Residue detection method of E.coliHCD residue detection kit

Abstract

The invention discloses an E.coliHCD residue detection kit residue detection method, which comprises the following steps: selecting target Escherichia coli host bacteria, carrying out culture expression to obtain Escherichia coli host cell protein, dissolving the Escherichia coli host cell protein by using a solvent pair solid, and carrying out freeze drying to obtain the E.coliHCD residue detection kit. Secondly, diluting a to-be-detected Escherichia coli host cell protein solution by using a diluent, wherein the concentrations of the diluted Escherichia coli host cell protein solution are 1.1 ng / mu L, 0.11 ng / mu L, 0.011 ng / mu L, 0.0011 ng / mu L and 0.00011 ng / mu L respectively; according to the method, a sample to be tested is obtained, the sample to be tested is converted into positively charged gaseous ions through an electrospray or MALDI technology, the gaseous ions enter a mass spectrometer to be dissociated, then the mass spectrum ratio is obtained, a mass-to-mass-charge ratio spectrogram is drawn, and by analyzing the mass-to-charge ratio spectrogram, the types and the number of the proteins can be obtained; the sample pretreatment method adopted by the invention is simple in process, low in cost and low in sample demand.

Description

Technical Field

[0001] The invention relates to the technical field related to analysis and detection, and in particular to a residue detection method of an E.coli HCD residue detection kit. Background Art

[0002] Escherichia coli (E.col) was the earliest host bacteria used for recombinant protein expression. The E. coli expression system has many advantages over other expression systems, and its value in the industrial application of biopharmaceuticals is as follows: 1) Simple structure, physiological, biochemical and genetic background knowledge, especially a clear understanding of the gene expression regulation mechanism; 2) Easy to culture on a large scale, short culture cycle and low cost; 3) Strong anti-pollution ability; 4) After a series of genetic modifications, it has developed into a safe genetic engineering experimental system with a variety of strains and vector systems; So far, E. coli is still considered the main tool in the field of protein expression.

[0003] Recombinant expression in E. coli is a relatively simple and cost-effective method for producing certain proteins and DNA. Many of these products are therapeutic agents for humans and animals and, therefore, must be highly purified. During the manufacture and purification of these products, there is the possibility of impurities from E. coli host cell proteins (HCP). These impurities can reduce the efficacy of therapeutic drugs and cause adverse toxicity or immune responses, so it is necessary to detect protein residues in E. coli. The pretreatment process of traditional HPLC is complicated and requires cumbersome derivatization labeling, which is time-consuming and labor-intensive. The derivatization reagents are highly toxic, the culture matrix interferes severely, and the single sample analysis test time is long, resulting in a small amount of sample analysis per unit time, low efficiency, and low detection throughput and automation. Summary of the invention

[0004] In order to solve the problem that the existing technology has E. coli protein residues, the main method is high performance liquid chromatography. The traditional high performance liquid chromatography has a complex pre-treatment process, requires cumbersome derivatization labeling, is time-consuming and labor-intensive, and the derivatization reagent is highly toxic, the culture matrix interferes seriously, and the single sample analysis test time is long, resulting in a small amount of sample detection and analysis per unit time, low efficiency, and low detection throughput and automation. The present invention provides an E. coli HCD residue detection kit residue detection method.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention discloses a method for detecting residues of an E. coli HCD residue detection kit, wherein the detection kit comprises a solvent and a diluent;

[0007] The detection method of the detection kit is as follows: select the target Escherichia coli host bacteria, culture and express them, obtain the Escherichia coli host cell protein, use a solvent to dissolve the solid Escherichia coli host cell protein, and then first dilute the Escherichia coli host cell protein solution to be detected with a diluent, and the concentrations after dilution are 1.1ng / μL, 0.11ng / μL, 0.011ng / μL, 0.0011ng / μL, and 0.00011ng / μL respectively; obtain a test sample, convert the test sample into positively charged gaseous ions through electrospray or MALDI technology, enter a mass spectrometer, dissociate, and then obtain a mass spectrum ratio, then draw a mass-to-charge ratio spectrum, and by analyzing the mass-to-charge ratio spectrum, the type and quantity of the protein can be obtained.

