CN110101847A

CN110101847A - The application of transferrins and the composition comprising transferrins

Info

Publication number: CN110101847A
Application number: CN201910432249.2A
Authority: CN
Inventors: 栗琳; 崔雅轩; 谢蓝; 张丽丽; 娄钰霞
Original assignee: Beijing Haosi Biotechnology Co Ltd
Current assignee: Jiangsu Haosi Muke Biotechnology Co ltd
Priority date: 2019-05-23
Filing date: 2019-05-23
Publication date: 2019-08-09
Also published as: CN113801219A

Abstract

The invention belongs to biologics application fields, more particularly to a kind of application of transferrins and composition comprising transferrins, comprising: transferrins and other components (human serum albumin, human immunoglobulin(HIg), clinical treatment senile dementia one of first-line drug or a variety of)；It preferably includes: transferrins, human serum albumin and human immunoglobulin(HIg), the more excellent mass ratio for three are as follows: 0.5-4 mass parts, 5-40 mass parts, 5-40 mass parts.The raw material for preparing of transferrins includes human plasma Cohn IV component, and the preparation method of transferrins successively includes: pretreatment fluid preparation, hydrophobic chromatography purification step, preferably includes concentration displacement step.Composition is applied to: the drug or/and intelligence development class replenishers or drug of preparation prevention or/and improvement or/and treatment Alzheimer's disease.Transferrins of the invention is combined with other plasma proteins, can be played the role of enhancing memory, be improved cognition.

Description

The application of transferrins and the composition comprising transferrins

Technical field

The invention belongs to biologics application fields, and in particular to a kind of application of transferrins and include transferrins Composition.

Background technique

Alzheimer disease (Alzheimer ' s disease, AD) is also known as senile dementia, is one kind by neuron loss Caused mesencephalic centre nervous system degenerative disease, the disease incidence of the disease is about 4%-8% in elderly population.1906, moral State's neuropathologist's Alzheimer reports 51 years old female patient that an example has the performance of progressive decrease of memory for the first time. 1910, this disease is named as by A Er with the psychiatrist Emil Kraepelin proposal that cerebral disorders of naming and classify are famous Ci Haimo disease.The common clinical manifestation of the disease is progressive memory and cognitive power decline, aphasis, psychomotor exception Deng causing to seriously affect to the normal life of patient, while also bringing heavy burden to family and society.2010, WHO report Road whole world dementia patients are 35,000,000, and China is 5,400,000.From 2010, it will double within whole world dementia total population every 20 years. " report of world's Alzheimer disease 2015 " points out that the number of patients to the year two thousand fifty, global AD will be increased to 1.3 hundred million.The hair of AD Interpretation of the cause, onset and process of an illness system is complicated, is related to different kinds of molecules signal path, only 4 kinds of the therapeutic agent listed at present (donepezil, galanthamine, Rivastigmine and Memantine), to delay in AD early stage symptom progress and improve based on early stage AD clinical condition, there is no so far Reverse or prevent the active drug of disease progression.

There is research to be injected into Aged Mice the blood plasma from young mice is duplicate in recent years, discovery blood plasma can lead to It overregulates the classical molecular pathway for participating in hippocampus cognition and improves memory function.And blood plasma composition is extremely complex, including protein, Lipid, inorganic salts, sugar, amino acid, metabolic waste and a large amount of water, there is no that determine be which ingredient tool in blood plasma so far It is improved memory, improves and treat the effect of senile dementia；And at present Chinese blood product company to the exploitation of Plasma Pheresis/Apheresis Plasma and Using needing to improve.

Therefore, in current scientific research and practice, needing to find out in blood plasma has improving studing ability and treatment old The ingredient of year property dementia, and the utilization rate of sufficiently raising Plasma Pheresis/Apheresis Plasma, develop new blood product.

Summary of the invention

Composition the purpose of the present invention is to provide a kind of application of transferrins and comprising transferrins.

The purpose of the present invention is what is be achieved through the following technical solutions:

Application of the transferrins in the drug of preparation prevention or/and improvement or/and treatment Alzheimer's disease；Or/and Application of the transferrins in terms of preparation intelligence development class replenishers or drug.

A kind of composition comprising transferrins, comprising: transferrins and other components, the other components include: people Blood albumin, human immunoglobulin(HIg), clinical treatment senile dementia one of first-line drug or a variety of；Preferably, described Composition includes: transferrins, human serum albumin and human immunoglobulin(HIg)；It is highly preferred that pressing mass fraction, the composition packet It includes: transferrins 0.5-4 mass parts, human serum albumin 5-40 mass parts, human immunoglobulin(HIg) 5-40 mass parts；Further preferably Ground, by mass fraction, the composition includes: 1 mass parts of transferrins, 8.33 mass parts of human serum albumin, people's immune globulin White 8.33 mass parts.

In a preferred embodiment, the first-line drug of the clinical treatment senile dementia includes: donepezil, Garland He is quick, Rivastigmine, one or more of Memantine.

In a preferred embodiment, the composition further includes pharmaceutically acceptable carrier.

In a preferred embodiment, the raw material for preparing of the transferrins includes human plasma Cohn IV component.

In a preferred embodiment, the preparation method of the transferrins successively includes: pretreatment fluid preparation step, is dredged Water chromatography purification step；It preferably, further include concentration displacement step.

In a preferred embodiment, in the pretreatment fluid preparation step, plasma C ohn IV component is successively passed through Primary redissolution processing, first time centrifugal treating, first time filtration treatment adjust pH and handle, removal impurity protein processing, and second Centrifugal treating, second of filtration treatment, obtains pretreatment fluid；In the hydrophobic chromatography purification step, the pretreatment fluid is passed through Buffer replacement Treatment is crossed, hydrophobic chromatography processing obtains purifying transferrins；Preferably, the purity of the purifying transferrins For more than or equal to 95%；Preferably, in the concentration displacement step, the purifying transferrins is subjected to hyperfiltration treatment, displacement Processing, obtains transferrins.

In a preferred embodiment, in the pretreatment fluid preparation step, the first time redissolution processing, using PBS Buffer redissolves, and the mass volume ratio of the plasma C ohn IV component and the PBS buffer solution is 1:(2-10), preferably 1: 3.5；Preferably, the PBS buffer solution are as follows: 10-50mmol/L PBS, pH6.0-8.0, more preferably 20mmol/L PBS, pH7.5；The first time centrifugal treating, revolving speed 4,000-15,000rpm, temperature be 0-10 DEG C, preferably 10,000rpm, 4 DEG C, retain supernatant and is used for the first time filtration treatment；The first time filtration treatment, uses aperture for 0.1-1.0 μm, excellent It is selected as 0.45 μm of filter membrane, takes filtrate for adjusting pH processing after filtering；The adjusting pH processing, by the pH value of the filtrate It is adjusted to 6.5-7.5, preferably 7.0；More preferably using 1mol/L sodium citrate solution as pH adjusting agent；The removal impurity Albumen processing is added protein precipitant into the filtrate after adjusting pH and removes impurity protein；It is highly preferred that the protein precipitant It is selected from: PEG4000 and/or ethanol solution；It is further preferred that the quality volume of the filtrate after the PEG4000 and adjusting pH Than for 10-20%, preferably 15%；The concentration expressed in percentage by volume of the ethanol solution be 90-98%, preferably 96%, temperature 0 It is 4 DEG C preferably subzero to subzero 10 DEG C；Second of centrifugal treating, revolving speed 4,000-10,000rpm, temperature 0-10 DEG C, preferably 10000rpm, retains supernatant and is used for second of filtration treatment by 4 DEG C；Second of filtration treatment, using hole Diameter is 0.1-1.0 μm, and preferably 0.45 μm of filter membrane, obtained filtrate is as pretreatment fluid.

