CN110885373A - Phaseolus vulgaris agglutinin egg yolk antibody and preparation method and application thereof - Google Patents
Phaseolus vulgaris agglutinin egg yolk antibody and preparation method and application thereof Download PDFInfo
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- CN110885373A CN110885373A CN201911198074.XA CN201911198074A CN110885373A CN 110885373 A CN110885373 A CN 110885373A CN 201911198074 A CN201911198074 A CN 201911198074A CN 110885373 A CN110885373 A CN 110885373A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
Abstract
The invention discloses a phaseolus vulgaris agglutinin egg yolk antibody and a preparation method and application thereof, wherein the method comprises the following steps: (1) treating phaseolus vulgaris agglutinin as immunogen to immunize egg laying hens for a period of time, and collecting eggs; (2) removing egg white to obtain egg yolk, diluting, adjusting the pH value to 5.2-5.6 by using caprylic acid, performing primary precipitation, centrifuging to obtain supernatant, adding 48-52% ammonium sulfate solution, performing secondary precipitation, centrifuging to remove supernatant, performing tertiary precipitation by adding 33-40% ammonium sulfate, centrifuging, removing supernatant to obtain white precipitate, namely the phaseolus vulgaris agglutinin egg yolk antibody crude product; (3) purification of phaseolus vulgaris agglutinin yolk antibody: collecting eluate of the obtained crude product of phaseolus vulgaris agglutinin yolk antibody by G100 molecular sieve and ion exchange column chromatography, dialyzing and concentrating to obtain the final product; the phaseolus vulgaris agglutinin yolk antibody can effectively recognize and combine phaseolus vulgaris agglutinin, and has excellent detoxifying effect on the phaseolus vulgaris agglutinin in animal experiments.
Description
Technical Field
The invention relates to the technical field of antibody preparation, in particular to a phaseolus vulgaris agglutinin egg yolk antibody and a preparation method and application thereof.
Background
The main cause of kidney bean poisoning is that kidney bean agglutinin is toxic to human body, and the poisoning symptoms, mainly nausea, vomiting and dizziness, appear about thirty to sixty minutes after eating the kidney bean agglutinin. The existing detoxification way of kidney bean poisoning is a general method of food poisoning, namely emetic, gastric lavage and intravenous fluid infusion, and has no specificity and poor patient sensitivity. At present, the method for avoiding the eating poisoning of the kidney beans is mainly a cooking method, namely the method of heating the kidney beans in boiling water for more than five minutes to completely destroy agglutinin and then cooking the kidney beans is the gold standard of the current kidney bean processing, but a lot of nutrients in the kidney beans are lost due to long-time heating. The existing method for avoiding the kidney bean agglutinin poisoning is a high-temperature heating method, which brings great trouble to the cooking of the kidney beans and also greatly reduces the nutritional value of the kidney beans. The food of kidney beans often has poisoning accidents caused by that the agglutinin is not damaged, no efficient specific antidote aiming at the kidney beans is available on the market, and the detoxification method is a general method for food poisoning. Low efficiency and certain uncomfortable feeling to the body.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a phaseolus vulgaris agglutinin egg yolk antibody, a preparation method and application thereof.
The invention is realized by the following steps:
one of the purposes of the invention is to provide a preparation method of a phaseolus vulgaris agglutinin yolk antibody, which comprises the following steps:
step 1, preparation of a phaseolus vulgaris agglutinin yolk antibody: treating phaseolus vulgaris agglutinin as immunogen to immunize egg laying hens for a period of time, and collecting eggs;
step 2, crude extraction of the phaseolus vulgaris agglutinin yolk antibody: removing egg white to obtain egg yolk, diluting, adjusting the pH value to 5.2-5.6 by using caprylic acid, performing primary precipitation, centrifuging to obtain supernatant, adding 48-52% ammonium sulfate solution, performing secondary precipitation, centrifuging to remove supernatant, performing tertiary precipitation by adding 33-40% ammonium sulfate, centrifuging, removing supernatant to obtain white precipitate, namely the phaseolus vulgaris agglutinin egg yolk antibody crude product;
and 3, purifying the phaseolus vulgaris agglutinin yolk antibody: and (3) passing the obtained crude phaseolus vulgaris agglutinin egg yolk antibody product through a G100 molecular sieve to collect macromolecular substances, collecting eluent through ion exchange column chromatography, dialyzing and concentrating to obtain the refined phaseolus vulgaris agglutinin egg yolk antibody.
