A method for site-specific modification of inflammatory markers with PEG and increasing protein stability

Abstract

The present invention discloses a method for PEG site-specific modification of inflammatory markers and increasing protein stability, comprising the following steps: a purified protein in procalcitonin, C-reactive protein, amyloid A or interleukin 6 is mixed with mPEG2000Mal in a 5mmol / L phosphate buffer having a pH value of 7.2-7.4 until the reaction is complete. The conjugate is purified by a chromatographic column, and then rinsed by a proportioning liquid until it is flushed flat; then the proportioning liquid is used for elution, so that the sample is collected when the absorbance of the product increases; the collected sample is placed in an ultrafiltration centrifuge tube and centrifuged by a centrifugal device. The present invention selects a peg with a molecular weight of 2000, because its molecular weight is relatively small, it can partially penetrate into the protein for modification, which can also reduce immunogenicity relatively less while increasing protein stability.

Description

Technical Field

[0001] The present invention relates to the technical field of protein modification, and in particular to a method for site-specific modification of inflammatory markers by PEG and increasing protein stability. Background Art

[0002] Quality control products are simply substances used to test the accuracy of hospital testing systems. Inflammatory markers such as procalcitonin, C-reactive protein, amyloid protein, etc. are markers produced when the human body has an inflammatory response and can be used to monitor whether the human body has infection and inflammatory response. When a hospital wants to confirm whether its testing system can detect inflammatory markers, it will use quality control products containing inflammatory markers to confirm the accuracy of the system.

[0003] However, since inflammatory markers are proteins in substance, their half-life at room temperature is short and they are prone to failure. In addition, if the concentrated samples are not effectively separated by centrifugation after reaction, the concentration of inflammatory markers will be incomplete, thus affecting the stability of subsequent use. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] In view of the above problems existing in the prior art, the present invention is proposed.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A method for site-specific modification of inflammatory markers with PEG and increasing protein stability, comprising the following steps:

[0008] Step 1: Mix one of the purified proteins of procalcitonin, C-reactive protein, amyloid A or interleukin-6 with mPEG2000Mal in a 5 mmol / L phosphate buffer with a pH of 7.2-7.4 until the reaction is complete.

[0009] Step 2: Purify the conjugate using a chromatographic column, and then wash it with a matching solution until it is flushed flat;

[0010] Step 3: Then use the matching solution to elute and collect the sample when the absorbance of the product increases;

[0011] Step 4: Place the collected sample in an ultrafiltration centrifuge tube, and then place it in a centrifuge device for balancing before centrifugation;

[0012] The centrifugal device comprises: a centrifuge unit, a rotor unit and a trigger unit;

[0013] Wherein, the centrifuge unit comprises: a centrifuge body, a control panel arranged on the front side of the centrifuge body, a warning light arranged on the control panel, and a cover rotatably arranged on the centrifuge body, a cavity is opened in the centrifuge body, and the rotor unit comprises: a rotor disk arranged in the cavity, a plurality of cavities opened on the rotor disk, and a hollow column arranged on the bottom side of the rotor disk, a spherical support is fixedly connected to the bottom side of the hollow column, a rotating seat is rotatably matched on the spherical support, and the bottom side of the rotating seat is driven to rotate by the centrifuge output shaft, and a plurality of balancing mechanisms are arranged on the bottom side of the rotor disk;

[0014] The trigger unit comprises: a plurality of support seats fixedly arranged on the inner side of the cavity, a horizontal rod arranged on the support seat, a first switch seat and a second switch seat are respectively arranged on the horizontal rod, and a film touch switch for controlling the turning on and off of the warning light is respectively arranged on the first switch seat and the second switch seat;

[0015] Step 5: After balancing, the sample is centrifuged in the centrifuge body (101), and the centrifugal temperature is maintained at 2-8°C and 3,000-4,000 r / min to be concentrated to 250-500 μL. The concentrated sample is desalted to obtain the PEG-site-modified inflammatory marker.

[0016] As a preferred embodiment of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, the plurality of lumens are distributed in a uniform annular array, and the plurality of balancing mechanisms are distributed in one-to-one correspondence with the lumens.

[0017] As a preferred embodiment of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, the balancing mechanism comprises: a balancing seat fixedly arranged on the bottom side of the rotor disk, a slideway opened in the balancing seat and concave toward the inner side of the rotor disk, and a counterweight ball placed on the slideway, wherein two openings are opened on the bottom side of the slideway.

