阅读说明：本技术 一种犬血清中白蛋白的提纯方法 (Purification method of albumin in dog serum ) 是由 石晶 赵健 陈宪平 陈放 付玉 侯强 董鹏 殷玉和 于 2019-10-16 设计创作，主要内容包括：本发明公开了一种犬血清中白蛋白的提纯方法,通过乙醇两步法从犬血清中分离提纯白蛋白,其中,步骤S1采用温度≤-20℃的低温乙醇法,步骤S2采用温度65～68℃的热乙醇法。本发明提供的犬血清中白蛋白的提纯方法,采用乙醇两步法即可获得高纯度的犬血白蛋白,是一种快速、简单、高纯度及高回收率的从犬血清中提纯白蛋白的工艺方法,可应用于犬白蛋白药物以及犬相关疾病治疗药物的制备,工艺方法简单,节约资源,成本低,适合规模化生产。(The invention discloses a purification method of albumin in dog serum, which is used for separating and purifying the albumin from the dog serum by an ethanol two-step method, wherein a low-temperature ethanol method with the temperature of less than or equal to-20 ℃ is adopted in the step S1, and a hot ethanol method with the temperature of 65-68 ℃ is adopted in the step S2. The method for purifying the albumin in the dog serum provided by the invention can obtain the high-purity dog blood albumin by adopting an ethanol two-step method, is a quick, simple, high-purity and high-recovery-rate process method for purifying the albumin from the dog serum, can be applied to the preparation of dog albumin medicaments and medicaments for treating dog-related diseases, has the advantages of simple process method, resource saving and low cost, and is suitable for large-scale production.)

1. The method for purifying the albumin in the dog serum is characterized by separating and purifying the albumin from the dog serum by an ethanol two-step method, wherein a low-temperature ethanol method with the temperature of less than or equal to-20 ℃ is adopted in the step S1, and a hot ethanol method with the temperature of 65-68 ℃ is adopted in the step S2, so that the high-purity dog serum albumin is finally obtained.

2. The method of claim 1, comprising the steps of:

step S1, Low temperature ethanol Process

Centrifuging the melted serum, filtering the supernatant through a clarification column core, placing the supernatant in an environment at the temperature of-8 to-12 ℃, adjusting the pH value to 6.8 to 7.2, slowly adding 95 percent ethanol at the temperature of less than or equal to-20 ℃ when the temperature is reduced to below 10 ℃, wherein the addition is 25 to 30 percent of the volume of the serum, stirring and uniformly mixing the mixture, and reacting for 14 to 18 hours; stirring uniformly, centrifuging, and obtaining supernate which is the crude extract of the dog blood albumin;

step S2, Hot ethanol Process

Taking the crude dog blood albumin extract obtained in the step S1, adding a sodium chloride solution, and mixing the two solutions until the final concentration of sodium chloride is 0.85-0.92% (w/v); pouring sodium caprylate powder into the container until the final concentration is 0.75-0.85% (w/v); adjusting the pH value to 6.0-6.2, and stirring for 40-60 minutes to obtain the water bath solution; subpackaging the water bath solution, sealing, and placing in a water bath kettle at 65-68 ℃ for over 1 hour in a water bath way; standing and cooling the liquid after water bath at-20 deg.C for 12-16 hr to obtain cooling liquid; centrifuging the cooling liquid with a desktop centrifuge at a centrifugal force of 5000g or more for 20-30 min, and collecting the supernatant; after centrifugal supernatant is ultrafiltered by 500KDa, the liquid under the membrane is the refined and pure stock solution of the dog blood albumin.

