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CN112142839B - Method suitable for extracting yolk antibody in industrialized large scale - Google Patents

Method suitable for extracting yolk antibody in industrialized large scale Download PDF

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CN112142839B
CN112142839B CN202011203157.6A CN202011203157A CN112142839B CN 112142839 B CN112142839 B CN 112142839B CN 202011203157 A CN202011203157 A CN 202011203157A CN 112142839 B CN112142839 B CN 112142839B
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yolk
yolk antibody
antibody
egg
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CN112142839A (en
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宋学宏
郭培红
孙丙耀
黄健
田丹阳
刘金龙
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Kunshan Beiruikang Biological Technology Co ltd
Shandong Best Care Biotechnology Co ltd
Suzhou University
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Abstract

本发明公开了一种适用于工业化大规模提取卵黄抗体的方法,特点是包括以下步骤:(1)将高免蛋清洗消毒后分离得卵黄,搅拌均匀,制得卵黄液;(2)将一种纳米碳点加入pH值为5.0~5.2的乙酸‑乙酸钠缓冲溶液中,制成含碳点的卵黄抗体提取缓冲液;(3)将卵黄液加入到卵黄抗体提取缓冲液中,搅拌均匀,室温静置沉淀取上清,再将沉淀经离心得上清液,合并两次所得上清液,即为卵黄抗体提取液,优点是提取率高、稳定性好、无污染且具有高效抑菌作用。

Figure 202011203157

The invention discloses a method suitable for industrialized large-scale extraction of egg yolk antibodies, which is characterized by comprising the following steps: (1) washing and sterilizing high-immunity eggs to separate egg yolks, stirring them evenly to prepare egg yolk liquid; (2) mixing a The nano-sized carbon dots are added to the acetic acid-sodium acetate buffer solution with a pH value of 5.0-5.2 to prepare the egg yolk antibody extraction buffer containing carbon dots; (3) the egg yolk solution is added to the egg yolk antibody extraction buffer, stirred evenly, Allow the precipitation to stand at room temperature to get the supernatant, then centrifuge the precipitation to obtain the supernatant, and combine the supernatant obtained twice, which is the egg yolk antibody extract. effect.

Figure 202011203157

Description

Method suitable for extracting yolk antibody in industrialized large scale
Technical Field
The invention relates to a yolk antibody extraction method, in particular to a method suitable for extracting yolk antibodies in an industrialized large scale.
Background
The yolk antibody is also called yolk immunoglobulin (IgY), is a specific antibody formed by transferring chicken serum IgG into yolk, has the immunocompetence similar to that of mammal IgG, but compared with the mammal IgG, the IgY has the advantages of better chemical stability, high yield, simple preparation, low cost, greenness, safety and the like, and is widely applied to the aspects of food, medicine, biological products and the like.
The egg yolk contains about 48% of water, about 17% of protein and about 30% of fat, and the fat is easy to combine with the protein to form lipoprotein, wherein the egg yolk antibody belongs to soluble egg yolk protein. The existing yolk antibody extraction methods are various, and the common methods comprise: 1. polyethylene glycol (PEG) precipitation method, which is the commonly used method for extracting yolk antibody in large scale at present. Its advantages are high solubility of PEG in water, low heat dissipation and short time to form protein deposit. However, PEG belongs to an organic solvent, so that no method is available for removing PEG in the extracting solution at present, the residual PEG has no definite idea whether the cultured animals are harmful or not, and simultaneously, the PEG is high in price and large in dosage, so that the cost is increased. 2. Ammonium sulfate precipitation. The method has the advantages of low requirements on equipment and conditions, low price of ammonium sulfate and mild property. The disadvantages are too large dosage of ammonium sulfate, poor buffer capacity, influence on the activity of the yolk antibody and environmental pollution caused by waste liquid of a large amount of ammonium sulfate. 3. Extraction with organic solvent such as octanoic acid or chloroform. The organic solvent in the method not only influences the activity of the antibody, but also is toxic and pollutes the environment. 4. The principle of the acidified water extraction method is that in an acidic pH and low-concentration ionic environment, lipoprotein in the yolk is coagulated and precipitated, so that the purpose of separating and extracting yolk antibody is achieved. This method has a large loss of yolk antibody. In addition, the acidified water has a poor stabilizing system, and the quality guarantee period of the extracted egg yolk antibody is short. Therefore, exploring a yolk antibody purification method with simple process, good fat removal effect, low cost, stable antibody and environmental protection is a technical difficulty which needs to be broken through by the technical personnel in the field.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for extracting the yolk antibody in an industrialized large scale, which has high extraction rate, good stability, no pollution and high-efficiency bacteriostatic action.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for extracting yolk antibodies in an industrialized large scale is characterized by comprising the following steps:
(1) washing and sterilizing high-immunity egg white, separating to obtain egg yolk, and stirring to obtain egg yolk liquid;
(2) adding a nano carbon dot into an acetic acid-sodium acetate buffer solution with the pH value of 5.0-5.2 to prepare a yolk antibody extraction buffer solution containing the carbon dot;
(3) adding the yolk solution into the yolk antibody extraction buffer solution, stirring uniformly, standing at room temperature for precipitation to obtain a supernatant, centrifuging the precipitate to obtain a supernatant, and combining the supernatants to obtain the yolk antibody extract.