[0008] As a preferred technical solution of the present invention, the Escherichia coli host cells need to be cultured before detection, wherein the culture method is to immunize animals with Escherichia coli host cell proteins to obtain anti-Escherichia coli host cell protein polyclonal antibodies; and then use an accurate coverage detection method to detect the purified anti-Escherichia coli host cell protein polyclonal antibodies.

[0009] As a preferred technical solution of the present invention, the method for obtaining Escherichia coli host cell protein is as follows: obtaining a broken solution of Escherichia coli host bacteria, placing it in a centrifuge tube, adding 5 mL of deionized water, placing it in a boiling water bath for heating denaturation for 30 minutes, cooling it to room temperature, adding 0.10 mol / L ammonium bicarbonate solution to the denatured solution to make the volume 10 mL, and then shaking it to obtain a sample working solution; filtering the sample working solution through a micron membrane to remove particulate matter in the sample working solution; placing the sample working solution in a centrifuge and centrifuging it at 10,000 g for 15 minutes; taking out the centrifuged sample working solution, and adding nuclease to remove nucleic acids in the sample working solution; adding a protein inhibitor to the sample working solution to inhibit protein hydrolysis in the sample working solution to obtain a mother liquor; and then purifying and enriching the mother liquor to obtain a test sample.

[0010] As a preferred technical solution of the present invention, the method for purifying and enriching the mother liquor to obtain the test sample comprises the following steps:

[0011] Step 1, dissolving or diluting the inorganic salt aqueous solution mother solution, and mixing to obtain a mixed solution;

[0012] Step 2, mixing the low eutectic solvent and the mixed solution in step 1 uniformly, and then centrifuging to obtain a liquid-liquid two-phase system, and collecting the lower phase extract;

[0013] Step 3, diluting the lower phase extract in step 4 with water, mixing evenly and centrifuging to obtain a liquid-solid two-phase system;

[0014] Step 4, dissolving the solid phase in step 3 with water, and performing ultrafiltration centrifugation to remove the residual low eutectic solvent components and inorganic salts to obtain a protein solution;

[0015] Step 5: freeze-dry the protein solution in step 4 to obtain a pure protein product.

[0016] As a preferred technical solution of the present invention, in the step 2, the low eutectic solvent and the mixed solution are mixed by oscillation or vortexing, wherein the oscillation rate is 400-10000 rpm, the oscillation time is 10-30 min, and the oscillation temperature is 30-40°C.

[0017] As a preferred technical solution of the present invention, the solvent is ethanol; the diluent is a mixture of acetonitrile: water: formic acid: 1.1 mol / L ammonium acetate solution, and the volume ratio thereof is 45-50:45-49.5:0.5-10.

[0018] The beneficial effects of the present invention are:

[0019] The residue detection method of the E. coli HCD residue detection kit selects a target E. coli host bacteria, performs culture expression, obtains the E. coli host cell protein, uses a solvent to dissolve the solid E. coli host cell protein, and then first dilutes the E. coli host cell protein solution to be detected with a diluent to obtain a test sample, converts the test sample into positively charged gaseous ions through electrospray or MALDI technology, enters a mass spectrometer, dissociates, and then obtains a mass spectrum ratio, draws a mass-to-charge ratio spectrum, and analyzes the mass-to-charge ratio spectrum to obtain the type and quantity of proteins, which can effectively reduce the sample demand, reduce the difficulty of sample pretreatment, increase the online separation degree of proteins, accurately identify the number and type of host cells, and the sample pretreatment method is simple in process, low in cost, and small in sample demand. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0021] In the attached picture:

[0022] Figure 1 The present invention is a schematic flow diagram of a residual detection method of an E. coli HCD residual detection kit. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0024] Example: Figure 1 As shown, the present invention provides an E. coli HCD residue detection kit residue detection method, wherein the detection kit comprises a solvent and a diluent;

