CN107987158B - Separation and purification method of medicinal bovine serum albumin - Google Patents
Separation and purification method of medicinal bovine serum albumin Download PDFInfo
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- CN107987158B CN107987158B CN201711460327.7A CN201711460327A CN107987158B CN 107987158 B CN107987158 B CN 107987158B CN 201711460327 A CN201711460327 A CN 201711460327A CN 107987158 B CN107987158 B CN 107987158B
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Abstract
The invention discloses a separation and purification method of medicinal bovine serum albumin, which comprises the following steps: taking bovine blood, placing the bovine blood in a centrifuge tube, detachably arranging the centrifuge tube in a sealed container, introducing carbon dioxide into the centrifuge tube, sealing, placing the sealed container in a freezing refrigerator, adjusting the temperature of the freezing refrigerator from 20 ℃ to minus 30 ℃ at a speed of 5 ℃/min, keeping the temperature for 10min, then heating to 20 ℃ at a speed of 5 ℃/min, keeping the temperature for 4min, repeating the steps for 3 times, taking out the frozen container, adding sodium citrate with the concentration of 3.8% in 10% of the total mass of the bovine blood into the centrifuge tube, introducing the carbon dioxide again, sealing, separating the centrifuge tube from the sealed container, controlling the temperature to be lower than 2 ℃ through a centrifuge, and separating and removing blood cells to obtain plasma. The method has the advantages of effectively avoiding the problem of pollution probability improvement caused by the use of multiple containers in the preparation process, and simultaneously improving the plasma yield and the purity of the medicinal bovine serum albumin.
Description
Technical Field
The invention relates to the technical field of protein extraction. More specifically, the invention relates to a separation and purification method of medicinal bovine serum albumin.
Background
The medicinal protein Bovine Serum Albumin (BSA), also called as the fifth component, is a globulin in Bovine serum, and comprises 583 amino acid residues, wherein 35 cysteines form 17 disulfide bonds, a free sulfhydryl group is located at the 34 th position of a peptide chain, the molecular weight is 66.446kDa, the isoelectric point is 4.7, the nitrogen content is 16%, the sugar content is 0.08%, and the fat content is only 0.2%. Bovine serum albumin mainly plays a role in maintaining osmotic pressure, a role in pH buffering, a role in a carrier, and a role in nutrition. In the serum-free culture of animal cells, the protein is added to play a role in physiological and mechanical protection and a role in a carrier, and meanwhile, the protein has wide application in advanced research, genetic engineering and medical research.
At present, there are many methods for producing bovine serum albumin, mainly including salting out method, organic solvent precipitation method, heat shock method, etc., wherein the salting out method mainly includes: separating serum from sanguis bovis Seu Bubali, performing fractional precipitation with ammonium sulfate, and refining by octanoic acid treatment; the organic solvent precipitation method is firstly used for producing human blood products, and various serum proteins can be produced by separating plasma proteins by utilizing the low-valence electrical constant property of ethanol and the different solubilities of the proteins in ethanol with different concentrations under the conditions of certain temperature, pH, ionic strength and concentration; the heat shock method comprises centrifuging anticoagulant of cattle blood to obtain plasma, heating, adding sodium caprylate, adjusting pH, adding ethanol, filtering, desalting, concentrating, and lyophilizing to obtain albumin in cattle blood serum; among them, the heat shock method is the most widely studied process at present, but how to avoid the pollution probability increase caused by using multiple containers in the whole preparation process in the preparation process, and the problems of improving the yield, the purity and the quality of the bovine serum albumin finished product are still urgently needed to be solved at present.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide a method for separating and purifying the medicinal bovine serum albumin, which effectively avoids the problem of pollution probability improvement caused by using a plurality of containers in the preparation process, and simultaneously improves the plasma yield and the purity rate of the medicinal bovine serum albumin.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for separating and purifying pharmaceutical bovine serum albumin, comprising the steps of:
step one, taking bovine blood and placing the bovine blood in a centrifugal tube, wherein the centrifugal tube is detachably arranged in a sealed container, introducing carbon dioxide into the centrifugal tube and then sealing the centrifugal tube, placing the sealed container in a freezing refrigerator, regulating the temperature of the freezing refrigerator to be reduced to minus 30 ℃ from 20 ℃ at a speed of 5 ℃/min and keeping the temperature for 10min, then heating to 20 ℃ at a speed of 5 ℃/min and keeping the temperature for 4min, repeating the steps for 3 times, taking out the frozen container, adding sodium citrate with the concentration of 3.8 percent, which is 10 percent of the total mass of the bovine blood, into the centrifugal tube, introducing the carbon dioxide again and then sealing the centrifugal tube, separating the centrifugal tube from the sealed container, controlling the temperature to be lower than 2 ℃ through a centrifugal machine;
wherein, centrifuging tube is inversion round platform form, sealed container includes:
the sealing plug can be disassembled and arranged at the opening at the top of the centrifugal tube, the top surface of the sealing plug is downwards sunken to form an accommodating groove, and the bottom surface of the accommodating groove penetrates through the accommodating groove to form a first through hole;
