CN113527480B - Specific egg yolk antibody for Eriocheir sinensis helicobacter, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a specific egg yolk antibody for Eriocheir sinensis helicobacter, a preparation method and application thereof, wherein the antibody is a specific IgY antibody for resisting S.eriocheiris, and is a specific antibody obtained by injecting crushed helicobacter into a hen body, and separating and purifying egg yolk of eggs. The specific antibody can be applied to the infection of Eriocheir sinensis against S.eriocheir by feeding or injecting, and the result shows that compared with the existing method for treating the trembling disease of Eriocheir sinensis, the method provided by the invention has the advantages of no environmental pollution, strong pertinence and remarkable effect. The invention successfully proves the potential effect of the anti-S.eriocheir specificity IgY in the S.eriocheir infection resisting process, can be applied to aquaculture to improve the yield of the eriocheir sinensis and the income of fishermen, and promotes the development of the eriocheir sinensis aquaculture industry.

Description

Specific egg yolk antibody for Eriocheir sinensis helicobacter, and preparation method and application thereof

Technical Field

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

Background

Eriocheir sinensis commonly called as river crab is one of important cultured varieties in Chinese aquaculture industry, and along with the rapid increase of international and domestic market demands, the industry of Eriocheir sinensis rapidly develops to meet the market demands. Along with the intensive development of the Eriocheir sinensis industry, the outbreak of diseases caused by various bacteria, viruses, parasites or physical and chemical factors has caused serious restrictions on the sustainable development of the industry, wherein tremble diseases caused by Eriocheir sinensis spiroplasma (Spiroplasma eriocheiris) are the most destructive diseases for the Eriocheir sinensis breeding industry, and a series of research results have been achieved for the pathogenesis, detection and prevention work of the diseases at present. At present, the treatment of the disease mainly depends on the use of antibiotics such as terramycin, but abuse of chemical drugs such as antibiotics can cause a series of problems such as drug residues, drug-resistant bacteria generation, environmental pollution, and damage to the immune system of aquatic animals. The spread of resistant bacteria greatly reduces the choice of available control measures and increases the difficulty of healing and causes death from infection, and therefore, there is a strong need for effective and pollution-free antibiotic alternatives.

At present, 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 preventing and treating S.eriocheiris infection of eriocheir sinensis so as to expect to use specific anti-S.eriocheiris IgY to prevent and treat trembling diseases of eriocheir sinensis, thereby increasing the survival rate of eriocheir sinensis in the S.eriocheir infection process and improving the yield of eriocheir sinensis and income of fishermen. Therefore, the invention provides a specific egg yolk antibody for the helicobacter pylori of Eriocheir sinensis and a preparation method and application thereof.

The invention is realized by the following technical scheme:

a specific egg yolk antibody for the spiroplasma Eriocheir sinensis is a specific IgY antibody against S.eriocheiris, which is obtained by injecting crushed spiroplasma into hen body, separating and purifying egg yolk.

A preparation method of a specific egg yolk antibody for the helicobacter pylori of Eriocheir sinensis comprises the following steps:

step 1) culturing S.eriocheiris in an R2 culture medium for 24 hours, centrifuging to remove supernatant, and re-suspending with PBS;

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

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

step 4) injecting hen 3 times, every 2 weeks, collecting eggs every day until the ninth week, extracting egg yolk, separating IgY from the yolk, and purifying.

An application of specific egg yolk antibody against Eriocheir sinensis helicobacter in inhibiting S.eriocheir growth is provided.

An application of a specific egg yolk antibody aiming at the helicobacter pylori of Eriocheir sinensis in inhibiting the growth of S.eriocheiris, comprising the following steps:

step a) IgY is separated from chicken yolk injected with 200-400 mug/mL S.eriocheiris and purified;

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

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

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

An application of specific egg yolk antibody against Eriocheir sinensis helicobacter sp in resisting S.eriocheir infection.