In a preferred embodiment, in the hydrophobic chromatography purification step, the buffer exchange processing is used HiTrap Desalting desalting column, the equilibration buffer of use are as follows: 10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration be 0.5-3mol/L, preferably 20mmol/L PBS, pH7.0, (NH₄)₂SO₄Concentration is 1.5mol/L；It is described to dredge Water Image processing, first by octyl drainage column described in the equilibration buffer, then it is described pungent with equilibration buffer elution Base drainage column, then eluted with elution buffer；The hydrophobic chromatography column is octyl drainage column, with Octyl Sepharose 4Fast Flow filling；The equilibration buffer are as follows: 10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 1.5-3mol/L, preferably 20mmol/L PBS, pH7.0, (NH₄)₂SO₄Concentration is 1.5mol/L；The elution buffer are as follows: 10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 0.9-1.4mol/L, preferably 20mmol/L PBS, pH7.0,(NH₄)₂SO₄Concentration is 1.2mol/L；Preferably, the hyperfiltration treatment, using 1KD-10KD, preferably 3KD ultrafiltration Film；The replacement Treatment, the displacement buffer of use are as follows: 10-50mmol/L PBS, pH7.0-7.4, preferably 20mmol/L PBS, pH7.2.

Application of the composition in the drug of preparation prevention or/and improvement or/and treatment Alzheimer's disease, or/ Application in terms of preparation intelligence development class replenishers or drug.

Compared with the prior art, the invention has the following beneficial effects:

1, in the present invention, transferrins can play the role of enhancing memory, improve cognitive function.Inventor's discovery turns Ferritin can remarkably promote the growth of nerve cell, and can significantly improve the ability of learning and memory of dementia animal model. Experimental result of nesting also indicates that the transferrins that the present invention extracts can be improved the ability of nesting of dementia animal model, prompts dull-witted The self-care ability of animal improves.

2, in the present invention, transferrins can be with one line medicine of other plasma proteins or/and clinical treatment senile dementia Object is used in combination, and further increases therapeutic effect.

3, the present invention also provides a kind of transferrins preparation method, which can be from currently used Nitschmann- Transferrins is extracted in waste Cohn IV component when Kistler or 6 method of cohn or cohn6+9 method produce blood product, Sufficiently, limited blood plasma is reasonably utilized, reduces the waste of blood plasma resource.

4, the preparation method of transferrins of the present invention is relatively simple, and can direct industrial amplification production, on a large scale Ground is applied to pharmaceuticals industry.

Detailed description of the invention

Fig. 1: (a): hydrophobic chromatography UV280 peak figure in embodiment 1.(b): SDS-PAGE detects different step in embodiment 1 The electrophoretogram of the obtained product containing albumen.

Fig. 2: the effect of the transferrins processing cultured primitive hippocampal neuron of light microscopic detection various concentration in detection example 1 Figure, part A are hippocampus neuron number histogram, and part B is hippocampal neuron projection length histogram.

Fig. 3: improve A after the transferrins processing cultured primitive hippocampal neuron of mtt assay detection various concentration in detection example 1 β_25-35Damage to cultured primitive hippocampal neuron.

Fig. 4: water maze Behaviors survey detects transferrins, transferrins+human serum albumin, turns iron egg in detection example 1 White+human immunoglobulin(HIg), human serum albumin+human immunoglobulin(HIg), transferrins+human serum albumin+human immunoglobulin(HIg) processing Nest building behavior score before and after mouse.

Fig. 5: water maze Behaviors survey detects transferrins, transferrins+human serum albumin, turns iron egg in detection example 1 White+human immunoglobulin(HIg), human serum albumin+human immunoglobulin(HIg), transferrins+human serum albumin+human immunoglobulin(HIg) processing The memory improvement situation of mouse.Wherein, part A is that mouse is appeared on the stage the time in the hiding platform training stage, and part B is that mouse is being surveyed Examination stage percentage of time locating for target quadrant.

Specific embodiment

In order to be preferably illustrated to technical characteristic and effect of the invention, below using specific embodiment to this hair It is bright to be described in detail, but the present invention is not limited thereto.In a first aspect, the present invention provide transferrins preparation prevention or/and Application in the drug of improvement or/and treatment Alzheimer's disease；Or/and transferrins is in preparation intelligence development class replenishers or drug The application of aspect.

The present invention demonstrates the influence that various concentration transferrins grows primary hippocampal neurons in cellular level, and leads to Improve A β after crossing the transferrins processing cultured primitive hippocampal neuron of mtt assay detection various concentration_25-35To originally culture hippocampus The damage of neuron, test result show that in the concentration range of 0.25-2mg/ml, transferrins promotes hippocampal synapse length The effect of growth is obvious, can significantly improve A β_25-35To the toxic damages of cultured primitive hippocampal neuron.In addition, inventor Transferrins and composition comprising transferrins are had detected to the note of 5 × FAD transgenosis (APP) mouse from animal test level Recalling power improves situation, the results showed that, transferrins and the composition comprising transferrins can be obviously improved Model of Dementia mouse Memory.

Second aspect, the present invention provides a kind of compositions comprising transferrins, comprising: transferrins and other groups Point, the other components include: human serum albumin, human immunoglobulin(HIg), clinical treatment senile dementia first-line drug in It is one or more；

Preferably, the composition includes: transferrins, human serum albumin and human immunoglobulin(HIg)；Three kinds of albumen joints The effect being used alone using any albumen can be dramatically increased.

It is highly preferred that press mass fraction, transferrins 0.5-4 mass parts (such as: can for 0.5,0.8,1,1.2,1.5, 1.8, arbitrary value or any numberical range between the two in 2,2.3,2.5,2.8,3,3.2,3.5,3.8,4 mass parts), people's blood Albumin 5-40 mass parts (such as: it can be 5,8,10,12,15,18,20,23,25,28,30,32,35,38,40 mass parts Middle arbitrary value or any numberical range between the two), human immunoglobulin(HIg) 5-40 mass parts (such as: can for 5,8,10, 12, arbitrary value or any numberical range between the two in 15,18,20,23,25,28,30,32,35,38,40 mass parts)；Into One step preferably, 1 mass parts of transferrins, 8.33 mass parts of human serum albumin, 8.33 mass parts of human immunoglobulin(HIg).

