CN114732895A

CN114732895A - Application of sporoderm protein in prevention and control of shrimp enterocytozoon infection in mixed feeding of shrimps

Info

Publication number: CN114732895A
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: 2022-07-12
Anticipated expiration: 2042-05-06
Also published as: CN114732895B

Abstract

The invention discloses application of sporoderm protein in prevention and control of shrimp liver enterocytozoon infection in mixed feeding of shrimps, and belongs to the technical field of disease prevention and control in aquaculture. The sporoderm protein SWP12 of the shrimp liver enterocytozoon is produced by prokaryotic expression, after the sporoderm protein is mixed with feed according to different concentrations to feed the prawn, the prawn fed with the feed with high-concentration protein (65mg/kg) is found, the prawn fed with the feed containing the shrimp liver enterocytozoon can resist the infection of the shrimp liver enterocytozoon by 100 percent in at least 10 days after the feeding of the SWP12 protein is stopped, and even within 21 days after the feeding of the SWP12 protein is stopped, the infection rate of the shrimp liver enterocytozoon is still obviously lower than that of the prawn without feeding or fed with the low (medium) concentration SWP12 protein, which shows that the feeding of the sporoderm protein SWP12 of the high-concentration shrimp liver enterocytozoon can obviously enhance the capability of the prawn against the shrimp liver enterocytozoon infection. The method is safer and more effective, and has better operability.

Description

Application of sporoderm protein in prevention and control of shrimp enterocytozoon infection in mixed feeding of shrimps

Technical Field

The invention belongs to the technical field of disease prevention and control in aquaculture, and particularly relates to preparation of sporoderm protein and application of sporoderm protein in prevention and control of shrimp liver enterocytozoon infection in shrimp mixing feeding.

Background

The Enterocytozoon hepaticae (abbreviated as EHP) belongs to Microsporidia (Microsporidia), Enterocytozoon (enterozoonidae) and Enterocytozoon (enterozoon), is a common intracellular parasitism microsporidian in the culture of litopenaeus vannamei, mainly parasitizes in hepatopancreatic cells, and causes a disease called enterocytozoosis of shrimps. The shrimps suffering from the shrimp enterohepatic cytozoosis mainly show growth retardation, the weight of the shrimps is only 10% -40% of that of healthy shrimps in the same batch, the time for feeding the shrimps to the market is prolonged, the feed consumption is larger than that of the normal shrimps to the market, but the yield is lower, more feed is consumed, the number of the shrimps cultured in the same pond in one year is reduced, and serious economic loss is caused to farmers.

The shrimp liver enterocytozoon has wide harm range, most of the shrimp liver enterozoon can be detected in pond water and bottom mud for culturing the litopenaeus vannamei, the shrimp liver enterozoon can be transmitted by multiple ways such as water flow with zoospore, sick shrimps, polluted feed and the like, and the prevention and control difficulty for cutting off the transmission way from the environment is very high. Therefore, until now, there is no effective prevention and control measure, and it is urgently needed to develop a new effective prevention and control technology from a new perspective.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of sporoderm protein SWP12 in preventing and controlling shrimp enterocytozoon infection of mixed feeding shrimps.

The purpose of the invention is realized by the following technical scheme:

the invention provides an application of sporoderm protein SWP12 in preparing a product for preventing and controlling shrimp liver enterocytozoon infection;

further, the spore wall protein SWP12 is applied to preparing products for preventing and controlling shrimp liver enterocytozoon infection of mixed feeding shrimps.

Preferably, the shrimp is litopenaeus vannamei, hereinafter referred to as prawn;

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

Preferably, the spore wall protein SWP12 is added in an amount of 60mg/kg or more, further 65. + -.5 mg/kg.

The invention also provides a sporoderm protein polyclonal antibody, wherein the polyclonal antibody is prepared by immunizing animals with sporoderm protein SWP 12;

the method comprises the following specific steps:

(1) firstly, carrying out prokaryotic expression on sporoderm protein SWP12, purifying, determining the protein concentration, and then carrying out animal immunization;

(2) collecting serum of the immunized animal: after 2 times of animal immunization, blood collection and detection are carried out, and the titer of antiserum to the spore wall protein SWP12 is determined until the titer is more than or equal to 1: 51200 collecting antiserum after final blood collection;

(3) and (5) purifying the antibody.

The animals are New Zealand rabbits;

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

A kit for diagnosing or detecting shrimp liver enterocytozoon comprises the spore wall protein polyclonal antibody.

