CN114732895B

CN114732895B - Application of spore wall protein in preventing and controlling shrimp liver and intestine cytozoon infection of feeding shrimp

Info

Publication number: CN114732895B
Application number: CN202210486990.9A
Authority: CN
Inventors: 张其中; 凃彦芳; 付耀武; 陈维丰
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2022-05-06
Filing date: 2022-05-06
Publication date: 2023-09-29
Anticipated expiration: 2042-05-06
Also published as: CN114732895A

Abstract

The invention discloses an application of spore wall protein in preventing and controlling shrimp liver and intestine cytozoon infection of feeding shrimp, belonging to the technical field of disease prevention and control of aquaculture. According to the invention, the spore wall protein SWP12 of the shrimp liver and intestine cyst is produced by prokaryotic expression, after the spore wall protein is mixed with feed according to different concentrations to feed the shrimp, the shrimp fed with the feed with high concentration protein (65 mg/kg) is found, when the feeding of the SWP12 protein is stopped for at least 10 days, the shrimp can resist the infection of the shrimp by 100 percent after the hepatopancreas infection containing the shrimp liver and intestine cyst is fed, even if the feeding of the SWP12 protein is stopped for less than 21 days, the infection rate of the shrimp liver and intestine cyst is still obviously lower than that of the shrimp which is not fed or fed with low (medium) concentration SWP12 protein, so that the shrimp liver and intestine cyst infection resistance of the shrimp can be obviously enhanced when the shrimp is fed with the high concentration SWP12. The method is safer and more effective and has better operability.

Description

Application of spore wall protein in preventing and controlling shrimp liver and intestine cytozoon infection of feeding shrimp

Technical Field

The invention belongs to the technical field of prevention and control of aquaculture diseases, and particularly relates to preparation of spore wall protein and application of spore wall protein in prevention and control of shrimp liver and intestine cyst infection of mixed feeding shrimp.

Background

Shrimp liver and intestinal cytozoon (Enterocytozoon hepatopenaei, abbreviated as EHP) belongs to Microsporum (Microsporida), enteromozoidae (Enteromozoidae) and Enteromozoon (Enteromozocon), and is a common intracellular parasitic microsporidian in Litopenaeus vannamei culture, and is mainly parasitic in hepatopancreatic cells, and the disease caused by the shrimp liver and intestinal cytozoon is called shrimp liver and intestinal cytozoon. The shrimps with the shrimp liver and intestine cytozoonosis mainly show growth retardation, the weight of the shrimps is only 10% -40% of that of healthy shrimps in the same batch, the time of feeding and marketing is prolonged, the feed consumption is larger than that of the shrimps on the market normally, but the yield is lower, the feed is consumed more, the number of shrimps cultured in the same pond in one year is reduced, and serious economic loss is caused to farmers.

The damage range of shrimp liver and intestinal cytozoon is very wide, most of pond water bodies and sediment for culturing litopenaeus vannamei can detect the shrimp, the shrimp can be transmitted through multiple ways such as water flow with spores of the shrimp bodies, sick shrimp, polluted feed and the like, and the prevention and control difficulty of cutting off the transmission way from the environment is very high. Therefore, so far, no effective prevention and control measures exist, and development of new effective prevention and control technologies from a new perspective is urgently needed.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide an application of spore wall protein SWP12 in preventing and controlling shrimp liver and intestine cyst infection of feeding shrimps.

The aim of the invention is achieved by the following technical scheme:

the invention provides an application of spore wall protein SWP12 in preparing a product for preventing and controlling shrimp liver, intestine and cyst insect infection;

furthermore, the spore wall protein SWP12 is applied to the preparation of products for preventing and controlling shrimp liver and intestine cyst insect infection of feeding shrimp.

Preferably, the shrimps are litopenaeus vannamei, hereinafter referred to as prawns;

the amino acid sequence of the spore wall protein SWP12 is shown in SEQ ID NO. 1; the nucleotide sequence of the coded spore wall protein SWP12 is shown as SEQ ID NO.2 or MN 604021.1.

Preferably, the addition amount of the spore wall protein SWP12 in the product is more than 60mg/kg, and further 65+/-5 mg/kg.

