Polypeptide synthesis method
阅读说明:本技术 一种多肽合成方法 (Polypeptide synthesis method ) 是由 沈红超 孔伟伟 苏生鹏 于 2019-11-08 设计创作,主要内容包括:本发明属于多肽合成技术领域,具体的说是一种多肽合成方法,该合成方法中所使用到的合成设备包括支架和合成罐;支架上端中心位置设有正反转电机,正反转电机的输出轴连接合成罐内设有的转动轴一端,转动轴另一端连接多个搅动板;合成罐外壁与合成罐内壁之间开设有加温腔;合成罐内壁设有一号板,一号板上开设多个出气孔,且出气孔通过一号板与一号板下端面设有的一号管道连通,且控制单元用于控制氮气通入一号管道和合成罐内废液从一号管道流出,控制单元左端设有废液流出的二号管道,控制单元右端设有氮气通入的三号管道;通过正反转电机、合成罐、一号管道、控制单元、二号管道和三号管道,实现合成罐内多肽的合成。(The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a polypeptide synthesis method, wherein synthesis equipment used in the synthesis method comprises a support and a synthesis tank; a positive and negative rotation motor is arranged at the central position of the upper end of the bracket, an output shaft of the positive and negative rotation motor is connected with one end of a rotating shaft arranged in the synthesis tank, and the other end of the rotating shaft is connected with the plurality of stirring plates; a heating cavity is arranged between the outer wall of the synthesis tank and the inner wall of the synthesis tank; the inner wall of the synthesis tank is provided with a first plate, the first plate is provided with a plurality of air outlet holes, the air outlet holes are communicated with a first pipeline arranged on the lower end face of the first plate through the first plate, the control unit is used for controlling nitrogen to be introduced into the first pipeline and waste liquid in the synthesis tank to flow out of the first pipeline, the left end of the control unit is provided with a second pipeline for the waste liquid to flow out, and the right end of the control unit is provided with a third pipeline for the nitrogen to flow in; the synthesis of the polypeptide in the synthesis tank is realized through a positive and negative rotating motor, the synthesis tank, a first pipeline, a control unit, a second pipeline and a third pipeline.)
1. A method of polypeptide synthesis, comprising: the synthesis method comprises the following steps:
s1: firstly drying alfalfa, breaking the wall, crushing the alfalfa, keeping the crushing granularity of the alfalfa between 120 plus materials and 150 meshes, filtering the alfalfa by using a microporous filter bag, simultaneously adding cellulase, and continuously extracting the crushed material containing the microporous filter bag body by using ultrasonic extraction equipment;
s2: transferring the ultrasonic extraction product in the S1 into a fermentation device, adding proteolytic enzyme into the fermentation device, controlling the fermentation temperature to be between 50 and 70 ℃, and controlling the decomposition time to be within 3 to 4 hours;
s3: after decomposition is finished, transferring the reaction of decomposition fermentation into centrifugal equipment, and carrying out layered separation on decomposition liquid in the centrifugal equipment by using a layered biomembrane to obtain an amino acid product;
s4: putting the amino acid product in the S3 into a synthesis device, adding polypeptide resin, Fmoc-protected amino acid, a condensing agent and a solvent, and introducing nitrogen to eliminate static electricity on synthesis equipment; after the reaction is finished, filtering waste liquid to obtain a polypeptide product to be synthesized;
wherein the synthesis device used in the step S3 comprises a bracket (1) and a synthesis tank (2); the lower end of the support (1) is provided with a controller (11), the controller (11) is used for controlling the synthesis device to work, a forward and reverse rotating motor (12) is arranged at the center of the upper end of the support (1), an output shaft of the forward and reverse rotating motor (12) is connected with one end of a rotating shaft (21) arranged in the synthesis tank (2), and the other end of the rotating shaft (21) is connected with a plurality of stirring plates (211); a heating cavity (22) is formed between the outer wall of the synthesis tank (2) and the inner wall of the synthesis tank (2), a water inlet pipe (221) is arranged on the left lower side wall of the heating cavity (22), the water inlet pipe (221) is communicated with the heating cavity (22), a water outlet pipe (222) is arranged on the right upper side wall of the heating cavity (22), and