Further, in the step (1), the stirring speed is 50-500 r/min, and the time is 10-20 min.
Further, in the step (2), the concentration of the nano carbon dots in the acetic acid-sodium acetate buffer solution is 5-25 mu g/ml.
Further, the nano carbon dots are zero-dimensional carbon-based nano materials with the size below 50nm, which are prepared by a one-step electrochemical method of vitamin C.
Further, the concentration of the acetic acid-sodium acetate buffer solution in the step (2) is 0.1-0.5M, and the pH value is 5.0-5.2.
Further, the mass-to-volume ratio of the yolk solution to the yolk antibody extraction buffer solution in the step (3) is 1: (5-10).
Further, the specific process of the step (3) is as follows: mixing yolk liquid according to the mass volume ratio of 1: (5-10), adding the yolk antibody into the yolk antibody extraction buffer solution in a ratio of 50-500 r/min, stirring for 10-20 min, and standing and precipitating for 8-15 h at room temperature; and sucking the supernatant, centrifuging the precipitate at 4000-12000 r/min for 10-30 min, taking the supernatant, and combining the supernatants to obtain the egg yolk antibody extracting solution.
Compared with the prior art, the invention has the advantages that:
1) compared with the traditional acidified water extraction method, the invention adds decomposable residue-free carbon dots prepared by vitamin C into the buffer system, further improves the yield of IgY and ensures that the obtained IgY has good stability. Solves the problems of high cost and high antibody loss in the method for extracting the yolk antibody in the prior art.
2) The yolk antibody can be extracted at room temperature due to the strong bacteriostatic action of the carbon dots. Therefore, compared with the traditional extraction method which needs to be precipitated at 4 ℃ overnight, the method has the advantages of simple equipment requirement and simple and convenient operation. Meanwhile, the carbon dots can be completely degraded into CO under visible light in the air2CO and H2O is non-toxic, so the precipitate can be developed into food or feed raw materials, the utilization rate is improved, and zero pollution is realized.
3) Compared with the traditional acidified water extraction method, the invention adopts 0.1-0.5M acetic acid-sodium acetate buffer solution to ensure that the activity of IgY is stable in a buffer system.
4) After the carbon point extraction process adopted by the invention is finished, the carbon point is completely degraded into CO within about 20 days under natural light2CO and H2And O, no residue, green and safe. Solves the technical problem that residual organic matters and high-dose preservatives in the extracting solution have potential risks to animals or human bodies in the conventional method.
In conclusion, the invention provides a method for extracting the yolk antibody in an industrialized large scale, which has simple steps, low cost and high efficiency; the added nano carbon dots have high-efficiency antibacterial action, improve the yield of the yolk antibody, avoid the defect that the yolk antibody needs to be precipitated at low temperature of 4 ℃ overnight when being extracted by traditional acidified water, and are green and pollution-free due to no or little addition of a preservative.
