A process for purifying a recombinant antigen of hepatitis c virus

Abstract

The utility model provides a kind of HCV recombination antigen purification process device, specifically relates to antigen purification field, filler in chromatography column is nickel column filler;The upper and lower sides of interlayer are respectively equipped with water inlet and water outlet;Water inlet is sequentially connected constant-current pump, pre-cooling water bucket by water inlet rubber tube;Water outlet is connected with pre-cooling water bucket by water outlet rubber tube;Protein purification complete system includes conductivity meter, electric ultraviolet light detector, PH detector;Constant-current A pump and constant-current B pump one end are connected with sample A barrel, sample B barrel respectively by import connection pipe, and constant-current A pump and constant-current B pump other end are connected with the liquid inlet of chromatography column by import connection pipe through mixer;The liquid outlet of chromatography column is connected with sample barrel by outlet connection pipe sequentially through conductivity meter, electric ultraviolet light detector, pH detector.The utility model can reduce the tedious operation in experimental process to make test process more succinct, while guaranteeing the production activity and purity of recombination antigen.

Description

Technical Field

[0001] This utility model belongs to the field of antigen purification, specifically relating to an HCV recombinant antigen purification process device. Background Technology

[0002] Hepatitis C virus (HCV) infection typically causes hepatitis C, and the main modes of transmission are blood, sexual contact, and mother-to-child transmission. According to the World Health Organization, the global HCV infection rate is approximately 3%, and HCV-related mortality, including deaths from liver failure and hepatocellular carcinoma, is expected to continue to rise, becoming a serious social and public health problem. HCV core antigen (HCV cAg) is a nucleocapsid polypeptide of HCV released into the plasma during viral assembly and can be detected in the early stages of HCV infection and throughout the entire infection process. Therefore, HCV core antigen testing can be used as an auxiliary diagnostic tool for early acute hepatitis C before HCV seroconversion, especially in HCV-infected individuals during the window period. It can also be used to assist in the diagnosis of whether infants born to HCV-positive mothers have the disease. Furthermore, HCV antigen screening can be used for immunocompromised or congenitally immunodeficient individuals, such as HIV-infected individuals, kidney disease patients on long-term dialysis, organ transplant recipients, or patients with congenital immunodeficiency, as antibody production may be affected. In the purification process of HCV antigen, the unstable raw materials of recombinant antigen often lead to a more complex experimental process in the production process, which increases the experimental cost, makes the process cumbersome, and is prone to uncontrollable random errors. Utility Model Content

[0003] The purpose of this invention is to provide an HCV recombinant antigen purification process device that can reduce cumbersome operations in the experimental process, making the experimental process simpler, while ensuring the production activity and purity of the recombinant antigen.

[0004] This utility model provides the following technical solution:

[0005] An HCV recombinant antigen purification process device includes a chromatography column for separating and purifying the antigen and a protein purification system; the packing material inside the chromatography column is a nickel column packing material; the chromatography column is provided with a jacket, and the upper and lower sides of the jacket are respectively provided with a water inlet and a water outlet; the water inlet is connected to a constant flow pump and a pre-cooling water tank in sequence through a water inlet rubber tube; the water outlet is connected to the pre-cooling water tank through a water outlet rubber tube.

[0006] The protein purification system includes a conductivity meter for detecting the salt content of reagents and cell lysate supernatant, an electro-ultraviolet spectrophotometer for detecting protein concentration, and a pH meter for detecting the pH value of the nickel column packing environment.

[0007] The protein purification system also includes a constant flow A pump and a constant flow B pump. One end of the constant flow A pump and the constant flow B pump are connected to the sample loading tank A and the sample loading tank B respectively through the inlet connecting tube. The other end of the constant flow A pump and the constant flow B pump are connected to the liquid inlet of the chromatography column through the mixer via the inlet connecting tube.

[0008] The outlet of the chromatography column is connected to the sample container in sequence via an outlet connecting pipe through a conductivity meter, an electro-ultraviolet spectrophotometer, and a pH meter.

