product that the comparative example is obtained is named as C1, and C1 electrophoresis results are as follows：25kD、48kD、67kD、71kD、 115kD、155kD、200kD；The work that product C1 suppresses primary hippocampal cells apoptosis is tested using with the identical method of test case 3 Property, promote the activity of Synaptic formation between hippocampal cell, its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 600-700/square centimeter, the about 420- of experimental group 2 500/square centimeter, and control group about 340-420/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 65- 75/square centimeter, about 45-52/square centimeter of experimental group 2, about 32-44/square centimeter of control group.

Comparative example 2

In addition to the cold sulphur ammonium settling step of step (IV) is eliminated, other operating procedures are same as Example 1.

The final product that the comparative example is obtained is named as C2, and C2 electrophoresis results are as follows：25kD、48kD、67kD、71kD、 80kD、115kD、155kD、200kD；Withered using product C2 suppression primary hippocampal cells are tested with the identical method of test case 3 The activity died, the activity for promoting Synaptic formation between hippocampal cell, its result are as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 620-710/square centimeter, the about 430- of experimental group 2 510/square centimeter, and control group about 350-430/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 68- 72/square centimeter, about 48-54/square centimeter of experimental group 2, about 33-45/square centimeter of control group.

Comparative example 3

In addition to step (V) and (VI) is eliminated, other operating procedures are same as Example 1.

The final product that the comparative example is obtained is named as C3, and C3 electrophoresis results are as follows：25kD、48kD、67kD、71kD、 115kD、155kD、200kD；The work that product C3 suppresses primary hippocampal cells apoptosis is tested using with the identical method of test case 3 Property, promote the activity of Synaptic formation between hippocampal cell, its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 580-660/square centimeter, the about 430- of experimental group 2 490/square centimeter, and control group about 320-410/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 68- 77/square centimeter, about 46-55/square centimeter of experimental group 2, about 36-45/square centimeter of control group.

Comparative example 4

In addition to step (VIII) and (IX) is eliminated, other operating procedures are same as Example 1.

The final product that the comparative example is obtained is named as C4, and C4 electrophoresis results are as follows：15kD、25kD、48kD、67kD、 71kD、115kD、155kD、180kD、200kD；Suppress primary hippocampal using product C3 is tested with the identical method of test case 3 The activity of Apoptosis, the activity for promoting Synaptic formation between hippocampal cell, its result are as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 630-720/square centimeter, the about 430- of experimental group 2 500/square centimeter, and control group about 350-440/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 68- 80/square centimeter, about 46-55/square centimeter of experimental group 2, about 33-45/square centimeter of control group.

Comparative example 5

In addition to step (IV), other operating procedures are same as Example 1.

Step (IV) includes following operation：

(IV) cold sulphur ammonium precipitation：

By the 0-4 DEG C of ammonium sulfate solution of the saturation of precooling and the low temperature ultrafiltration product according to volume ratio 1:9.5 mixing, then at 0 DEG C 12h is placed, become real to precipitation automatic sedimentation, obtain cold sulphur ammonium precipitated product.

The final product that the comparative example is obtained is named as C5, and C5 electrophoresis results are as follows：28kD、45kD、55kD、120kD、 180kD、200kD；Suppress the activity of primary hippocampal cells apoptosis, rush using product C5 is tested with the identical method of test case 3 Enter the activity of Synaptic formation between hippocampal cell, its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 300-380/square centimeter, the about 410- of experimental group 2 510/square centimeter, and control group about 320-430/square centimeter.The Synaptic formation quantity of experimental group 1 reaches about 30- 35/square centimeter, about 48-55/square centimeter of experimental group 2, about 34-48/square centimeter of control group.

Comparative example 6

In addition to the anion exchange step of step (VII) is different from embodiment, other operating procedures and the phase of embodiment 1 Together.The wash-out of step (VII) is that gradient elution is eluted rather than step level, only have collected an eluent without repeatedly being divided Stage collects eluent.

Anion exchange step is as follows in this comparative example：By centrifugation product, 200 microlitres are added to anion-exchange column In Source Q, gradient elution is carried out with the Tris hydrochloride buffers (200mM, pH8.0) containing 500mM sodium chloride, wherein cloudy The filler of ion exchange column is Q Sepharose (being purchased from GE Healthcare), and particle mean size is 3 microns, anion-exchange column Volume is 25 milliliters, and elution speed is 4ml/min, and loading speed is 3ml/ml；Start to collect when elution volume is 35 milliliters Eluent, stops collecting eluent when elution volume is 45 milliliters；Elution fractions being collected into, as anion exchange Product.