the opening and closing mechanism comprises a first guide pipe coaxially and fixedly arranged on the bottom surface of the accommodating groove, a first elastic plug filled at the lower part in the first guide pipe, a first spring fixedly connected with the top surface of the first elastic plug, a first gland in screwed connection with the top end of the first spring, a second guide pipe coaxially and fixedly arranged on the top surface of the first gland, a second elastic plug filled at the lower part in the second guide pipe, a second spring fixedly connected with the top surface of the second elastic plug, and a second gland in screwed connection with the top end of the second spring, wherein a second through hole penetrates through the top surface of the first gland, a third through hole penetrates through the top surface of the second gland, a first elastic hole vertically penetrates through the first elastic plug under the second through hole on the first elastic plug, a second elastic hole vertically penetrates through the second elastic plug under the third through hole on the second elastic plug, and a second elastic hole is arranged on the second elastic plug, the first through hole, the second through hole and the third through hole are equal in distance from the central axis of the accommodating groove;
the heating pipe is detachably sleeved outside the centrifugal pipe to form a water storage cavity wrapping the outer wall of the centrifugal pipe between the heating pipe and the outer wall of the centrifugal pipe, a ring-shaped rubber plug is plugged at the top end of the water storage cavity to seal the water storage cavity, a fourth through hole penetrates through the rubber plug, an air guide pipe is detachably communicated with the fourth through hole, one end, away from the fourth through hole, of the air guide pipe is a hard pipe body, and a through hole is formed in the upper portion of the outer side wall of the heating pipe;
the charging mechanism comprises a truncated cone-shaped movable plug arranged on the through hole in a plugging manner, a movable rod hinged with the small end of the movable plug positioned on the outer side of the heating pipe, and a charging bag sleeved on the outer side of the movable rod and provided with an opening fixedly arranged on the circumferential side wall of the heating pipe positioned on the through hole, wherein a mixture of citric acid and sodium bicarbonate with the mass ratio of 1:3 is placed in the charging bag;
in the first step, the step of introducing carbon dioxide into the centrifugal tube and then sealing specifically comprises the following steps: cover in proper order and establish and rotatory second gland, first gland, make first perforation, second perforation, the perforation intercommunication of third, the one end of air duct pass in proper order open and shut the mechanism and with centrifuging tube intercommunication, rotatory movable rod, to sealed through-hole behind the reinforced bag internal mixture of water storage cavity, to let in carbon dioxide in the centrifuging tube, take out the air duct tip, and rotatory second gland, first gland make first perforation, second perforation, third perforation do not communicate to sealed centrifuging tube.
Preferably, the method for separating and purifying pharmaceutical bovine serum albumin further comprises: one end of the vent pipe is communicated and fixedly connected with the lower end of the first through hole, a plurality of vent holes are formed in the circumferential direction of the vent pipe, and the aperture of each vent hole is gradually reduced along the direction close to the first through hole;
the sealing plug top surface undercut forms the holding tank, runs through the holding tank bottom surface forms the fifth perforation, the sealing plug establishes the holding stopper in the holding tank.
Preferably, the method for separating and purifying pharmaceutical bovine serum albumin further comprises:
placing a centrifugal tube in a sealed container, arranging a heating device in a water storage cavity, controlling the temperature of a water body in the water storage cavity to be 68-72 ℃, adding sodium caprylate with the volume of 0.15-0.20% of the blood plasma into the centrifugal tube, introducing carbon dioxide into the centrifugal tube through a vent tube for 10-12min, adding a hydrochloric acid solution with the concentration of 1mol/L, adjusting the pH to 5-5.5, continuously introducing the carbon dioxide for 5-6min, adding ethanol with the concentration of 80% and the mass of 6% of the blood plasma, continuously introducing the carbon dioxide for 10-12min, centrifuging, and taking a supernatant;
and step three, placing the supernatant in an ultrafiltration machine, desalting by using a filter membrane with the molecular weight of 18000, concentrating to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain the medicinal bovine serum albumin.
Preferably, a sixth perforation is formed through the top surface of the rubber plug, the fifth perforation and the sixth perforation are communicated through a tube body, a one-way valve for allowing fluid to flow to the fifth perforation along the sixth perforation is arranged on the tube body, and an exhaust fan is arranged between the sixth perforation and the one-way valve on the tube body;
and in the process of introducing carbon dioxide in the step two, the exhaust fan works.
Preferably, the inner cavities of the first guide pipe and the second guide pipe respectively comprise an up-and-down communicated cylindrical cavity and an inverted round table-shaped cavity, wherein the first elastic plug is filled in the inverted round table-shaped cavity of the first guide pipe, and the second elastic plug is filled in the inverted round table-shaped cavity of the second guide pipe.
Preferably, the freeze-drying in the third step is specifically as follows: cooling the concentrated solution from room temperature to-40 deg.C at a speed of 20 deg.C/min and maintaining for 8h, heating to-25 deg.C at a speed of 20 deg.C/min and maintaining for 15-16h, heating to 0 deg.C at a speed of 5 deg.C/min and maintaining for 1-2h, and heating to 20 deg.C at a speed of 5 deg.C/min.
The invention at least comprises the following beneficial effects:
first, fresh bovine blood is through freezing treatment in advance, thrombin activity can be effectively improved, the addition of later stage sodium citrate is cooperated, thrombin and fibrinogen coagulation sedimentation in plasma are effectively avoided to result in albumin coprecipitation, plasma content is improved, simultaneously in the whole centrifugal process, carbon dioxide is introduced, when a certain high-pressure environment is caused, fibrinogen in plasma is effectively avoided to form fibrin sedimentation, albumin coprecipitation is also caused, plasma content is further improved, an anaerobic environment is provided in the whole preparation process, aerobic bacteria breeding pollution of bovine blood is effectively avoided, layered freeze-drying operation is favorable for improving bovine serum albumin finished product phase, and drying effect is improved, the obtained bovine serum albumin is white-like powder, and purity is higher than 94% through detection discovery.