The invention also provides a spore wall protein polyclonal antibody, which is prepared by immunizing animals with spore wall protein SWP12;

the method comprises the following specific steps:

(1) Prokaryotic expression is carried out on the spore wall protein SWP12, the protein concentration is measured after purification, and then animal immunization is carried out;

(2) Serum from immunized animals was collected: after 2 times of immunization on animals, blood is taken, and the titer of antiserum against the spore wall protein SWP12 is detected, and when the titer is more than or equal to 1:51200 final collection of antisera;

(3) And (5) purifying the antibody.

The animal is New Zealand rabbit;

the spore wall protein polyclonal antibody is applied to preparation of a reagent or a kit for diagnosing or detecting shrimp liver and intestine cyst insects.

A kit for diagnosing or detecting shrimp liver and intestine cytozoon contains the sporoderm protein polyclonal antibody.

The invention obtains a spore wall protein (Spore wall protein, SWP 12) coding region from shrimp liver and intestine cyst insect genome clone, which is 732bp, codes 244 amino acids, and the amino acid sequence contains a heparin binding motif. The amino acid sequence of the spore wall protein SWP12 is shown as SEQ ID NO.1, and subcellular localization of the protein by utilizing a laser confocal technology shows that SWP12 is mainly distributed on spore walls.

After the spore wall protein SWP12 is added into shrimp feed and healthy prawns are fed, an EHP toxicity attack experiment is performed. The method specifically comprises the following steps:

s1: prokaryotic expression and purification of spore wall protein SWP12

Prokaryotic expression spore wall protein SWP12, and after purification, the concentration of the purified recombinant protein was determined.

S2: preparation of polyclonal antibodies, antibody titer determination and specificity assay

After immunization of New Zealand rabbits with purified SWP12 according to a standard immunization method, blood was collected, the polyclonal antibody titer was determined by ELISA, and the polyclonal antibody specificity was detected by Western blotting.

S3: feeding SWP12 protein to healthy prawns for a period of time, and detecting the condition of the EHP infected prawns by EHP virus attack prawns.

The healthy prawns are respectively fed with common feed (without adding SWP 12), feed (65 mg/kg feed) with BSA, feed (15 mg/kg feed) with low-concentration SWP12 protein, feed (40 mg/kg feed) with medium-concentration SWP12 protein, feed (65 mg/kg feed) with high-concentration SWP12 protein for 3 times a day, continuous feeding for 14 days, and immediate stopping feeding of SWP12 protein; then, feeding the shrimp groups with the EHP-containing sick shrimp hepatopancreas to attack toxin, and continuously feeding for 7 days; then, each group is changed to be added with common feed for 24 days, shrimp samples are taken on the 3 rd, 7 th, 14 th, 21 th and 24 th days of changing to be added with common feed, hepatopancreatic DNA is extracted, and the presence or absence of EHP in the shrimp samples is detected by using a Taqman probe fluorescent quantitative PCR technology.

The main mechanism of the invention is as follows:

the protein on the spore wall of the shrimp liver and intestine cytozoon is a molecule which is firstly contacted with a host cell when the shrimp infects the host cell, and the spore wall protein molecule plays an important mediating role in infection, including the functions of recognition, adhesion and the like; at the same time, these wall proteins are also the earliest molecules that cause the host to produce a rejection (immune) response, and the host immune response (only innate immune response in shrimp) caused by them can reduce or even eliminate infection of host cells by the insect. Therefore, a large amount of spore wall proteins can be produced and added into the feed to feed the shrimps, and immune molecules are stimulated to be produced by the spore wall proteins, so that the shrimps are prevented from being infected by the shrimp liver and intestinal cytozoon. This is a new idea to prevent this parasitic infection. Based on the thought, cloning a shrimp liver and intestine cyst spore wall protein gene, expressing the protein by a prokaryotic method, adding the protein into feed, feeding litopenaeus vannamei for a period of time, and detecting the shrimp liver and intestine cyst infection resisting effect.

Compared with the prior art, the invention has the following advantages and effects:

(1) According to the invention, the spore wall protein SWP12 of the shrimp liver and intestine cyst is produced by prokaryotic expression, after the spore wall protein is mixed with feed according to different concentrations to feed the shrimp, the shrimp fed with the feed with high concentration protein (65 mg/kg) is found, when the feeding of the SWP12 protein is stopped for at least 10 days, the shrimp can resist the infection of the shrimp by 100 percent after the hepatopancreas infection containing the shrimp liver and intestine cyst is fed, even if the feeding of the SWP12 protein is stopped for less than 21 days, the infection rate of the shrimp liver and intestine cyst is still obviously lower than that of the shrimp which is not fed or fed with low (medium) concentration SWP12 protein, so that the shrimp liver and intestine cyst infection resistance of the shrimp can be obviously enhanced when the shrimp is fed with the high concentration SWP12.

