阅读说明：本技术 一种针对中华绒螯蟹螺原体的特异性卵黄抗体及其制备方法和应用 (Specific egg yolk antibody for eriocheir sinensis spiroplasma as well as preparation method and application of specific egg yolk antibody ) 是由 曹晓慧 刘鸿丽 顾伟 王文 孟庆国 于 2021-06-16 设计创作，主要内容包括：本发明公开了一种针对中华绒螯蟹螺原体的特异性卵黄抗体及其制备方法和应用,所述抗体为抗S.eriocheiris的特异性IgY抗体,是由破碎后的螺原体注射进母鸡体内,从鸡蛋的卵黄中分离并纯化得到的特异性抗体。该特异性抗体可通过饲料饲喂或注射等方式应用于中华绒螯蟹抵抗S.eriocheiris的感染中,结果证明与现有的治疗中华绒螯蟹颤抖病的方法相比,本发明的方法对环境无污染、针对性强、效果显著。本发明成功地证明了抗S.eriocheiris特异性IgY在中华绒螯蟹抵抗S.eriocheiris感染过程中的潜在作用,并可以应用于水产养殖中以提高中华绒螯蟹的产量和渔民的收入,促进中华绒螯蟹养殖产业的发展。(The invention discloses a specific egg yolk antibody for eriocheir sinensis spiroplasma and a preparation method and application thereof, wherein the antibody is a specific IgY antibody for resisting S.eriocheiris, and is obtained by injecting crushed spiroplasma into a hen body, and separating and purifying egg yolk of eggs. The specific antibody can be applied to the eriocheir sinensis to resist the infection of the S.eriocheiris in the modes of feed feeding or injection and the like, and the result proves that compared with the existing method for treating the tremble disease of the eriocheir sinensis, the method disclosed by the invention has the advantages of no pollution to the environment, strong pertinence and obvious effect. The invention successfully proves the potential effect of the anti-S.eriochelis specific IgY in the process of resisting S.eriochelis infection of the Eriocheir sinensis, and can be applied to aquaculture to improve the yield of the Eriocheir sinensis and the income of fishermen and promote the development of the Eriocheir sinensis aquaculture industry.)

1. A specific egg yolk antibody aiming at the eriocheir sinensis spiroplasma is characterized in that the antibody is a specific IgY antibody for resisting S.eriocheiris, and the specific antibody is obtained by injecting crushed spiroplasma into a hen body, separating and purifying egg yolk of an egg.

2. The method for preparing the specific egg yolk antibody against the eriocheir sinensis spiroplasma of claim 1, which is characterized by comprising the following steps of:

step 1) culturing s.eriocheiris in R2 medium for 24h, centrifuging to remove supernatant, and resuspending with PBS;

step 2) carrying out ultrasonic crushing on the S.eriocheiris subjected to the resuspension in the step 1), and storing at 20 ℃ for later use;

step 3) intramuscular injection is carried out on the hens, 1mL of S.eriocheiris crushed in the step 2) is injected, 200-400 mu g/mL of S.eriocheiris is injected, and specific antibody IgY is produced;

and 4) injecting the hens for 3 times at an interval of 2 weeks, collecting eggs every day until the ninth week, extracting egg yolks of the eggs, separating IgY from the egg yolks, and purifying to obtain the IgY.

3. Use of a specific egg yolk antibody against spirochete eriocheiriae of claim 1 to inhibit the growth of s.

4. Use according to claim 3, characterized in that it comprises the following steps:

step a), separating and purifying IgY from the yolk of the S.eriocheiris injected by 200-400 mu g/mL;

step b) diluting the IgY obtained in the step a) according to the titer of 1: 1-30000 to obtain specific IgY;

step c) inoculating S.eriocheiris into an R2 culture medium, adding the specific IgY obtained in the step b), mixing the IgY and the culture medium according to the ratio of 1: 4-100, and observing the growth condition of the S.eriocheiris after culturing for 0-48 h.