3. The method of claim 1 or 2, wherein the amount of ethanol added in step S1 is 27% by volume of the serum.

4. The method of claim 1 or 2, wherein the reaction time in step S1 is 16 hours.

5. The method of claim 2, wherein the pH of the solution is adjusted to 7.0 in step S1.

6. The method of claim 2, wherein the final concentration of sodium chloride in step S2 is 0.9% (w/v).

7. The method of claim 2, wherein the final concentration of sodium caprylate in step S2 is 0.8% (w/v).

8. The method of claim 1, comprising the steps of:

step S1, Low temperature ethanol Process

Centrifuging the melted serum at 16000rpm, filtering the supernatant with 0.45um clear column core, placing in-10 deg.C environment, adjusting pH to 7.0, slowly adding 95% ethanol with temperature less than or equal to-20 deg.C when the temperature is below 10 deg.C, adding 27% of the volume of serum, stirring, and reacting for 16 h; stirring uniformly, centrifuging at 16000rpm, and collecting supernatant as crude extract of dog blood albumin;

step S2, Hot ethanol Process

Adding sodium chloride solution into the dog blood albumin crude extract obtained in the step S1, and mixing the two solutions to obtain the final sodium chloride concentration of 0.9% (w/v); after pouring sodium caprylate powder to make the final concentration at 0.8% (w/v); adjusting the pH value to 6.1, and stirring for 50 minutes to obtain water bath liquid; subpackaging the water bath solution, sealing, and placing in a water bath kettle at 65-68 ℃ for over 1 hour in a water bath way; standing and cooling the liquid after water bath at-20 deg.C for 14 hr to obtain cooling liquid; centrifuging the cooling liquid with a desktop centrifuge at a centrifugal force of 5000g or more for 20 minutes, and collecting the supernatant; after centrifugal supernatant is ultrafiltered by 500KDa, the liquid under the membrane is the refined and pure stock solution of the dog blood albumin.

9. The method of claim 8, wherein the pH of the supernatant is adjusted in step S1 or S2 with a buffered HAC-NaAC solution at pH 4.0.

10. The method of claim 8, wherein the concentration of the sodium chloride solution in step S2 is 0.71M L with water for injection at 40-55 ℃.

Technical Field

The invention relates to the technical field of biological products, in particular to a method for purifying albumin in canine serum.

Background

With the increasing abundance of people's lives in the process of economic development, more and more people raise pets to accompany people and families. The number of dogs is the largest among pet species, and the number of dogs in China is up to 1.2 hundred million according to incomplete statistics. With the increasing number of dogs, the occurrence of dog diseases is inevitable, the use of dog blood albumin is common and effective in the dog disease treatment process, but the dog blood albumin products in the market are different, and the situation that the serum is taken as the dog blood albumin is insufficient.

In the human serum albumin purification process, a low-temperature ethanol five-step or seven-step method is generally adopted to separate and purify various components in human plasma step by step, the operation is complicated, the steps are multiple, the reagent consumption is high, the cost is high, although the human serum albumin purification process can be used for reference, albumin and globulin in dog serum are mainly used for pharmacy, and other components cannot form products at present, so if the low-temperature ethanol component-by-component separation of the human serum albumin is simulated one by one, the resource waste and the cost increase are caused, and the human serum albumin separation and purification method cannot be suitable for the separation and purification of the dog serum albumin and is more unfavorable for the marketization of the dog serum albumin. Therefore, a rapid, simple, high purity and high recovery process for the purification of albumin from canine serum is urgently needed.

Disclosure of Invention

The invention aims to provide a rapid and simple method for purifying albumin in dog serum.

In order to achieve the purpose, the technical scheme provided by the invention is as follows:

a method for purifying albumin in dog serum comprises the step of separating and purifying albumin from the dog serum by an ethanol two-step method, wherein a low-temperature ethanol method with the temperature of less than or equal to-20 ℃ is adopted in the step S1, a hot ethanol method with the temperature of 65-68 ℃ is adopted in the step S2, and the high-purity dog serum albumin is finally obtained.

Preferably, the method for purifying albumin in canine serum comprises the following steps:

step S1, Low temperature ethanol Process

Filtering the molten serum and supernatant by a clarification column core, placing the filtered supernatant in an environment at the temperature of-8 to-12 ℃, adjusting the pH value to 6.8 to 7.2, slowly adding 95 percent ethanol at the temperature of less than or equal to-20 ℃ when the temperature is reduced to below 10 ℃, wherein the addition is 25 to 30 percent of the volume of the serum, stirring and uniformly mixing, and reacting for 14 to 18 hours; stirring uniformly, centrifuging, and obtaining supernate which is the crude extract of the dog blood albumin;