An application of a specific egg yolk antibody aiming at Eriocheir sinensis helicobacter sp in resisting S.eriocheir infection of Eriocheir sinensis, comprising the following steps:

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

step B-1), mixing the specific IgY prepared in the step A) with feed, wherein the mixing mass ratio is 1:4, and using egg white as an adhesive to prepare mixed feed;

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

An application of a specific egg yolk antibody aiming at Eriocheir sinensis helicobacter sp in resisting S.eriocheir infection of Eriocheir sinensis, comprising the following steps:

step A) IgY is separated from chicken yolk injected with 400 mug/mL of S.eriocheiris and purified; diluting the separated and purified IgY in a ratio 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;

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

An application of specific egg yolk antibody against Eriocheir sinensis helicobacter in aquaculture.

The beneficial effects of the invention are as follows:

the use of IgY for prophylaxis and disease control has certain advantages in terms of cost effectiveness, convenience, yield, no environmental pollution, etc., and the passive immunization of IgY has also been widely used for controlling diseases of aquatic animals. Thus, anti-s.eriocheirisigy antibodies extracted from egg yolk of pre-immunized laying hens are a promising alternative to the treatment of trembling diseases of eriocheir sinensis. The invention successfully proves the potential effect of the anti-S.eriocheir specificity IgY in the S.eriocheir infection resisting process, can be applied to aquaculture to improve the yield of the eriocheir sinensis and the income of fishermen, and promotes the development of the eriocheir sinensis aquaculture industry.

Drawings

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

FIG. 2 is a graph showing the determination of the Optical Density (OD) of ELISA plate wells at 450nm with an antibody titer of 1:10,000 against IgY produced by injection of 400. Mu.g/mL of antigen;

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

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

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

in fig. 4: e is injection PBS of Eriocheir sinensis; f is eriocheiri injected into Eriocheir sinensis; g is eriocheir sinensis injection S+common IgY; h is eriocheir sinensis injection S.eriocheir+ specific IgY.

FIG. 5 shows the copy number of S.eriocheir sinensis in blood lymphocytes after feeding a feed containing IgY;

FIG. 6 shows the copy number of S.eriocheir sinensis in the blood lymphocytes of Eriocheir sinensis after IgY injection;

FIG. 7 shows the morphological changes of S.eriocheiris in Eriocheir sinensis blood lymphocytes after feeding with a feed containing IgY;

FIG. 8 shows the morphological changes of S.eriocheir sinensis in blood lymphocytes after IgY injection.

Detailed Description

The invention will be described in further detail with reference to the drawings and the specific embodiments.

Example 1

A specific egg yolk antibody for the spiroplasma Eriocheir sinensis is a specific IgY antibody against S.eriocheiris, which is obtained by injecting crushed spiroplasma into hen body, separating and purifying egg yolk.

A preparation method of a specific egg yolk antibody for the helicobacter Eriocheir sinensis comprises the following specific steps:

(1) S. eriocheiris was cultured in R2 medium for 24h and the supernatant was removed by centrifugation and resuspended in PBS.

(2) The resuspended S.eriocheiris was sonicated (500W, 6min, 5s sonicated, 9s apart) and kept in a refrigerator at 20deg.C for further use.

(3) 8 hens were grouped into 4 groups and given intramuscular injections, as shown in figure 1, as follows:

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

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

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

group D: specific antibody IgY was produced by injecting 1mL of 400. Mu.g/mL of crushed S.eriocheiris.

(4) 3 injections were performed for each chicken, with eggs collected daily for 2 weeks intervals until the ninth week; extracting ovum gallus Domesticus flavus, separating IgY from ovum gallus Domesticus flavus, and purifying with ammonium sulfate.

Example 2 selection of optimal antigen concentration and optimal dilution

ELISA assays were performed on specific IgY (1:1) and three IgY dilutions (1:30,000, 1:20,000 and 1:10,000) isolated and purified from chicken yolk injected with 200. Mu.g/mL, 300. Mu.g/mL and 400. Mu.g/mL of S.eriocheiris, as shown in FIG. 1. The dilutions were pbst+5% foetal calf serum (pbst=pbs (pH 7.4,0.01M) +0.05% tween-20.). The titers of specific IgY were determined by measuring the Optical Density (OD) of ELISA plate wells at 450/630nm using an enzyme-labeled instrument. The results show that the best specific IgY obtained is IgY produced by injection of 400. Mu.g/mL antigen, with a titer of 1:10,000.