5. The use according to claim 4, wherein in the egg yolk of the chicken of step a) the concentration of injected S.erochelis is 400 μ g/mL; the titer of the step b) is 1: 10000; step c) the ratio of IgY to medium is 1: 4.

6. The use of a specific egg yolk antibody against the spiroplasma eriocheir sinensis of claim 1 for the protection of eriocheir sinensis against s.

7. Use according to claim 6, characterized in that it comprises the following steps:

step A) separating and purifying IgY from the yolk of chickens injected with 400 mug/mL of S.eriocheiris; diluting the separated and purified IgY in a ratio of titer of 1:10000 to obtain specific IgY;

step B-1) mixing the specific IgY prepared in the step A) with feed in a mixing mass ratio of 1:4, and preparing mixed feed by using egg white as an adhesive;

and C-1) exposing the mixed feed prepared in the step B-1) to the sun until the mixed feed is dried, feeding the eriocheir sinensis, wherein the feeding rate is 1% per day, and observing the result after feeding for 15 days.

8. Use according to claim 6, characterized in that it comprises the following steps:

step A) separating and purifying IgY from the yolk of chickens injected with 400 mug/mL of S.eriocheiris; diluting the separated and purified IgY in a ratio of titer of 1:10000 to obtain specific IgY;

step B-2) mixing the specific IgY prepared in the step A) with PBS according to the volume ratio of 1:4 to prepare an injection;

and C-2) injecting the injection prepared in the step B-2) into the eriocheir sinensis, and observing the result after 15 days.

9. The use of a specific egg yolk antibody against the spiroplasma Eriocheir sinensis of claim 1 in aquaculture.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a specific egg yolk antibody for a Eriocheir sinensis spiroplasma and a preparation method and application thereof.

Background

The eriocheir sinensis is commonly called river crab, is one of important breeding varieties in the aquaculture industry of China, and with the rapid increase of the international and domestic market demands, the industry thereof also develops rapidly to meet the market demands. With the intensive development of the eriocheir sinensis industry, the disease outbreak caused by various bacteria, viruses, parasites or physical and chemical factors severely restricts the sustainable development of the eriocheir sinensis industry, wherein the shiver disease caused by the eriocheir sinensis Spiroplasma (spiroplama eriocheiris) is the most destructive disease for the eriocheir sinensis breeding industry, and a series of research results on the pathogenic mechanism, detection and prevention work of the disease are obtained at present. At present, the treatment of the disease mainly depends on the use of antibiotics such as oxytetracycline, but the abuse of chemical drugs such as antibiotics can cause a series of problems such as drug residues, drug-resistant bacteria generation, environmental pollution, aquatic animal immune system damage and the like. The spread of drug-resistant bacteria can greatly reduce the choice of available preventive measures, increase the difficulty of healing and lead to death caused by infection, and therefore, an effective and pollution-free antibiotic substitute is urgently needed.

Currently, there is no study on the use of specific IgY against s.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to verify the effect of IgY in the prevention and treatment of S.eriocheiris infection of Eriocheir sinensis, so as to expect that the specific anti-S.eriocheiris IgY is used for preventing and treating the shaking disease of the Eriocheir sinensis, further increase the survival rate of the Eriocheir sinensis in the S.eriocheiris infection process, and improve the yield of the Eriocheir sinensis and the income of fishermen. Therefore, the invention provides a specific egg yolk antibody for the Eriocheir sinensis spiroplasma and a preparation method and application thereof.

The invention is realized by the following technical scheme:

a specific egg yolk antibody aiming at the Eriocheir sinensis spiroplasma is a specific IgY antibody for resisting S.eriocheiris, and is obtained by injecting crushed spiroplasma into a hen body, separating and purifying egg yolk of eggs.