step S2, Hot ethanol Process

Taking the crude dog blood albumin extract obtained in the step S1, adding a sodium chloride solution, and mixing the two solutions until the final concentration of sodium chloride is 0.85-0.92% (w/v); pouring sodium caprylate powder into the container until the final concentration is 0.75-0.85% (w/v); adjusting the pH value to 6.0-6.2, and stirring for 40-60 minutes to obtain the water bath solution; subpackaging the water bath solution, sealing, and placing in a water bath kettle at 65-68 ℃ for over 1 hour in a water bath way; standing and cooling the liquid after water bath at-20 deg.C for 12-16 hr to obtain cooling liquid; centrifuging the cooling liquid with a desktop centrifuge at a centrifugal force of 5000g or more for 20-30 min, and collecting the supernatant; after centrifugal supernatant is ultrafiltered by 500KDa, the liquid under the membrane is the refined and pure stock solution of the dog blood albumin.

Preferably, in the method for purifying albumin in canine serum, the addition amount of ethanol in step S1 is 27% of the serum volume.

Preferably, in the above method for purifying albumin in canine serum, the reaction time in step S1 is 16 hours.

Preferably, in the above method for purifying albumin in canine serum, the pH is adjusted to 7.0 in step S1.

Preferably, in the above method for purifying albumin in canine serum, the final concentration of sodium chloride in step S2 is 0.9% (w/v).

Preferably, in the above method for purifying albumin in canine serum, the final concentration of sodium caprylate in step S2 is 0.8% (w/v).

Preferably, the method for purifying albumin in canine serum comprises the following steps:

step S1, Low temperature ethanol Process

Centrifuging the melted serum at 16000rpm, filtering the supernatant with 0.45um clear column core, placing in-10 deg.C environment, adjusting pH to 7.0, slowly adding 95% ethanol with temperature less than or equal to-20 deg.C when the temperature is below 10 deg.C, adding 27% of the volume of serum, stirring, and reacting for 16 h; stirring uniformly, centrifuging at 16000rpm, and collecting supernatant as crude extract of dog blood albumin;

step S2, Hot ethanol Process

Adding sodium chloride solution into the dog blood albumin crude extract obtained in the step S1, and mixing the two solutions to obtain the final sodium chloride concentration of 0.9% (w/v); then pouring sodium caprylate powder into the mixture until the final concentration is 0.8% (w/v); adjusting the pH value to 6.1, and stirring for 50 minutes to obtain water bath liquid; subpackaging the water bath solution, sealing, and placing in a water bath kettle at 65-68 ℃ for over 1 hour in a water bath way; standing and cooling the liquid after water bath at-20 deg.C for 14 hr to obtain cooling liquid; centrifuging the cooling liquid with a desktop centrifuge at a centrifugal force of 5000g or more for 20 minutes, and collecting the supernatant; after centrifugal supernatant is ultrafiltered by 500KDa, the liquid under the membrane is the refined and pure stock solution of the dog blood albumin.

Preferably, in the above method for purifying albumin in canine serum, the pH of the supernatant is adjusted by using HAC-NaAC buffer solution with pH 4.0 in step S1 or S2.

Preferably, in the above method for purifying albumin in canine serum, step S2 is performed by using water for injection at 40-55 ℃ to prepare 0.71M l sodium chloride solution.

Compared with the prior art, the invention has the technical effects that:

the method for purifying the albumin in the dog serum provided by the invention can obtain the high-purity (more than or equal to 95%) dog blood albumin by adopting an ethanol two-step method, namely a low-temperature ethanol method and a high-temperature ethanol method, is a quick, simple, high-purity and high-recovery-rate process method for purifying the albumin from the dog serum, can be applied to the preparation of dog albumin medicaments and medicaments for treating related diseases of dogs, has the advantages of simple process method, resource conservation and low cost, and is suitable for large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.

FIG. 1 shows the result of SDS-PAGE provided in example 1 of the present invention; wherein, lane 1: protein Marker, lane 2: albumin concentrate, lane 3: globulin protein.

