CN115282934A - Nano antibody fluorescent affinity column and method for purifying HbeAg antigen by using same - Google Patents
Nano antibody fluorescent affinity column and method for purifying HbeAg antigen by using same Download PDFInfo
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- CN115282934A CN115282934A CN202210922358.4A CN202210922358A CN115282934A CN 115282934 A CN115282934 A CN 115282934A CN 202210922358 A CN202210922358 A CN 202210922358A CN 115282934 A CN115282934 A CN 115282934A
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a nano antibody fluorescent affinity column and a method for purifying HbeAg antigen by using the same. The nanometer antibody fluorescence affinity column uses agarose to construct a base frame, and the agarose base frame is coupled with a nanometer antibody containing anti-His protein, a fluorescent group and/or a fluorescent dye. According to the invention, the gel column is combined with the specific nano antibody containing the anti-His protein, so that the target HbeAg antigen can be effectively and efficiently purified, the impurity interference is reduced, the purification effect is excellent, and the detection precision after purification is high.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a nano antibody fluorescent affinity column and a method for purifying HbeAg antigen by using the same.
Background
Hepatitis B E antigen, abbreviated as HBeAg in English, is a soluble protein in hepatitis B virus core particles. In general, HBeAg is buried inside HBcAg, and when HBcAg is cleaved, HBeAg dissolves into serum from the hepatocyte nucleus, and it appears later than HBsAg but disappears earlier than HBsAg, so it is the 2 nd serological antigen marker that human body infected with HBV appears following HBsAg. It has a prominent identifying feature with a His-tag.
However, the prior art prefers to use Ni filler for purifying the protein with the His tag, but the Ni filler is used for purifying the protein with the His tag, because the His tag is not exposed completely in the protein expression folding process, the His tag is not firmly combined with the filler, and the specificity of the Ni column for the His tag is not strong enough, so that the protein can be easily eluted together with the hybrid protein in FT or low-concentration imidazole, and the purity of the obtained protein is low. In addition, when imidazole is used for elution, because imidazole also has a certain background absorption peak in A280, whether the eluted solution contains the target protein cannot be intuitively judged.
Therefore, the existing purification effect and efficiency are poor, and improvement is needed.
Disclosure of Invention
In order to solve the problems that the existing HbeAg antigen has poor purification effect, and the purity is reduced due to the fact that impurities are easily introduced, the invention provides a nano antibody fluorescent affinity column and a method for purifying the HbeAg antigen by using the same.
The invention aims to:
1. providing an affinity column that can be effectively used for HbeAg antigen purification;
2. the binding specificity and firmness of the affinity column are improved;
3. impurities can be effectively identified, and a good purification effect is ensured;
4. the method can be effectively used for purifying the HbeAg antigen and improving the detection precision after purification.
In order to achieve the purpose, the invention adopts the following technical scheme
A nanometer antibody fluorescence affinity column is characterized in that a base frame is constructed by agarose, and a nanometer antibody containing anti-His protein, a fluorescent group and/or a fluorescent dye are coupled on the agarose base frame.
As a preference, the first and second liquid crystal compositions are,
the nano antibody fluorescent affinity column is constructed by adopting the following method:
activating agarose gel by an ethylene oxide activation method, pre-activating a filler by epoxy, coupling a nano antibody containing anti-His protein on the activated agarose gel in a manner of coupling sulfydryl with ligand, and constructing a base frame in a manner of coupling a fluorescent group with the ligand and then connecting the fluorescent group with the filler; thus obtaining the nanometer antibody fluorescent affinity column.
As a matter of preference,
the nano antibody containing the anti-His protein is obtained by a mammal expression system by using a phage display technology;
the phage display technique was performed in the following specific procedures:
1) Cell recovery;
2) Transfecting cells;
3) Detecting the transfected cells;
4) Counting the cells;
5) The supernatant was collected by centrifugation.
As a matter of preference,
the fluorescent group includes: FAM or TET;
the fluorescent dye includes: phycoerythrin or fluorescein isothiocyanate or Gel-Green dye.
As a preference, the first and second liquid crystal compositions are,
the pH value of the nano antibody fluorescence affinity column is 4-13.
A method for purifying an HbeAg antigen,
the method comprises the following steps:
and taking an expressed HbeAg sample, crushing the expressed HbeAg sample, taking a supernatant containing target protein, removing impurities, and then loading the sample through a nano antibody fluorescent affinity column, wherein the flow rate of the loaded sample is 1.0mL/min, the temperature is 2-8 ℃, and after the loading is finished, eluting and collecting eluent, namely completing the purification of the HbeAg antigen.