the water outlet pipe (222) is communicated with the heating cavity (22); a number plate (23) is arranged on the inner wall of the synthesis tank (2), a plurality of air outlet holes (231) are formed in the number plate (23), the air outlet holes (231) are communicated with a first pipeline (24) arranged on the lower end face of the number plate (23) through the number plate (23), and the lower end of the first pipeline (24) penetrates through the bottom of the synthesis tank (2) and is communicated with the control unit (3); the control unit (3) is positioned on a bracket (25) arranged below the synthesis tank (2), the control unit (3) is used for controlling nitrogen to be introduced into a first pipeline (24) and waste liquid in the synthesis tank (2) to flow out from the first pipeline (24), a second pipeline (26) for waste liquid to flow out is arranged at the left end of the control unit (3), and a third pipeline (27) for nitrogen to be introduced is arranged at the right end of the control unit (3); the synthesis of the polypeptide in the synthesis tank (2) is realized through a forward and reverse rotating motor (12), the synthesis tank (2), a first pipeline (24), a control unit (3), a second pipeline (26) and a third pipeline (27).
2. A method of synthesizing a polypeptide according to claim 1, wherein: the control unit (3) comprises a control valve (31), a control valve core (32), a control hand wheel (33), a control valve core outlet (34) and a control valve core inlet (35); the central position of the control valve core (32) is connected with a control hand wheel (33) at the front end of the control valve (31), a cavity (321) is arranged in the control valve core (32), a sealing ball (322) is arranged in the cavity (321), the sealing ball (322) is connected with the inner wall of the cavity (321) through a first spring (323), a control valve core outlet (34) is formed in the outer wall of the control valve core (32), the control valve core outlet (34) penetrates through the cavity (321) in the control valve core (32), a control valve core inlet (35) is formed in the outer wall of the control valve core (32), the control valve core inlet (35) penetrates through the cavity (321) in the control valve core (32), and the direction of the control valve core inlet (35) is vertical to the direction of the control valve core; the nitrogen is introduced into the synthesis tank (2) and the waste liquid in the synthesis tank (2) flows out through the control unit (3).
3. A method of synthesizing a polypeptide according to claim 2, wherein: the control valve core (32) is provided with a first groove (324), the first groove (324) is opposite to the control valve core outlet (34), the first groove (324) is provided with a second spring (325) and an elastic sealing gasket (326), and the outer ring of the sealing gasket (326) is fixedly connected with the notch of the first groove (324); the third pipeline (27) is further sealed through the cooperation between the second spring (325) and the sealing gasket (326).
4. A method of synthesizing a polypeptide according to claim 1, wherein: a second groove (212) is formed in the lower end face of the rotating shaft (21), and balls (213) are arranged at the lower end in the second groove (212); the upper end surface of the first plate (23) is provided with a screw rod (232), and when the screw rod (232) is in an initial position, the upper end part of the screw rod (232) is connected with a ball (213); a plurality of sliding grooves (28) are arranged on the inner side wall of the synthesis tank (2); the outer ring of the first plate (23) is provided with sliding blocks (233) the number of which corresponds to that of the sliding grooves (28); the first plate (23) moves up and down in the synthesis tank (2) through the matching among the ball (213), the screw rod (232), the sliding groove (28) and the sliding block (233).
5. A method of synthesizing a polypeptide according to claim 4, wherein: the air outlet hole (231) at the upper edge of the first plate (23) is obliquely formed towards the side wall direction of the synthesis tank (2); the side wall of the synthesis tank (2) is cleaned through the air outlet (231) which is obliquely arranged.
