阅读说明：本技术 一种菜豆凝集素卵黄抗体及其制备方法与应用 (Phaseolus vulgaris agglutinin egg yolk antibody and preparation method and application thereof ) 是由 韩雪 陈泽才 陈禅友 徐勋 陈高 于 2019-11-29 设计创作，主要内容包括：本发明公开了一种菜豆凝集素卵黄抗体及其制备方法与应用,所示方法包括：(1)将菜豆凝集素经处理后作为免疫原来免疫产蛋母鸡一段时间后收集鸡蛋；(2)去蛋清得蛋黄,稀释后用辛酸调pH至5.2～5.6进行第一次沉淀后离心取上清,加48％～52％硫酸铵溶液进行第二次沉淀后离心去除上清后,再加以33％～40％硫酸铵进行第三次沉淀,离心、去除上清后得到白色沉淀即为菜豆凝集素卵黄抗体粗品；(3)菜豆凝集素卵黄抗体的纯化：将得到的菜豆凝集素卵黄抗体粗品经G100分子筛、离子交换柱层析收集洗脱液,透析浓缩后即得；该菜豆凝集素卵黄抗体可有效识别和结合菜豆凝集素,并在动物实验中体现有优良的对菜豆凝集素的解毒作用。(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.)

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.

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