As a matter of preference,
the impurity removal comprises filtration.
As a matter of preference,
the dissociation liquid used for elution is 2-8 mol/L urea solution.
As a preference, the first and second liquid crystal compositions are,
the elution process is connected with an accounting protein detector, and the elution is finished when no protein is detected;
the elution process is connected with a fluorescence detector, and the fluorescence detector is used for detecting whether the ligand falls off.
As a matter of preference,
the eluate is subjected to protein gel electrophoresis.
Because current Ni filler column is when purification HbeAg antigen, the Ni post is relatively weak to His label's specificity entrapment effect, and the entrapment effect is relatively poor, leads to HbeAg to lose easily and leads to lou examining in the actual purification process, has improved the detection threshold value during the specific use in other words, can't effectively realize the early HbeAg antigen detection of sickening to need adopt imidazole when the cooperation Ni post elutes, imidazole elutes and has produced great background noise, the easy false positive scheduling problem that appears. The present invention prepares a gel column by adopting a specific manner. The gel column can be effectively combined with the anti-His protein-containing nano antibody, has obviously better specific trapping and enriching effects on the His label than a Ni column, can reduce impurity interference, greatly reduces the purification threshold value when the HbeAg antigen is purified, is equivalent to reduce the detection threshold value when in actual use, and can realize more effective and wider detection.
In the technical scheme of the invention, urea is used for elution, so that the background noise interference generated by eluent can be reduced, and meanwhile, the ligand can be detected by a fluorescence detector through the fluorescence of the purification column, so that the interference of the falling of the ligand on the detection result is reduced and avoided, and the detection precision is improved.
The invention has the beneficial effects that:
according to the invention, the gel column is combined with the specific nano antibody containing the anti-His protein, so that the target HbeAg antigen can be effectively and efficiently purified, the impurity interference is reduced, the purification effect is excellent, and the detection precision after purification is high.
Detailed Description
The present invention is described in further detail below with reference to specific examples. Those skilled in the art will be able to implement the invention based on these teachings. Moreover, the embodiments of the present invention described in the following description are generally only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.
Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available or available to those skilled in the art; unless otherwise specified, the methods used in the examples of the present invention are all those known to those skilled in the art.
Example 1
A nanometer antibody fluorescence affinity column, the nanometer antibody fluorescence affinity column uses agarose to construct a base frame, and a nanometer antibody containing anti-His protein and a fluorescent group and/or a fluorescent dye are coupled on the agarose base frame;
specifically, the nano antibody fluorescent affinity column is constructed by adopting the following method:
and (3) mixing the following components in percentage by mass: 1, mixing epichlorohydrin and agarose gel, dropwise adding a 50wt% NaOH solution with the same volume as the epichlorohydrin at a constant speed within 1h, reacting for 3h at 70 ℃, namely, activating the agarose gel through ethylene oxide groups to obtain an activated matrix, filling the activated matrix in a tubular carrier, and soaking the activated matrix in thioglycolic acid, deionized water and ammonium persulfate, wherein the dosage ratio of the thioglycolic acid, the deionized water, the ammonium persulfate and the activated matrix is 1.5mL:10mL, 0.3g:10mL, reacting for 8 hours at 60 ℃, removing the solvent, sequentially cleaning with 30wt% acetone solution, 70wt% acetone solution and 100wt% acetone solution, cleaning with 100wt% dioxane for 3 times to obtain an intermediate matrix, and finally adding dioxane, N-hydroxysuccinimide and supernate containing the anti-His protein nano antibody into the intermediate matrix for infiltration, wherein the dosage ratio of the dioxane, the N-hydroxysuccinimide, the supernate containing the anti-His protein nano antibody and the intermediate matrix is 10mL:0.5g:0.5g:10mL, oscillating for 12h at 25 ℃, namely realizing the coupling of the nano antibody containing His protein to the agarose gel to obtain antibody coupling gel, and then mixing the gel with a volume ratio of 1mL:10mL of: mixing Gel-Green dye, deionized water and antibody coupling Gel according to the proportion of 10mL, infiltrating and dyeing the antibody coupling Gel, and finally cleaning 3 times by using 100wt% dioxane, 3 times by using 100wt% methanol and 3 times by using 100wt% acetone in sequence to obtain the nano antibody fluorescent affinity column.