6. A method of synthesizing a polypeptide according to claim 1, wherein: the stirring plate (211) is made of elastic materials, and the stirring plate (211) is provided with a stirring blade (214); the mixed liquid in the synthesis tank (2) is further stirred by the stirring blade (214).
Technical Field
The invention belongs to the technical field of polypeptide synthesis, and particularly relates to a polypeptide synthesis method.
Background
The polypeptide is a compound formed by connecting α -amino acids together by peptide bonds, and is an intermediate product of protein hydrolysis, a compound formed by dehydration condensation of two amino acid molecules is called dipeptide, and by analogy, tripeptide, tetrapeptide, pentapeptide and the like are also available, and a compound formed by dehydration condensation of 10-100 amino acid molecules is usually called polypeptide.
Polypeptide synthesis is a solid phase synthesis sequence generally from the N-terminus (amino terminus) to the C-terminus (carboxyl terminus). The synthesis of polypeptides in the past has been carried out in solution and is referred to as a liquid phase synthesis. Since Merrifield successfully develops a solid-phase polypeptide synthesis method in 1963, the solid-phase method becomes a common technology in polypeptide and protein synthesis by continuous improvement and perfection, and the method has incomparable advantages compared with the classical liquid-phase synthesis method, thereby greatly reducing the difficulty of product purification in each step. Polypeptide synthesis is broadly divided into two categories: solid phase synthesis and liquid phase polypeptide synthesis.
The polypeptide synthesizer adopts a mild reaction method to synthesize the polypeptide, and can be generally divided into a pure nitrogen bubbling method and a shaking type. The pure nitrogen bubbling method adopts a nitrogen bubbling reaction mode to stir reactants, namely, a reactor on a synthesizer is fixed, nitrogen is discharged from the lower part of the reactor to the upper part of the reactor through the reactor, and a solid phase and a liquid phase are mixed by bubbles generated in the process.
The polypeptide synthesis equipment has the advantages that nitrogen is discharged upwards from the lower part of the reactor to stir the reaction, and when the synthesis process is stopped, the nitrogen supply is closed, so that the waste liquid is easy to cause counter flow and enters a nitrogen supply pipeline to cause pollution; meanwhile, polypeptide solid phase is generated during synthesis of the polypeptide, and the polypeptide solid phase has adhesiveness and can adhere to synthesis equipment, so that the polypeptide synthesis is unsuccessful.
Disclosure of Invention
Aiming at the defects of the prior art and solving the problems that the waste liquid in the polypeptide synthesis equipment flows back and enters a nitrogen pipeline to cause pollution and the polypeptide solid phase is adhered to the polypeptide synthesis equipment, the invention provides a polypeptide synthesis method.
The technical scheme adopted by the invention for solving the technical problems is as follows: the invention relates to a polypeptide synthesis method, which comprises the following steps:
s1: firstly drying alfalfa, breaking the wall, crushing the alfalfa, keeping the crushing granularity of the alfalfa between 120 plus materials and 150 meshes, filtering the alfalfa by using a microporous filter bag, simultaneously adding cellulase, and continuously extracting the crushed material containing the microporous filter bag body by using ultrasonic extraction equipment;
s2: transferring the ultrasonic extraction product in the S1 into a fermentation device, adding proteolytic enzyme into the fermentation device, controlling the fermentation temperature to be between 50 and 70 ℃, and controlling the decomposition time to be within 3 to 4 hours;
s3: after decomposition is finished, transferring the reaction of decomposition fermentation into centrifugal equipment, and carrying out layered separation on decomposition liquid in the centrifugal equipment by using a layered biomembrane to obtain an amino acid product;
s4: putting the amino acid product in S3 into a synthesis device, adding polypeptide resin, Fmoc-protected amino acid, a condensing agent and a solvent for reaction, introducing nitrogen gas to eliminate static electricity on synthesis equipment, and filtering waste liquid after the reaction is finished to obtain a polypeptide product to be synthesized;