Secondly, the centrifugal tube is integrated with the heating container through the arrangement of a sealing cover opening and closing mechanism, the sealing performance of the whole sealing plug and the pressure resistance of the centrifugal tube are not weakened while the opening requirements are met through the arrangement of a first perforation, a second perforation and a third perforation which are adjustable and communicated or not communicated, the relative positions of a first pressing cover and a first guide tube can be moved up and down to adjust the pressing degree of a first spring and a second spring, and the sealing degree of an elastic hole in a first elastic plug and the sealing degree of an elastic hole in a second elastic plug are further adjusted and controlled; the feeding mechanism is arranged to facilitate the operation and control of adding the mixture into the water storage cavity so that the mixture can generate carbon dioxide under the action of water, and the feeding mechanism and the water storage cavity are operated and isolated at the same time, so that the whole device is simple and convenient to operate and has an obvious effect; the setting of breather pipe when providing the anaerobic environment again, makes more, the great intraductal liquid of process centrifugation of bubble to traditional stirring is replaced in the flow of bubble, when reaching the stirring effect, avoids mechanical stirring to the destruction of target product, reduces the target product extraction rate.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 shows a schematic structural view of a sealed container according to the present invention;
fig. 2 shows a schematic view of a sealing plug according to the invention;
fig. 3 shows a schematic structural view of the charging mechanism according to the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
< example 1>
A separation and purification method of medicinal bovine serum albumin comprises the following steps:
step one, taking 100ml of fresh cattle blood of a yellow cattle and placing the fresh cattle blood into a centrifugal tube 1, detachably arranging the centrifugal tube 1 in a sealed container 2, introducing carbon dioxide into the centrifugal tube 1 and sealing the centrifugal tube, placing the sealed container 2 in a freezing refrigerator, adjusting the temperature of the freezing refrigerator from 20 ℃ to minus 30 ℃ at the speed of 5 ℃/min and keeping the temperature for 10min, then heating the centrifugal tube to 20 ℃ at the speed of 5 ℃/min and keeping the temperature for 4min, repeating the steps for 3 times, taking out the centrifugal tube, adding sodium citrate with the concentration of 3.8 percent in 10 percent of the total mass of the cattle blood into the centrifugal tube 1, introducing carbon dioxide again and sealing the centrifugal tube, separating the centrifugal tube 1 from the sealed container 2, controlling the temperature to be lower than 2 ℃ through a centrifugal machine, controlling the rotating speed of the centrifugal machine to be 3500r/min, centrifuging for 30min, and separating and;
wherein, as shown in fig. 1-3, centrifuge tube 1 is the shape of an inverted round platform, sealed container 2 includes:
the sealing plug 3 is detachably sealed (specifically, the sealing plug can be in threaded connection and matched with a sealing ring) and is plugged at an opening at the top of the centrifuge tube 1, the top surface of the sealing plug 3 is recessed downwards to form an accommodating groove 30, and the bottom surface of the accommodating groove 30 penetrates through to form a first through hole 31;
the opening and closing mechanism 4 comprises a first guide tube 40 coaxially and fixedly arranged on the bottom surface of the accommodating groove 30, a first elastic plug 41 filled at the lower part of the first guide tube 40, a first spring 42 fixedly connected with the top surface of the first elastic plug 41, a first gland 43 screwed at the top end of the first spring 42, a second guide tube 44 coaxially and fixedly arranged on the top surface of the first gland 43, a second elastic plug 45 filled at the lower part of the second guide tube 44, a second spring 46 fixedly connected with the top surface of the second elastic plug 45, and a second gland 47 screwed at the top end of the second spring 46, wherein a second through hole 430 penetrates through the top surface of the first gland 43, a third through hole 470 penetrates through the top surface of the second gland 47, a first elastic hole 410 vertically penetrates through the first elastic plug 41 and is arranged right below the second through hole 430 on the first elastic plug 41, and a second elastic hole 420 vertically penetrates through the second elastic plug 45 and is arranged right below the third through hole 470 on the second elastic plug 45, the first through hole 31, the second through hole 430 and the third through hole 470 are at equal distances from the central axis of the containing groove 30, the internal cavities of the first guide tube 40 and the second guide tube 44 respectively comprise a cylindrical cavity and an inverted circular truncated cone-shaped cavity which are communicated up and down, wherein the first elastic plug 41 is filled in the inverted circular truncated cone-shaped cavity of the first guide tube 40, the second elastic plug 45 is filled in the inverted circular truncated cone-shaped cavity of the second guide tube 44, the opening and closing mechanism 4 can not weaken the sealing performance of the whole sealing plug 3 and the pressure resistance of the centrifuge tube 1 through the arrangement of the first through hole 31, the second through hole 430 and the third through hole 470 which can be adjusted and communicated or not communicated, and the relative position of the first pressing cover 43 and the first guide tube 40 can be moved up and down through the screw connection, so as to adjust the first spring 42, The compression degree of the second spring 46 further regulates and controls the sealing degree of the first elastic hole on the first elastic plug 41 and the sealing degree of the second elastic hole on the second elastic plug 45;