(2) According to the invention, after feeding SWP12 protein for 14 days, the interval time of stopping feeding the protein is not more than 10 days, and then the SWP12 protein is continuously fed for 14 days, so that feeding, stopping feeding and feeding are circulated in turn until the prawns come into the market; or, feeding SWP12 protein continuously until the prawns come into the market; can ensure that the shrimp liver and intestinal cytozoon infection is completely prevented in the period, and compared with other prevention and control methods (such as physical and chemical prevention and control methods) for eliminating the shrimp liver and intestinal cytozoon in water environment, the method is safer and more effective and has better operability.

Drawings

FIG. 1 shows the result of SDS-PAGE electrophoresis of magnetic bead affinity purified spore wall protein SWP12; wherein M: standard protein molecular weight; lane 1: ultrasonically crushing recombinant engineering bacteria to obtain supernatant; lane 2: washing liquid after purifying the protein by the magnetic beads; lane 3: supernatant after inclusion body dissolution; lanes 4-14:11 SWP12 protein samples eluted with different imidazole concentration eluent working solutions; lanes 15-17: SWP12 protein samples eluted with three complete imidazole eluents.

FIG. 2 is a Western blotting analysis of the specificity of polyclonal antibodies against the sporozoite protein SWP12; wherein M: standard protein molecular weight; 1: immunoblots of recombinant protein SWP12.

FIG. 3 is a laser confocal localization of SWP12 protein distribution on the EHP spore wall; wherein a is an experimental group; b is a negative control group; DAPI was used for nuclear staining, diI was used for cell membrane staining, FITC fluorescence-labeled goat anti-rabbit secondary antibody was used to bind to the corresponding rabbit anti-SWP 12 primary antibody, and mere was a superimposed image of two fluorescence channel images showing SWP12 on the spore wall.

FIG. 4 shows that the EHP infection rate is detected at different time after feeding prawns with different SWP12 protein feed for 14 days and then feeding prawns containing shrimp liver and intestine cytozoon for 7 days; wherein, the letters represent whether the difference between different groups is significant in the same time point, the difference between groups containing the same letters is not significant (p > 0.05), and the difference between groups of different letters is significant (p < 0.05). Control group: general feed, BSA group: the feed supplemented with BSA (65 mg/kg) was fed, the low-concentration SWP12 group (15 mg/kg feed), the medium-concentration SWP12 group (40 mg/kg feed) and the high-concentration SWP12 group (65 mg/kg feed).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. It should be understood that the specific examples described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The test methods for specific experimental conditions are not noted in the examples below, and are generally performed under conventional experimental conditions or under experimental conditions recommended by the manufacturer. The materials, reagents and the like used, unless otherwise specified, are those obtained commercially.

The construction of real-time fluorescent quantitative PCR detection methods for shrimp liver and intestine cyst (Enterocytozoon hepatopenaei) and detection of shrimp samples [ J ]. Development of fishery science, 2016,37 (02): 119-126 ", are disclosed in the documents" Liu Zhen, zhang Qingli, mo Xiaoyuan, ma Fang, huang ".

The amino acid sequence of the shrimp liver and intestine cyst wall protein SWP12 is shown in SEQ ID NO. 1.

Example 1:

1. construction of spore wall protein SWP12 prokaryotic expression plasmid

According to the published spore wall protein SWP12 gene of shrimp liver and intestine cyst (NCBI accession number is MN 604021.1), carrying out codon optimization on the spore wall protein SWP12 gene, constructing the codon optimized nucleotide sequence as shown in SEQ ID No.2 on a plasmid pET-28a (+) (conventional commercial product) to obtain a recombinant expression plasmid pET28a-SWP12; wherein the gene SWP12 is inserted between the BamHI and Xho I cleavage sites of plasmid pET-28a (+). Transferring the recombinant expression plasmid pET28a-SWP12 into engineering bacteria escherichia coli Rosetta (conventional commercial product), sequencing and verifying, culturing a large amount of engineering bacteria, and storing the strain at-80 ℃ to obtain recombinant engineering bacteria Rosetta/pET28a-SWP12.