The first-line drug of the clinical treatment senile dementia includes: donepezil, galanthamine, Rivastigmine, U.S. dollar One or more of just.

Transferrins of the present invention, human serum albumin, human immunoglobulin(HIg) are source of people albumen, in following embodiment Human serum albumin, the human immunoglobulin(HIg) used is commercial product, and human serum albumin purity is not less than 96%, human immunoglobulin(HIg) Purity is not less than 90%.

The composition further includes pharmaceutically acceptable carrier.Above-mentioned pharmaceutically acceptable carrier includes: pharmaceutically Acceptable propellant, buffer, gelatin, monosaccharide, polysaccharide, amino acid, chelating agent, sugar alcohol, polyethylene glycol and surface-active One of agent is a variety of.

Several ingredients in the composition, can use individually, and be used in conjunction with again after can also mixing.

The transferrins can be the human transferrin using any method preparation at present, preferably with the following method The transferrins of preparation uses human plasma Cohn IV component as raw material, and the SDS-PAGE of obtained transferrins is denaturalized glue The band that high-visible 1 molecular weight is about 79kD in electrophoretogram, human plasma Cohn IV component refer to by Nitschmann- Waste --- Cohn IV component when Kistler method or 6 method of cohn or cohn 6+9 method production human blood product.

Either clinically the treatment of AD or the thinking in AD new drug development are all from single access or mechanism at present Incision, and the clinical pathology mechanism of AD further relates to cholinergic damage, abnormalities of sugar/lipid metabolism, oxygen radical damage in addition to mutated gene Wound, immune inflammation etc..Contain a large amount of albumin and immunoglobulin in blood plasma, clinical test shows that human serum albumin can remove AD trouble The A β to dissociate in person's blood plasma, to improve cognitive ability to a certain extent.There is natural itself resist in healthy population blood The anti-A β of body, these antibody are mainly made of immunoglobulin M, and results of animal shows that intravenous injection of immunoglobulin can be improved The cognitive ability of animal, transferrins have expression in intracerebral, and there is data to suggest that in the white matter of the cortex of AD patient's multiple regions Transferrin content be remarkably decreased.The Cohn IV component that the present invention is discarded from current existing blood product preparation process In separated by hydrophobic chromatography method and identify a kind of plasma protein-transferrins, and find that this albumen can promote mind The cognitive ability of Model of Dementia animal, including learning and memory ability are grown and improved through first cell.

Above-mentioned transferrins specific is the preparation method is as follows: successively the following steps are included: prepared by pretreatment fluid, hydrophobic chromatography Purifying, concentration displacement.

Step 1: prepared by pretreatment fluid: the purpose of this step is the albumen redissolved in human plasma Cohn IV components precipitate Matter removes filter aid, and removes some foreign proteins after redissolving.

Step by step one, first time redissolution processing: Cohn IV constituent solid is dissolved into PBS buffer solution, is uniformly mixed, It obtains redissolving liquid for the first time.

The mass volume ratio (w/v) of above-mentioned human plasma Cohn IV component and PBS buffer solution is 1:(2-10), preferably 1: 3.5；Above-mentioned PBS buffer solution are as follows: 10-50mmol/L PBS, pH6.0-8.0, preferably 20mmol/L PBS, pH7.5.

Two, first time centrifugal treating step by step: above-mentioned first time is redissolved into liquid centrifugation, retains supernatant, as the first time Supernatant.

The revolving speed of above-mentioned centrifugation be 4,000-15,000rpm, temperature be 0-10 DEG C, preferably 10,000rpm, 4 DEG C.

Three, first time filtration treatment step by step: by above-mentioned first time supernatant with 0.1-1.0 μm, preferably 0.45 μm Membrane filtration obtains filtrate, as first time filtered fluid.

Step by step four, it adjusts pH processing: the sodium citrate solution of 1mol/L being added in above-mentioned first time filtered fluid, obtains Filtered fluid after adjusting pH value, pH value 6.5-7.5, preferably 7.0.

Step by step five, removal impurity protein processing: the filtered fluid after stirring above-mentioned adjusting pH value slowly adds thereto simultaneously Entering protein precipitant, (change herein refers in fact: supernatant precipitating has apparent line of demarcation, and supernatant becomes in fact to the change of precipitating automatic sedimentation Bright, precipitating quantity no longer increases), supernatant is taken later, as removal impurity protein liquid, in case the centrifugation of next step.

Above-mentioned protein precipitant is preferably Macrogol 4000 (PEG4000), PEG4000 and the filtered fluid after adjusting pH value Mass volume ratio be 10-20%, preferably 15%.

Above-mentioned protein precipitant may be that concentration expressed in percentage by volume is 90-98%, preferably 96%, and temperature is subzero 10 DEG C, preferably subzero 4 DEG C of ethanol solution；Due to the meeting heat production in precipitation process using ethanol solution, so ethanol solution is wanted Keep low temperature.

This can effectively remove a part of impurity protein step by step, while can improve the safety of product with precipitate virus Property.

Six, second of centrifugal treating step by step: above-mentioned removal impurity protein liquid is centrifuged, and retains supernatant, as second Secondary supernatant.

The revolving speed of above-mentioned centrifugation is 4000-15000rpm, and temperature is 0-10 DEG C, preferably 10000rpm, 4 DEG C.

Seven, second of filtration treatment step by step: by above-mentioned second of supernatant with 0.1-1.0 μm, preferably 0.45 μm Membrane filtration obtains filtrate, as human plasma CohnIV component pretreatment fluid.

Step 2: hydrophobic chromatography purifies: the purpose of this step is to remove it from human plasma Cohn IV component pretreatment fluid His protein impurities, isolated purifying transferrins.

Step by step one, buffer exchange processing:

This step by step in, select HiTrap Desalting desalting column, replace the pretreatment of above-mentioned human plasma Cohn IV component Liquid makes its initial salt concentration and pH and equilibration buffer (10-50mmol/L PBS, pH6.0-8.0,0.5-3mol/L (NH₄)₂SO₄, preferably 20mmol/L PBS, pH7.0,1.5mol/L (NH₄)₂SO₄) identical.Specific operation are as follows: (1) by HiTrap Desalting desalting column in advance with the equilibration buffer of 5-10 times of column volume (10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 0.5-3mol/L, preferably 20mmol/L PBS, pH7.0, wherein (NH₄)₂SO₄Concentration is 1.5mol/L) It balances, until the pH value of the liquid of conductivity detector display outflow is with the equilibration buffer consistent.It (2) again will be suitable Above-mentioned human plasma CohnIV component pretreatment fluid is fed to HiTrap Desalting column.(3) again with equilibration buffer (including 10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 0.5-3mol/L, preferably 20mmol/L PBS, PH7.0, wherein (NH₄)₂SO₄Concentration is 1.5mol/L) HiTrap Desalting column is eluted, it is received according to UV280 peak figure Collect protein solution, obtains the Cohn IV protein solution of displacement buffer.