wherein the synthesis device used in S3 includes a holder and a synthesis tank; the lower end of the support is provided with a controller for controlling the operation of the controller and the synthesis device, the central position of the upper end of the support is provided with a forward and reverse rotating motor, the output shaft of the forward and reverse rotating motor is connected with one end of a rotating shaft arranged in the synthesis tank, and the other end of the rotating shaft is connected with a plurality of stirring plates; a heating cavity is formed between the outer wall of the synthesis tank and the inner wall of the synthesis tank, a water inlet pipe is arranged on the left lower side wall of the heating cavity and communicated with the heating cavity, a water outlet pipe is arranged on the right upper side wall of the heating cavity and communicated with the heating cavity; the inner wall of the synthesis tank is provided with a first plate, the first plate is provided with a plurality of air outlet holes, the air outlet holes are communicated with a first pipeline arranged on the lower end surface of the first plate through the first plate, and the lower end of the first pipeline penetrates through the bottom of the synthesis tank and is communicated with the control unit; the control unit is positioned on a bracket below the synthesis tank and is used for controlling nitrogen to be introduced into the first pipeline and waste liquid in the synthesis tank to flow out of the first pipeline, the left end of the control unit is provided with a second pipeline for the waste liquid to flow out, and the right end of the control unit is provided with a third pipeline for introducing nitrogen; the synthesis of the polypeptide in the synthesis tank is realized through a positive and negative rotating motor, the synthesis tank, a first pipeline, a control unit, a second pipeline and a third pipeline; when the device is used, firstly, water is introduced into a heating cavity from a water inlet pipe, the water temperature is controlled to be 60-80 ℃, then a control unit controls a third pipeline to introduce nitrogen, the nitrogen is introduced into a first pipeline through the control unit and is discharged through an air outlet hole in a first plate, then amino acid products, peptide resin, amino acid protected by Fmoc, a condensing agent and a solvent are added into a synthesis tank, then a controller starts a forward and reverse motor to rotate, an output shaft of the forward and reverse motor drives a rotating shaft to rotate, the rotating shaft drives a stirring plate to rotate, the stirring plate rotates and stirs mixed liquid in the synthesis tank, and when the nitrogen is introduced, firstly, the mixed liquid in the synthesis tank is blown, and stirring and mixing of the stirring plate are assisted; in the second aspect, during the synthesis of the polypeptide, the polypeptide solid phase carrier formed in the synthesis tank generates a clustering phenomenon under the action of static electricity, so that the polypeptide solid phase cannot be fully mixed with the liquid phase, the introduction of nitrogen can eliminate the static electricity in the synthesis tank, and the pressure of the nitrogen blown into the synthesis tank is increased through the control unit, so that the static electricity elimination efficiency in the synthesis tank is accelerated; in the third aspect, the polypeptide solid phase carrier has viscosity, if the polypeptide solid phase carrier is adhered to the inner side wall of the synthesis tank and is difficult to remove, nitrogen is discharged from the air outlet, and the nitrogen can impact the polypeptide solid phase carrier adhered to the inner side wall of the synthesis tank; after the reaction of the mixed liquid in the synthesis tank is finished, the control unit controls the nitrogen to stop introducing into the synthesis tank, the control unit controls the second pipeline to be communicated with the first pipeline, waste liquid in the synthesis tank flows into the first pipeline through the air outlet hole in the first plate, the waste liquid is discharged from the second pipeline, and residual solid in the synthesis tank is a polypeptide product.