The final product that the comparative example is obtained is named as C6, and C6 electrophoresis results are as follows：25kD、48kD、67kD、71kD、 115kD；Suppress the activity of primary hippocampal cells apoptosis, promotion hippocampus using product C6 is tested with the identical method of test case 3 The activity of Synaptic formation between cell, its result is as follows：

The quantity of the primary hippocampal cells of experimental group 1 reaches about 400-550/square centimeter, the about 420- of experimental group 2 510/square centimeter, the Synaptic formation quantity of about 360-440/square centimeter experimental group 1 of control group reaches about 45-55 About 35-45/square centimeter of individual/square centimeter, about 40-50/square centimeter of experimental group 2, and control group.

Claims

It is 1. a kind of to strengthen the mixture from blood plasma remembered, it is characterised in that：The SDS-PAGE denaturation glue electricity of the mixture Swimming at least includes 7 band, and molecular size range is respectively：25kD、48kD、67kD、71kD、115kD、155kD、200kD.
2. mixture according to claim 1, it is characterised in that：The blood plasma derives from mammal；It is highly preferred that described Blood plasma derives from the mankind.
3. mixture according to claim 1, it is characterised in that：Analyzed by protein spectrum, at least contained in the mixture There are 67 kinds of albumen；Preferably, the mixture also includes and the protein bound small molecule；It is highly preferred that 67 hatching egg Include in vain：

14-3-3 albumen ζ/triangle, alpha-2-anti fibrinolysins, annexin, Annexin A1, apolipoprotein (a), double work( Energy 3 ' 5 '-phosphosulfate of phosphosulfate synthase 1, BPI is folded and contains family member 1, carbohydrate sulfotransferase 12, carboxylic first Base sulphur 12, cathepsin D's light chain, plasma thromboplastin antecedent, coiled-coil domain protein 10 4, complement C1r sub-component samples albumen, Complement C2b, supplemental component C8 β chains, CFI light chain, cornea chain albumen, skin from albumen, mycin, desmoglein 1, E3 ubiquitin protein ligase HUWE1, E3 ubiquitin protein ligases midline-1, eukaryotic translation initiation factor γ 1, aliphatic acid knot Hop protein, myosin B, filaggrin 2, Gas2 samples albumen 2, GPI, heat shock are homologous 71kDa albumen, Hemopexin, hemoglobin, hepatocyte growth factor-like protein, keratin, immunoglobulin-like are more Peptide 1, immunoglobulin-like polypeptide 5, the hypotype of fibrinogen-α chains, with reference to ferrihemoglobin, humanized's kallikrein Associated proteins, II types keratin, Lysine-tRNA ligase, malic dehydrogenase, neutrophil leucocyte alexin 1, peroxide Enzyme body memebrane protein pmp34, phosphoribosylformylglycinamidine cyclo-ligase, plasma kallikrein, plasminogen, platelet basic Albumen, immunoglobulin receptor polymer, g protein coupled receptor 149, leucine tRNA ligases, mitochondria, Protein S 100- The abundant saliva acidity phosphorylation egg of A8, Protein S 100-A9, retinol-binding proteins (blood plasma, isotype CRA_b), proline White 1/2, serum ferritin, serpin, signal recognition particle subunit SRP72, sapiens's Solute Carrier family 12 (sodium/ Potassium/chlorion transport protein, member 2, hypotype cra_a), oxter gland androgen regulatory protein 3B, upper storage reservoir albumen, spt20 it is homologous Transcription factor 1, three peptidyl peptidases 2, tubulin poly glutamy enzyme ttll11, Ugl-Y3, cell propagation raise the factor, dimension life Plain D associated proteins, WD repeat to contain protein 19.
4. the preparation method of any described mixtures of a kind of claim 1-3, it is characterised in that：The preparation method includes successively Following steps：It is plasma collection, cold sulphur ammonium precipitation, SD inactivations, centrifugation, anion exchange, concentration for the first time, molecular sieve, saturating Analysis, second concentration step；Preferably, after the plasma collection procedure, before the cold sulphur ammonium settling step, also including low Warm filtration step and low temperature ultrafiltration step.
5. preparation method according to claim 4, it is characterised in that：In the cold sulphur ammonium settling step, will be described low Warm ultrafiltration product or blood plasma stand after mixing with the ammonium sulfate solution of saturation, collect supernatant；Again by the supernatant and saturation Stood after ammonium sulfate solution mixing, retain precipitation, obtain cold sulphur ammonium precipitated product；