2. Prokaryotic expression and purification of spore wall protein SWP12

2.1 prokaryotic expression

Coating recombinant engineering bacteria Rosetta/pET28a-SWP12 on a plate containing kanamycin, culturing for about 14h, picking single colony on the plate, adding into 50mL LB liquid medium containing 50 μL kanamycin (100 mg/mL), shaking at 37deg.C for about 1 hr until OD ₆₀₀ Between=0.4 and 0.6. 1mL is taken as an uninduced control group, the rest bacterial liquid is added with IPTG for induction until the final concentration of the IPTG is 0.6mmol/L, and the bacterial liquid is placed at 37 ℃ for shake cultivation for 8 hours. Taking out the bacterial liquid, centrifuging and collecting bacterial cells. The SWP12 protein is found to exist mainly in the form of inclusion bodies through SDS-PAGE electrophoresis detection and ultrasonic disruption, and the molecular weight of the protein is about 28 kDa.

2.2 protein purification

Recombinant protein SWP12 expression was induced in large amounts, cells were collected, and after cell precipitation in 1 XPBS buffer, the cells were subjected to ice bath and ultrasonication (5 mPBS was added per 100mL of cell pellet). Centrifuging to collect the crushed precipitate and the crushed supernatant, wherein inclusion bodies are in the precipitate. The inclusion bodies were solubilized with 2mL of Binding Buffer (Binding Buffer), incubated at room temperature for 30 minutes, centrifuged and the supernatant (i.e., the target protein) after solubilization of the inclusion bodies was collected.

After the magnetic beads are pretreated with a Binding Buffer, 2mL of a target protein dissolved in the Binding Buffer is added into a pretreated centrifuge tube containing the magnetic beads, and the target protein is mixed with the magnetic beads by shaking vigorously for a plurality of times, gently reversing and uniformly mixing for 20 minutes at room temperature. After carefully discarding the supernatant (flow-through), 2mL of Washing solution (Washing buffer) was added to the centrifuge tube containing only the beads, and the tube was turned over several times, resuspended, magnetically separated, and the Washing solution was collected, and the Washing solution was repeated once more, and the Washing solution was collected and combined twice, and was called "Washing solution after purification of protein by magnetic beads". Adding 1mL of eluent (Elution Buffer) with different imidazole concentrations into a tube with the magnetic beads removed from the cleaning liquid, eluting SWP12 bound on the magnetic beads, collecting an eluted SWP12 protein sample, wherein the eluent (Elution Buffer) with different imidazole concentrations is a mixed working solution of a Binding Buffer and an eluent (Elution Buffer) according to a certain ratio, starting the cleaning by taking 1mL of the Binding Buffer as the eluent working solution, and collecting the eluted liquid each time, sequentially adding 0.1mL of the eluent and correspondingly reducing 0.1mL of the Binding Buffer, and keeping the volume of the eluent working solution to be 1mL unchanged until the eluent working solution is the eluent completely. And eluting three times by using complete imidazole eluent without Binding Buffer, and collecting the three eluents respectively. SDS-PAGE verification is carried out on the supernatant (see the lane 1 in FIG. 1) obtained after ultrasonic disruption of the recombinant engineering bacteria, the flushing liquid (see the lane 2 in FIG. 1) obtained after purification of proteins by magnetic beads, the supernatant (see the lane 3 in FIG. 1) obtained after dissolution of inclusion bodies, SWP12 protein samples (see the lanes 4-14 in FIG. 1) eluted by working solutions with different imidazole concentrations and SWP12 protein samples (see the lanes 15-17 in FIG. 1) eluted by three complete imidazole eluents, and the obtained target protein has single band and the result is shown in FIG. 1.

The purified protein was added to a dialysis bag and the dialysate was dialyzed overnight with 8M urea. The dialysate was collected and lyophilized (the protein dry powder was dissolved in 1mL of PBS buffer) to obtain a large amount of the target protein with high purity. The concentration of the purified recombinant protein was determined using Lowry kit, and 1mL of dry protein powder dissolved in PBS buffer was measured at a concentration of 0.561mg/mL.

Wherein, binding Buffer:8M Urea (Urea), 50mM NaH ₂ PO ₄ 5-50 mM Imidazole (Imidazole), 100mM Tris-HCl. And the pH was adjusted to 8.0.