A preparation method of a specific egg yolk antibody aiming at the Eriocheir sinensis spiroplasma comprises the following steps:

step 1) culturing s.eriocheiris in R2 medium for 24h, centrifuging to remove supernatant, and resuspending with PBS;

step 2) carrying out ultrasonic crushing on the S.eriocheiris subjected to the resuspension in the step 1), and storing at 20 ℃ for later use;

step 3) intramuscular injection is carried out on the hens, 1mL of S.eriocheiris crushed in the step 2) is injected, 200-400 mu g/mL of S.eriocheiris is injected, and specific antibody IgY is produced;

and 4) injecting the hens for 3 times at an interval of 2 weeks, collecting eggs every day until the ninth week, extracting egg yolks of the eggs, separating IgY from the egg yolks, and purifying to obtain the IgY.

An application of a specific egg yolk antibody aiming at the Eriocheir sinensis spiroplasma in inhibiting the growth of S.

The application of a specific egg yolk antibody aiming at the eriocheir sinensis spiroplasma in the aspect of inhibiting the growth of S.eriocheiris comprises the following steps:

step a), separating and purifying IgY from the yolk of the S.eriocheiris injected by 200-400 mu g/mL;

step b) diluting the IgY obtained in the step a) according to the titer of 1: 1-30000 to obtain specific IgY;

step c) inoculating S.eriocheiris into an R2 culture medium, adding the specific IgY obtained in the step b), mixing the IgY and the culture medium according to the ratio of 1: 4-100, and observing the growth condition of the S.eriocheiris after culturing for 0-48 h.

Preferably, in the egg yolk of the chicken of step a), the concentration of injected s.eriocheiris is 400 μ g/mL; the titer of the step b) is 1: 10000; step c) the ratio of IgY to medium is 1: 4.

An application of a specific egg yolk antibody aiming at the Eriocheir sinensis spiroplasma in resisting S.

The application of the specific egg yolk antibody aiming at the eriocheir sinensis spiroplasma in resisting S.eriocheiris infection of the eriocheir sinensis comprises the following steps:

step A) separating and purifying IgY from the yolk of chickens injected with 400 mug/mL of S.eriocheiris; diluting the separated and purified IgY in a ratio of titer of 1:10000 to obtain specific IgY;

step B-1) mixing the specific IgY prepared in the step A) with feed in a mixing mass ratio of 1:4, and preparing mixed feed by using egg white as an adhesive;

and C-1) exposing the mixed feed prepared in the step B-1) to the sun until the mixed feed is dried, feeding the eriocheir sinensis, wherein the feeding rate is 1% per day, and observing the result after feeding for 15 days.

The application of the specific egg yolk antibody aiming at the eriocheir sinensis spiroplasma in resisting S.eriocheiris infection of the eriocheir sinensis comprises the following steps:

step A) separating and purifying IgY from the yolk of chickens injected with 400 mug/mL of S.eriocheiris; diluting the separated and purified IgY in a ratio of titer of 1:10000 to obtain specific IgY;

step B-2) mixing the specific IgY prepared in the step A) with PBS according to the volume ratio of 1:4 to prepare an injection;

and C-2) injecting the injection prepared in the step B-2) into the eriocheir sinensis, and observing the result after 15 days.

An application of a specific egg yolk antibody aiming at the Eriocheir sinensis spiroplasma in aquaculture.

The invention has the following beneficial effects:

IgY has certain advantages in cost-effectiveness, convenience, yield and no environmental pollution when used for prevention and disease control, and the IgY passive immunization method has also been widely applied to control of aquatic animal diseases. Therefore, an anti-s.eriocheirisis igy antibody extracted from the egg yolk of pre-immunized laying hens is a promising alternative for the treatment of crab tremble disease. The invention successfully proves the potential effect of the anti-S.eriochelis specific IgY in the process of resisting S.eriochelis infection of the Eriocheir sinensis, and can be applied to aquaculture to improve the yield of the Eriocheir sinensis and the income of fishermen and promote the development of the Eriocheir sinensis aquaculture industry.