The invention obtains a sporoderm protein (Spore wall protein 12, SWP12) coding region from the genome clone of the shrimp liver enterocytozoon, which has 732bp in total, codes 244 amino acids, and has a heparin binding motif in the amino acid sequence. The amino acid sequence of the spore wall protein SWP12 is shown in SEQ ID NO.1, and subcellular localization of the protein by using a laser confocal technology shows that SWP12 is mainly distributed on the spore wall.

The spore wall protein SWP12 is added into shrimp feed and fed to healthy prawn, and then EHP challenge experiment is carried out. The method specifically comprises the following steps:

s1: prokaryotic expression and purification of sporoderm protein SWP12

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

S2: preparation of polyclonal antibodies, determination of antibody titers and specificity testing

After the new zealand rabbit was immunized with purified SWP12 according to the standard immunization method, blood was collected, the titer of the polyclonal antibody was determined by ELISA, and the specificity of the polyclonal antibody was detected by Western blotting.

S3: feeding the SWP12 protein with a mixed feed to healthy prawns for a period of time, attacking the prawns with EHP, and detecting the condition of infecting the prawns with EHP.

Dividing healthy prawns into five groups, respectively feeding common feed (not added with SWP12), feed added with BSA (65mg/kg feed), feed added with low-concentration SWP12 protein (15mg/kg feed), feed added with medium-concentration SWP12 protein (40mg/kg feed) and feed added with high-concentration SWP12 protein (65mg/kg feed), feeding for 3 times every day, continuously feeding for 14 days, and immediately stopping feeding SWP12 protein; feeding the hepatopancreas of the sick shrimps containing EHP to the shrimps for counteracting the toxicity, and continuously feeding for 7 days; then, common feed is added into each group for 24 days, shrimp samples are taken on the 3 rd, 7 th, 14 th, 21 th and 24 th days of the common feed, hepatopancreas DNA is extracted, and the existence of EHP in the shrimp samples is detected by using Taqman probe fluorescence quantitative PCR technology.

The main mechanism of the invention is as follows:

the protein on the spore wall of the shrimp liver enterocytozoon is a molecule which is firstly contacted with a host cell when the shrimp liver enterocytozoon infects the host cell, and the spore wall protein molecule plays an important mediating role in infection, including the functions of identification, attachment and the like; at the same time, these muramyl proteins are the earliest molecules that caused the host to mount an rejection (immune) response, and the host immune response caused by them (only the innate immune response in shrimp) could reduce or even eliminate the infection of the host cells by the insect. Therefore, a large amount of sporoderm protein can be produced and added into the feed to feed the shrimps, so that immune molecules can be generated by the sporoderm protein, and the shrimps are prevented from being infected by the liver enterocytozoon of the shrimps. This is a new idea for preventing this parasitic infection. Based on the thought, a shrimp liver enterocytozoon spore wall protein gene is cloned, the protein is expressed by pronucleus, the protein is added into feed to feed litopenaeus vannamei for a period of time, and the effect of the shrimp on resisting shrimp liver enterocytozoon infection is detected.

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

(1) the sporoderm protein SWP12 of the shrimp liver enterocytozoon is produced by prokaryotic expression, after the sporoderm protein is mixed with feed according to different concentrations to feed the prawn, the prawn fed with the feed with high-concentration protein (65mg/kg) is found, the prawn fed with the feed containing the shrimp liver enterocytozoon can resist the infection of the shrimp liver enterocytozoon by 100 percent in at least 10 days after the feeding of the SWP12 protein is stopped, and even within 21 days after the feeding of the SWP12 protein is stopped, the infection rate of the shrimp liver enterocytozoon is still obviously lower than that of the prawn without feeding or fed with the low (medium) concentration SWP12 protein, which shows that the feeding of the sporoderm protein SWP12 of the high-concentration shrimp liver enterocytozoon can obviously enhance the capability of the prawn against the shrimp liver enterocytozoon infection.

(2) After 14 days of feeding the SWP12 protein, the interval time for stopping feeding the protein is not more than 10 days, and then continuously feeding the SWP12 protein for 14 days, so that the feeding, stopping feeding and re-feeding alternately circulate until the prawns come into the market; or continuously feeding the SWP12 protein until the prawns are listed; can ensure that the shrimp liver enterocytozoon 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 enterocytozoon in the water environment, the method is safer, more effective and more operable.

Drawings

FIG. 1 shows the SDS-PAGE result of magnetic bead affinity purified spore wall protein SWP 12; wherein, M: a standard protein molecular weight; lane 1: supernatant liquid after the recombinant engineering bacteria are subjected to ultrasonic crushing; lane 2: washing liquid after protein purification by magnetic beads; lane 3: supernatant after the inclusion body is dissolved; lanes 4-14: samples of SWP12 protein eluted from 11 different imidazole concentration eluent working solutions; lanes 15-17: three samples of SWP12 protein eluted with a complete imidazole eluent.