Elution Buffer:8M Urea (Urea), 50mM NaH ₂ PO ₄ 500mM Imidazole (Imidazole), 100mM Tris-HCl. And the pH was adjusted to 8.0.

3. Preparation of spore wall protein SWP12 polyclonal antibody, potency determination and specificity test

Preparation and potency determination of 3.1SWP12 polyclonal antibodies

Taking 2mL of New Zealand rabbit ear vein blood raised for one week to prepare negative serum, standing at 37 ℃ for 1 hour, separating out the serum, subpackaging into different centrifuge tubes, and storing at-80 ℃. The lyophilized protein powder was dissolved in 1M PBS buffer to prepare a 1mg/mL concentration as an antigen solution. The antigen solution was mixed with an equal volume of Freund's complete adjuvant, a first immunization was performed, serum was collected from the ear vein and titers were determined. Two weeks later, booster immunization was performed, the antigen amount was 500 μg/mL, blood was collected from the New Zealand rabbit heart, polyclonal antibody serum was collected, and the titer was found to reach 1 by indirect ELISA detection: 51200 and packaging, and placing in a refrigerator at-80deg.C.

Specificity test of 3.2SWP12 polyclonal antibody

The recombinant expressed SWP12 protein is transferred onto a PDVF membrane after SDS-PAGE, and dried, and the specificity of the polyclonal antibody is tested by Western blotting by using a rabbit antibody (marked as SWP12 polyclonal antibody) of the recombinant SWP12 protein as a primary antibody, specifically as follows: SWP12 polyclonal antibody was prepared with TBST solution at 1: diluting with 5000 volume ratio, standing at 37deg.C for 2 hr, washing with TBST solution for 4 times (5 min each time), and rinsing with TBS solution for 1 time (5 min); the TBST solution was used at 1: diluting goat anti-rabbit antibody IgG-HRP with a volume ratio of 5000, standing and incubating the secondary antibody at 37 ℃ for 2 hours, washing the membrane by TBST, adding a color development liquid on the membrane, and developing in a dark place; after the color development strip is clear, the membrane is washed 3 times with deionized water to terminate the color development, and the membrane is photographed and stored. As a result, as shown in FIG. 2, a clear band was found at about 29kDa, which was consistent with the size of the target band, indicating that the polyclonal antibody was able to specifically bind to the target protein.

4. Laser confocal positioning of spore wall protein SWP12 distributed in shrimp liver, intestine and cyst insect

And (3) dripping 10 mu L of purified shrimp liver and intestine cytozoospores onto an anti-drop slide glass, uniformly coating, and drying for 10 minutes at room temperature. 300. Mu.L of 5% paraformaldehyde was added dropwise to cover shrimp liver and intestine cytozoospores, and the mixture was fixed for 15 minutes. Carefully discarding the supernatant, slowly washing the surface of the glass slide three times by using a pipette to absorb 0.1M PBS buffer solution, slowly dripping 0.5% Triton-100 to cover and permeabilize for 15 minutes, and changing the permeability of the cell membrane; carefully discard the liquid on the surface of the slide, add 1% BSA dropwise and block for 1 hour at room temperature; carefully blotting the blocking solution with absorbent paper, adding SWP12 polyclonal antibody diluted 500-fold with 1% BSA and negative control serum, respectively, and incubating overnight at 4 ℃; discarding the surface liquid of the slide, sucking the PBST solution, and slowly washing the slide for three times; adding FITC fluorescence-marked goat anti-rabbit secondary antibody diluted 500 times by 1% BSA in a dark place, and placing in a wet box for incubation for one hour in a dark place; removing liquid on the surface of the glass slide, and slowly flushing the glass slide with PBST solution for three times; 10. Mu.M DiI (cell membrane red fluorescent dye) was added and incubated for 15 minutes at room temperature; discarding the supernatant, and slowly washing the glass slide for three times by using a PBST solution; adding 5 mug/mL DAPI, incubating for 15 minutes at normal temperature, discarding the supernatant, and slowly washing the glass slide three times by using PBST solution; after the glass slide is slightly dried, two drops of anti-fluorescence quenching sealing tablets are dripped in the center of a sample area, a cover glass is covered, and a nail oil sealing sheet is coated on the edge of the cover glass; observed under a laser confocal scanning microscope. The results are shown in FIG. 3, where blue, green and red fluorescent signals appear in the experimental groups, and the green and red fluorescent signals form a circle, respectively, and when the green and red fluorescent signals overlap, the yellow fluorescent light appears to be apparent, indicating that SWP12 protein is distributed on the spore wall of shrimp liver and intestine cyst. In the negative control group, there was no green fluorescence, and after overlapping, there was no yellow fluorescence.