As shown in FIG. 2, igY produced by injection of 400. Mu.g/mL antigen has an antibody titer of 1:10,000. Wherein each group was repeated 3 times (n=3). ELISA experiments are carried out by using specific IgY which is injected with 400 mu g/mL of antigen for 1:10,000 dilution and the antigen concentrations are respectively 100, 50 and 20g/mL, and the results show that when the antigen concentrations of IgY which is injected with 400 mu g/mL of antigen are respectively 100, 50 and 20g/mL, the OD value at 450/630nm can reach 0.8 at the fourth week, and the lowest antigen concentration of IgY is 20g/mL. The above results indicate that the specific IgY after injection of 400. Mu.g/mL of antigen produced and diluted 10,000 is the IgY that reacted the antigen most rapidly, and that the OD value reached 0.8 even if the antigen concentration was only 20g/mL at the fourth week after the first immunization.

Example 3S. growth inhibition experiment of eriocheiris

The effect of specific IgY on inhibition of s. Inoculating S.eriocheiris into the R2 culture medium, adding the purified IgY, and mixing the IgY with the culture medium according to three different ratios: growth of S.eriocheiris was observed after culturing for 0h, 12h, 24h and 48h at 1:100, 1:10 and 1:4. The experiments were divided into four groups, namely a negative control group, a positive control group, a general IgY group and an s. The inhibition of S.eriocheiris growth was determined by comparing the color change of the R2 medium between groups, the samples whose color change was consistent with the positive control were IgY concentrations that did not inhibit S.eriocheiris growth, while the samples that showed similar color to the negative control were IgY concentrations that optimally inhibited S.eriocheiris growth. The results show that a 1:4 ratio is most suitable for the use of S.eriocheiris-specific IgY.

Example 4 infection experiments and IgY use

The IgY neutralization experiment and application prove the effect of the specific IgY on the Eriocheir sinensis against S.eriocheir infection. anti-S.eriocheir IgY is expected to have an effect on Eriocheir sinensis survival during S.eriocheir infection due to its specificity and sensitivity to S.eriocheir, and the optimal concentration of the specific IgY to be injected or fed is determined.

The Eriocheir sinensis (50+/-3) g is purchased, and cultured in 10L water tanks with circulating water function, ultraviolet sterilization and water temperature control system for 1 week before experiment, 10 Eriocheir sinensis are cultured in each water tank, and the PCR detection and electron microscope negative dyeing observation prove that the Eriocheir sinensis used in the experiment is negative.

1. Application in feed

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. IgY is mixed with the feed in a ratio of 1:4 (w/w), the feed is exposed to sunlight until dry, the feeding rate is 1% per day, and the feeding time is 15 days. Experiments were performed in 4 groups: taking Eriocheir sinensis which does not infect helicobacter and is fed with commercial feed as a negative control (A); eriocheiris was injected and eriocheir sinensis fed commercial feed daily as positive control (B); injecting S.eriocheiris, and feeding the feed containing common IgY every day to obtain a control group (C); s. eriocheiris was injected and the experimental group (D) was fed daily with feed containing specific IgY.

As shown in FIG. 3, the survival rates of Eriocheir sinensis in the four groups at the end of the experiment after 15 days were 57% (A), 3% (B), 3% (C) and 43% (D), respectively. The results show that the specific IgY has a significant effect on the survival rate in the process of infecting Eriocheir sinensis by the helicobacter. The specific IgY for resisting S.eriocheiris can help eriocheir sinensis to survive better, and the effect is higher than the survival rate of the ill river crabs fed with commercial feed or fed with common IgY.

2. Application of injection

An additional 4 different sets of Eriocheir sinensis were subjected to IgY injections as shown in FIG. 1. The grouping is as follows: eriocheir sinensis injected with PBS was used as negative control (E); injecting eriocheir sinensis as positive control (F); eriocheir sinensis and ordinary IgY mixture are injected as a control group (G); eriocheir sinensis with S.eriocheir-specific IgY mixture was injected as experimental group (H). The injectate is made of a mixture of IgY and PBS with a mixing volume ratio of 1:4.

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

3. Results and mechanism study

The experimental results of the above 1 and 2 show that the survival rate of eriocheir sinensis added with anti-s.