Preferably, the control unit comprises a control valve, a control valve core, a control hand wheel, a control valve core outlet and a control valve core inlet; the central position of the control valve core is connected with a control hand wheel at the front end of the control valve, a cavity is arranged in the control valve core, a sealing ball is arranged in the cavity, the sealing ball is connected with the inner wall of the cavity through a first spring, a control valve core outlet is formed in the outer wall of the control valve core, the control valve core outlet penetrates through the cavity in the control valve core, a control valve core inlet is formed in the outer wall of the control valve core, the control valve core inlet penetrates through the cavity in the control valve core, and the direction of the control valve core inlet is perpendicular to the direction of; nitrogen is introduced into the synthesis tank and the waste liquid in the synthesis tank flows out through the control unit; when the device is used, the control unit controls the nitrogen to be introduced into the synthesis tank, the control hand wheel rotates 90 degrees anticlockwise to drive the control valve core to rotate, then the inlet of the control valve core is communicated with the third pipeline, the outlet of the control valve core is communicated with the first pipeline, the nitrogen passes through the third pipeline, the acting force of the nitrogen pushes the sealing ball to move, so that the inlet of the control valve core is opened, and the third pipeline is communicated with the first pipeline; let in nitrogen gas in the synthetic jar when needs stop, when the waste liquid discharge in the synthetic jar, the hand wheel anticlockwise rotation 90 degrees of controling once more, control valve core import and pipeline intercommunication No. one this moment, control valve core export and pipeline intercommunication No. two, under the effect of waste liquid pressure in the synthetic jar, the sealing ball is pushed down to the waste liquid for control valve core import is opened, pipeline and pipeline intercommunication No. two this moment, and final waste liquid flows in following the pipeline No. two.
Preferably, the control valve core is provided with a first groove, the first groove is opposite to the outlet of the control valve core, the first groove is provided with a second spring and an elastic sealing gasket, and the outer ring of the sealing gasket is fixedly connected with the notch of the first groove; the third pipeline is further sealed through the matching between the second spring and the sealing gasket; during the use, when control case import and pipeline intercommunication No. one, during control case export and pipeline intercommunication No. two, sealed the pad and be located No. three pipeline pipe orifices departments, under the effect of No. two spring elastic forces, No. two springs make sealed pad tightly attached at No. three pipeline pipe orifices departments, further sealed No. three pipeline orificial, avoid No. three pipelines of nitrogen gas and control case contact department to spill over to prevent nitrogen gas's waste, reduced the synthetic cost of polypeptide.
Preferably, the lower end face of the rotating shaft is provided with a second groove, and the lower end in the second groove is provided with a ball; the upper end surface of the first plate is provided with a screw rod, and the upper end part of the screw rod is connected with the ball when the screw rod is in an initial position; a plurality of sliding grooves are formed in the inner side wall of the synthesis tank; the outer ring of the first plate is provided with sliding blocks corresponding to the number of the sliding grooves; the first plate moves up and down in the synthesis tank through the matching among the ball, the screw rod, the sliding groove and the sliding block; when the device is used, when the forward and reverse rotating motor rotates forwards, the rotating shaft also rotates forwards along with the forward and reverse rotating motor, the screw rod moves upwards in the second groove under the constraint between the screw rod and the ball, and the sliding block on the first plate is constrained by the sliding groove, so that the screw rod drives the first plate to move upwards along the side wall of the synthesis tank; when the forward and reverse rotation motor rotates reversely, the screw rod pushes the first plate to move downwards along the side wall of the synthesis tank; when the first plate moves up and down along the side wall of the synthesis tank, the polypeptide solid phase adhered to the side wall of the synthesis tank is cleaned in time, so that the reaction efficiency of the mixed liquid in the synthesis tank is improved.
Preferably, the air outlet hole at the upper edge of the first plate is obliquely formed towards the side wall of the synthesis tank; the side wall of the synthesis tank is cleaned through the air outlet holes which are obliquely formed; during the use, when nitrogen gas discharged from a board venthole, nitrogen gas was blown to the mixing liquid drum in the synthesis jar, and the supplementary stirring board stirs the stirring and mixes, and the venthole that the slope of a board edge was seted up, after the discharge nitrogen gas, nitrogen gas impact on synthesis jar inside wall, further cleared up the polypeptide solid phase of adhesion on the synthesis jar side wall.