5. Shrimp liver and intestine cytozoon infection resisting effect of feeding spore wall protein SWP12

5.1 feeding the spore wall protein SWP12

Healthy Litopenaeus vannamei was divided into a blank control group (feeding a normal feed), an irrelevant protein group (feeding a feed with BSA added at a content of 65mg/kg feed), a low-concentration protein group (feeding a feed with SWP12 protein added at a content of 15mg/kg feed), a medium-concentration protein group (feeding a feed with SWP12 protein added at a content of 40mg/kg feed), and a high-concentration protein group (feeding a feed with SWP12 protein added at a content of 65mg/kg feed), each group was provided with three parallel controls. The feeding amount per day is about 10% of the shrimp weight, three times per day, and 14 days continuously, and the feeding of the recombinant SWP12 protein is stopped immediately.

The common feed is penaeus monodon compound feed produced by Jiangmen and sea feed limited company, and the main raw materials comprise: imported fish meal, cuttlefish powder, shrimp meal, high gluten flour, yeast powder, minerals and microelements, vitamins and mildew preventive.

5.2EHP toxicity attack experiment

After feeding for 14 days, all groups were changed to feed EHP-infected sick shrimp hepatopancreas for 7 days continuously.

5.3 feeding ordinary feed, sampling and sample treatment

After the toxin attacking stage is finished, all groups are changed into common feed, 5 time points are taken out from all groups (10 th, 14 th, 21 st, 28 th and 31 th days after stopping feeding the recombinant SWP12 protein respectively) at the 3 rd, 7 th, 14 th, 21 st and 24 th days, 5 shrimps are taken out from each parallel, the hepatopancreas of the dissected shrimps is taken out from the dissected shrimps, and the dissected shrimps are placed in a freezing tube, quick frozen by liquid nitrogen and then placed in a refrigerator at the temperature of minus 80 ℃.

5.4 protective Effect of feeding the recombinant SWP12 protein on shrimp against shrimp liver and intestine Cytophilus infection

As a result of artificial infection of 7 days of EHP in the shrimp fed with the recombinant SWP12 protein for 14 days, as shown in FIG. 4, it was found that 100% of the shrimps in the high concentration group (65 mg/kg feed) were not infected with EHP within 10 days of stopping feeding the recombinant SWP12 protein, whereas EHP infection was detected in each group after 14 days of stopping feeding the recombinant SWP12 protein (i.e., 7 days after stopping the challenge), but the infection rate of the high concentration group was 6.67%, which was significantly lower than the infection rate (33.33% -53.33%) of the other groups (p < 0.05), and the infection rate of each group was increased with time to the highest on the 31 th day (24 days after stopping the challenge). The infection rates of the BSA group, the low-concentration protein group and the medium-concentration protein group are not obviously different from those of the control group. The result shows that the litopenaeus vannamei can enhance the EHP infection resistance by feeding the litopenaeus vannamei with high-concentration protein (65 mg/kg) mixed with feed, and 100% prevention of the shrimp liver and intestine cytozoon infection can be ensured within at least 10 days after stopping feeding the recombinant SWP12 protein; when the feeding of the recombinant SWP12 protein is stopped for 21 days, the infection rate of the shrimp liver and intestine cyst is obviously lower than that of the shrimp which is not fed or fed with the SWP12 protein with low (medium) concentration, which indicates that the feeding of the SWP12 protein with high concentration can obviously enhance the capability of resisting the shrimp liver and intestine cyst infection of the shrimp.