Drawings

FIG. 1 is a schematic flow diagram of the present invention;

FIG. 2 shows the Optical Density (OD) of ELISA plate wells measured at 450nm at an antibody titer of 1:10,000 for IgY produced by injection of 400. mu.g/mL antigen;

FIG. 3 shows the survival rate of Eriocheir sinensis after feeding with IgY-containing feed;

in fig. 3: a is a commercial feed group fed with Eriocheir sinensis injected with PBS; b is a commercial feed group fed after injecting S.eriocheiris to the eriocheir sinensis; c, feeding the eriocheir sinensis with a feed group containing common IgY after S.eriocheiris injection; d, feeding a specific IgY feed group after injecting S.eriocheiris to the eriocheir sinensis;

FIG. 4 shows the survival rate of Eriocheir sinensis after IgY injection;

in fig. 4: e is eriocheir sinensis PBS injection; f is eriocheir sinensis injection S.eriocheirii; g is eriocheir sinensis injected S.eriocheiris + common IgY; h is eriocheir sinensis injected S.eriocheiris + specific IgY.

FIG. 5 shows the copy number of the S.eriocheiris in the hemolymph cells of Eriocheir sinensis after the feed containing IgY is fed;

FIG. 6 shows the copy number of S.eriocheiris in Eriocheir sinensis hemolymphocytes after IgY injection;

FIG. 7 shows the morphological changes of eriocheiris in eriocheir sinensis hemolymphocytes after the feed containing IgY is fed;

fig. 8 is a morphological change of s.eriocheiris in eriocheir sinensis hemolymphocytes after IgY injection.

Detailed Description

The invention is described in further detail below with reference to specific embodiments and with reference to the following drawings.

Example 1

A specific egg yolk antibody aiming at the Eriocheir sinensis spiroplasma is a specific IgY antibody for resisting S.eriocheiris, and is obtained by injecting crushed spiroplasma into a hen body, separating and purifying egg yolk of eggs.

A preparation method of a specific egg yolk antibody aiming at the eriocheir sinensis spiroplasma comprises the following specific steps:

(1) eriocheiris was cultured in R2 medium for 24h, then centrifuged to remove the supernatant, and resuspended in PBS.

(2) The resuspended s.erochelis was sonicated (500W, 6min, 5s sonicated at 9s intervals) and stored in a refrigerator at 20 ℃ until needed.

(3) The 8 hens were divided into 4 groups and injected intramuscularly as shown in figure 1, as follows:

group A: injecting 1mL PBS as a control group to generate non-specific IgY/normal IgY;

group B: injecting 1mL of S.eriocheiris after 200 mug/mL of disruption to produce specific antibody IgY;

group C: injecting 1mL of S.eriocheiris after 300 mug/mL of disruption to produce specific antibody IgY;

group D: specific antibody IgY was produced by injecting 1mL of disrupted S.eriocheiris at 400. mu.g/mL.

(4) Injecting each chicken for 3 times at an interval of 2 weeks, and collecting eggs every day until the ninth week; extracting egg yolk, separating IgY from the yolk, and purifying with ammonium sulfate.

Example 2 selection of optimal antigen concentration and optimal dilution

ELISA assays were performed on specific IgY (1:1) isolated and purified from yolk of chickens injected with 200. mu.g/mL, 300. mu.g/mL and 400. mu.g/mL of S.eriocheiris, and three dilutions of IgY (1:30,000, 1:20,000 and 1:10,000), as shown in FIG. 1. The diluent was PBST + 5% fetal bovine serum (PBST ═ PBS (pH 7.4,0.01M) + 0.05% Tween-20.). The titer of specific IgY was determined by measuring the Optical Density (OD) of ELISA plate wells at 450/630nm using a microplate reader. The results showed that the best specific IgY was obtained by injecting 400. mu.g/mL of antigen to produce IgY with a titer of 1:10,000.