[0018] As a preferred embodiment of the method for site-specifically modifying inflammatory markers with PEG and increasing protein stability described in the present invention, when the rotor disk is in a balanced state, the counterweight ball is located on one side close to the center of the rotor disk; when the rotor disk is in an unbalanced state, the balance seat is tilted, and the counterweight ball rolls to the side away from the center of the rotor disk.

[0019] As a preferred solution of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, wherein: a rotating shaft is rotatably connected at the center of the rotor disk, a knob is fixedly connected to the upper end of the rotating shaft, a threaded rod is fixedly connected to the bottom end of the rotating shaft, and a rectangular threaded sleeve is threadedly connected to the threaded rod;

[0020] The rectangular threaded sleeve is slidably inserted into the rotor disk, and the bottom end of the rectangular threaded sleeve passes through the spherical support. A positioning hole matching the bottom end of the spherical support is opened on the inner side of the rotating seat.

[0021] As a preferred scheme of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, one end of the plurality of horizontal rods is commonly fixedly connected to a slip ring, a conical groove is provided on the inner side of the slip ring, open grooves are symmetrically provided on the outer side of the hollow column, a top block is fixedly connected to the rectangular threaded sleeve, and the top block passes through the open groove, a conical disk is also fixedly connected to the outer side of the hollow column, and the conical groove cooperates with the conical disk.

[0022] As a preferred scheme of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, one end of the horizontal rod is slidably connected in the support seat, and a spring is fixedly connected to the inner side of the support seat, and the upper end of the spring is fixedly connected to the horizontal rod.

[0023] As a preferred embodiment of the method of the present invention for site-specific modification of inflammatory markers with PEG and increasing protein stability, the balancing work is implemented as follows:

[0024] S1: Place the ultrafiltration tube containing the sample in the lumen;

[0025] S2: The adjusting knob drives the threaded rod to rotate, so that the rectangular threaded sleeve is retracted into the spherical support. At the same time, the top block presses against the bottom side of the conical groove to drive the slip ring to rise. The horizontal rod drives the first switch seat and the second switch seat close to the opening, and waits for the warning light to light up.

[0026] S3: If the sample proportions in the rotor disk are out of balance, causing the rotor disk to lose balance, the spherical support will deflect in the rotating seat, causing the weight ball to roll at the tilted position. When the weight ball rolls outward and presses over the membrane touch switch on the first switch seat, the warning light will light up. The staff will judge the balancing status of the sample by observing the warning light. If the balance requirement is not met, the sample needs to be replaced.

[0027] As a preferred embodiment of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, the ratio of liquids used in step 2 and step 3 are respectively a proportional mixture of liquid A and liquid B, wherein liquid A comprises: 20 mmol / L Tris-HCl, 1 mmol / L EDTA· Na2, 0.01% NaN3, and liquid B comprises: 20 mmol / L Tris-HCl, 1 mmol / L EDTA· Na2, 0.01% NaN3, and 1000 mmol / L NaCl.

[0028] As a preferred embodiment of the method for site-specific modification of inflammatory markers with PEG and increasing protein stability described in the present invention, the ratio of liquid A to liquid B in the mixing liquid in step 2 is 4:1, and the ratio of liquid A to liquid B in the mixing liquid in step 3 is 2:1.

[0029] Beneficial effects of the present invention:

[0030] The present invention uses mPEG2000Mal for modification, which has the following advantages: mPEG2000Mal can specifically bind to the thiol group of the protein without reacting with other groups, and the thiol group is the group with the smallest relative content on the protein, which can increase the stability of the protein while reducing the immunogenicity relatively less; at the same time, PEG with a molecular weight of 2000 is selected because its molecular weight is relatively small and can partially penetrate into the interior of the protein for modification, which can also increase the stability of the protein while reducing the immunogenicity relatively less.