the heating pipe 5 is detachably sleeved outside the centrifugal pipe 1 to form a water storage cavity 50 for coating the outer wall of the centrifugal pipe 1 with the outer wall of the centrifugal pipe 1, the inner side wall of the heating pipe 5 is positioned in the water storage cavity 50 and can be provided with a plurality of supporting rods for supporting the centrifugal pipe 1 to realize detachable connection between the centrifugal pipe 1 and the sealed container 2, the top end of the water storage cavity 50 is plugged with an annular rubber plug 51 for sealing the water storage cavity 50, a fourth through hole 52 is formed in the rubber plug 51, an air guide pipe is detachably communicated with the fourth through hole 52, one end of the air guide pipe, which is far away from the fourth through hole 52, is a hard pipe body, and a through hole 53 is;
the feeding mechanism 6 comprises a truncated cone-shaped movable plug 60 plugged on the through hole 53, a movable rod 61 hinged to a small end of the movable plug 60 positioned on the outer side of the heating pipe 5 (namely the diameter of one end of the movable plug 60 positioned in the water storage cavity 50 is larger than that of one end of the movable plug 60 positioned outside the water storage cavity 50), and a feeding bag 62 sleeved on the outer side of the movable rod 61 and provided with an opening fixedly arranged on the circumferential side wall of the heating pipe 5 positioned in the through hole 53, wherein a mixture of citric acid and sodium bicarbonate with the mass ratio of 1:3 is placed in the feeding bag 62, the feeding mechanism 6 is convenient to control and add the mixture into the water storage cavity 50, so that the mixture generates carbon dioxide under the action of water, and meanwhile, the feeding mechanism 6 and the water storage cavity 50 are controlled and isolated 2, and the whole device is simple and convenient to control and has obvious effect;
in the first step, the step of introducing carbon dioxide into the centrifugal tube 1 and then sealing specifically comprises the following steps: sequentially covering and rotating the second gland 47 and the first gland 43 to enable the first perforation 31, the second perforation 430 and the third perforation 470 to be communicated, sequentially enabling one end of the air duct to pass through the opening and closing mechanism 4 and be communicated with the centrifuge tube 1, rotating the movable rod 61, adding the mixture in the feeding bag 62 into the water storage cavity 50, sealing the through hole 53, introducing carbon dioxide into the centrifuge tube 1, drawing out the end part of the air duct, and rotating the second gland 47 and the first gland 43 to enable the first perforation 31, the second perforation 430 and the third perforation 470 not to be communicated so as to seal the centrifuge tube 1;
further comprising: one end of the vent pipe 7 is fixedly connected with the lower end of the first through hole 31, a plurality of vent holes are formed in the circumferential direction of the vent pipe 7, the aperture of each vent hole is gradually reduced along the direction close to the first through hole 31, and when the sealing plug 3 is plugged on the centrifugal tube 1, the distance between the bottom end of the vent pipe 7 and the bottom end of the centrifugal tube 1 is small;
the top surface of the sealing plug 3 is recessed downwards to form a receiving groove 8, a fifth through hole 80 is formed by penetrating through the bottom surface of the receiving groove 8, and the sealing plug 3 in the receiving groove 8 is provided with a receiving plug 81.
Further comprising:
secondly, placing the centrifuge tube 1 in a sealed container 2, arranging a heating device in the water storage cavity 50, controlling the temperature of the water in the water storage cavity 50 to be 70 ℃, adding sodium caprylate with the volume of 0.17 percent of the plasma volume into the centrifuge tube 1, meanwhile, after carbon dioxide is introduced into the centrifugal tube 1 through the vent pipe 7 for 12min, hydrochloric acid solution with the concentration of 1mol/L is added, the pH value is adjusted to 5.3, after carbon dioxide is continuously introduced for 5min, ethanol with the concentration of 80 percent and the mass of 6 percent of blood plasma is added, after carbon dioxide is continuously introduced for 10-12min, centrifugation is carried out, supernatant is taken, the vent pipe 7 is arranged, and while an anaerobic environment is provided, so that more bubbles and larger area of liquid pass through the liquid in the centrifugal tube 1, the flow of the bubbles replaces the traditional stirring, while the stirring effect is achieved, the damage of mechanical stirring to the target product is avoided, and the extraction rate of the target product is reduced;
and step three, placing the supernatant in an ultrafiltration machine, desalting by using a filter membrane with the molecular weight of 18000, concentrating to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain the medicinal bovine serum albumin.
Wherein, the sealed container 2 further comprises a base arranged below the heating pipe 5 to support the heating pipe 5.