[0025] The detection method of the detection kit is as follows: select the target Escherichia coli host bacteria, culture and express them, obtain the Escherichia coli host cell protein, use a solvent to dissolve the solid Escherichia coli host cell protein, and then first dilute the Escherichia coli host cell protein solution to be detected with a diluent, and the concentrations after dilution are 1.1ng / μL, 0.11ng / μL, 0.011ng / μL, 0.0011ng / μL, and 0.00011ng / μL respectively; obtain a test sample, convert the test sample into positively charged gaseous ions by electrospray or MALDI technology, enter a mass spectrometer, dissociate, and then obtain a mass spectrum ratio, then draw a mass-to-charge ratio spectrum, and analyze the mass-to-charge ratio spectrum to obtain the type and quantity of the protein;

[0026] Before detection, the E. coli host cells need to be cultured, wherein the culture method is to immunize animals with E. coli host cell proteins to obtain anti-E. coli host cell protein polyclonal antibodies; then use an accurate coverage detection method to detect the purified anti-E. coli host cell protein polyclonal antibodies:

[0027] The method for obtaining Escherichia coli host cell protein is as follows: obtaining a broken solution of Escherichia coli host bacteria, placing it in a centrifuge tube, adding 5 mL of deionized water, placing it in a boiling water bath for heating and denaturation for 30 minutes, cooling it to room temperature, adding 0.10 mol / L ammonium bicarbonate solution to the denatured solution to make the volume 10 mL, and then shaking it to obtain a sample working solution; filtering the sample working solution through a micron membrane to remove particulate matter in the sample working solution; placing the sample working solution in a centrifuge and centrifuging it at 10000g for 15 minutes; taking out the centrifuged sample working solution, and adding nuclease to remove nucleic acids in the sample working solution; adding a protein inhibitor to the sample working solution to inhibit protein hydrolysis in the sample working solution to obtain a mother solution; and then purifying and enriching the mother solution to obtain a test sample;

[0028] The method for purifying and enriching the mother liquor to obtain the test sample comprises the following steps:

[0029] Step 1, dissolving or diluting the inorganic salt aqueous solution mother solution, and mixing to obtain a mixed solution;

[0030] Step 2, mixing the low eutectic solvent and the mixed solution in step 1 uniformly, and then centrifuging to obtain a liquid-liquid two-phase system, and collecting the lower phase extract;

[0031] Step 3, diluting the lower phase extract in step 4 with water, mixing evenly and centrifuging to obtain a liquid-solid two-phase system;

[0032] Step 4, dissolving the solid phase in step 3 with water, and performing ultrafiltration centrifugation to remove the residual low eutectic solvent components and inorganic salts to obtain a protein solution;

[0033] Step 5: freeze-dry the protein solution in step 4 to obtain a pure protein product.

[0034] In the step 2, the low eutectic solvent and the mixed solution are mixed by oscillation or vortexing, wherein the oscillation rate is 400-10000 rpm, the oscillation time is 10-30 min, and the oscillation temperature is 30-40° C.;

[0035] The solvent is ethanol; the diluent is a mixture of acetonitrile: water: formic acid: 1.1 mol / L ammonium acetate solution, and the volume ratio thereof is 45-50:45-49.5:0.5-10.

[0036] The residue detection method of the E. coli HCD residue detection kit selects a target E. coli host bacteria, performs culture expression, obtains the E. coli host cell protein, uses a solvent to dissolve the solid E. coli host cell protein, and then first dilutes the E. coli host cell protein solution to be detected with a diluent, and the concentrations after dilution are 1.1 ng / μL, 0.11 ng / μL, 0.011 ng / μL, 0.0011 ng / μL, and 0.00011 ng / μL, respectively; obtains a test sample, converts the test sample into positively charged gaseous ions through electrospray or MALDI technology, enters a mass spectrometer, dissociates, and then obtains a mass spectrum ratio, draws a mass-to-charge ratio spectrum, and analyzes the mass-to-charge ratio spectrum to obtain the type and quantity of protein, and the sample pretreatment method is simple in process, low in cost, and small in sample demand.