[0031] The rolling of the counterweight ball in the slideway triggers the first switch seat and different switches on the first switch seat, thereby controlling the lighting state of the warning light. When the warning light is on, the rotor disk is unbalanced, the sample on the ultrafiltration tube is not balanced, or the ultrafiltration tube sample in the tube cavity is not symmetrically placed, reminding the staff to correct it in time, thereby ensuring the balance of the centrifuge during high-speed rotation, ensuring sufficient centrifugation of the sample, and ensuring complete concentration of the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0033] Figure 1 A schematic diagram of a method for site-specific modification of inflammatory markers with PEG and increasing protein stability proposed by the present invention;

[0034] Figure 2 It is a structural schematic diagram of the centrifugal device in step 4;

[0035] Figure 3 is an overall schematic diagram of a centrifugal device;

[0036] Figure 4 It is a structural schematic diagram of a centrifugal device rotor unit and a trigger unit;

[0037] Figure 5 for Figure 3 Schematic diagram of the structure at an upward viewing angle;

[0038] Figure 6 for Figure 4 Front structural cross-sectional view;

[0039] Figure 7 for Figure 5 A schematic diagram of the enlarged structure of

[0040] Figure 8 Schematic diagram of the rolling of the weighted ball in the balancing mechanism under different conditions.

[0041] In the figure: 100-centrifuge unit, 101-centrifuge body, 101a-cavity, 102-control panel, 103-warning light, 104-machine cover, 200-rotor unit, 201-rotor disk, 201a-tube cavity, 202-knob, 203-hollow column, 203a-opening groove, 204-rotating seat, 205-centrifuge output shaft, 206-top block, 207-conical disk, 208-spherical support, 209-rectangular threaded sleeve, 210-threaded rod, 211-balancing mechanism, 211a-balancing seat, 211b-counterweight ball, 211c-slide, 211d-opening, 300-trigger unit, 301-support seat, 302-horizontal rod, 303-spring, 304-slip ring, 304a-conical groove, 305-first switch seat, 306-second switch seat. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0043] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0044] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0045] Secondly, the present invention is described in detail with reference to the schematic diagram. When describing the embodiments of the present invention in detail, for the sake of convenience, the cross-sectional diagrams showing the device structure will not be partially enlarged according to the general scale, and the schematic diagrams are only examples, which should not limit the scope of protection of the present invention. In addition, in actual production, the three-dimensional dimensions of length, width and depth should be included.

[0046] Reference Figure 1-Figure 8 , as one embodiment of the present invention, provides a method for site-specific modification of inflammatory markers with PEG and increasing protein stability, the method comprising:

[0047] Step 1: Mix one of the purified proteins of procalcitonin, C-reactive protein, amyloid A or interleukin-6 with mPEG2000Mal in 5 mmol / L phosphate buffer with a pH of 7.2-7.4 until the reaction is complete and can be detected by HPLC.

[0048] Step 2: Purify the conjugate using a chromatographic column, and then wash it with a matching solution until it is flushed flat;

[0049] Step 3: Then use the matching solution to elute and collect the sample when the absorbance of the product increases;

[0050] It should be explained here that the proportioning liquids used in step 2 and step 3 are respectively a mixed solution of liquid A and liquid B in proportion, wherein liquid A includes: 20 mmol / L Tris-HCl, 1 mmol / L EDTA· Na2, 0.01% NaN3, and liquid B includes: 20 mmol / L Tris-HCl, 1 mmol / L EDTA· Na2, 0.01% NaN3, 1000 mmol / L NaCl, and the ratio of liquid A to liquid B in the proportioning liquid in step 2 is 4:1, and the ratio of liquid A to liquid B in the proportioning liquid in step 3 is 2:1.

[0051] Step 4: Place the collected samples in an ultrafiltration centrifuge tube, and then place it in a centrifuge device for balancing before centrifugation. If balancing is not performed, the rotor of the centrifuge will be deformed due to unbalanced force over time, thus affecting the centrifugation effect of the sample.

[0052] Step 5: After balancing, the sample is centrifuged in the centrifuge body 101, and the temperature is maintained at 2-8°C and 3,000-4,000 r / min to be concentrated to 250-500 μL. The concentrated sample is desalted to obtain the PEG-site-modified inflammatory marker.