The S.eriocheiris copy number in Eriocheir sinensis blood cells at different time points after helicobacter 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 showed that the copy number of the spiroplasma in the eriocheir sinensis haemolymph after feeding (figure 5) or injecting (figure 6) the anti-s-eriocheir sinensis specific IgY was significantly reduced compared to the control group fed or injected with PBS and normal IgY, indicating that the number of s-eriocheir sinensis in eriocheir sinensis could be effectively inhibited regardless of whether feeding or injecting the anti-s-eriocheir specific IgY, which is consistent with the eriocheir sinensis survival results.

TABLE 1 Artificial primer sequence Listing

In order to investigate the mechanism of inhibition of S.eriocheiris by anti-S.eriocheiris specific IgY, S.eriocheiris was treated with specific IgY and negatively stained and the effect of anti-S.eriocheiris specific IgY on the morphology of S.eriocheiris was observed using transmission electron microscopy, as shown in FIGS. 7 and 8. FIGS. 7 (Group A) and 8 (Group D) show normal feed feeding of Eriocheir sinensis after injection of the spiroplasma, and normal spiroplasma in Eriocheir sinensis blood lymphocytes after injection of PBS, and show complete cell membrane and regular morphology. Fig. 7 (Group B) and fig. 8 (Group E) show that the spiroplasma structure of the eriocheir sinensis after injection of the spiroplasma is slightly different and irregular from that of the normal spiroplasma structure by feeding the eriocheir sinensis with normal IgY and the spiroplasma in the eriocheir sinensis blood lymphocytes after injection of the normal IgY, specifically shown by the arrows in fig. 7 (Group B) and fig. 8 (Group E). FIGS. 7 (Group C) and 8 (Group F) show the altered S.eriocheir morphology following treatment with anti-S.eriocheir specific IgY, in an irregular, agglomerated, split or membranous form, as shown in FIG. 7 (Group C), FIG. 8 (Group F), after administration of specific IgY to Eriocheir sinensis by feeding Eriocheir sinensis with specific IgY and administration of specific IgY. Therefore, the specific IgY treatment causes great damage to the morphological structure of the S.eriocheiris, thereby affecting the growth, propagation and infection of the S.eriocheiris.

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 environmental pollution, strong pertinence and remarkable effect. Compared with PBS or nonspecific IgY, the specific IgY of the invention can obviously improve the survival rate of Eriocheir sinensis, reduce the copy number of the S.eriocheir, change the morphological structure of the S.eriocheir, and lead the membrane to split or dissolve.

Sequence listing

<110> university of Nanjing teachers and students

<120> a specific egg yolk antibody against Eriocheir sinensis helicobacter, and preparation method and application thereof

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<170> SIPOSequenceListing 1.0

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<213> Artificial sequence (Artificial Sequence)

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Claims (3)

1. The application of the specific egg yolk antibody aiming at the molluscum sinensis in preparing feed or medicine for preventing Spiroplasma eriocheiris infection of the eriocheir sinensis is characterized in that the antibody is a specific IgY antibody for resisting S.eriocheiris, and the specific antibody is obtained by injecting the crushed molluscum into a hen body, separating and purifying egg yolk;

the preparation method comprises the following steps:

step 1) culturing S.eriocheiris in an R2 culture medium for 24 hours, centrifuging to remove supernatant, and re-suspending with PBS; step 2) carrying out ultrasonic crushing on the S.eriocheiris subjected to the re-suspension in the step 1), and preserving at 20 ℃ for later use; step 3) intramuscular injection is carried out on the hen, 1mL of 200-400 mug/mL S.eriocheiris crushed in the step 2) is injected, and specific antibody IgY is produced;

step 4) injecting hen 3 times, every 2 weeks, collecting eggs every day until the ninth week, extracting egg yolk, separating IgY from the yolk, and purifying.

2. The use according to claim 1, characterized in that the preparation method of the feed comprises the following steps:

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

and B-1) mixing the specific IgY prepared in the step A) with commercial feed, wherein the mixing mass ratio is 1:4, and using egg white as an adhesive.

3. The use according to claim 1, wherein the method of preparing the medicament comprises the steps of:

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

and B-2) mixing the specific IgY prepared in the step A) with PBS according to the volume ratio of 1:4.

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