Step by step two, hydrophobic chromatography processing:

This step by step in, select octyl drainage column (Octyl Sepharose 4Fast Flow filling).

Specific operation are as follows: (1) in advance with the equilibration buffer of 5-10 times of column volume (10-50mmol/L PBS, pH6.0- 8.0, wherein (NH₄)₂SO₄Concentration is 1.5-3mol/L, preferably 20mmol/L PBS, pH7.0, wherein (NH₄)₂SO₄Concentration is Octyl drainage column 1.5mol/L) is balanced, until the pH value and the equilibration buffer of the liquid of conductivity detector display outflow Unanimously.(2) the Cohn IV protein solution of suitable above-mentioned displacement buffer is then fed to octyl drainage column.(3) again with flat Weigh buffer (10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 1.5-3mol/L, preferably 20mmol/ L PBS, pH7.0, wherein (NH₄)₂SO₄Concentration is 1.5mol/L) eluted, make target component sufficiently with octyl drainage column knot It closes, liquid (also known as penetrating liquid) is flowed through according to the collection of UV280 peak figure.(4) again with elution buffer (10-50mmol/L PBS, PH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 0.9-1.4mol/L, preferably 20mmol/L PBS, pH7.0, wherein (NH₄)₂SO₄Concentration is 1.2mol/L) it is eluted, obtained eluent is to purify transferrins.

As preferred embodiment, above-mentioned preparation method further include:

Step 3: concentration displacement step:

One, concentration step by step: above-mentioned purifying transferrins is used into 1KD-10KD, the preferably ultrafiltration membrane of 3KD is dense Contracting, obtains concentrate.The chromatography process of above-mentioned steps two can dilute sample, this purpose step by step is that reach sample concentration can With the degree used.

Two, replacement Treatment step by step:, will be in above-mentioned concentrate using displacement buffer by the method for dialysis or ultrafiltration In high salt concentration (mainly ammonium sulfate) displacement to displacement buffer, the albumen on the protein solution or ultrafiltration membrane in dialysis membrane is taken Solution is transferrins.Originally purpose step by step is the buffer for the high salt concentration (mainly ammonium sulfate) for containing concentrate It replaces in PBS buffer solution.

The displacement buffer wherein used is 10-50mmol/L PBS, pH7.0-7.4, preferably 20mmol/L PBS, pH7.2。

When using dialysis: preferred above-mentioned displacement buffer is as elution buffer, dialysis time 20-72h, preferably For 24 hours, dialysis volume ratio is 1:(100-10000), preferably 1:500 retains the protein solution in dialysis membrane, as turns iron egg It is white.

When using ultrafiltration: preferred above-mentioned displacement buffer is 1-10KD as Ultrafiltration buffer, the specification of ultrafiltration membrane, preferably For 3KD, pressure 0.1-0.3MPa, preferably 0.1MPa, retain the protein solution on ultrafiltration membrane, as transferrins.

Mass volume ratio involved in the present invention (w/v) refers to: the ratio between the quality (g) and liquid volume (ml) of solid Example, such as: the mass volume ratio (w/v) of above-mentioned human plasma Cohn component and PBS buffer solution is 1:3.5, refers to human plasma Cohn IV When component measures 1g, PBS buffer solution then measures 3.5ml.

The preparation of transferrins of the present invention, identification and application are illustrated below by embodiment.In following embodiment Specific experimental condition and method is such as not specified in the molecular biology manipulations being related to, and please refers to the chief editor such as SambrookJ, scientific publication Society, 2017, the specification of Molecular Cloning:A Laboratory guide (fourth edition) or corresponding product.Primary hippocampal used in the following embodiment Cell is 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 transferrins, comprising the following steps:

S1: the preparation of human plasma Cohn IV component pretreatment fluid:

(1) it redissolves for the first time: waste material --- human plasma when by by Nitschmann-Kistler method production blood product Cohn IV constituent solid is dissolved into 20mmol/L PBS solution (pH7.5), after 1:3.5 (w/v) dissolution, is stirred with magnetic force Device is mixed, is uniformly mixed, obtains redissolving liquid for the first time.

(2) it is centrifuged for the first time: first time being redissolved into liquid in 10000rpm, 4 DEG C of centrifugations obtain first time supernatant.

(3) it filters for the first time: by 0.45 μm of membrane filtration of first time supernatant, obtaining first time filtered fluid.

(4) it adjusts pH: pH value is transferred to 7.0 with 1mol/L sodium citrate by first time filtered fluid.

(5) it removes impurity protein: the PEG4000 of 15% (w/v) being added in first time filtered fluid, needs in the same of stirring When be slowly added to, it is real that PEG4000 is completely dissolved rear change to be precipitated, supernatant is taken, as removal impurity protein liquid.

(6) be centrifuged for second: by removal impurity protein liquid in 10000rpm, 4 DEG C of centrifugations obtain second of supernatant.

(7) it filters for second: by second of supernatant, 0.45 μm of membrane filtration, it is pre- to obtain human plasma Cohn IV component Treatment fluid.

S2: the purifying of human plasma Cohn IV component pretreatment fluid hydrophobic chromatography:

(1) buffer exchange is handled:

A) by HiTrap Desalting desalting column (1 × 8cm) in advance with the equilibration buffer of 5-10 times of column volume (20mmol/L PBS,pH7.0,1.5mol/L(NH₄)₂SO₄) balance, until the pH of the liquid of conductivity detector display outflow It is worth consistent with the equilibration buffer.

B) suitable above-mentioned human plasma CohnIV component pretreatment fluid is fed to HiTrap Desalting column.

C) equilibration buffer (20mmol/L PBS, pH7.0,1.5mol/L (NH is used₄)₂SO₄) to HiTrap Desalting Column is eluted, and is collected protein solution according to UV280 peak figure, is obtained the Cohn IV protein solution of displacement buffer.

(2) hydrophobic chromatography is handled:

A) in advance with the equilibration buffer of 5-10 times of column volume (20mmol/L PBS, pH7.0,1.5mol/L (NH₄)₂SO₄) It balances octyl drainage column (Octyl Sepharose 4Fast Flow filling), until the liquid of conductivity detector display outflow PH value it is consistent with the equilibration buffer.

B) the Cohn IV protein solution of suitable above-mentioned displacement buffer is fed to octyl drainage column.

C) equilibration buffer (20mmol/L PBS, pH7.0,1.5mol/L (NH is used₄)₂SO₄) eluted, according to UV280 Peak figure collection penetrates liquid.

D) elution buffer (20mmol/L PBS, pH7.0,1.2mol/L (NH is used₄)₂SO₄) eluted, according to UV280 Peak figure collects eluent, as purifying transferrins.

S3: concentration displacement:

(1) it is concentrated: utilizing 3KD ultrafiltration membrane, purifying transferrins is concentrated to get concentrate.

(2) replace: by concentrate by the way of dialysis, the time is volume ratio 1:500 for 24 hours, and elution buffer is 20mmol/L PBS (pH7.2) takes in dialysis membrane protein solution as transferrins.