Preferably, the laying hens are immunized 3 times in the step 1, and the immunogen used by the first needle (day 1) is emulsion formed by emulsifying a phaseolin standard with an equal volume of Freund's complete adjuvant, and the emulsion is injected into the laying hens subcutaneously on the back; the immunogen used in the second needle (day 7) and the third needle (day 21) was an emulsion of phaseolin standard with an equal volume of Freund's incomplete adjuvant.
Preferably, the eggs are collected 45 days after the first injection of the immunogen in step 1.
Preferably, the temperature of the first precipitation, the second precipitation and the third precipitation in the step 2 is 4 ℃; the centrifugation speed is 8000-10000 r/min; the centrifugation time is 20-60 min.
Preferably, the step 3 of sieving the G100 molecular sieve comprises the following specific steps: balancing a chromatographic column by using Buffer I, dissolving the crude phaseolus vulgaris agglutinin egg yolk antibody by using Buffer I solution, loading the solution on the chromatographic column, eluting by using Buffer I, and collecting eluted macromolecular substances; the Buffer I solution is as follows: 20mmol/L Tris, 20mmol NaCl.
Preferably, the ion exchange column chromatography in step 3 specifically comprises the following steps: dissolving macromolecular substances collected by a G100 molecular sieve by using a Buffer I balanced ion exchange column with 3 times of column volume for 3 times; then gradient elution is carried out on the NaCl gradient concentration eluent to collect the eluent respectively, and then dialysis concentration is carried out.
The NaCl gradient concentration eluent contains 20mmol/L Tris, and the NaCl concentration is respectively 100-1000 mmol/L.
The second purpose of the invention is to provide the phaseolus vulgaris agglutinin yolk antibody prepared by the method.
The invention also aims to provide the application of the phaseolus vulgaris agglutinin yolk antibody in preparing a phaseolus vulgaris antidote.
Compared with the prior art, the invention has the following advantages and effects:
1. the preparation method of the phaseolus vulgaris agglutinin egg yolk antibody provided by the invention comprises the steps of treating phaseolus vulgaris agglutinin to serve as an immunogen to immunize laying hens for a period of time, collecting eggs, and performing crude extraction (by caprylic acid and ammonium sulfate precipitation) and purification (after molecular sieve and ion exchange chromatography) to obtain the egg yolk antibody with the purity of more than 95%.
2. The phaseolus vulgaris agglutinin egg yolk antibody provided by the invention is (1) extracted from an egg of an immunized chicken, a serum antibody is compared, the serum antibody is derived from blood and contains various uncertain components (including viruses, bacteria and the like) which are not suitable for oral administration, the egg yolk antibody is derived from egg yolk, bacteria and viruses cannot enter the egg yolk due to the placenta barrier effect, and the egg is used as a daily food and has high oral administration safety, so the egg yolk antibody extracted from the egg yolk is suitable for oral administration. (2) The egg yolk antibody preparation cost is lower than that of a serum antibody, and the egg yolk antibody can be maintained for one year after one laying hen is boosted according to the calculation of the laying hen, one egg contains 400mg of egg yolk antibody, and one chicken can produce 40-50 g of egg yolk antibody for one year, which is equal to the serum antibody yield of 38 rabbits in the same period. Thus, egg yolk antibody costs are much lower than serum antibody costs. (3) The yolk antibody is heat-resistant, acid-resistant, stomach protein-resistant and trypsin-resistant, has the storage life of 10 years, is more stable than serum antibody, and is approved by USDA and FDA for human beings.
3. The bean agglutinin yolk antibody provided by the invention can specifically recognize and combine bean agglutinin; can effectively relieve the damage degree of mouse liver and kidney caused by gavage of mouse kidney bean agglutinin.