Drawings
FIG. 1 shows the results of circular dichroism analysis of the influence of carbon points of different concentrations on the structure of IgY;
FIG. 2 is the SDS-PAGE electrophoresis preliminary detection of IgY yield of yolk antibody extracted from different concentration carbon spots and different proportion buffers after primary precipitation by 19 wt% ammonium sulfate solution. Wherein: m is a protein Marker, lanes 1-3 are respectively provided with a carbon spot concentration of 0 mug/mL, lanes 4-6 are respectively provided with a carbon spot concentration of 6.25 mug/mL, lanes 7-9 are respectively provided with a carbon spot concentration of 12.5 mug/mL, and lanes 10-12 are respectively provided with a carbon spot concentration of 25 mug/mL; meanwhile, lanes 1, 4, 7, 10 also show a 1:5 ratio of yolk to buffer by mass/volume, lanes 2, 5, 8, 11 also show a 1: lanes 3, 6, 9, 12 also show the egg yolk to buffer mass to volume ratio of 1: 9;
FIG. 3 shows the results of measuring the antibody titer in the anti-Scophthalmus maximus IL-1 beta yolk antibody extract by ELISA;
FIG. 4 shows the results of ELISA assay for antibody titer in egg yolk antibody extract against crucian gill hemorrhage virus (CyHV-2);
FIG. 5 shows that the mass-to-volume ratio of the egg yolk for resisting crucian gill hemorrhage virus (CyHV-2) to the buffer solution is 1:9, standing and precipitating the yolk antibodies extracted from the carbon spots with different concentrations for 15 hours at the temperature of 4 ℃ and at room temperature, and then obtaining the total bacterial count in the crude extract.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiments
Example 1
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 100r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding yolk antibody extraction buffer solution into yolk solution at a mass-to-volume ratio of 1:5, stirring at 100r/min
Stirring for 20min, standing at room temperature for precipitation for 8 h; sucking the supernatant with vacuum pump, centrifuging at 12000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 2
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 200r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.2M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:7, stirring at 200r/min for 20min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, centrifuging at 8000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 3
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:9, stirring at 500r/min for 10min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, centrifuging the precipitate at 5000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 4
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:5, stirring at 400r/min for 10min, and standing and precipitating at room temperature for 9 h; sucking the supernatant with vacuum pump, centrifuging at 12000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 5
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 400r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.2M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:7, stirring at 300r/min for 10min, and standing and precipitating at room temperature for 10 h; sucking the supernatant with vacuum pump, centrifuging at 9000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 6
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 400r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 200r/min for 30min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, centrifuging the precipitate at 4000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 7
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 200r/min for 15min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:5, stirring at 400r/min for 10min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, centrifuging at 12000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 8
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 100r/min for 20min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.2M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:7, stirring at 300r/min for 30min, and standing and precipitating at room temperature for 10 h; sucking the supernatant with vacuum pump, centrifuging at 7000r/min for 30min to obtain supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 9
Extraction of anti-turbot IL-1 beta yolk antibody
1. Washing anti-turbot IL-1 beta hyperimmune egg with normal temperature tap water for 1min, and disinfecting the washed egg with 0.25mg/L chlorine dioxide water solution for 1 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.3M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 300r/min for 15min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, precipitating, centrifuging at 6000r/min for 20min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 10
Extraction of yolk antibody against crucian gill hemorrhage virus (CyHV-2)
1. Washing high-immunity eggs for resisting crucian gill hemorrhage virus (CyHV-2) with normal temperature tap water for 1min, and disinfecting and cleaning eggs with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 200r/min for 15min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 6.25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-volume ratio of 1:9, stirring at 400r/min for 10min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, centrifuging the precipitate at 5000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 11
Extraction of yolk antibody against crucian gill hemorrhage virus (CyHV-2)