[0053] The centrifugal device of the above steps comprises: a centrifuge unit 100, a rotor unit 200 and a trigger unit 300;

[0054] The centrifuge unit 100 includes: a centrifuge body 101, a control panel 102 arranged on the front side of the centrifuge body 101, a warning light 103 arranged on the control panel 102, and a cover 104 rotatably arranged on the centrifuge body 101, a cavity 101a is opened in the centrifuge body 101, and the rotor unit 200 includes: a rotor disk 201 arranged in the cavity 101a, a plurality of cavities 201a opened on the rotor disk 201, and a hollow column 203 arranged on the bottom side of the rotor disk 201, a spherical support 208 is fixedly connected to the bottom side of the hollow column 203, a rotating seat 204 is rotatably matched on the spherical support 208, and the bottom side of the rotating seat 204 is driven to rotate by the centrifuge output shaft 205, and a plurality of balancing mechanisms 211 are arranged on the bottom side of the rotor disk 201.

[0055] The multiple cavities 201a here are distributed in a circular uniform array, and the multiple balancing mechanisms 211 are distributed one-to-one with the cavities 201a, that is, each balancing mechanism 211 corresponds to a cavity 201a. When the samples in the multiple cavities 201a are not completely balanced or are placed asymmetrically, the balancing mechanisms 211 at the corresponding positions will be tilted. The balancing mechanism 211 includes: a balancing seat 211a fixedly arranged on the bottom side of the rotor disk 201, a slideway 211c opened in the balancing seat 211a and concave toward the inner side of the rotor disk 201, and a counterweight ball 211b placed on the slideway 211c. Two openings 211d are opened on the bottom side of the slideway 211c. The size of the opening 211d here is only enough for the switch seat to pass through, and the counterweight ball 211b cannot pass through. When the balancing mechanism 211 is skewed, it will trigger the counterweight ball 211b to roll.

[0056] When the rotor disk 201 maintains a balanced state, the counterweight ball 211b is located on one side close to the center of the rotor disk 201. When the rotor disk 201 is in an unbalanced state, the balance seat 211a is tilted, and the counterweight ball 211b rolls to the side away from the center of the rotor disk 201. When the rotor disk 201 rotates at a high speed, the counterweight ball 211b will press against the side wall of the slideway 211c.

[0057] In addition, a rotating shaft is rotatably connected at the center of the rotor disk 201, a knob 202 is fixedly connected to the upper end of the rotating shaft, a threaded rod 210 is fixedly connected to the bottom end of the rotating shaft, and a rectangular threaded sleeve 209 is threadedly connected to the threaded rod 210, the rectangular threaded sleeve 209 is slidably inserted into the rotor disk 201, and the bottom end of the rectangular threaded sleeve 209 passes through the spherical support 208, and a positioning hole matching the bottom end of the spherical support 208 is opened on the inner side of the rotating seat 204. When the centrifuge is working at high speed, the rectangular threaded sleeve 209 needs to be inserted into the positioning hole of the rotating seat 204 to ensure the torque transmission of the centrifuge output shaft 205, and when performing balancing work, the rotating seat 204 is separated from the positioning hole to ensure that the rotor disk 201 is in a rotatable state.

[0058] The trigger unit 300 includes: a plurality of support seats 301 fixedly arranged on the inner side of the cavity 101a, and a horizontal rod 302 arranged on the support seat 301. Specifically, one end of the horizontal rod 302 is slidably connected in the support seat 301, and a spring 303 is fixedly connected to the inner side of the support seat 301. The upper end of the spring 303 is fixedly connected to the horizontal rod 302. The horizontal rod 302 can slide up and down, and the spring 303 is used to control the lifting and resetting of the horizontal rod 302. A first switch seat 305 and a second switch seat 306 are respectively arranged on the horizontal rod 302. The first switch seat 305 and the second switch seat 306 are respectively provided with a film touch switch for controlling the turning on and off of the warning light 103. One end of each horizontal rod 302 is fixedly connected to a slip ring 304, a conical groove 304a is provided on the inner side of the slip ring 304, an open groove 203a is symmetrically provided on the outer side of the hollow column 203, a top block 206 is fixedly connected to the rectangular threaded sleeve 209, and the top block 206 passes through the open groove 203a, and a conical disk 207 is also fixedly connected to the outer side of the hollow column 203, the conical groove 304a cooperates with the conical disk 207, so that the up and down movement of the top block 206 can support the slip ring 304, drive the horizontal rod 302 to slide up and down, and the horizontal rod 302 can be tightly attached to the conical disk 207 under the elastic force of the spring 303, thereby playing an effect of assisting in stabilizing the rotation of the hollow column 203.