< example 2>
A separation and purification method of medicinal bovine serum albumin comprises the following steps:
step one, taking 100ml of fresh cattle blood of a cattle, placing the fresh cattle blood into a centrifugal tube 1, detachably arranging the centrifugal tube 1 in a sealed container 2, introducing carbon dioxide into the centrifugal tube 1, sealing, placing the sealed container 2 in a freezing refrigerator, adjusting the temperature of the freezing refrigerator from 20 ℃ to minus 30 ℃ at a speed of 5 ℃/min, keeping for 10min, then heating to 20 ℃ at a speed of 5 ℃/min, keeping for 4min, repeating for 3 times, taking out, adding sodium citrate with a concentration of 3.8 percent, which is 10 percent of the total mass of the cattle blood, into the centrifugal tube 1, introducing carbon dioxide again, sealing, separating the centrifugal tube 1 from the sealed container 2, controlling the temperature to be lower than 2 ℃ through a centrifugal machine, and separating and removing blood cells to obtain plasma;
wherein, as shown in fig. 1-3, centrifuge tube 1 is the shape of an inverted round platform, sealed container 2 includes:
the sealing plug 3 is detachably arranged at the opening at the top of the centrifuge tube 1, the top surface of the sealing plug 3 is recessed downwards to form an accommodating groove 30, and the bottom surface of the accommodating groove 30 penetrates through to form a first through hole 31;
the opening and closing mechanism 4 comprises a first guide tube 40 coaxially and fixedly arranged on the bottom surface of the accommodating groove 30, a first elastic plug 41 filled at the lower part of the first guide tube 40, a first spring 42 fixedly connected with the top surface of the first elastic plug 41, a first gland 43 screwed at the top end of the first spring 42, a second guide tube 44 coaxially and fixedly arranged on the top surface of the first gland 43, a second elastic plug 45 filled at the lower part of the second guide tube 44, a second spring 46 fixedly connected with the top surface of the second elastic plug 45, and a second gland 47 screwed at the top end of the second spring 46, wherein a second through hole 430 penetrates through the top surface of the first gland 43, a third through hole 470 penetrates through the top surface of the second gland 47, a first elastic hole 410 vertically penetrates through the first elastic plug 41 and is arranged right below the second through hole 430 on the first elastic plug 41, and a second elastic hole 420 vertically penetrates through the second elastic plug 45 and is arranged right below the third through hole 470 on the second elastic plug 45, the first through hole 31, the second through hole 430 and the third through hole 470 have the same distance from the central axis of the accommodating groove 30;
the heating pipe 5 is detachably sleeved outside the centrifugal pipe 1 to form a water storage cavity 50 for coating the outer wall of the centrifugal pipe 1 with the outer wall of the centrifugal pipe 1, a ring-shaped rubber plug 51 is plugged at the top end of the water storage cavity 50 to seal the water storage cavity 50, a fourth through hole 52 is formed by penetrating through the rubber plug 51, an air guide pipe is detachably communicated with the fourth through hole 52, one end, away from the fourth through hole 52, of the air guide pipe is a hard pipe body, and a through hole 53 is formed in the upper portion of the outer side wall of the heating pipe 5;
the charging mechanism 6 comprises a truncated cone-shaped movable plug 60 plugged on the through hole 53, a movable rod 61 hinged with the movable plug 60 at the small end positioned on the outer side of the heating pipe 5, and a charging bag 62 sleeved on the outer side of the movable rod 61 and fixedly provided with an opening on the circumferential side wall of the heating pipe 5 positioned on the through hole 53, wherein a mixture of citric acid and sodium bicarbonate with the mass ratio of 1:3 is placed in the charging bag 62;
in the first step, the step of introducing carbon dioxide into the centrifugal tube 1 and then sealing specifically comprises the following steps: cover in proper order and establish and rotatory second gland 47, first gland 43, make first perforation 31, the perforation 430 of second, the perforation 470 intercommunication of third, the one end of air duct pass in proper order open and shut mechanism 4 and with centrifuging tube 1 intercommunication, rotatory movable rod 61, to sealed through-hole 53 behind the mixture in the water storage chamber 50 addition reinforced bag 62, to let in carbon dioxide in the centrifuging tube 1, take out the air duct tip to rotatory second gland 47, first gland 43 make first perforation 31, the perforation 430 of second, the perforation 470 of third do not communicate, with sealed centrifuging tube 1.
Further comprising: one end of the vent pipe 7 is fixedly connected with the lower end of the first through hole 31, a plurality of vent holes are formed in the circumferential direction of the vent pipe 7, and the aperture of each vent hole is gradually reduced along the direction close to the first through hole 31;
the top surface of the sealing plug 3 is recessed downwards to form a receiving groove 8, a fifth through hole 80 is formed by penetrating through the bottom surface of the receiving groove 8, and the sealing plug 3 in the receiving groove 8 is provided with a receiving plug 81.
Further comprising:
secondly, the centrifugal tube 1 is arranged in the sealed container 2, a heating device is arranged in the water storage cavity 50, the temperature of the water body in the water storage cavity 50 is controlled to be 70 ℃, sodium caprylate with the volume of 0.17% of the blood plasma is added into the centrifugal tube 1, meanwhile, hydrochloric acid solution with the concentration of 1mol/L is added into the centrifugal tube 1 after carbon dioxide is introduced into the centrifugal tube 1 for 10-12min through the vent pipe 7, the pH value is adjusted to be 5.35, after the carbon dioxide is continuously introduced for 5.5min, ethanol with the concentration of 80% of the blood plasma mass is added, after the carbon dioxide is continuously introduced for 11min, centrifugation is carried out, and supernatant is taken out, wherein the heating device specifically comprises a heating part (a heating pipe 5 which can be arranged in the water storage cavity 50), and a temperature adjusting part (a temperature sensor which is arranged inside the water storage cavity 50 to detect the temperature information of the water body in, A control part connected with the heating part and the temperature adjusting part (the control part regulates and controls the work of the heating part according to the received new type);
and step three, placing the supernatant in an ultrafiltration machine, desalting by using a filter membrane with the molecular weight of 18000, concentrating to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain the medicinal bovine serum albumin.
A sixth perforation is formed by penetrating through the top surface of the rubber plug 51, the fifth perforation 80 is communicated with the sixth perforation through a pipe body, a one-way valve for allowing fluid to flow to the fifth perforation 80 along the sixth perforation is arranged on the pipe body, an exhaust fan is arranged between the sixth perforation and the one-way valve on the pipe body, and the use of mixture raw materials is greatly reduced due to the arrangement of the exhaust fan and the one-way valve;
and in the process of introducing carbon dioxide in the step two, the exhaust fan works.