As shown in FIG. 2, the antibody titer of IgY produced by injection of 400. mu.g/mL of antigen was 1:10,000. Where each group was repeated 3 times (n-3). After specific IgY generated by injecting 400 mu g/mL antigen is diluted by 1:10,000, ELISA experiments are carried out by using the antigen concentrations of 100, 50 and 20g/mL respectively, and the result shows that the OD value at 450/630nm can reach 0.8 in the fourth week when the antigen concentrations of the IgY generated by injecting 400 mu g/mL antigen are 100, 50 and 20g/mL respectively, and the lowest antigen concentration of the IgY is 20 g/mL. The above results indicate that the specific IgY produced by injecting 400. mu.g/mL antigen and diluted to 10,000 is the IgY that reacts most rapidly with the antigen, and the OD value can reach 0.8 even if the antigen concentration is only 20g/mL in the fourth week after the first immunization.

Example 3 growth inhibition experiment of s

The effect of specific IgY on inhibiting the growth of S.eriocheiris was examined by growth inhibition. R2 medium was inoculated with s.eriocheiris and purified IgY was added, mixed with the medium in three different ratios: growth of s. eriocheiris was observed at 1:100, 1:10 and 1:4 after 0h, 12h, 24h and 48h of culture. The experiment was divided into four groups, namely a negative control group, a positive control group, a normal IgY group and an s. The s.eriochelis growth inhibition effect was determined by comparing the color change of R2 medium between groups, the samples with color change consistent with the positive control were the IgY concentrations that did not inhibit s.eriochelis growth, while the samples showing similar color to the negative control were the IgY concentrations that best inhibited s.eriochelis growth. The results show that a ratio of 1:4 is most suitable for the application of s.

Example 4 infection experiments and IgY applications

The IgY neutralization experiment and application are to prove the function of specific IgY in resisting S. Eriocheiris IgY is expected to affect survival of eriocheir sinensis during s.eriocheiris infection due to its specificity and sensitivity to s.eriocheiris, and determines the optimal concentration of injected or fed specific IgY.

Purchasing 50 +/-3 g of Chinese mitten crabs, culturing for 1 week in 10L of water tanks with a circulating water function, an ultraviolet sterilization and water temperature control system before an experiment, and culturing 10 Chinese mitten crabs in each water tank, wherein PCR detection and electron microscope negative dye observation prove that the Chinese mitten crabs used in the experiment are negative in S.

1. Application in feed

The IgY feed for oral administration is a commercial feed mixed with general IgY or specific IgY, and egg white is used as a binder, as shown in fig. 1. The ratio of the IgY to the feed is 1:4(w/w), the feed is exposed to the sun until the feed is dry, the feeding rate is 1% per day, and the feeding time is 15 days. The experiments were carried out in 4 groups: taking Eriocheir sinensis which is not infected with spiroplasma and is fed with commercial feed as a negative control (A); eriocheir sinensis injected with s.eriocheiris and fed with commercial feed daily as a positive control (B); injecting s.eriocheiris, feeding feed containing common IgY daily as control group (C); eriocheiris injected, feed containing specific IgY fed daily was the experimental group (D).

As shown in FIG. 3, the survival rates of Eriocheir sinensis in the four groups were 57% (A), 3% (B), 3% (C) and 43% (D), respectively, at the end of the experiment after 15 days. The result shows that the specific IgY has obvious influence on the survival rate in the process of infecting the Eriocheir sinensis with the spiroplasma. Eriocheiris resistant specific IgY can help eriocheir sinensis to better survive, and the survival rate of the eriocheir sinensis is higher than that of the diseased river crab fed with commercial feed or common IgY.