Preferably, the temperature of the ammonium sulfate solution of the saturation is 0-4 DEG C；

Preferably, the volume ratio of the ammonium sulfate solution of the saturation and the low temperature ultrafiltration product or blood plasma is 1：(9-10)；

Preferably, the volume ratio of the ammonium sulfate solution of the saturation and the supernatant is 1：(1-5)；

Preferably, the dwell temperature is 0-10 DEG C, and time of repose is 8-16 hours.
6. the preparation method according to claim 4 or 5, it is characterised in that：In the plasma collection procedure, using anti-freezing Pipe collects blood, then by the way that supernatant is collected by centrifugation, obtains blood plasma；

Preferably, in the centrifugation, centrifugal force is 500-1300g, and the time is 10-40 minutes, and centrifuging temperature is 0-5 DEG C.
7. according to any described preparation methods of claim 4-6, it is characterised in that：In the low temperature ultrafiltration step, by institute State filtrate carries out film bag ultrafiltration by applying pressure, obtains low temperature ultrafiltration product；

Preferably, the film bag molecular cut off is 3kD-10kD；

Preferably, the temperature from ultrafiltration is 0-15 DEG C, and pressure is 1-20MPa；

Preferably, the buffer solution that the ultrafiltration is used is in phosphate buffer, Tris- hydrochloride buffers, HBS buffer solutions Kind；It is highly preferred that the concentration of the Tris- hydrochloride buffers be 0.2-300mM, pH value be 7.0-9.2, the phosphate buffer Concentration be 1.0-500mM, pH value be 6.0-9.0, the concentration of the HBS buffer solutions is 0.5-250mM, pH value is 6.4-8.4； It is further preferred that it is 250mM, the Tris- hydrochloride buffers that pH value is 8.0 that the buffer solution is concentration.
8. according to any described preparation methods of claim 4-7, it is characterised in that：In the filter at low temperature step, in pressure The blood plasma is filtered under the conditions of power, is obtained filtrate；

Preferably, the aperture of filter membrane is that, not less than 0.22 micron, the aperture of more preferably described filter membrane is that 0.22 micron and 0.45 are micro- Rice；

Preferably, the temperature of the filtering is 0-15 DEG C, and pressure is 1-20MPa.
9. according to any described preparation methods of claim 4-8, it is characterised in that：In the SD inactivation steps, will be described Molecular weight retention product stands after being suspended again with SD inactivators, obtains inactivating product；

Preferably, the dwell temperature is 4~37 DEG C, and the time is 6~20 hours；More preferably described dwell temperature is 20 DEG C, Time is 10 hours；

Preferably, the SD inactivators contain 0.3~2% N- butyl triphosphate, 0.5~2% tween；

In the step with centrifugal separation, supernatant will be retained after the inactivation product centrifugation, obtain centrifugation product；It is preferred that Ground, in the centrifugation, centrifugal force is 2000-10000g, and the time is 10-30 minutes, and temperature is 0-10 DEG C.
10. according to any described preparation methods of claim 4-9, it is characterised in that：In the anion exchange step, will The centrifugation product carries out step level wash-out in adding anion-exchange column, collects the eluent for obtaining every time, after mixing To anion exchanged product；

Preferably, in the step level wash-out, first eluted with first time elution buffer, obtain first time eluent；Again with second Secondary elution, obtains waste liquid；Third time elution is used again, obtains third time eluent；Merging the first time washes De- liquid and third time eluent, obtain anion exchanged product；

It is highly preferred that the sodium chloride containing 50-80mM in the first time eluent buffer solution, second eluent buffering Liquid contains the sodium chloride of 250-300mM, and the third time eluent buffer solution contains the sodium chloride of 450-500mM；It is highly preferred that The first time buffer elution liquid, second buffer elution liquid, third time buffer elution liquid are concentration for 0.2-200mM, pH It is worth the Tris- hydrochloride buffers for 7.0-9.2.
11. according to any described preparation methods of claim 4-10, it is characterised in that：In the first time concentration step, will The anion exchanged product is concentrated, and obtains first time enriched product；