[0059] The above step 4, the implementation method of balancing work is:

[0060] S1: placing the ultrafiltration tube containing the sample in the tube cavity 201a;

[0061] S2: The adjusting knob 202 drives the threaded rod 210 to rotate, so that the rectangular threaded sleeve 209 is retracted into the spherical support 208. At the same time, the top block 206 abuts against the bottom side of the conical groove 304a to drive the slip ring 304 to rise. The horizontal rod 302 drives the first switch seat 305 and the second switch seat 306 to approach the opening 211d, and waits for the warning light 103 to light up.

[0062] S3: If the sample ratio in the rotor disk 201 is out of balance, causing the rotor disk 201 to lose balance, the spherical support 208 deflects in the rotating seat 204, causing the weight ball 211b to roll at the tilted position. When the weight ball 211b rolls outward and presses over the membrane touch switch on the first switch seat 305, the warning light 103 lights up. The staff judges the balancing state of the sample by observing the warning light 103. If the balance requirement is not met, the sample needs to be replaced.

[0063] After the balancing is completed, the rotor disk 201 recovers its balance, and the weight ball 211b presses over the membrane touch switch on the second switch seat 306, and the warning light 103 is turned off. At this time, the rectangular threaded sleeve 209 must be reset to the positioning hole of the rotating seat 204 in time before the centrifugal operation can continue.

[0064] The use of mPEG2000Mal for modification in the present invention has the following advantages: mPEG2000Mal can specifically bind to the thiol group of the protein without reacting with other groups, and the thiol group is the group with the least relative content on the protein, which can increase the stability of the protein while reducing the immunogenicity relatively less; at the same time, PEG with a molecular weight of 2000 is selected because its molecular weight is relatively small and can partially penetrate into the interior of the protein for modification, which can also increase the stability of the protein while reducing the immunogenicity relatively less. The rolling of the weighted ball 211b in the slide 211c triggers different switches on the first switch seat 305 and the second switch seat 306, thereby controlling the lighting state of the warning light 103. When the warning light 103 is lit, the rotor disk 201 is unbalanced, the sample on the ultrafiltration tube is not balanced or the ultrafiltration tube sample in the tube cavity 201a is not symmetrically placed, reminding the staff to correct it in time, thereby ensuring the balance of the centrifuge during high-speed rotation, ensuring sufficient centrifugation of the sample, and ensuring complete concentration of the sample.