[0009] Preferably, the chromatography column is made of transparent glass. Rubber caps are fitted at both the upper and lower ends of the inner layer of the chromatography column, and an inlet connecting pipe and an outlet connecting pipe are inserted into each rubber cap to form an inlet and an outlet.

[0010] Preferably, the inlet and outlet connecting pipes of the chromatography column are equipped with regulating valves for controlling the inlet and outlet, respectively.

[0011] Preferably, the inlet and outlet connecting pipes of the interlayer are equipped with regulating valves for controlling the flow rate of precooling water.

[0012] Preferably, the detection ultraviolet wavelength of the electro-ultraviolet photometer is set to 280nm.

[0013] Preferably, the mixer is equipped with a column position valve and a regulating valve in the downstream direction of the liquid flow.

[0014] Preferably, the conductivity meter is equipped with a column position valve and a regulating valve in the upstream direction of the liquid flow.

[0015] The beneficial effects of this utility model are:

[0016] This apparatus consists of two parts: a protein purification system and a nickel column chromatography column. The protein purification system allows for gradient loading. Since HCV recombinant antigen purification involves different concentrations of imidazole reagent, gradient loading significantly reduces the steps involved in reagent preparation, greatly improving purification efficiency and simplifying the process. The nickel column chromatography column uses a jacketed tube design, allowing circulating pre-cooling water to be introduced into the outer jacket. This keeps the unstable recombinant antigen at a low temperature during purification, ensuring its activity and reducing the inconvenience of manual cooling. This apparatus simplifies experimental procedures, ensuring the production activity and purity of the recombinant antigen, and is of great significance. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the present invention and form part of the specification. They are used together with the embodiments of the present invention to explain the present invention, but do not constitute a limitation thereof. In the drawings:

[0018] Figure 1This is a schematic diagram of the purification device.

[0019] Figure 2 This is a schematic diagram of a partial structure of the purification device;

[0020] Figure 3 Diagram of the chromatography column structure;

[0021] Figure 4 This is a structural diagram of the precooling device;

[0022] The following are labeled in the diagram: 1. Sample loading tank; 2. Sample loading tank A; 3. Sample loading tank B; 4. Constant flow pump A; 5. Constant flow pump B; 6. Mixer; 7. Conductivity meter; 8. Electro-UV spectrophotometer; 9. pH meter; 10. Protein purification system; 11. Inlet connecting tube; 12. Nickel column packing; 13. Outlet rubber tube; 14. Outlet connecting tube; 15. Inlet rubber tube; 16. Chromatography column; 17. Constant flow pump; 18. Pre-cooling water tank. Detailed Implementation

[0023] This application aims to provide a technical device structure connection. Those skilled in the art, in conjunction with this application, will recognize that the circuitry, automation, and integrated structural connections involved are all easily implemented using existing technologies and will not be elaborated further. Figures 1 to 4 The system includes a chromatography column 16 for separating and purifying antigens and a protein purification system 10. The chromatography column 16 is filled with nickel-coated packing material 12. The entire chromatography column 16 is made of transparent glass. Rubber caps are fitted at both ends of the inner layer of the chromatography column 16, with an inlet connector 11 and an outlet connector 14 connected to each cap. The inlet connector 11 and outlet connector 14 on the chromatography column 16 are equipped with regulating valves for controlling the inlet and outlet. The chromatography column 16 has an outer jacket with an inlet and outlet on its upper and lower sides, respectively. The inlet is connected to a constant flow pump 17 and a pre-cooling water tank 18 via an inlet rubber tube 15. The outlet is connected to the pre-cooling water tank 18 via an outlet rubber tube 13. Regulating valves for controlling the pre-cooling water flow are installed on the inlet connector 15 and outlet connector 13 on the jacket.

[0024] The protein purification system 10 includes a conductivity meter 7 for detecting the salt content of reagents and cell lysate supernatant, an electro-ultraviolet spectrophotometer 8 for detecting protein concentration, and a pH meter 9 for detecting the pH value of the nickel column packing environment.