< example 3>
A separation and purification method of medicinal bovine serum albumin comprises the following steps:
step one, taking 100ml of fresh cattle blood of a cattle, placing the fresh cattle blood into a centrifugal tube 1, detachably arranging the centrifugal tube 1 in a sealed container 2, introducing carbon dioxide into the centrifugal tube 1, sealing, placing the sealed container 2 in a freezing refrigerator, adjusting the temperature of the freezing refrigerator from 20 ℃ to minus 30 ℃ at a speed of 5 ℃/min, keeping for 10min, then heating to 20 ℃ at a speed of 5 ℃/min, keeping for 4min, repeating for 3 times, taking out, adding sodium citrate with a concentration of 3.8 percent, which is 10 percent of the total mass of the cattle blood, into the centrifugal tube 1, introducing carbon dioxide again, sealing, separating the centrifugal tube 1 from the sealed container 2, controlling the temperature to be lower than 2 ℃ through a centrifugal machine, and separating and removing blood cells to obtain plasma;
wherein, as shown in fig. 1-3, centrifuge tube 1 is the shape of an inverted round platform, sealed container 2 includes:
the sealing plug 3 is detachably arranged at the opening at the top of the centrifuge tube 1, the top surface of the sealing plug 3 is recessed downwards to form an accommodating groove 30, and the bottom surface of the accommodating groove 30 penetrates through to form a first through hole 31;
the opening and closing mechanism 4 comprises a first guide tube 40 coaxially and fixedly arranged on the bottom surface of the accommodating groove 30, a first elastic plug 41 filled at the lower part of the first guide tube 40, a first spring 42 fixedly connected with the top surface of the first elastic plug 41, a first gland 43 screwed at the top end of the first spring 42, a second guide tube 44 coaxially and fixedly arranged on the top surface of the first gland 43, a second elastic plug 45 filled at the lower part of the second guide tube 44, a second spring 46 fixedly connected with the top surface of the second elastic plug 45, and a second gland 47 screwed at the top end of the second spring 46, wherein a second through hole 430 penetrates through the top surface of the first gland 43, a third through hole 470 penetrates through the top surface of the second gland 47, a first elastic hole 410 vertically penetrates through the first elastic plug 41 and is arranged right below the second through hole 430 on the first elastic plug 41, and a second elastic hole 420 vertically penetrates through the second elastic plug 45 and is arranged right below the third through hole 470 on the second elastic plug 45, the first through hole 31, the second through hole 430 and the third through hole 470 have the same distance from the central axis of the accommodating groove 30;
the heating pipe 5 is detachably sleeved outside the centrifugal pipe 1 to form a water storage cavity 50 for coating the outer wall of the centrifugal pipe 1 with the outer wall of the centrifugal pipe 1, a ring-shaped rubber plug 51 is plugged at the top end of the water storage cavity 50 to seal the water storage cavity 50, a fourth through hole 52 is formed by penetrating through the rubber plug 51, an air guide pipe is detachably communicated with the fourth through hole 52, one end, away from the fourth through hole 52, of the air guide pipe is a hard pipe body, and a through hole 53 is formed in the upper portion of the outer side wall of the heating pipe 5;
the charging mechanism 6 comprises a truncated cone-shaped movable plug 60 plugged on the through hole 53, a movable rod 61 hinged with the movable plug 60 at the small end positioned on the outer side of the heating pipe 5, and a charging bag 62 sleeved on the outer side of the movable rod 61 and fixedly provided with an opening on the circumferential side wall of the heating pipe 5 positioned on the through hole 53, wherein a mixture of citric acid and sodium bicarbonate with the mass ratio of 1:3 is placed in the charging bag 62;
in the first step, the step of introducing carbon dioxide into the centrifugal tube 1 and then sealing specifically comprises the following steps: cover in proper order and establish and rotatory second gland 47, first gland 43, make first perforation 31, the perforation 430 of second, the perforation 470 intercommunication of third, the one end of air duct pass in proper order open and shut mechanism 4 and with centrifuging tube 1 intercommunication, rotatory movable rod 61, to sealed through-hole 53 behind the mixture in the water storage chamber 50 addition reinforced bag 62, to let in carbon dioxide in the centrifuging tube 1, take out the air duct tip to rotatory second gland 47, first gland 43 make first perforation 31, the perforation 430 of second, the perforation 470 of third do not communicate, with sealed centrifuging tube 1.
Further comprising: one end of the vent pipe 7 is fixedly connected with the lower end of the first through hole 31, a plurality of vent holes are formed in the circumferential direction of the vent pipe 7, and the aperture of each vent hole is gradually reduced along the direction close to the first through hole 31;
the top surface of the sealing plug 3 is recessed downwards to form a receiving groove 8, a fifth through hole 80 is formed by penetrating through the bottom surface of the receiving groove 8, and the sealing plug 3 in the receiving groove 8 is provided with a receiving plug 81.
Further comprising:
secondly, placing a centrifugal tube 1 in a sealed container 2, arranging a heating device in the water storage cavity 50, controlling the temperature of water in the water storage cavity 50 to be 70 ℃, adding sodium caprylate with the volume of 0.17% of plasma into the centrifugal tube 1, introducing carbon dioxide into the centrifugal tube 1 through a vent pipe 7 for 11min, adding a hydrochloric acid solution with the concentration of 1mol/L, adjusting the pH to 5.25, continuously introducing the carbon dioxide for 5.5min, adding ethanol with the concentration of 80% and the mass of 6% of the plasma, continuously introducing the carbon dioxide for 11min, centrifuging, and taking supernatant;
and step three, placing the supernatant in an ultrafiltration machine, desalting by using a filter membrane with the molecular weight of 18000, concentrating to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain the medicinal bovine serum albumin.