[0065] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for site-specific modification of inflammatory markers with PEG and increasing protein stability, characterized in that: The following steps are involved: Step 1: Mix one of the purified proteins of procalcitonin, C-reactive protein, amyloid A or interleukin-6 with mPEG2000Mal in a 5 mmol / L phosphate buffer with a pH of 7.2-7.4 until the reaction is complete; Step 2: Purify the conjugate using a chromatographic column, and then wash it with a matching solution until it is flushed flat; Step 3: Then use the matching solution to elute and collect the sample when the absorbance of the product increases; Step 4: Place the collected sample in an ultrafiltration centrifuge tube, and then place it in a centrifuge device for balancing before centrifugation; The centrifugal device comprises: a centrifuge unit (100), a rotor unit (200) and a trigger unit (300); The centrifuge unit (100) comprises: a centrifuge body (101), a control panel (102) arranged on the front side of the centrifuge body (101), a warning light (103) arranged on the control panel (102), and a cover (104) rotatably arranged on the centrifuge body (101); a cavity (101a) is provided in the centrifuge body (101); and the rotor unit (200) comprises: a rotor arranged in the cavity (101a); A sub-disk (201), a plurality of cavities (201a) provided on the rotor disk (201), and a hollow column (203) provided on the bottom side of the rotor disk (201); a spherical support (208) is fixedly connected to the bottom side of the hollow column (203); a rotating seat (204) is rotatably matched on the spherical support (208); and the bottom side of the rotating seat (204) is driven to rotate by the centrifuge output shaft (205); and a plurality of balancing mechanisms (211) are provided on the bottom side of the rotor disk (201); The trigger unit (300) comprises: a plurality of support seats (301) fixedly arranged inside the cavity (101a), a horizontal rod (302) arranged on the support seat (301), a first switch seat (305) and a second switch seat (306) respectively arranged on the horizontal rod (302), and a membrane touch switch for controlling the turning on and off of the warning light (103) respectively arranged on the first switch seat (305) and the second switch seat (306); Step 5: After balancing, the sample is centrifuged in the centrifuge body (101), and the centrifugal temperature is maintained at 2-8°C and 3,000-4,000 r / min to be concentrated to 250-500 μL. The concentrated sample is desalted to obtain the PEG-site-modified inflammatory marker; The balancing mechanism (211) comprises: a balancing seat (211a) fixedly arranged on the bottom side of the rotor disk (201), a slideway (211c) provided in the balancing seat (211a) and recessed toward the inside of the rotor disk (201), and a small counterweight ball (211b) arranged on the slideway (211c), wherein the bottom side of the slideway (211c) has two openings (211d); when the rotor disk (201) is in a balanced state, the small counterweight ball (211b) is located at a side position close to the center of the rotor disk (201); when the rotor disk (201) is in an unbalanced state, the balancing seat (211a) is tilted, and the small counterweight ball (211b) rolls to a side position away from the center of the rotor disk (201); A rotating shaft is rotatably connected at the center of the rotor disk (201); a knob (202) is fixedly connected to the upper end of the rotating shaft; a threaded rod (210) is fixedly connected to the bottom end of the rotating shaft; and a rectangular threaded sleeve (209) is threadedly connected to the threaded rod (210); The rectangular threaded sleeve (209) is slidably inserted into the rotor disk (201), and the bottom end of the rectangular threaded sleeve (209) passes through the spherical support (208), and a positioning hole matching the bottom end of the spherical support (208) is opened on the inner side of the rotating seat (204); A plurality of horizontal rods (302) are commonly fixedly connected at one end with a slip ring (304), a tapered groove (304a) is provided on the inner side of the slip ring (304), an open groove (203a) is symmetrically provided on the outer side of the hollow column (203), a top block (206) is fixedly connected to the rectangular threaded sleeve (209), and the top block (206) passes through the open groove (203a), a tapered disk (207) is also fixedly connected to the outer side of the hollow column (203), and the tapered groove (304a) matches the tapered disk (207); One end of the horizontal rod (302) is slidably connected in the support seat (301), and a spring (303) is fixedly connected to the inner side of the support seat (301), and the upper end of the spring (303) is fixedly connected to the horizontal rod (302).

2. The method for site-specific modification of inflammatory markers with PEG and increasing protein stability according to claim 1, characterized in that: The plurality of lumens (201a) are distributed in a circular uniform array, and the plurality of balancing mechanisms (211) are distributed in a one-to-one correspondence with the lumens (201a).

3. The method for site-specific modification of inflammatory markers with PEG and increasing protein stability according to claim 1, characterized in that: The balancing work implementation method is: S1: placing the ultrafiltration tube containing the sample in the tube cavity (201a); S2: The adjusting knob (202) drives the threaded rod (210) to rotate, so that the rectangular threaded sleeve (209) is retracted into the spherical support (208), and at the same time, the top block (206) abuts against the bottom side of the conical groove (304a) to drive the slip ring (304) to rise, and the horizontal rod (302) drives the first switch seat (305) and the second switch seat (306) to approach the opening (211d), and waits for the warning light (103) to light up; S3: If the sample ratio is out of balance in the rotor disk (201), causing the rotor disk (201) to lose balance, the spherical support (208) deflects in the rotating seat (204), causing the weight ball (211b) to roll at the tilted position. When the weight ball (211b) rolls outward and presses over the membrane touch switch on the first switch seat (305), the warning light (103) is lit. The staff can judge the balance state of the sample by observing the warning light (103). If the balance requirement is not met, the sample needs to be replaced.

4. The method for site-specific modification of inflammatory markers with PEG and increasing protein stability according to claim 1, characterized in that: The proportioned liquids used in step 2 and step 3 are respectively a proportional mixture of liquid A and liquid B, wherein liquid A includes: 20 mmol / L Tris-HCl, 1 mmol / L EDTA· Na2, 0.01% NaN3, and liquid B includes: 20mmol / L Tris-HCl, 1 mmol / L EDTA· Na2, 0.01% NaN3, 1000 mmol / L NaCl.

5. The method for site-specific modification of inflammatory markers with PEG and increasing protein stability according to claim 4, characterized in that: The ratio of liquid A to liquid B in the proportioning liquid in step 2 is 4:1, and the ratio of liquid A to liquid B in the proportioning liquid in step 3 is 2:1.

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