S4: sterilizing:

Above-mentioned transferrins is used into filtration sterilization, filter membrane is the pvdf membrane of 0.22 μm of low protein adsorption, and pressure is 5bars, the transferrins after being sterilized.

Embodiment 2

The present embodiment is transferrins (TF solution) and the human serum albumin after the sterilizing that 1 step S4 of embodiment is obtained (HSA), human immunoglobulin(HIg) (lgG) is combined.

Transferrins after sterilizing is mixed with human serum albumin, human immunoglobulin(HIg), obtains TF+HSA+lgG mixed liquor, The mass ratio of TF, HSA, IgG are 1:8.33:8.33 in the mixed liquor.

Embodiment 3

The present embodiment is transferrins (TF solution) and the human serum albumin after the sterilizing that 1 step S4 of embodiment is obtained (HSA) it is combined.

Transferrins after sterilizing is mixed with human serum albumin, obtains TF+HSA mixed liquor, TF, HSA in the mixed liquor Mass ratio be 1:8.33.

Embodiment 4

The present embodiment is transferrins (TF solution) and the human immunoglobulin(HIg) after the sterilizing that 1 step S4 of embodiment is obtained (lgG) it is combined.

Transferrins after sterilizing is mixed with human immunoglobulin(HIg), obtains TF+lgG mixed liquor, TF in the mixed liquor, The mass ratio of IgG is 1:8.33.

Detect example 1

It detects 1-1:SDS-PAGE and detects transferrins.

Transferrins prepared by embodiment 1 carries out SDS-PAGE denaturation gel electrophoresis identification, comprising the following steps:

(1) respectively from redissolution Cohn IV (first time of S1 (1) redissolves liquid i.e. in embodiment 1), Cohn IV pretreatment fluid (the human plasma Cohn IV component pretreatment fluid that step S1 (7) is obtained i.e. in embodiment 1), hydrophobic chromatography eluting peak (i.e. embodiment The purifying transferrins that step S2 (2) is obtained in 1) it is inner take out sample respectively, BCA method measures total protein concentration.

(2) above-mentioned redissolution Cohn IV, Cohn IV pretreatment fluid, hydrophobic chromatography eluting peak are diluted with water to respectively 1.25mg/ml is mixed with 5 × albumen sample-loading buffer (being purchased from Beijing Suo Laibao Science and Technology Ltd), is as needed to carry out electricity The sample of swimming.

(3) electrophoresis Sample is warming up to 100 DEG C, heating makes protein denaturation in 5 minutes, and sample is put into ice immediately after On, waiting starts race SDS-PAGE denaturation after five minutes and goes back virgin rubber.

The preparation method that virgin rubber is gone back in the denaturation is as follows: the acrylamide Acrylamide (being purchased from Sigma) of 30 (w/v) % takes 2.0ml, 1.5M Tris- hydrochloride buffer (pH 8.8, be purchased from Sigma) takes 1.3ml, the SDS of 10 (w/v) % takes 0.05ml, 10% (w/v) ammonium persulfate (being purchased from Sigma) takes 0.05ml, TEMED (purchased from Sigma) that 0.002ml and water is taken to take 1.6ml, Total 5ml, is frozen into separation gel in room temperature 0.5-1h after mixing, using about 1ml isopropanol sealing, flattens；After solidification, by isopropyl Alcohol is outwelled, and prepare concentration glue: the acrylamide Acrylamide (being purchased from Sigma) of 30 (w/v) % takes 0.17ml, 1.0M Tris- Hydrochloride buffer (pH 6.8 is purchased from Sigma) takes 0.13ml, the SDS of 10 (w/v) % takes 0.01ml, 10% (w/v) ammonium persulfate (being purchased from Sigma) takes 0.01ml, TEMED (purchased from Sigma) that 0.001ml and water is taken to take 0.68ml, amounts to 1ml.It is inserted after encapsulating Enter glue comb, is frozen into concentration glue in room temperature 0.5-1h.

When running glue, the albumen applied sample amount of each swimming lane is 15 micrograms, sets and runs glue voltage as 100V, starts to run glue, runs glue Time is 1 hour.

(4) after running through glue, with coomassie brilliant blue staining liquid (dyeing liquor the preparation method comprises the following steps: by 2.5g Coomassie brilliant blue R-250 is dissolved in 95% ethanol solution of 500ml, then the acetic acid solution that 100ml 85% is added is supplemented to distilled water 1000ml, this dye liquor, which is put, to be kept 4 DEG C of at least six moons stablizing) glue is dyed.

Testing result is referring to Fig. 1: (a): hydrophobic chromatography UV280 peak figure.(b): swimming lane M: Protein Marker；Swimming lane 1- 3: include: 1 in the main band of swimming lane 1-3: redissolution Cohn IV, 2:CohnIV pretreatment fluid, 3: hydrophobic chromatography eluting peak. It can be seen that the other compositions in raw blood plasma are almost completely removed by hydrophobic chromatography step, having obtained molecular weight is about The purifying transferrins (purity > 95%) of 79KD.

Detect 1-2:

In the extracting method of embodiment 1, the purifying abridged table of 3 stage products is as follows:

Wherein, the measurement and calculation of total yield are as follows: determined first with ELISA method and redissolve target egg in Cohn IV Total initial amount is defined as 100% using the total content as total initial amount by white total content；And then divided with ELISA method Not Ce Ding Cohn IV pretreatment fluid, in hydrophobic chromatography eluting peak target protein total content, then the target egg with this 2 products For white total content divided by the total content of target protein in above-mentioned redissolution Cohn IV, the percentage obtained is total yield.Above-mentioned target Albumen selects human serum transferrin (human serotransferrin, HTF), contains 679 amino acid residues, molecular weight Size is 79570Da, referring to " Transferrin as a Metal Ion Mediator " (Hongzhe Sun, Hongyan Li,and,and Peter J.Sadler*Chemical Reviews 1999 99(9),2817-2842DOI:10.1021/ cr980430w)。

The measure and calculation mode of purity are as follows: measure the SDS- of above-mentioned 3 stage products respectively using image analysis software The gray-scale intensity of each band of the result of PAGE gel electrophoresis, and calculate the gray value (ash of each band of each product The sum of spend intensity), then the gray value of the target stripe of each product is obtained divided by the sum of the gray value of total band of the product Percentage out is purity.

Detection 2: primary hippocampal neurons culture and plasma protein fraction processing.

(1) using 0.1% poly-L-lysine be coated with 48 well culture plates, be put into 37 DEG C of incubators be coated with 2 hours with On.

(2) 0 day neonatal mice is dissected in the HBSS-buffer of ice, takes out hippocampus.It is cleaned 3 times with 1 × PBS.Add 0.25%trypsin, 37 DEG C digest 15 minutes.

(3) it is terminated and is digested with complete DMEM, and inhaled hippocampal cell 16-20 times with 1ml rifle featheriness, be without obvious tissue block Can, then with 40mM strainer filtering, 1000rpm is centrifuged 3min.