A sixth perforation is formed through the top surface of the rubber plug 51, the fifth perforation 80 is communicated with the sixth perforation through a pipe body, a one-way valve for allowing fluid to flow to the fifth perforation 80 along the sixth perforation is arranged on the pipe body, and an exhaust fan is arranged between the sixth perforation and the one-way valve on the pipe body;
and in the process of introducing carbon dioxide in the step two, the exhaust fan works.
The freeze-drying in the third step is specifically as follows: the concentrated solution is reduced to-40 ℃ from room temperature at the speed of 20 ℃/min and kept for 8h, then is increased to-25 ℃ at the speed of 20 ℃/min and kept for 15.5h, then is increased to 0 ℃ at the speed of 5 ℃/min and kept for 1.5h, and finally is increased to 20 ℃ at the speed of 5 ℃/min.
< comparative example 1>
A separation and purification method of medicinal bovine serum albumin comprises the following steps:
step one, taking bovine blood, placing the bovine blood in a centrifugal tube of a conventional low-temperature centrifuge, adding sodium citrate with the concentration of 3.8 percent, wherein the concentration of 10 percent of the total mass of the bovine blood, into the centrifugal tube, controlling the temperature to be lower than 2 ℃ through the centrifuge, and separating and removing blood cells to obtain plasma.
< comparative example 2>
A separation and purification method of medicinal bovine serum albumin is disclosed, wherein the first step and the third step are the same as the example 1, and the second step specifically comprises the following steps:
and step two, placing the centrifugal tube 1 in a sealed container 2, arranging a heating device in the water storage cavity 50, controlling the temperature of the water body in the water storage cavity 50 to be 70 ℃, adding sodium caprylate with the volume of 0.17% of the plasma volume into the centrifugal tube 1, mechanically stirring, adding a hydrochloric acid solution with the concentration of 1mol/L, adjusting the pH value to be 5.4, continuously stirring, adding ethanol with the concentration of 80% of the plasma mass of 6%, centrifuging, and taking the supernatant.
< comparative example 3>
A separation and purification method of medicinal bovine serum albumin is disclosed, wherein the first step and the second step are the same as the example 3, and the third step specifically comprises the following steps: placing the supernatant in an ultrafiltration machine, desalting with a filter membrane with molecular weight of 18000, concentrating to obtain concentrated solution, cooling to-40 deg.C at 20 deg.C/min from room temperature, maintaining for 20h, and heating to 20 deg.C. Obtaining the medicinal bovine serum albumin.
Analysis of results
1: comparing the plasma contents obtained in the first step in example 1 with that obtained in comparative example 1, wherein 52ml of plasma is obtained from 100ml of bovine blood in example 1, and the plasma content in comparative example 1 is only 47ml, and the thrombin activity can be effectively improved mainly due to the pre-freezing treatment in example 1, and the generation of albumin coprecipitation caused by coagulation and precipitation of thrombin and fibrinogen in the plasma can be effectively avoided by matching with the addition of sodium citrate in the later period, so that the plasma content is improved; in the whole centrifugation process, carbon dioxide is introduced to cause a certain high-pressure environment, meanwhile, the albumin coprecipitation caused by the fibrinogen forming fibrin deposition in the plasma is effectively avoided, and the plasma content is further improved; furthermore, carbon dioxide is introduced twice to provide an anaerobic environment, so that aerobic bacteria in the blood of the cattle are effectively prevented from breeding and polluting.
2: comparing the content of the supernatant obtained in the second step with that of the supernatant in comparative example 1 in example 1, wherein the volume of the supernatant obtained in example 1 is larger than that of the supernatant in comparative example 1, and the content of albumin in the supernatant obtained in the second step in example 1 and comparative example 2, respectively, were further measured by the bromocresol chloride method, it was found that the content of albumin in the supernatant obtained in example 1 is larger than that in the supernatant in comparative example 1.
3: comparing the bovine serum albumin obtained in the example 3 with the bovine serum albumin obtained in the comparative example 3, wherein the finished bovine serum albumin obtained in the example 3 is white-like powder with a water content of 3.2%, and the finished bovine serum albumin obtained in the comparative example 3 is an uneven massive white-like substance with a water content of 6%, so that the hierarchical freeze-drying operation is beneficial to improving the phase of the finished bovine serum albumin and improving the drying effect.