Preferably, the stirring plate is made of elastic materials, and the stirring plate is provided with stirring blades; further stirring the mixed liquid in the synthesis tank by stirring blades; when the polypeptide synthesis tank is used, the rotating shaft drives the stirring plate to rotate, then when the stirring plate rotates mixed liquid, the stirring blades rotate under the action of the mixed liquid, and simultaneously, the rotation of the stirring blades can further promote the stirring amplitude of the mixed liquid in the synthesis tank, so that the synthesis of polypeptide is accelerated; when a board is close to stirring the board gradually, stir the board and slowly attach face on a board, to the clearance of a board face, in time to the venthole clearance, let out the more effectual discharge nitrogen gas of gas pocket.
The invention has the technical effects and advantages that:
1. according to the polypeptide synthesis method, the control unit can control the entering of nitrogen and the entering amount, and the control unit can switch the first pipeline to be communicated with the second pipeline, so that waste liquid in the synthesis tank is discharged, and the waste liquid does not enter the nitrogen pipe to cause pollution; the heating cavity is internally communicated with hot water to heat the mixed liquid in the synthesis tank, so that the reaction efficiency of the mixed liquid is improved.
2. According to the polypeptide synthesis method, the balls arranged in the rotating shaft and the lead screw on the first plate are matched, so that the first plate is pressed on the inner side wall of the synthesis tank to slide up and down, the swing amplitude of mixed liquid in the synthesis tank is increased, the reaction efficiency of the mixed liquid is accelerated, and meanwhile, the first plate moves up and down to timely clean a polypeptide solid phase adhered to the inner side wall of the synthesis tank; and the nitrogen discharged from the air outlet hole obliquely arranged at the edge of the first plate impacts on the inner side wall of the synthesis tank, and the inner side wall of the synthesis tank is further cleaned.
Drawings
The invention will be further explained with reference to the drawings.
FIG. 1 is a flow diagram of a synthetic process of the present invention;
FIG. 2 is a perspective view of a synthesis apparatus used in the present invention;
FIG. 3 is an enlarged view of a portion of FIG. 2 at A;
FIG. 4 is an enlarged view of a portion of FIG. 2 at B;
FIG. 5 is a cross-sectional view of the control unit;
in the figure: the device comprises a support 1, a
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
As shown in FIGS. 1-5, the synthesis method of the polypeptide of the present invention comprises the following steps:
s1: firstly drying alfalfa, breaking the wall, crushing the alfalfa, keeping the crushing granularity of the alfalfa between 120 plus materials and 150 meshes, filtering the alfalfa by using a microporous filter bag, simultaneously adding cellulase, and continuously extracting the crushed material containing the microporous filter bag body by using ultrasonic extraction equipment;
s2: transferring the ultrasonic extraction product in the S1 into a fermentation device, adding proteolytic enzyme into the fermentation device, controlling the fermentation temperature to be between 50 and 70 ℃, and controlling the decomposition time to be within 3 to 4 hours;
s3: after decomposition is finished, transferring the reaction of decomposition fermentation into centrifugal equipment, and carrying out layered separation on decomposition liquid in the centrifugal equipment by using a layered biomembrane to obtain an amino acid product;
s4: putting the amino acid product in S3 into a synthesis device, adding polypeptide resin, Fmoc-protected amino acid, a condensing agent and a solvent for reaction, introducing nitrogen gas to eliminate static electricity on synthesis equipment, and filtering waste liquid after the reaction is finished to obtain a polypeptide product to be synthesized;
wherein the synthesis device used in S3 includes a holder 1 and a
As an embodiment of the present invention, the control unit 3 includes a
As an embodiment of the present invention, the
In one embodiment of the present invention, a
As an embodiment of the present invention, the
In an embodiment of the present invention, the
When the device is used, firstly, water is introduced into the
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
- 上一篇:一种医用注射器针头装配设备
- 下一篇:一种蛋白高效分离装置