Drawings
FIG. 1 is an SDS-PAGE pattern of a phaseolus vulgaris agglutinin yolk antibody obtained after purification in the method for preparing the phaseolus vulgaris agglutinin yolk antibody according to the embodiment of the present invention;
FIG. 2 shows the results of the potency measurement when Phaseolus vulgaris agglutinin yolk antibody IgY and agglutinin PHA are added simultaneously;
FIG. 3 shows the result of potency measurement when lectin PHA is added for 15min and then phaseolus vulgaris lectin yolk antibody IgY is added;
FIG. 4 is a graph showing the change in the appearance of the internal organs of a mouse in the gavage test of the mouse in Experimental example 2; wherein (A) is a cut-away view of a mouse; (B) the figure is a drawing of the viscera;
FIG. 5 is a section of liver tissue of a mouse in the gavage test of the mouse in Experimental example 2;
FIG. 6 is a section of mouse kidney tissue in the mouse gavage test in Experimental example 2.
Detailed Description
Example 1
First, preparation of yolk antibody
100ug of phaseolus vulgaris agglutinin standard substance is taken and emulsified with Freund's adjuvant with the same volume, the back of the phaseolus vulgaris agglutinin standard substance is injected with egg-laying hens subcutaneously, Freund's complete adjuvant is used for the first injection, Freund's incomplete adjuvant is used for 2 and 3 injections to strengthen immunity, and eggs are collected 25-30 days after the first injection of immunogen.
Diluting lyophilized soybean agglutinin with sterilized normal saline to appropriate concentration, adding equal volume of Freund complete adjuvant FCA or Freund incomplete adjuvant FICA, mixing adjuvant and immunizing antigen by syringe double-push method to obtain water-in-oil emulsion,
second, crude extraction of yolk antibody
Removing egg white from the eggs, diluting the egg yolk by 9 times according to the weight of distilled water, adjusting the pH value to 5.4 by caprylic acid, standing overnight at 4 ℃, centrifuging at 9000r/min for 20min, precipitating the supernatant with 50% ammonium sulfate solution at 4 ℃ overnight, centrifuging at 9000r/min for 30min, removing the supernatant, performing secondary precipitation again with 40% ammonium sulfate at 4 ℃ overnight, centrifuging at 9000r/min for 30min, and removing the supernatant to obtain white precipitate which is crude IgY.
Thirdly, preparation of egg yolk antibody
G100 molecular sieve: dissolving the IgY crude product by using a Buffer I solution, carrying out Buffer I equilibrium chromatography column, (the Buffer1 solution is 20mmol/L Tris, 20mmol NaCl, and the total volume is 150ml), loading a sample on the column, eluting by using Buffer I, and collecting a macromolecular part which does not enter into gel.
Ion exchange column chromatography: 3 times the column volume of a Buffer I equilibrium chromatography column, (Buffer1 solution: 20mmol/LTris, 20mmol NaCl, total volume 150ml), the crude IgY was dissolved with Buffer I solution and loaded onto the column 3 times. And eluting the IgY by NaCl gradient concentration eluent, wherein the eluent contains 20mmol/LlTris, the NaCl concentration is respectively from 100-1000mmol/L, respectively collecting the eluent, and dialyzing and concentrating to obtain the purified IgY. After the IgY purified by caprylic acid, ammonium sulfate crude extraction, molecular sieve and ion exchange column is analyzed by SDS-PAGE denaturing electrophoresis, two main bands of 65KD and 25KD appear, which is consistent with the properties of the IgY, and the figure shows that the purity of the IgY reaches more than 95%.
Example 2
Except for the crude extraction step of the yolk antibody, the rest of the steps are the same as those in example 1, specifically:
first, yolk antibody was prepared as in example 1
Second, crude extraction of yolk antibody
Removing egg white from the eggs, diluting the egg yolk by 9 times according to the weight of distilled water, adjusting the pH value to 5.2 by caprylic acid, standing overnight at 4 ℃, centrifuging for 20min at 8000r/min, precipitating the supernatant by 52% ammonium sulfate solution at 4 ℃ for standing overnight, centrifuging for 30min at 8000r/min, removing the supernatant, performing secondary precipitation again by 37% ammonium sulfate, standing overnight at 4 ℃, centrifuging for 30min at 8000r/min, and removing the supernatant to obtain white precipitate which is crude IgY.
Thirdly, the egg yolk antibody was prepared as in example 1. The yolk antibody with the purity of 94 percent is obtained.