4 the purity of the finished bovine serum albumin obtained in example 3 was 94.5% as measured by a cellulose acetate membrane method.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (4)
1. A separation and purification method of medicinal bovine serum albumin is characterized by comprising the following steps:
step one, taking bovine blood and placing the bovine blood in a centrifugal tube, wherein the centrifugal tube is detachably arranged in a sealed container, introducing carbon dioxide into the centrifugal tube and then sealing the centrifugal tube, placing the sealed container in a freezing refrigerator, regulating the temperature of the freezing refrigerator to be reduced to minus 30 ℃ from 20 ℃ at a speed of 5 ℃/min and keeping the temperature for 10min, then heating to 20 ℃ at a speed of 5 ℃/min and keeping the temperature for 4min, repeating the steps for 3 times, taking out the frozen container, adding sodium citrate with the concentration of 3.8 percent, which is 10 percent of the total mass of the bovine blood, into the centrifugal tube, introducing the carbon dioxide again and then sealing the centrifugal tube, separating the centrifugal tube from the sealed container, controlling the temperature to be lower than 2 ℃ through a centrifugal machine;
wherein, centrifuging tube is inversion round platform form, sealed container includes:
the sealing plug can be disassembled and arranged at the opening at the top of the centrifugal tube, the top surface of the sealing plug is downwards sunken to form an accommodating groove, and the bottom surface of the accommodating groove penetrates through the accommodating groove to form a first through hole;
the opening and closing mechanism comprises a first guide pipe coaxially and fixedly arranged on the bottom surface of the accommodating groove, a first elastic plug filled at the lower part in the first guide pipe, a first spring fixedly connected with the top surface of the first elastic plug, a first gland in screwed connection with the top end of the first spring, a second guide pipe coaxially and fixedly arranged on the top surface of the first gland, a second elastic plug filled at the lower part in the second guide pipe, a second spring fixedly connected with the top surface of the second elastic plug, and a second gland in screwed connection with the top end of the second spring, wherein a second through hole penetrates through the top surface of the first gland, a third through hole penetrates through the top surface of the second gland, a first elastic hole vertically penetrates through the first elastic plug under the second through hole on the first elastic plug, a second elastic hole vertically penetrates through the second elastic plug under the third through hole on the second elastic plug, and a second elastic hole is arranged on the second elastic plug, the first through hole, the second through hole and the third through hole are equal in distance from the central axis of the accommodating groove;
the heating pipe is detachably sleeved outside the centrifugal pipe to form a water storage cavity wrapping the outer wall of the centrifugal pipe between the heating pipe and the outer wall of the centrifugal pipe, a ring-shaped rubber plug is plugged at the top end of the water storage cavity to seal the water storage cavity, a fourth through hole penetrates through the rubber plug, an air guide pipe is detachably communicated with the fourth through hole, one end, away from the fourth through hole, of the air guide pipe is a hard pipe body, and a through hole is formed in the upper portion of the outer side wall of the heating pipe;
the charging mechanism comprises a truncated cone-shaped movable plug arranged on the through hole in a plugging manner, a movable rod hinged with the small end of the movable plug positioned on the outer side of the heating pipe, and a charging bag sleeved on the outer side of the movable rod and provided with an opening fixedly arranged on the circumferential side wall of the heating pipe positioned on the through hole, wherein a mixture of citric acid and sodium bicarbonate with the mass ratio of 1:3 is placed in the charging bag;
in the first step, the step of introducing carbon dioxide into the centrifugal tube and then sealing specifically comprises the following steps: sequentially covering and rotating a second gland and a first gland to enable the first perforation, the second perforation and the third perforation to be communicated, sequentially penetrating one end of the air duct through an opening and closing mechanism and communicating with the centrifuge tube, rotating a movable rod, adding a mixture in a charging bag into the water storage cavity, sealing the through hole, introducing carbon dioxide into the centrifuge tube, drawing out the end part of the air duct, and rotating the second gland and the first gland to enable the first perforation, the second perforation and the third perforation to be not communicated so as to seal the centrifuge tube;
further comprising: one end of the vent pipe is communicated and fixedly connected with the lower end of the first through hole, a plurality of vent holes are formed in the circumferential direction of the vent pipe, and the aperture of each vent hole is gradually reduced along the direction close to the first through hole;
the top surface of the sealing plug is recessed downwards to form an accommodating groove, a fifth through hole is formed by penetrating through the bottom surface of the accommodating groove, and an accommodating plug is arranged in the accommodating groove;
further comprising:
placing a centrifugal tube in a sealed container, arranging a heating device in a water storage cavity, controlling the temperature of a water body in the water storage cavity to be 68-72 ℃, adding sodium caprylate with the volume of 0.15-0.20% of the blood plasma into the centrifugal tube, introducing carbon dioxide into the centrifugal tube through a vent tube for 10-12min, adding a hydrochloric acid solution with the concentration of 1mol/L, adjusting the pH to 5-5.5, continuously introducing the carbon dioxide for 5-6min, adding ethanol with the concentration of 80% and the mass of 6% of the blood plasma, continuously introducing the carbon dioxide for 10-12min, centrifuging, and taking a supernatant;
and step three, placing the supernatant in an ultrafiltration machine, desalting by using a filter membrane with the molecular weight of 18000, concentrating to obtain a concentrated solution, and freeze-drying the concentrated solution to obtain the medicinal bovine serum albumin.
2. The method for separating and purifying the pharmaceutical bovine serum albumin as claimed in claim 1, wherein a sixth perforation is formed through the top surface of the rubber stopper, the fifth perforation and the sixth perforation are communicated through a tube body, the tube body is provided with a check valve for allowing fluid to flow to the fifth perforation along the sixth perforation, and an exhaust fan is arranged between the sixth perforation and the check valve on the tube body;
and in the process of introducing carbon dioxide in the step two, the exhaust fan works.
3. The method for separating and purifying the pharmaceutical bovine serum albumin according to claim 1, wherein the inner cavities of the first guide tube and the second guide tube each comprise a cylindrical cavity and an inverted circular truncated cone-shaped cavity which are vertically communicated, wherein the first elastic plug is filled in the inverted circular truncated cone-shaped cavity of the first guide tube, and the second elastic plug is filled in the inverted circular truncated cone-shaped cavity of the second guide tube.
4. The method for separating and purifying the medicinal bovine serum albumin according to claim 1, wherein the lyophilization in the third step is specifically as follows: cooling the concentrated solution from room temperature to-40 deg.C at a speed of 20 deg.C/min and maintaining for 8h, heating to-25 deg.C at a speed of 20 deg.C/min and maintaining for 15-16h, heating to 0 deg.C at a speed of 5 deg.C/min and maintaining for 1-2h, and heating to 20 deg.C at a speed of 5 deg.C/min.
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