2. Use in injection

Another 4 groups of different Eriocheir sinensis were injected with IgY as shown in FIG. 1. The grouping is as follows: eriocheir sinensis injected with PBS was used as a negative control (E); eriocheir sinensis injected with s.eriocheiris was used as a positive control (F); eriocheir sinensis injected with a mixture of s.eriocheiris and common IgY was used as a control group (G); eriocheir sinensis injected with a mixture of s.eriocheiris and anti-s.eriocheiris specific IgY was the experimental group (H). The injection is prepared from a mixture of IgY and PBS, and the mixing volume ratio is 1: 4.

As shown in fig. 4, the results after 15 days of feeding showed that the survival rates of the four groups were 70% (E), 5% (F), 15% (G) and 50% (H), respectively. Experimental results show that the survival rate of the Eriocheir sinensis injected with the specific IgY is higher than that of the Eriocheir sinensis injected with the IgY group and the ordinary IgY group.

3. Results and mechanism study

The experimental results of the above 1 and 2 show that the survival rate of eriocheir sinensis to which anti-s.eriocheiris specific IgY is added using an injection or feeding method is remarkably increased during s.eriocheiris infection.

The copy number of S.eriocheiriis in Eriocheir sinensis blood cells at different time points after spiroplasma infection was determined by DNA extraction and real-time PCR using primers Se-QF (shown as SEQ ID NO. 1) and Se-QR (shown as SEQ ID NO. 2), as shown in FIGS. 5 and 6. The results show that the copy number of the spiroplasma in hemolymph of the eriocheir sinensis is obviously reduced after the anti-S.eriocheir specific IgY is fed (figure 5) or injected (figure 6) compared with the control group fed or injected with PBS and common IgY, which indicates that the amount of the S.eriocheir in the eriocheir sinensis can be effectively inhibited by the anti-S.eriocheir specific IgY whether the eriocheir sinensis is fed or injected, and the results are consistent with the survival rate results of the eriocheir sinensis.

TABLE 1 sequence listing of artificial primers

To investigate the inhibition mechanism of s.eriochelis by the anti-s.eriochelis specific IgY, the specific IgY was negatively stained after s.eriochelis treatment and the effect of the anti-s.eriochelis specific IgY on s.eriochelis morphological structure was observed using transmission electron microscopy, as shown in fig. 7 and 8. FIGS. 7(Group A) and 8(Group D) show that normal feed feeding is performed on Eriocheir sinensis after injecting the spiroplasma and normal spiroplasma is present in hemolymph cells of Eriocheir sinensis after injecting PBS, and the cell membrane is intact and the morphology is regular. FIGS. 7(Group B) and 8(Group E) show that the structure of the spiroplasma is slightly different and irregular compared with the normal spiroplasma, as shown by the arrows in FIGS. 7(Group B) and 8(Group E), when the eriocheir sinensis is fed with the spiroplasma containing the normal IgY and the hemolymph of the eriocheir sinensis after the injection of the normal IgY. Fig. 7(Group C) and 8(Group F) show that when the eriocheir sinensis is fed with the specific IgY-containing sample after the spiroplasma injection and the normal spiroplasma in the eriocheir sinensis hemolymphocytes after the specific IgY injection, the s.eriocheir morphological structure after the s.eriocheir-resistant specific IgY treatment is changed and shows an irregular, clustered, cleaved or membrane-solubilized form, as shown in fig. 7(Group C) and 8(Group F). Specific IgY treatment therefore causes a great disruption to the morphological structure of s.erochelis, which in turn affects the growth, reproduction and infection of s.erochelis.

In conclusion, compared with the existing method for treating the tremble disease of the Eriocheir sinensis, the method disclosed by the invention has the advantages of no pollution to the environment, strong pertinence and obvious effect. Compared with PBS or nonspecific IgY, the specific IgY for resisting S.eriocheiris can obviously improve the survival rate of Eriocheir sinensis, reduce the copy number of S.eriocheiris, change the morphological structure of S.eriocheiris and crack or dissolve the membrane of S.eriocheiris.

Sequence listing

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