[0037] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting residues of an E. coli HCD residue detection kit, characterized in that: The detection kit comprises a solvent and a diluent; The detection method of the detection kit is as follows: select the target Escherichia coli host bacteria, culture and express them, obtain the Escherichia coli host cell protein, use a solvent to dissolve the solid Escherichia coli host cell protein, and then first dilute the Escherichia coli host cell protein solution to be detected with a diluent, and the concentrations after dilution are 1.1ng / μL, 0.11ng / μL, 0.011ng / μL, 0.0011ng / μL, and 0.00011ng / μL respectively; obtain a test sample, convert the test sample into positively charged gaseous ions through electrospray or MALDI technology, enter a mass spectrometer, dissociate, and then obtain a mass spectrum ratio, then draw a mass-to-charge ratio spectrum, and by analyzing the mass-to-charge ratio spectrum, the type and quantity of the protein can be obtained.

2. The method for detecting the residue of an E.coli HCD residue detection kit according to claim 1, characterized in that: Before detection, the E. coli host cells need to be cultured, wherein the culture method is to immunize animals with E. coli host cell proteins to obtain anti-E. coli host cell protein polyclonal antibodies; and then use an accurate coverage detection method to detect the purified anti-E. coli host cell protein polyclonal antibodies.

3. The method for detecting the residue of an E.coli HCD residue detection kit according to claim 2, characterized in that: The method for obtaining Escherichia coli host cell protein is as follows: obtaining a broken solution of the Escherichia coli host bacteria, placing it in a centrifuge tube, adding 5 mL of deionized water, heating it in a boiling water bath for denaturation for 30 minutes, cooling it to room temperature, adding 0.10 mol / L ammonium bicarbonate solution to the denatured solution to make it 10 mL, and then shaking it to obtain a sample working solution; filtering the sample working solution through a micron membrane to remove particulate matter in the sample working solution; The sample working solution is placed in a centrifuge and centrifuged at 10,000 g for 15 minutes; the sample working solution after centrifugation is taken out, and nuclease is added to remove nucleic acids in the sample working solution; a protein inhibitor is added to the sample working solution to inhibit protein hydrolysis in the sample working solution to obtain a mother solution; and then the mother solution is purified and enriched to obtain a test sample.

4. The method for detecting the residue of an E.coli HCD residue detection kit according to claim 3, characterized in that: The method for purifying and enriching the mother liquor to obtain the test sample comprises the following steps: Step 1, dissolving or diluting the inorganic salt aqueous solution mother solution, and mixing to obtain a mixed solution; Step 2, mixing the low eutectic solvent and the mixed solution in step 1 uniformly, and then centrifuging to obtain a liquid-liquid two-phase system, and collecting the lower phase extract; Step 3, diluting the lower phase extract in step 4 with water, mixing evenly and centrifuging to obtain a liquid-solid two-phase system; Step 4, dissolving the solid phase in step 3 with water, and performing ultrafiltration centrifugation to remove the residual low eutectic solvent components and inorganic salts to obtain a protein solution; Step 5: freeze-dry the protein solution in step 4 to obtain a pure protein product.

5. The method for detecting the residue of an E.coli HCD residue detection kit according to claim 4, characterized in that: In the step 2, the low eutectic solvent and the mixed solution are mixed by oscillation or vortexing, wherein the oscillation rate is 400-10000 rpm, the oscillation time is 10-30 min, and the oscillation temperature is 30-40°C.

6. The method for detecting the residue of an E.coli HCD residue detection kit according to claim 1, characterized in that: The solvent is ethanol; the diluent is a mixture of acetonitrile: water: formic acid: 1.1 mol / L ammonium acetate solution, and the volume ratio thereof is 45-50:45-49.5:0.5-10.

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