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Sequence listing

<110> and university of south China

Application of spore wall protein in preventing and controlling shrimp liver, intestine and cyst insect infection of feeding shrimp

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 244

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> amino acid sequence of spore wall protein SWP12

<400> 1

Met Glu Ala Pro Lys Lys Ile Ser Thr Thr Lys Ile Arg Glu Thr Leu

1 5 10 15

Lys Asn Thr Gln Lys Lys Ile Ala Gln Lys Phe Lys Thr Ile Asp Tyr

20 25 30

Val Asn Thr Asp Leu Asn Pro Gly Phe Leu Glu Leu Glu Glu Glu Phe

35 40 45

Lys Asn Ile Arg Asn Thr Ala Lys Met Leu Lys Asp Cys Leu Leu Thr

50 55 60

Phe Lys Asn Tyr Glu Tyr Gly His Ser Ile Leu Lys Asn Val Tyr Asn

65 70 75 80

Gly Phe Glu Trp Val Glu Lys Lys Leu Asn Thr Glu Ile Val Ser Lys

85 90 95

Lys Glu Leu Tyr Gly Ser Leu Ala Glu Ala Gly Thr Asn Ile Ala Lys

100 105 110

Phe Thr His Asp Lys Asn Arg Lys Glu Leu Ala Ile Ala Phe Gln Asn

115 120 125

Ser Tyr Leu Ala Ile Ser Asp Tyr Lys Lys Ser Phe Asn Ser Glu Val

130 135 140

Lys Gln Leu Ile Leu Asn Ile Asp Val Leu Leu Ser Lys Ala Glu Glu

145 150 155 160

Ile Ser Ser Lys Arg Lys Gln Ile Arg Thr Ile Arg Tyr Asp Leu Glu

165 170 175

Met Ala Ile Leu Asp Asp Asn Tyr Asp Asn Asp Leu Val Lys Ser Glu

180 185 190

Arg Lys Lys Leu Ser Gly Glu Cys Lys Gln Cys Met Ser Glu Met Asn

195 200 205

Glu Phe Ile Lys Asp Lys Ser Ile Gly Lys Ile Ile Lys Lys Phe Gln

210 215 220

Lys Leu His Cys Lys Phe Tyr Arg Gln Ile Tyr Asp Glu Leu Asp Val

225 230 235 240

Ile Glu His Phe

<210> 2

<211> 732

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nucleotide sequence encoding teichoic protein SWP12

<400> 2

atggaagcgc cgaaaaagat cagcaccacc aaaatccgtg aaaccctgaa aaacacccag 60

aaaaagatcg cgcagaaatt caaaaccatc gattacgtta acaccgatct gaatccgggt 120

ttcctggaac tggaagaaga attcaaaaac atccgtaaca ccgcgaaaat gctgaaagat 180

tgcctgctga ccttcaaaaa ctacgaatac ggccactcta tcctgaaaaa cgtttacaac 240

ggtttcgaat gggttgagaa aaaactgaac accgaaatcg tgagcaaaaa agaactgtac 300

ggtagcctgg cggaagcggg caccaacatc gcgaaattca cccacgacaa aaaccgtaaa 360

gaactggcga tcgcgttcca gaactcttac ctggcgatca gcgattacaa gaaatctttc 420

aacagcgaag ttaaacagct gatcctgaac atcgatgttc tgctgtcgaa agcggaagaa 480

atcagctcta aacgtaaaca gatccgcacc atccgttatg atctggagat ggctatcctg 540

gatgataact acgacaacga cctggtgaaa agcgaacgta aaaaactgtc cggcgaatgc 600

aaacagtgca tgagtgaaat gaacgaattc attaaagata aaagcatcgg caaaatcatc 660

aaaaaattcc agaaactgca ctgcaaattc taccgtcaga tctacgacga actggacgtg 720

atcgaacact tc 732

Claims

1. The application of the spore wall protein SWP12 as the only active ingredient in preparing a product for preventing and controlling shrimp liver, intestine and cyst insect infection is characterized in that: the amino acid sequence of the spore wall protein SWP12 is shown as SEQ ID NO.1, and the object infected by shrimp liver and intestine cytozoon is Litopenaeus vannamei.

2. The use according to claim 1, characterized in that:

the spore wall protein SWP12 is used as the only active ingredient in the preparation of a product for preventing and controlling shrimp liver and intestine cyst insect infection of the mixed feeding shrimp.

3. Use according to any one of claims 1-2, characterized in that:

the nucleotide sequence of the coded spore wall protein SWP12 is shown as SEQ ID NO. 2.

4. Use according to any one of claims 1-2, characterized in that:

the adding amount of the spore wall protein SWP12 in the product is more than 60 mg/kg.

5. The use according to claim 4, characterized in that:

the adding amount of the spore wall protein SWP12 in the product is 65+/-5 mg/kg.