1. Washing high-immunity eggs for resisting crucian gill hemorrhage virus (CyHV-2) with normal temperature tap water for 1min, and disinfecting and cleaning eggs with 0.25mg/L chlorine dioxide water solution for 1 min; spraying sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 100r/min for 20min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 12.5 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 300r/min for 30min, and standing and precipitating at room temperature for 12 h; sucking the supernatant with vacuum pump, precipitating, centrifuging at 6000r/min for 30min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
Example 12
Extraction of yolk antibody against crucian gill hemorrhage virus (CyHV-2)
1. Washing high-immunity eggs for resisting crucian gill hemorrhage virus (CyHV-2) with normal temperature tap water for 1min, and disinfecting and cleaning eggs with 0.25mg/L chlorine dioxide water solution for 2 min; spraying the sterilized egg with tap water for 2min, removing disinfectant, separating egg shell, egg white and yolk in an egg white and yolk separator, collecting yolk, weighing, and stirring at 300r/min for 10min to obtain yolk liquid;
2. adding the carbon dots of the novel zero-dimensional carbon-based nano material into an acetic acid-sodium acetate buffer solution with the concentration of 0.1M and the pH value of 5.1 to prepare a yolk antibody extraction buffer solution with the carbon dot concentration of 25 mug/mL;
3. adding a yolk antibody extraction buffer solution into the yolk solution according to the mass-to-volume ratio of 1:9, stirring at 300r/min for 15min, and standing and precipitating at room temperature for 15 h; sucking the supernatant with vacuum pump, precipitating, centrifuging at 7000r/min for 25min, collecting supernatant, and mixing the supernatants to obtain yolk antibody extractive solution.
In the above embodiments, the carbon dot preparation method comprises: the zero-dimensional carbon-based nano material with the size below 50nm is prepared from vitamin C by a one-step electrochemical method. The zero-dimensional carbon-based nano carbon dots with different concentrations are added into the purified IgY protein solution, and the influence of the carbon dots with different concentrations on the structure of the IgY protein is analyzed through circular dichroism chromatography, so that the protein structure and the stability of the IgY are basically not influenced when the concentration of the carbon dots is lower than 25 mu g/mL, the peak height between 190 and 210nm is slightly reduced when the concentration of the carbon dots reaches 50 mu g/mL, and the stability of the secondary structure of the IgY is slightly lower (see figure 1). Therefore, the nano carbon dots with low concentration below 25 mug/mL can be used for extracting the yolk antibody.
Second, result analysis
1. And (3) determining the protein content of the crude yolk antibody against the turbot IL-1 beta. The content of water-soluble protein in the yolk antibody extract was measured by using BCA protein concentration assay kit (purchased from Beijing Solebao Biotech Co., Ltd.), and the results are shown in Table 1.
TABLE 1 efficiency of extracting water-soluble protein from yolk (mg/g yolk) with different concentrations of carbon dots and different ratios of buffers
Figure BDA0002756100530000081
Figure BDA0002756100530000091
Note: all data with different shoulder letters indicated significant differences (P < 0.05).
As is clear from Table 1, the concentration of the water-soluble protein in the yolk antibody extract of each test group increased as the concentration of the carbon dots increased. Comprehensively comparing, when the carbon dot concentration is 25 mug/mL, the yolk antibody extraction effect of the group with the buffer ratio of 1:9 is the best, the extremely significant difference (P < 0.01) is achieved, and then the group with the buffer ratio of 1:7 (P <0.05) is achieved; protein extraction efficiency also reached significant levels (P <0.05) when the carbon spot concentration was 12.5. mu.g/mL and the buffer ratio was 1: 9. It is shown that the addition of carbon dots aids in the extraction of water-soluble proteins.
2. And (3) visually measuring the amount of IgY in the crude extract of the anti-turbot IL-1 beta yolk antibody. Because a certain amount of impure protein exists in the egg yolk antibody extracting solution, in order to further visually detect the extraction effect of IgY in the anti-turbot IL-1 beta egg yolk antibody extracting solution, the egg yolk antibody extracting solution is precipitated by using an ammonium sulfate solution (national drug group, AR level, CAS: 7783-20-2) with the mass-volume ratio of 19%, then the egg yolk antibody extracting solution is re-suspended by using PBS (0.1M, pH 7.2) buffer solution with the same volume as the egg yolk antibody extracting solution, and then SDS-PAGE electrophoresis is carried out to identify the antibody extraction effect, and the result is shown in figure 2. Wherein M: protein Marker, lanes 1-3: carbon spot concentration of 0. mu.g/mL, lanes 4-6 (examples 1-3): carbon spot concentration of 6.25. mu.g/mL, lanes 7-9 (examples 4-6), respectively: carbon spot concentrations of 12.5. mu.g/mL, lanes 10-12 (examples 7-9), respectively: respectively, the carbon dot concentration is 25 mug/mL; the yolk to buffer mass/volume ratio in lanes 1, 4, 7, 10 was 1:5, the yolk to buffer mass/volume ratio in lanes 2, 5, 8, 11 was 1:7, and the yolk to buffer mass/volume ratio in lanes 3, 6, 9, 12 was 1: 9.