Wherein, the nano antibody containing anti-His protein is obtained by a mammal expression system by using a phage display technology; the phage display technique was performed in the following specific procedures:
1) Cell recovery;
2) Cell transfection;
3) Detecting the transfected cells;
4) Counting the cells;
5) The supernatant was collected by centrifugation.
The anti-His protein-containing nano antibody can also be prepared by directly purchasing the existing His-tag antibody, and the concentration of the prepared anti-His protein-containing nano antibody solution is 5000-50000 IU/mL.
The pH value of the nano antibody fluorescent affinity column prepared in this embodiment is detected, and the pH value of the nano antibody fluorescent affinity column meets the standard of 4 to 13.
Example 2
A method for purifying HbeAg antigen comprises the steps of taking Escherichia coli expressing HbeAg, crushing the Escherichia coli, taking 1mL of supernatant, filtering through filter cloth to remove solid impurities, loading the Escherichia coli at a flow rate of 1.0mL/min at 4 ℃, enabling the Escherichia coli to pass through a nano antibody fluorescence affinity column (the volume of a filler is 10 mL) prepared in example 1, eluting the column with 2mol/L of urea aqueous solution, connecting the column to a nucleic acid protein detector and a fluorescence detector in an elution process until no protein is detected, finishing elution until no protein is detected, judging that a ligand falls off in the elution process if the fluorescence reaction occurs, collecting an eluent sample which is finished in elution and has no fluorescence reaction (ligand falls off), carrying out gel electrophoresis analysis on the eluent sample to calculate the HbeAg antigen protein content in the supernatant, carrying out 20 times of purification and gel electrophoresis analysis tests, and taking the average value of 20 times of calculation results and recording the result as C (HbeAg) 1 。
The same 1mL supernatant was used and subjected to Abbott ARCHITECT i2000 luminescence immunoassayThe analyzer detects the content of HbeAg antigen protein in the supernatant, and the average value of 20 detection results is recorded as C (HbeAg) 2 。
With C (HbeAg) 2 As a standard result, a result deviation Rb is calculated.
The calculation formula of the result deviation is as follows:
the calculation result shows that Rb is less than 0.1 percent. The method has the advantages that HbeAg antigen protein can be effectively and efficiently separated and purified by purification, the method is used for performing simple, efficient and low-cost gel electrophoresis content analysis, and the detection precision of the gel electrophoresis content analysis can be effectively improved.
Comparative example 1
A method for purifying HbeAg antigen comprises the steps of taking Escherichia coli expressing HbeAg, crushing the Escherichia coli, taking 1mL of supernatant, filtering with filter cloth to remove solid impurities, loading the Escherichia coli at a flow rate of 1.0mL/min at 4 ℃, passing the Escherichia coli through a Ni column which is conventionally used for HbeAg antigen protein separation, eluting with 2.0mol/L of imidazole aqueous solution, collecting an eluted eluate sample, carrying out gel electrophoresis on the eluate sample to analyze the HbeAg antigen protein content in the supernatant, carrying out 20 times of purification and gel electrophoresis analysis tests, taking 20 times of calculation result average value to record C (HbeAg) 1 ′。
With C (HbeAg) obtained in example 2 2 As a standard result, a result deviation Rb' is calculated.
The calculation formula of the result deviation is as follows:
the calculation results show that Rb' =2.82%, a large deviation of results occurs, and a large deviation occurs in 20 experiments, mainly due to imidazole interference, resulting in a large detection error.