(4) supernatant is abandoned after being centrifuged, full DMEM is added and cell is resuspended.And neuron is planted in tissue culture plate, it plants Density is 3 × 10⁴cells/cm²。

Serum-free Neurobasal culture medium changes liquid entirely within (5) second days；Third day processing: by the transferrins after sterilizing (the step S4 of embodiment 1 is obtained) is added in different cultivation plate holes, and (its purity is by final concentration of 0.25mg/ml 96.5%), 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml, and using 1 × PBS as control；5th day: utilizing cell-IQ Phase contrast microscope is taken pictures.

Testing result is referring to fig. 2: where part A is hippocampus neuron number histogram, and part B is hippocampal neuron Projection length histogram.In part A, PBS processing: 1.00 ± 0.07AU (arbitrary unit), 0.25mg/ml processing: 1.21 ± 0.07AU (p < 0.05vs.PBS processing), 0.5mg/ml processing: 1.33 ± 0.09AU (p < 0.01vs.PBS processing), 1mg/ml processing: 1.25 ± 0.12AU, 2mg/ml processing: 1.19 ± 0.09AU, 4mg/ml processing: 0.75 ± 0.09AU (p < 0.05vs.PBS processing).In part B, PBS processing: 1.00 ± 0.06AU, 0.25mg/ml processing are (at p < 0.001vs.PBS Reason): 1.76 ± 0.18AU, 0.5mg/ml processing: 1.74 ± 0.09AU (p < 0.001vs.PBS processing), 1mg/ml processing: 1.84 ± 0.17AU (p < 0.001vs.PBS processing), 2mg/ml processing: 1.72 ± 0.10AU (p < 0.001vs.PBS processing), 4mg/ml Processing: 1.14 ± 0.12AU.

It is described above in the concentration range of 0.25-0.5mg/ml, transferrins to promote hippocampal neuron growth Obviously；In the concentration range of 0.25-2mg/ml, transferrins promotes the effect of hippocampal neuron enation obvious.

Detection 3: the protective effect that transferrins damages primary hippocampal neurons caused by A β can significantly improve A β_25-35To the toxic damages of cultured primitive hippocampal neuron.4 amyloid A β has cytotoxicity, if can antagonism A β nerve Poison is expected to achieve the purpose that anti-AD.

(1)Aβ_25-35It is dissolved in the distilled water of sterilizing, makes its final concentration of 1mg/ml, place 7 days and carried out always at 37 DEG C Change, the A β of aging_25-35It is frozen after packing in -20 DEG C.

(2) using 0.1% poly-L-lysine be coated with 96 well culture plates, be put into 37 DEG C of incubators be coated with 2 hours with On.

(3) 0 day neonatal mice is dissected in the HBSS-buffer of ice, takes out hippocampus.It is cleaned 3 times with 1 × PBS.Add 0.25%trypsin, 37 DEG C digest 15 minutes.

(4) it is terminated and is digested with complete DMEM, and inhaled hippocampal cell 16-20 times with 1ml rifle featheriness, be without obvious tissue block Can, then with 40mM strainer filtering, 1000rpm is centrifuged 3min.

(5) supernatant is abandoned after being centrifuged, full DMEM is added and cell is resuspended.And neuron is planted in tissue culture plate, it plants Density is 2 × 10⁵cells/cm²。

(6) second days serum-free Neurobasal culture mediums (containing 10 μM of cytarabines) change liquid entirely；5th day, the 8th day nothing Serum N eurobasal culture medium partly changes liquid.

After (7) the 8th days change liquid, the transferrins (the step S4 of embodiment 1 is obtained) after sterilizing is added to different In cultivation plate hole, final concentration of 0.125mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml, 2mg/ml, 4mg/ml.

In Fig. 3, leftmost Control is added the control (negative control) of 1 × PBS be A β is not added, and second from left to right Column is the control (model comparison) that A β and 1 × PBS is added, the two comparisons are that whether modeling is successful for detection AD cell model；It is left 6 columns for playing 3-8 are the transferrins groups that A β and various concentration is added, for studying the effect of the drug in AD cell model.

After (8) 37 DEG C are incubated for 24 hours, the A β of aging is added_25-35, final concentration of 30 μM.

After (9) 37 DEG C are incubated for 24 hours, add the MTT of 10 μ l 5mg/ml.

(10) 37 DEG C be incubated for 4 hours after, remove supernatant, be added 150 μ l DMSO on enzyme-linked immunosorbent assay instrument to measure wave Long 570nm, reference wavelength 630nm measure light absorption value.

Testing result is referring to Fig. 3: in figure from left to right, it is Bar1-8 that 1-8 column is numbered respectively, and Bar2 shows 30 μM of agings A β_25-35After being incubated for 24 hours with primary hippocampal neurons, cell MTT reduction is substantially reduced compared with Bar1, cell survival rate 70.7 ± 8.5% (p=0.0088) are reduced to, and Bar6-8 pre-processes 24 with the transferrins of 1mg/ml, 2mg/ml, 4mg/ml Hour can be obviously improved A β_25-35To the toxic damages of cell, and with the increase of drug dose, this improvement result is brighter It is aobvious, wherein the transferrins of 4mg/ml it is most significant (cell survival rate be respectively 108.6 ± 7.4%, p=0.0022,134.1 ± 15.7%, p=0.0009,139.2 ± 16.8%, p=0.0008).

It is described above in the concentration range of 1-4mg/ml, transferrins can significantly improve A β_25-35To originally culture sea The toxic damages of horse neuron.

Detection 4: nest building behavior experiment detection:

(1) kitchen paper is cut into scrunch after thin strip, 1cm or so high sawdust padding is added in empty cage, on padding The even kitchen paper for being sprinkled into 3-4g and shredding is put into 5 × FAD transgenosis (APP) mouse or wild type (WT) control mice.

Observation, which is nested, after (2) 48 hours situation and scores.Standards of grading are as follows:

0 point: mouse did not move material of nesting, when material of nesting holding is put into as former state.

1 point: although material of nesting was moved by mouse, not having obviously to form the position of nest in cage, material of nesting dispersion In cage.

2 points: having the position of obvious nest, nest is flat, and centre does not have significant depressions.

3 points: having the position of obvious nest, be slightly recessed among nest, 1/2 after highly arching lower than back of mice.

4 points: having the position of obvious nest, there is recess among nest, 1/2 after highly arching for back of mice.

5 points: having the position of obvious nest, there is recess among nest, be highly higher than 1/2 after back of mice is arched.

(3) mouse is divided into following seven groups, every group of 8 mouse.