As shown in FIG. 2, the SDS-PAGE electrophoresis of the yolk antibody shows the heavy IgY chain of about 66kDa, the light IgY chain of about 25kDa, and two hetero-protein bands of about 40 kDa. From the analysis of antibody purity (i.e., band gray scale), the antibody extraction effect was the best when the carbon spot concentration was 6.25. mu.g/mL in the same buffer solution ratio group, and the difference was not significant in the other groups, which were the second group with a concentration of 12.5. mu.g/mL. The result is inconsistent with the result of the content of the water-soluble protein in the yolk antibody extracting solution, which shows that the water-soluble hybrid protein of part of non-yolk antibodies is improved when the yolk antibody is extracted by the high-concentration carbon points, and the extraction of the yolk antibody IgY is more facilitated by the low-concentration carbon points.
3. And (3) measuring the titer of the IgY in the crude extract of the anti-turbot IL-1 beta yolk antibody and the anti-crucian gill hemorrhagic disease virus (CyHV-2) yolk antibody. The antibody titer in the yolk antibody extracting solution is determined by adopting an indirect ELISA method, namely recombinant proteins of turbot IL-1 beta and crucian gill hemorrhage virus CyHV-2(ORF72, ORF66, ORF81 and ORF82 tandem genes, namely CyHV-2-D4ORF) are respectively used as coating antigens, yolk antibody extracting solutions of anti-turbot IL-1 beta and anti-CyHV-2-D4 ORF with different dilutions are respectively used as primary antibodies, and the antibody titer in the yolk antibody extracting solution is determined by adopting a 1: and detecting the antibody titer by using HRP goat anti-chicken IgY diluted by 2000 as a secondary antibody, wherein a negative control is a yolk antibody extracted from a common egg under the same condition, and a blank control is PBS. The judgment standard is as follows: and (2) P/N (OD value to be measured-blank control OD value)/(negative control OD value-blank control OD value), and when P/N is more than or equal to 2.1, the maximum dilution of the antibody is the antibody titer. The results are shown in FIGS. 3 and 4, respectively.
As can be seen from the ELISA results in FIG. 3, when the ratio of yolk to the buffer solution was 1:5, the antibody titer in the yolk antibody extract of each group was increased from 1:16000 to 1:32000 in the control group by adding carbon dots to the acetic acid-sodium acetate buffer solution; when the ratio of the yolk to the buffer solution is 1:7, the antibody titer of the groups with the carbon spot concentration of 6.25 mug/mL and 12.5 mug/mL is improved from 1:16000 to 1:32000, but the antibody titer of the group with the carbon spot concentration of 25 mug/mL is not different from that of the group without the carbon spot; when the ratio of yolk to the buffer solution was 1:9, the antibody titer of the control group was 1:8000, the antibody titer of the group with a carbon spot concentration of 25. mu.g/mL was 1:16000, and the antibody titers of the groups with carbon spot concentrations of 6.25. mu.g/mL and 12.5. mu.g/mL reached 1: 32000. this result indicates that the addition of carbon dots contributes to the extraction of IgY, and that the extraction of IgY from low-concentration carbon dots is more effective.