Example 3
Method for purifying HbeAg antigenPreparing 500IU/mL HbeAg antigen protein solution as a test sample, taking 1mL of HbeAg antigen protein solution as a test sample, loading the test sample at 4 ℃ at a flow rate of 1.0mL/min, passing the test sample through the nano-antibody fluorescent affinity column (the filler volume is 10 mL) prepared in example 1, eluting the test sample with 2mol/L of urea aqueous solution, connecting the test sample to a nucleic acid protein detector and a fluorescence detector in the elution process until no protein is detected, completing the elution until no protein is detected, judging that ligand falls off in the elution process if the fluorescence reaction occurs, collecting an eluent sample which is completely eluted and has no fluorescence reaction (ligand falls off), performing gel electrophoresis on the eluent sample to analyze the HbeAg antigen protein content in the supernatant, performing 20 times of purification and gel electrophoresis analysis tests, and taking the average value of 20 times of calculation results as C (HbeAg) 3 。
A method for purifying an HbeAg antigen,
preparing 500IU/mL HbeAg antigen protein solution as a base sample, mixing 1mL and 100 muL suspicious interferents (the positive samples of jaundice and rheumatoid factors are mixed by 1:1, and 500 IU/mL) to be used as a test sample, loading the test sample at the flow rate of 1.0mL/min at 4 ℃, passing the test sample through the nano-antibody fluorescence affinity column (the filler volume is 10 mL) prepared in example 1, then eluting the test sample by using 2mol/L urea aqueous solution, connecting the test sample to a nucleic acid protein detector and a fluorescence detector in the elution process until no protein is detected, judging that ligand is fallen off in the elution process if the fluorescence reaction is generated, collecting an eluent sample which is eluted and has no fluorescence reaction (ligand is fallen off), carrying out gel electrophoresis analysis on the eluent sample to calculate the HbeAg antigen protein content in the supernatant, carrying out 20 times of purification and gel electrophoresis analysis tests, and taking the average value of 20 times of calculation results to record the sample as C (HbeAg) 4 。
With C (HbeAg) 3 As a standard result, a result deviation Rb ″ is calculated.
The calculation formula of the result deviation is as follows:
the calculation result shows that Rb' is less than 0.1 percent, so that the affinity column and the purification method can effectively overcome the interference of a suspicious interfering substance on the test result, have high specificity and separation effect on the antigen protein containing the His label (HbeAg antigen protein), and can effectively separate and purify the HbeAg antigen protein.
According to the above examples and test results, the invention can realize effective and efficient purification of the target HbeAg antigen by combining the gel column with the specific nano antibody containing the anti-His protein, reduce impurity interference, and has the advantages of excellent purification effect and high detection precision after purification.
Claims (10)
1. A nanometer antibody fluorescence affinity column is characterized in that,
the nanometer antibody fluorescence affinity column uses agarose to construct a base frame, and the agarose base frame is coupled with a nanometer antibody containing anti-His protein, a fluorescent group and/or a fluorescent dye.
2. The nanobody fluorescent affinity column of claim 1,
the nano antibody fluorescent affinity column is constructed by adopting the following method:
activating agarose gel by an ethylene oxide activation method, pre-activating a filler by epoxy, coupling a nano antibody containing anti-His protein on the activated agarose gel in a manner of coupling sulfydryl with ligand, and constructing a base frame in a manner of coupling a fluorescent group with the ligand and then connecting the fluorescent group with the filler; thus obtaining the nanometer antibody fluorescent affinity column.
3. The nanobody fluorescent affinity column of claim 1 or 2,
the nano antibody containing the anti-His protein is obtained by a mammal expression system by using a phage display technology;
the phage display technique was performed in the following specific procedures:
1) Cell recovery;
2) Cell transfection;
3) Detecting the transfected cells;
4) Counting the cells;
5) The supernatant was collected by centrifugation.
4. The nanobody fluorescent affinity column of claim 1 or 2,
the fluorescent group includes: FAM or TET;
the fluorescent dye includes: phycoerythrin or fluorescein isothiocyanate or Gel-Green dye.
5. The nanobody fluorescent affinity column of claim 1 or 2,
the pH value of the nano antibody fluorescence affinity column is 4-13.
6. A method for purifying HbeAg antigen,
the method comprises the following steps:
and taking an expressed HbeAg sample, crushing the expressed HbeAg sample, taking a supernatant containing target protein, removing impurities, and then loading the sample through a nano antibody fluorescent affinity column, wherein the flow rate of the loaded sample is 1.0mL/min, the temperature is 2-8 ℃, and after the loading is finished, eluting and collecting eluent, namely completing the purification of the HbeAg antigen.
7. The method of claim 6, wherein the HbeAg antigen is purified,
the impurity removal comprises filtration.
8. The method of purifying HbeAg antigen according to claim 6,
the dissociation liquid used for elution is 2-8 mol/L urea solution.
9. The method of purifying HbeAg antigen according to claim 6 or 8,
the elution process is connected with an accounting protein detector, and the elution is finished when no protein is detected;
the elution process is connected with a fluorescence detector, and the fluorescence detector is used for detecting whether the ligand falls off.
10. The method of purifying HbeAg antigen according to claim 6,
the eluate is subjected to protein gel electrophoresis.
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