First group (WT_PBS): wild type (WT) control mice, tail vein inject 20mmol/L PBS (pH7.2) 100μl；This group is blank control；

Second group (TG_PBS): 5 × FAD transgenosis (APP) mouse, the tail vein of the group 5 × FAD transgenic mice are infused Penetrate 100 μ l of PBS；This group is model comparison；

Third group (TG_TF): the tail vein of 5 × FAD transgenic mice, the group 5 × FAD transgenic mice injects sterilizing 100 μ l (TF containing 4.5mg) of transferrins (the step S4 of embodiment 1 is obtained) afterwards；

4th group (TG_TF+lgG): the tail vein of 5 × FAD transgenic mice, the group 5 × FAD transgenic mice is injected Transferrins+human immunoglobulin(HIg) mixed liquor after the sterilizing of embodiment 4 totally 100 μ l (IgG containing 37.5mg, 4.5mg TF)；

5th group (TG_TF+HSA): the tail vein of 5 × FAD transgenic mice, the group 5 × FAD transgenic mice is injected Transferrins+human serum albumin mixed liquor after the sterilizing of embodiment 3 totally 100 μ l (HSA containing 37.5mg, 4.5mg TF)；

6th group (HSA+lgG): the tail vein of 5 × FAD transgenic mice, the group 5 × FAD transgenic mice injects people Blood albumin+human immunoglobulin(HIg) mixed liquor totally 100 μ l (HSA containing 37.5mg and 37.5mg IgG)；

7th group (TG_TF+HSA+lgG): the tail vein of 5 × FAD transgenic mice, the group 5 × FAD transgenic mice is equal Transferrins+human serum albumin+human immunoglobulin(HIg) mixed liquor (TF+HSA+ of embodiment 2 after injecting the sterilizing of embodiment 2 LgG) totally 100 μ l (HSA containing 37.5mg, 37.5mg IgG, 4.5mg TF)；

Above-mentioned 7 groups are injected once every three days, and co-injection 8 times；Nest building behavior is carried out according still further to (1) and (2) step later Experiment.

Test result is shown in Fig. 4.First group (WT_PBS) is scored at 3.63 ± 0.21, and second group (TG_PBS) is scored at 0.88 ± 0.32, third group (TG_TF) is scored at 3.25 ± 0.16, and the 4th group (TG_TF+lgG) is scored at 3.56 ± 0.15, and the 5th group (TG_TF+HSA) 3.50 ± 0.13 are scored at, the 6th group (HSA+lgG) is scored at 3.06 ± 0.15, the 7th group of (TG_TF+HSA+ LgG 3.81 ± 0.19) are scored at.It can be seen that for Alzheimer's disease model mice, injection transferrins, transferrins+ Human immunoglobulin(HIg), transferrins+human serum albumin, human serum albumin+human immunoglobulin(HIg), transferrins+human serum albumin+ Human immunoglobulin(HIg) can significantly improve nest building behavior；Individually injection transferrins is than injection human serum albumin+immune globulin The white ability of nesting, joint injection transferrins+human serum albumin or the transferrins+human immunoglobulin(HIg) of can be improved is than individually note Penetrating transferrins can be improved the ability of nesting, moreover, injection transferrins+human serum albumin+immunoglobulin can be further Raising is nested ability.

The ability of nesting of mouse is transformed into the integrated management ability that the mankind refer to the mankind to daily life, it can be seen that, injection Transferrins can be obviously improved the integrated management ability of Alzheimer's disease model mice daily life.

Detect 5:Morris water maze test (detection of water maze Behaviors survey).

(1) mouse is divided into following seven groups, every group of 10 mouse.

First group (WT_PBS): wild type (WT) mouse, as control, 100 μ l of mouse tail vein injection PBS；This group is Blank control；

Second group (TG_PBS): 5 × FAD transgenic mice, each 100 μ l of mouse tail vein injection PBS；This group is model Control；

Third group (TG_TF): 5 × FAD transgenic mice, the transferrins after each mouse tail vein injection sterilizing (are implemented What the step S4 of example 1 was obtained) 100 μ l (TF containing 4.5mg)；

4th group (TG_TF+HSA): 5 × FAD transgenic mice, after the sterilizing of each mouse tail vein injection embodiment 3 Transferrins+human serum albumin mixed liquor totally 100 μ l (HSA containing 37.5mg, 4.5mg TF)；

5th group (TG_TF+IgG): 5 × FAD transgenic mice, after the sterilizing of each mouse tail vein injection embodiment 4 Transferrins+human immunoglobulin(HIg) mixed liquor totally 100 μ l (IgG containing 37.5mg, 4.5mg TF)；

6th group (TG_HSA+IgG): 5 × FAD transgenic mice, each mouse tail vein injection human serum albumin+people are immune The mixed liquor (HSA+lgG) of globulin totally 100 μ l (HSA containing 37.5mg and 37.5mg IgG)；

7th group (TG_TF+HSA+IgG): 5 × FAD transgenic mice, the sterilizing of each mouse tail vein injection embodiment 2 Totally 100 μ l (contain transferrins+human serum albumin+human immunoglobulin(HIg) mixed liquor (TF+HSA+lgG of embodiment 2) afterwards 37.5mg HSA,37.5mg IgG,4.5mg TF)；

Above-mentioned 7 groups are injected once every three days, and co-injection 8 times；Morris water maze training is carried out later.

(2) water maze laboratory records the spatial discrimination of preclinical length measurement mouse.Water maze device is by diameter The platform of 120cm, the round pool of high 50cm and moveable diameter 10cm forms, the top in pond equipped with camera and with electricity The connection of brain automatic recording instrument, water temperature keep (22 ± 3) DEG C.

(3) the are trained for hiding platform training for 1-5 days, and platform is placed in fixed position and lower than water surface 1cm, and titanium white is added Powder keeps platform invisible, and mouse head is gently put into one of 4 quadrants in pond towards pool wall at random, tests every mouse trip every time Swim 60s, it is allowed to find underwater platform, it is allowed to stop 5s；If mouse fails to find platform after entering water, the time it will be recorded as 60s, And it is artificially placed on to 20s on platform, every mouse is repeated 4 times test daily.

(4) after last time orientation navigation experiment for 24 hours, platform is withdrawn from, tracker moves rail into the water by mouse Mark 60s, test mouse 60s in motion profile, incubation period and pass through original placement platform position quadrant number and time conduct Spatial memory forms index, and compare between group.

Note: keeping the indoor environments such as experiment indoor light, laying for goods consistent in entire test process, to exclude environment Interference.

Test result is shown in Fig. 5.Wherein, part A is that mouse is appeared on the stage the time in the hiding platform training stage, and part B is mouse In test phase percentage of time locating for target quadrant.

In part A, in the hiding platform training stage, 3 before WT_PBS group and TG_PBS group learning curve (fleeing from incubation period) Its significant difference, behind reach unanimity, WT_PBS group, TG_TF group, TG_TF+HSA group, TG_TF+IgG group, TG_HSA+IgG Group, TG_TF+HSA+IgG group learning curve (fleeing from incubation period) are close.All hiding put down is remembered to all mouse by association before test The position of platform.