[0025] The protein purification system also includes a constant flow A pump 4 and a constant flow B pump 5. One end of the constant flow A pump 4 and the constant flow B pump 5 are connected to the sample loading A tank 2 and the sample loading B tank 3 respectively through the inlet connecting pipe. The other end of the constant flow A pump 4 and the constant flow B pump 5 are connected to the liquid inlet of the chromatography column 16 through the inlet connecting pipe 11 and the mixer 6. The downstream direction of the liquid flow of the mixer 6 is equipped with a column position valve and a regulating valve.

[0026] The liquid outlet of the chromatography column 16 passes through the outlet connection pipe 14 and then through the conductivity meter 7, wherein the conductivity meter 7 is equipped with a column position valve and a regulating valve in the upstream direction of the liquid flow; the electro-ultraviolet photometer 8, wherein the detection ultraviolet wavelength of the electro-ultraviolet photometer 8 is set to 280nm; and the pH meter 9 is connected to the sample container 1.

[0027] The structure of this device will be further explained in conjunction with practical applications. This device includes a sample loading tank A (2), a sample loading tank B (3), a sample unloading tank (1), a protein purification system 10, and a chromatography column. The packing material inside the chromatography column is a nickel column. The protein purification system 10 is connected to sample loading tanks A (2) and B (3), and the chromatography column is connected to sample unloading tank 1. The protein purification system 10 is also connected to the chromatography column. The supernatant from the lysed bacterial cells in sample loading tanks A (2) and B (3) flows sequentially through the chromatography column and the protein purification system 10 via constant flow pumps A and B, undergoing purification before entering sample unloading tank 1. The structure and function of the protein purification system 10 and the chromatography column 16 will be further described below.

[0028] The protein purification system 10 includes a constant flow A pump 4, a constant flow B pump 5, a mixer 6, a conductivity meter 7, an electro-ultraviolet spectrophotometer 8, and a pH meter 9. The inlet of the constant flow pump 4 is connected to the sample loading tank B 3, the inlet of the constant flow pump 5 is connected to the sample loading tank A 2, and the inlet is connected to the mixer 6 through a connecting tube. The outlet of the mixer 6 is connected to the chromatography column through a connecting tube 11. The chromatography column is connected to the conductivity meter 7, the conductivity meter 7 is connected to the electro-ultraviolet spectrophotometer 8 through a connecting tube, and the electro-ultraviolet spectrophotometer 8 is connected to the pH meter 9 through a connecting tube.

[0029] The mixer 6 is used to ensure the chemical composition of the sample solution is uniform, and also to ensure the reagents are mixed evenly during gradient loading. The conductivity meter 7 detects the salt content of the reagents and the supernatant of the cell disruption. It has a maximum absorption peak at a wavelength of 280 nm, and its light absorption value is proportional to the protein concentration. Therefore, the detection wavelength of the electro-UV photometer 8 is set to A280. The pH meter 9 is used to detect whether the equilibration buffer has balanced the pH value of the nickel column packing environment.

[0030] The chromatography column 16 has a double-layered, hollow, transparent glass tube with openings at the top and bottom. The inner and outer layers are not connected. This glass tube and double-layered structure facilitates observation of the purification process. Rubber caps are fitted at both ends of the inner tube, which is filled with nickel column packing 12. The lower inlet of the outer double layer is connected to an inlet rubber tube 15, which is equipped with a constant flow pump 17 and connected to a pre-cooling water tank 18. The upper outlet of the outer double layer is connected to an outlet rubber tube 13, the other end of which is connected to the pre-cooling water tank 18.

[0031] Based on the above structure, the usage process of this utility model will be described in detail below:

[0032] 1. After ultrasonic disruption of the bacterial resuspension, the mixture is slowly filtered through filter paper on ice, and the supernatant is collected. The bacterial cell processing process is not related to the device described in this utility model, and therefore will not be elaborated upon.