Example 3
Except for the crude extraction step of the yolk antibody, the rest of the steps are the same as those in example 1, specifically:
first, yolk antibody was prepared as in example 1
Second, crude extraction of yolk antibody
Removing egg white from the eggs, diluting the egg yolk by 9 times according to the weight of distilled water, adjusting the pH value to 5.6 by caprylic acid, standing overnight at 4 ℃, centrifuging for 20min at 10000r/min, precipitating the supernatant by 48% ammonium sulfate solution at 4 ℃ for standing overnight, centrifuging for 30min at 10000r/min, removing the supernatant, performing secondary precipitation by 33% ammonium sulfate again, standing overnight at 4 ℃, centrifuging for 30min at 10000r/min, and removing the supernatant to obtain white precipitate which is crude IgY.
Thirdly, the egg yolk antibody was prepared as in example 1. The yolk antibody with the purity of 93 percent is obtained.
Comparative example 1
The procedure is as in example 1 except that the pH is adjusted to 5 by caprylic acid in the crude extraction step of the yolk antibody. The yolk antibody with the purity of 82% is obtained.
Comparative example 2
The procedure is as in example 1 except that the pH is adjusted to 6 by caprylic acid in the crude extraction step of the yolk antibody. The yolk antibody with the purity of 81 percent is obtained.
EXAMPLE 1 potency assay
The titer of the phaseolus vulgaris lectin yolk antibody IgY prepared in example 1 was measured.
FIG. 2 shows the results of the potency measurement when Phaseolus vulgaris agglutinin yolk antibody IgY and agglutinin PHA are added simultaneously; of the 14 wells, the IgY amount of the top row of 1 to 7 wells is 25ug, and the PHA amount of the top row of 1 to 7 wells is: 12.5ug 6.25ug, 3.13ug, 1.56ug, 0.78ug, 0.37ug, 0.19 ug. The IgG concentration in the lower row from 1 to 7 wells was 25 ug. PHA concentrations from 1 to 7 wells were: PHA amounts from 1 to 7 wells were: 12.5ug 6.25ug, 3.13ug, 1.56ug, 0.78ug, 0.37ug, 0.19 ug.
After conversion, 1ug of PHA was incubated with blood cells and the antibody was added thereto, and 7.9ug of IgY or 32ug of IgG was required to completely release the blood coagulation effect of 1ug of PHA. From the quantitative results, it was confirmed that IGY has a stronger ability to release PHA coagulation than IgG at a stage where PHA has not been brought into contact with blood cells to produce coagulation.
FIG. 3 shows the result of potency measurement when lectin PHA is added to blood cells for coagulation reaction (15min) and then phaseolus vulgaris lectin yolk antibody IgY is added; 1ug of PHA was added to each of 14 wells, and the resultant was subjected to a hemagglutination reaction with blood cells (reaction time: 15 minutes); of the 14 wells, the IgY amount of the top row of 1 to 7 wells is 25ug, and the PHA amount of the top row of 1 to 7 wells is: 12.5ug, 6.25ug, 3.13ug, 1.56ug, 0.78ug, 0.37ug, 0.19 ug. The lower row of 1 to 7 wells each had 25ug of IgG. PHA concentrations from 1 to 7 wells were: 12.5ug, 6.25ug, 3.13ug, 1.56ug, 0.78ug, 0.37ug, 0.19 ug.
After conversion, it was found that 1ug of PHA was aggregated with blood cells (15 minutes), and 32ug of IgY or 67ug of IgG was required to completely release 1ug of PHA for coagulation. From the quantitative results, the PHA and blood cells which have produced the blood coagulation effect have stronger PHA coagulation-releasing ability than IgG.
In conclusion, the lectin yolk antibody can effectively reduce the blood coagulation titer of the kidney bean lectin in vitro, and the blood coagulation effect of the lectin is obviously reduced after the lectin antibody is added.
Experimental example 2 gavage protection experiment for mice
1. The phaseolus vulgaris agglutinin yolk antibody IgY prepared in example 1 was subjected to a mouse gavage test by a conventional method. Grouping: (1) intragastric administration PHA group; (2) a gastric perfusion normal saline group; (3) PHA, 15min later IgY group; (4) PHA first, 1h later IgY group.