In part B, each group percentage of time locating for target quadrant is successively are as follows: first group (WT_PBS :) 28.62 ± 2.52, second group (TG_PBS): 19.27 ± 2.37 (p < 0.05vs.WT_PBS), third group (TG_TF): 26.76 ± 2.85, the Four groups (TG_TF+HSA): 28.23 ± 2.33, the 5th group (TG_TF+IgG): 28.69 ± 2.61, the 6th group (TG_HSA+IgG): 25.18 ± 2.01, the 7th group (TG_TF+HSA+IgG): 32.00 ± 2.06, it is seen that Alzheimer's disease model mice is come It says, injection transferrins, human serum albumin+human immunoglobulin(HIg), transferrins+human serum albumin+human immunoglobulin(HIg) can Enough improve 5 × FAD transgenosis (APP) mouse time locating for target quadrant.

It can be seen that injection transferrins, transferrins+human immunoglobulin(HIg), transferrins+human serum albumin, people's blood Albumin+human immunoglobulin(HIg), transferrins+human serum albumin+human immunoglobulin(HIg) can be obviously improved Alzheimer The human-subject test of disease model mice；Individually injection transferrins is mentioned than injection human serum albumin+human immunoglobulin(HIg) effect Height, joint injection transferrins+human serum albumin or transferrins+human immunoglobulin(HIg) are than individually injection transferrins to A Er Zi Haimo disease model mice human-subject test slightly improves, moreover, injection transferrins+human serum albumin+human immunoglobulin(HIg) Effect further increases.

Claims

1. application of the transferrins in the drug of preparation prevention or/and improvement or/and treatment Alzheimer's disease；Or/and turn Application of the ferritin in terms of preparation intelligence development class replenishers or drug.

2. a kind of composition comprising transferrins, it is characterised in that: the composition includes: transferrins and other components, The other components include: one of human serum albumin, human immunoglobulin(HIg), the first-line drug of clinical treatment senile dementia Or it is a variety of；

Preferably, the composition includes: transferrins, albumin and immunoglobulin；

It is highly preferred that pressing mass fraction, the composition includes: transferrins 0.5-4 mass parts, human serum albumin 5-40 mass Part, human immunoglobulin(HIg) 5-40 mass parts；

It is further preferred that pressing mass fraction, the composition includes: 1 mass parts of transferrins, 8.33 mass of human serum albumin Part, 8.33 mass parts of human immunoglobulin(HIg).

3. composition according to claim 2, it is characterised in that: the first-line drug packet of the clinical treatment senile dementia It includes: donepezil, galanthamine, Rivastigmine, one or more of Memantine.

4. composition according to claim 2, it is characterised in that: the composition further includes pharmaceutically acceptable load Body.

5. according to any one of the claim 2-4 composition, it is characterised in that: the raw material for preparing of the transferrins includes people Plasma C ohn IV component.

6. according to any one of the claim 2-4 composition, it is characterised in that: the preparation method of the transferrins is successively wrapped It includes: pretreatment fluid preparation step, hydrophobic chromatography purification step；It preferably, further include concentration displacement step.

7. composition according to claim 6, it is characterised in that:

In the pretreatment fluid preparation step, plasma C ohn IV component is successively passed through into first time redissolution processing, is centrifuged for the first time Processing, first time filtration treatment adjust pH processing, and removal impurity protein is handled, second of centrifugal treating, at second of filtering Reason, obtains pretreatment fluid；

In the hydrophobic chromatography purification step, the pretreatment fluid is handled by buffer exchange, hydrophobic chromatography processing obtains Purify transferrins；Preferably, the purity of the purifying transferrins is more than or equal to 95%；

Preferably, in the concentration displacement step, the purifying transferrins is subjected to hyperfiltration treatment, replacement Treatment, is turned Ferritin.

8. composition according to claim 7, it is characterised in that: in the pretreatment fluid preparation step,

The first time redissolution processing, is redissolved using PBS buffer solution, the plasma C ohn IV component and the PBS buffer solution Mass volume ratio is 1:(2-10), preferably 1:3.5；Preferably, the PBS buffer solution are as follows: 10-50mmol/L PBS, PH6.0-8.0, more preferably 20mmol/L PBS, pH7.5；

The first time centrifugal treating, revolving speed 4,000-15,000rpm, temperature be 0-10 DEG C, preferably 10,000rpm, 4 DEG C, retain supernatant and is used for the first time filtration treatment；

The first time filtration treatment, uses aperture for 0.1-1.0 μm, preferably 0.45 μm of filter membrane, takes filtrate to be used for after filtering The adjusting pH processing；

The adjusting pH processing, is adjusted to 6.5-7.5, preferably 7.0 for the pH value of the filtrate；More preferably use 1mol/L lemon Lemon acid sodium solution is as pH adjusting agent；

The removal impurity protein processing, is added protein precipitant into the filtrate after adjusting pH and removes impurity protein；More preferably Ground, the protein precipitant are selected from: PEG4000 and/or ethanol solution；It is further preferred that after the PEG4000 and adjusting pH Filtrate mass volume ratio be 10-20%, preferably 15%；The concentration expressed in percentage by volume of the ethanol solution is 90-98%, excellent It is selected as 96%, temperature is 0 to subzero 10 DEG C, 4 DEG C preferably subzero；

Second of centrifugal treating, revolving speed 4,000-10,000rpm, temperature are 0-10 DEG C, preferably 10000rpm, 4 DEG C, Retain supernatant and is used for second of filtration treatment；

Second of filtration treatment, uses aperture for 0.1-1.0 μm, preferably 0.45 μm of filter membrane, and obtained filtrate is as pre- Treatment fluid.

9. composition according to claim 7, it is characterised in that: in the hydrophobic chromatography purification step,

The buffer exchange processing, using HiTrap Desalting desalting column, the equilibration buffer of use are as follows: 10- 50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration be 0.5-3mol/L, preferably 20mmol/L PBS, pH7.0,(NH₄)₂SO₄Concentration is 1.5mol/L；

Hydrophobic chromatography processing, first by octyl drainage column described in the equilibration buffer, then with the equilibration buffer The octyl drainage column is eluted, then is eluted with elution buffer；

The hydrophobic chromatography column is octyl drainage column, is loaded with 4 Fast Flow of OctylSepharose；

The equilibration buffer are as follows: 10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 1.5-3mol/L, Preferably 20mmol/L PBS, pH7.0, (NH₄)₂SO₄Concentration is 1.5mol/L；

The elution buffer are as follows: 10-50mmol/L PBS, pH6.0-8.0, wherein (NH₄)₂SO₄Concentration is 0.9-1.4mol/ L, preferably 20mmol/L PBS, pH7.0, (NH₄)₂SO₄Concentration is 1.2mol/L；

Preferably, the hyperfiltration treatment, using 1KD-10KD, preferably 3KD ultrafiltration membrane；

The replacement Treatment, the displacement buffer of use are as follows: 10-50mmol/L PBS, pH7.0-7.4, preferably 20mmol/L PBS, pH7.2.

10. any one of the claim 2-9 composition is in preparation prevention or/and improves or/and treat Alzheimer's disease Application in drug；Or/and any one of claim 2-9 composition answering in terms of preparation intelligence development class replenishers or drug With.