[0033] 2. Pack an appropriate volume of Ni column packing material into the inner column tube, ensuring a smooth and tight column bed is formed after the packing material settles. Confirm that the inner column tube is sealed. Connect the outer column tube interlayer to the inlet rubber tubes 15. Install a constant flow pump 17 on the inlet rubber tubes 15 and connect them to a pre-cooling water tank 18. Inlet the constant flow pump 17 into the outer column tube interlayer, and connect the upper outlet rubber tube 13 to the pre-cooling water tank 18. Connect the inner column tube to the protein purification system 10 and set the parameters. Set the loading speed according to the actual column packing volume. For reference, for a 16ml Ni column chromatography column, the loading speed is 1ml / min. Set the column position valve from bypass to positive flush. Set the UV spectrophotometer 8 to on with the detection parameter A280. Set the pH meter 9 to on. Use the conductivity meter 7 for real-time monitoring. The mixer 6 is closed during linear loading and open during gradient loading. Pre-set the reagent ratios for gradient loading in the system. Wash the column with 5-10 column volumes of purified water to remove ethanol, and equilibrate the Ni column with equilibration buffer. Load the solution obtained in step 1) onto the Ni column, equilibrate with different equilibration buffers after loading, and then dissociate using a dissociation buffer.

[0034] 3. After dissociation, the sample is collected in sample container 1 and placed in dialysis buffer for dialysis treatment at a ratio of 1:50. The buffer is changed 3 times, with an interval of no less than 4 hours between dialysis steps. The subsequent steps are not related to the apparatus of this invention and will not be described further.

[0035] The above are merely preferred embodiments of the present utility model and are not intended to limit the present utility model. Although the present utility model has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present utility model should be included within the protection scope of the present utility model.

Claims

1. An apparatus for purifying HCV recombinant antigen, characterized in that: This includes chromatography columns for separating and purifying antigens and protein purification systems; The chromatography column is filled with nickel column packing; the chromatography column is equipped with an outer jacket, with an inlet and an outlet on the upper and lower sides of the jacket respectively; the inlet is connected to a constant flow pump and a precooling water tank in sequence through an inlet rubber tube; the outlet is connected to the precooling water tank through an outlet rubber tube. The protein purification system includes a conductivity meter for detecting the salt content of reagents and cell lysate supernatant, an electro-ultraviolet spectrophotometer for detecting protein concentration, and a pH meter for detecting the pH value of the nickel column packing environment. The protein purification system also includes a constant flow A pump and a constant flow B pump. One end of the constant flow A pump and the constant flow B pump are connected to the sample loading tank A and the sample loading tank B respectively through the inlet connecting tube. The other end of the constant flow A pump and the constant flow B pump are connected to the liquid inlet of the chromatography column through the mixer via the inlet connecting tube. The outlet of the chromatography column is connected to the sample container in sequence via an outlet connecting pipe through a conductivity meter, an electro-ultraviolet spectrophotometer, and a pH meter.

2. The HCV recombinant antigen purification process apparatus according to claim 1, characterized in that: The chromatography column is made of transparent glass. Rubber caps are fitted at both the top and bottom of the inner layer of the column. An inlet and an outlet connecting pipe are inserted into each rubber cap to form an inlet and an outlet.

3. The HCV recombinant antigen purification process apparatus according to claim 1, characterized in that: The inlet and outlet connecting pipes of the chromatography column are equipped with regulating valves for controlling the inlet and outlet of the liquid.

4. The HCV recombinant antigen purification process apparatus according to claim 1, characterized in that: The inlet and outlet connecting pipes of the interlayer are equipped with regulating valves for controlling the flow rate of precooling water.

5. The HCV recombinant antigen purification process apparatus according to claim 1, characterized in that: The detection ultraviolet wavelength of the electro-ultraviolet photometer is set to 280nm.

6. The HCV recombinant antigen purification process apparatus according to claim 1, characterized in that: The mixer is equipped with a column position valve and a regulating valve in the downstream direction of the liquid flow.

7. The HCV recombinant antigen purification process apparatus according to claim 1, characterized in that: The conductivity meter is equipped with a column position valve and a regulating valve in the upstream direction of the liquid flow.

Images