FIG. 2 is a SDS-PAGE result provided in comparative example 1 of the present invention; lane 1: prestained protein Marker (Thermo Fisher # 26616); lane 2: (ii) canine serum; lane 3: centrifuging the supernatant in a water bath; lane 4: water bath centrifugal precipitation; lane 5: discharging with a 500KDa ultrafiltration membrane; lane 8: performing ultrafiltration membrane liquid discharge by a low-temperature ethanol method; lane 9: 10KDa concentrated solution.

FIG. 3 is a SDS-PAGE result provided in comparative example 2 of the present invention; lane 1: a protein Marker; lane 2: (ii) canine serum; lane 3: 20% ethanol supernatant; lane 5: centrifuging the supernatant in 20% ethanol water bath; lane 6: centrifuging in 20% ethanol water bath; lane 8: centrifuging supernatant in 20% ethanol water bath, and ultrafiltering the concentrated solution.

Detailed Description

The method for purifying albumin in dog serum disclosed by the invention is used for separating and purifying albumin from the dog serum by an ethanol two-step method, wherein a low-temperature ethanol method with the temperature of less than or equal to-20 ℃ is adopted in the step S1, and a hot ethanol method with the temperature of 65-68 ℃ is adopted in the step S2, so that the high-purity dog blood albumin is finally obtained. Specifically, the method comprises the following steps:

step S1, Low temperature ethanol Process

Centrifuging the melted serum, filtering the supernatant through a clarification column core, and then placing the supernatant in an environment at the temperature of-8 to-12 ℃, and adjusting the pH value to 6.8 to 7.2, preferably 7.0; when the temperature is reduced to below 10 ℃, slowly adding 95 percent ethanol with the temperature less than or equal to minus 20 ℃, wherein the adding amount is 25 to 30 percent of the volume of the serum, and the optimal selecting amount is 27 percent; uniformly stirring and reacting for 14-18 hours, preferably 16 hours; stirring uniformly, centrifuging, and obtaining supernate which is the crude extract of the dog blood albumin;

step S2, Hot ethanol Process

Taking the crude dog blood albumin extract obtained in the step S1, adding a sodium chloride solution, and mixing the two solutions until the final concentration of sodium chloride is 0.85-0.92% (w/v); pouring sodium caprylate powder into the container until the final concentration is 0.75-0.85% (w/v); adjusting the pH value to 6.0-6.2, preferably 6.1, and stirring for 40-60 minutes to obtain the water bath solution; subpackaging the water bath solution, sealing, and placing in a water bath kettle at 65-68 ℃ for water bath for more than 1 hour, preferably 66 ℃, and carrying out water bath for 2 hours; standing and cooling the liquid after water bath at-20 deg.C for 12-16 hr to obtain cooling liquid; centrifuging the cooling liquid with a desktop centrifuge at a centrifugal force of 5000g or more for 20-30 min, and collecting the supernatant; after centrifugal supernatant is ultrafiltered by 500KDa, the liquid under the membrane is the refined and pure stock solution of the dog blood albumin.

In step S1, the pH value is adjusted to 6.8-7.2, the pH value is the isoelectric point of globulin, the globulin is easy to aggregate and separate out at the pH value, and the pH value is preferably 7.0 through tests; the addition of ethanol in step S1 can destroy the hydrated membrane of protein when dissolving to reduce the solubility of protein, including globulin; and in the step S1, the solubility of the protein is also reduced by reacting for 14-18 h in the environment of-8 to-12 ℃, so that the globulin is more rapidly aggregated and precipitated, and the reaction time is preferably 16 h.

In step S2 of the present invention, the final concentration of sodium chloride is 0.85-0.92% (w/v), preferably 0.9% (w/v), and the solubility of albumin is the greatest in a sodium chloride solution of this concentration; the final concentration of the sodium caprylate is 0.75-0.85% (w/v), preferably 0.8% (w/v), and the sodium caprylate and the albumin are combined reversibly, so that the heat resistance of the albumin can be increased; adjusting the pH value to 6.0-6.2, and stirring for 40-60 minutes to fully dissolve albumin and combine with sodium caprylate; performing water bath for more than 1 hour in a water bath kettle at the temperature of 65-68 ℃ during water bath to thermally denature proteins except the albumin protected by the sodium caprylate; standing at-20 deg.C, cooling for 12-16 hr to aggregate denatured protein, and centrifuging.

In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.