2. Observation of changes in the appearance of the mouse viscera: as shown in FIG. 4, the appearance of the mouse internal organs was visually observed, the liver of the PHA group was significantly different from that of the normal saline group, the liver of the PHA group was significantly lighter in color, the liver of the PHA group was whitish, and the hand-touch elasticity was not large, and the antibody-protected group (including the PHA-first after 15min IgY group; and the PHA-first after 1h IgY group) was as much in color as the normal saline group.
3. The case section results are shown in fig. 5 and 6: the section shows that PHA causes obvious damage to the liver and kidney of mice, and both the cytoplasm hollowing and the cell nucleus degeneration become large. The antibody protection group after PHA intragastric administration obviously protects the liver and kidney, and the liver and kidney section and the normal saline group have no difference.
As shown in FIG. 5, the phaseolus vulgaris agglutinin egg yolk antibody prepared by the invention can effectively relieve the mouse liver injury (hepatic cell cytoplasm hollow) caused by PHA; as is clear from FIG. 6, the phaseolus vulgaris agglutinin egg yolk antibody prepared by the present invention can effectively alleviate the PHA-induced renal injury (tubular cytoplasm cavitation) in mice.
The invention is not to be considered as limited to the particular embodiments shown, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A preparation method of a phaseolus vulgaris agglutinin yolk antibody is characterized by comprising the following steps:
step 1, preparation of a phaseolus vulgaris agglutinin yolk antibody: treating phaseolus vulgaris agglutinin as immunogen to immunize egg laying hens for a period of time, and collecting eggs;
step 2, crude extraction of the phaseolus vulgaris agglutinin yolk antibody: removing egg white to obtain egg yolk, diluting, adjusting the pH value to 5.2-5.6 by using caprylic acid, performing primary precipitation, centrifuging to obtain supernatant, adding 48-52% ammonium sulfate solution, performing secondary precipitation, centrifuging to remove supernatant, performing tertiary precipitation by adding 33-40% ammonium sulfate, centrifuging, removing supernatant to obtain white precipitate, namely the phaseolus vulgaris agglutinin egg yolk antibody crude product;
and 3, purifying the phaseolus vulgaris agglutinin yolk antibody: and (3) passing the obtained crude phaseolus vulgaris agglutinin egg yolk antibody through a molecular sieve to collect macromolecular substances, collecting eluent through ion exchange column chromatography, dialyzing and concentrating to obtain the refined phaseolus vulgaris agglutinin egg yolk antibody.
2. The method according to claim 1, wherein the laying hens are immunized 3 times in the step 1, and the first injection is performed by back subcutaneous injection of the laying hens with an emulsion of a phaseolin standard emulsified with an equal volume of Freund's complete adjuvant; the immunogen used in the second and third needles is an emulsion of a phaseolus vulgaris agglutinin standard and an equal volume of Freund's incomplete adjuvant.
3. The method of claim 2, wherein the collection of eggs is initiated 45 days after the injection of the first needle of immunogen in step 1.
4. The method according to claim 2, wherein the first precipitation, the second precipitation and the third precipitation in step 2 are all performed at 4 ℃; the centrifugation speed is 8000-10000 r/min; the centrifugation time is 20-60 min.
5. The preparation method of claim 2, wherein the step 3 of sieving the G100 molecular sieve comprises the following specific steps: balancing a chromatographic column by using Buffer I, dissolving the crude phaseolus vulgaris agglutinin egg yolk antibody by using Buffer I solution, loading the solution on the chromatographic column, eluting by using Buffer I, and collecting eluted macromolecular substances; the Buffer I solution is as follows: 20mmol/L Tris, 20mmol NaCl.
6. The preparation method of claim 2, wherein the ion exchange column chromatography in the step 3 comprises the following specific steps: dissolving macromolecular substances collected by a G100 molecular sieve by using a Buffer I balanced ion exchange column with 3 times of column volume for 3 times; then gradient elution is carried out on the NaCl gradient concentration eluent to collect the eluent respectively, and then dialysis concentration is carried out.
7. The method according to claim 6, wherein the gradient NaCl concentrations elute contains 20mmol/L Tris and has a NaCl concentration of 100 to 1000 mmol/L.
8. A phaseolus vulgaris agglutinin yolk antibody prepared by the method of any one of claims 1 to 7.
9. Use of a phaseolus vulgaris lectin yolk antibody as claimed in claim 8 in the preparation of a phaseolus vulgaris antidote.
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