The antibody titer in the egg yolk antibody extract against crucian gill hemorrhage virus (CyHV-2-D4ORF) was determined by ELISA method, and the results are shown in FIG. 4. When the ratio of yolk to buffer was 1:9, the antibody titers of the control group were 1:8000, and those of the groups with a carbon spot concentration of 6.25. mu.g/mL, 12.5. mu.g/mL and 25. mu.g/mL were 1:16000, 1:16000 and 1:8000, respectively. The results further verify that the effect of extracting IgY from the low-concentration carbon spots is best and the titer is obviously improved.
4. And (3) measuring the bacterial content in the crude egg yolk antibody extract for resisting crucian gill hemorrhage virus (CyHV-2). The total bacterial content of the carbon-point-treated egg yolk antibody extract against crucian gill hemorrhage virus (CyHV-2) was determined by plating with LB solid plates (example 10-12). Mixing yolk antibody extractive solution and yolk original fat precipitate, coating 100 μ L, culturing for 24 hr, and counting colony number.
The influence of carbon points with different concentrations on the total bacteria content in the egg yolk antibody extracting solution for resisting crucian gill hemorrhage virus (CyHV-2-D4ORF) is measured by an LB solid plate coating method, and the difference of the bacteria content of 15h of standing precipitation at room temperature and 4 ℃ when the processing parameters are the same. The results are shown in FIG. 5. The results show that: when other parameters are the same, the bacteria content of the antibody liquid extracted at room temperature is higher than that of the antibody liquid extracted at 4 ℃; after the carbon dots are added into the buffer solution, the content of bacteria in the yolk antibody extracting solution is obviously reduced (P is less than 0.05), and the colony number is reduced along with the increase of the concentration of the carbon dots until the growth of the bacteria is completely inhibited. The method can realize the extraction of the yolk antibody at room temperature under the condition of adding carbon points with certain concentration, and the carbon points can be degraded to cause no toxicity, so that byproducts after the yolk antibody is extracted can be further developed and utilized. Therefore, the technological parameters can be changed from 4 ℃ to room temperature after the nano material is added, and the method is simple and convenient. The utilization rate is improved.
And (4) conclusion: comprehensively evaluating the influence of carbon dots with different concentrations and buffer solutions with different proportions on the yolk antibody extraction effect, the invention finally determines that when the carbon dot concentration is 6.25-12.5 mug/mL, the ratio of the yolk solution to the buffer solution is 1: 5-1: 9, the yolk antibody can achieve better extraction effect. Further, when high concentrations of antibody are desired, the antibody can be further concentrated by other technical means.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.

Claims (3)

1. A method for extracting yolk antibodies in an industrialized large scale is characterized by comprising the following steps:
(1) washing and sterilizing high-immunity egg white, separating to obtain egg yolk, and stirring to obtain egg yolk liquid;
(2) adding a nano carbon dot into an acetic acid-sodium acetate buffer solution with the pH value of 5.0-5.2 to prepare a yolk antibody extraction buffer solution containing the carbon dot, wherein the concentration of the nano carbon dot in the acetic acid-sodium acetate buffer solution is 12.5 mu g/ml, the nano carbon dot is a zero-dimensional carbon-based nano material with the size below 50nm, which is prepared by vitamin C through a one-step electrochemical method, and the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.5M;
(3) adding the yolk solution into the yolk antibody extraction buffer solution, stirring uniformly, standing at room temperature for precipitation to obtain a supernatant, centrifuging the precipitate to obtain a supernatant, and combining the supernatants to obtain a yolk antibody extract, wherein the mass volume ratio of the yolk solution to the yolk antibody extraction buffer solution is 1: (7-9).
2. The method for extracting yolk antibody in industrial large scale according to claim 1, wherein the method comprises the following steps: in the step (1), the stirring speed is 50-500 r/min, and the time is 10-20 min.
3. The method for extracting yolk antibody in large scale is characterized in that the specific process of step (3) is as follows: mixing yolk liquid according to the mass volume ratio of 1: (7-9), adding the yolk antibody into the yolk antibody extraction buffer solution in a ratio of 50-500 r/min, stirring for 10-20 min, and standing and precipitating for 8-15 h at room temperature; and sucking the supernatant, centrifuging the precipitate at 4000-12000 r/min for 10-30 min, taking the supernatant, and combining the supernatants to obtain the egg yolk antibody extracting solution.
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