Preferably, the volume of the dialysis product is the enriched product not less than 50 times, more preferably 50-500 times；It is more excellent Selection of land, the volume of the first time enriched product is 500 microlitres -5 milliliters；

Preferably, the concentration is centrifuged using concentration tube, and the concentration tube allows the material of 1.0-2.5KD to pass through, it is described from In the heart, centrifugal force is 1000-3000g, and temperature is 2-8 DEG C；

Preferably, the concentration is to use concentration cup pressurization, and the concentration cup allows the material of 1.0-2.5KD to pass through, described to add The pressure of pressure is 1-15MPa.
12. according to any described preparation methods of claim 4-11, it is characterised in that：In the molecular sieve step, will be described First time enriched product is eluted in molecular sieve, obtains molecular sieve product；

Preferably, the sodium chloride containing 150-500mM in the elution buffer of the wash-out；

Preferably, the elution buffer is Tris- hydrochloride buffers, and its concentration is 0.2-100mM, and its pH value is 7.0-9.2； It is highly preferred that the elution buffer is Tris- hydrochloride buffers, its concentration is 20mM, and its pH value is 8.0；

Preferably, elution volume when starting to collect eluent is 40-50ml, and elution volume when stopping collecting eluent is 70-80ml。
13. according to any described preparation methods of claim 4-12, it is characterised in that：In the dialysis step, will be described Molecular sieve product is dialysed, and is collected the product in Dialysis container and is obtained product of dialysing；

Preferably, the elution buffer for using of dialysing is in Tris- hydrochloride buffers, phosphate buffer, HBS buffer solutions It is a kind of；It is highly preferred that the concentration of the Tris- hydrochloride buffers be 0.2-300mM, pH value be 7.0-9.2, the phosphoric acid buffer The concentration of liquid is that 1.0-500mM, pH value are 6.0-9.0, and the concentration of the HBS buffer solutions is 0.5-250mM, pH value is 6.4- 8.4；It is further preferred that the elution buffer is 1mM, the Tris hydrochloride buffers that pH value is 8；

Preferably, the time of the dialysis is 20-72h；

Preferably, the dialysis volume ratio of the dialysis is 1:(100-10000).
14. according to any described preparation methods of claim 4-13, it is characterised in that：In the concentration step, will be described Dialysis product carries out second concentration, obtains second enriched product, as described mixture；

Preferably, the volume of the dialysis product is the enriched product not less than 50 times, more preferably 50-200 times；

Preferably, the concentration is centrifuged using concentration tube, and the concentration tube allows the material of 1.0-3.5KD to pass through, it is described from In the heart, centrifugal force is 500-3000g；

Preferably, the concentration is to use concentration cup pressurization, and the concentration cup allows the material of 1.0-3.5KD to pass through, described to add The pressure of pressure is 1-20MPa.
15. a kind of pharmaceutical compositions, comprising any described mixtures of claim 1-3 and pharmaceutically acceptable carrier.
16. pharmaceutical compositions according to claim 15, it is characterised in that the pharmaceutically acceptable carrier is：Medicine Acceptable buffer solution, protein, gelatin, monose, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surface on One or more in activating agent.
17. pharmaceutical compositions according to claim 16, it is characterised in that described pharmaceutical composition includes following component：1 Any described mixtures of claim 1-3 of times volume, the 8.5wt%NaCl of 9 times of volumes or the PBS of 1.5M, pH7.0；

Preferably, albumin, glucose and glutamine are also included in described pharmaceutical composition；It is highly preferred that the white egg The white quality percent by volume in described pharmaceutical composition is 2%, quality of the glucose in described pharmaceutical composition Percent by volume is 1%, and quality percent by volume of the glutamine in described pharmaceutical composition is 3%.
A kind of 18. sustained release preparations, it is any described comprising any described mixtures of claim 1-3 or claim 15-17 Pharmaceutical composition and pharmaceutically acceptable biocompatible substance；Preferably, the formulation of the sustained release preparation is lipid Body, microballoon, hydrogel, Osmotic minipumps or microcapsules.
19. a kind of kits, comprising any described mixtures of claim 1-3, any described medicines of claim 15-17 Composition, or sustained release preparation described in claim 18.
Any described mixtures of 20. claim 1-3, claim 15-17 any described pharmaceutical composition, claim Kit described in sustained release preparation, claim 19 described in 18, prevention, improve or/and treatment senile dementia in or Application in enhancing memory medicine.