FIG. 2 is a Western blotting analysis of the polyclonal antibody specificity of the anti-sporin SWP 12; wherein, M: a standard protein molecular weight; 1: immunoblot band of recombinant protein SWP 12.

FIG. 3 is a confocal laser localization of the distribution of SWP12 protein on EHP spore walls; wherein a is an experimental group; b is a negative control group; DAPI for nuclear staining, DiI for cell membrane staining, FITC fluorescently labeled goat anti-rabbit secondary antibody for binding to the corresponding rabbit anti-SWP 12 primary antibody, and Merge is a superposition of the two fluorescence channel images showing SWP12 on the spore wall.

FIG. 4 shows that EHP infection rates were measured at different times after feeding prawns with feeds of different amounts of SWP12 protein for 14 days and then feeding prawns with shrimp liver enterocytozoon for 7 days to challenge the liver and pancreas; wherein, the alphabet shows whether the difference between different groups is significant or not at the same time point, the difference between groups containing the same letter is not significant (p is more than 0.05), and the difference between different groups containing different letters is significant (p is less than 0.05). Control group: general feed was fed, BSA group: the feed supplemented with BSA (65mg/kg), low-concentration SWP12 group (15mg/kg feed), medium-concentration SWP12 group (40mg/kg feed), and high-concentration SWP12 group (65mg/kg feed) were fed.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the invention.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

The Enterocytozoon of shrimp used in the examples is disclosed in the documents "Liuzhen, Zhang Qingli, Wanxiao, Mafang, Huang 20514the establishment of real-time fluorescent quantitative PCR detection method for Enterocytozoon of shrimp (Enterocytozon hepatopenae i) and the detection of samples of shrimps [ J ]. advanced fishery science, 2016,37(02): 119-126.".

The amino acid sequence of the shrimp liver enterocytozoon sporoderm protein SWP12 is shown in SEQ ID NO. 1.

Example 1:

1. construction of sporoderm protein SWP12 prokaryotic expression plasmid

According to the disclosed sporoderm protein SWP12 gene (NCBI accession number is MN604021.1) of the shrimp liver enterocytozoon, codon optimization is carried out on the sporoderm protein SWP12 gene, the nucleotide sequence subjected to codon optimization is shown as SEQ ID No.2, and the codon optimized nucleotide sequence is constructed on plasmid pET-28a (+) (a conventional commercial product), so as to obtain a recombinant expression plasmid pET28a-SWP 12; wherein the gene SWP12 was inserted between the BamH I and Xho I cleavage sites of plasmid pET-28a (+). The recombinant expression plasmid pET28a-SWP12 is transferred into engineering bacteria Escherichia coli Rosetta (a conventional commercial product), after sequencing verification, a large amount of engineering bacteria are cultured, and the strains are stored at-80 ℃, and are marked as recombinant engineering bacteria Rosetta/pET28a-SWP 12.

2. Prokaryotic expression and purification of sporoderm protein SWP12

2.1 prokaryotic expression

The recombinant engineered bacterium Rosetta/pET28a-SWP12 was spread on a kanamycin-containing plate and cultured for about 14 hours, a single colony on the plate was picked up and added to 50mL of LB liquid medium containing 50. mu.L of kanamycin (100mg/mL), and the mixture was shake-cultured at 37 ℃ for about 1 hour to OD₆₀₀0.4-0.6. Taking 1mL as an uninduced control group, adding IPTG (isopropyl-beta-thiogalactoside) into the other bacterial liquid for induction until the final concentration of IPTG is 0.6mmol/L, and placing the bacterial liquid in a shaking table at 37 ℃ for culturing for 8 hours. The bacterial solution was removed, centrifuged and the cells collected. Through SDS-PAGE electrophoresis detection and ultrasonic disruption, the SWP12 protein is mainly found to exist in the form of inclusion bodies, and the molecular weight of the protein is about 28 kDa.

2.2 protein purification

The expression of the recombinant protein SWP12 was induced in large quantities, and the cells were collected, suspended in 1 XPBS buffer and then subjected to ice-bath and ultrasonication (5 mL PBS per 100mL of the cell suspension). The disrupted precipitate and the disrupted supernatant were collected by centrifugation, and inclusion bodies were included 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) from the solubilization of the inclusion bodies was collected.

After the magnetic beads are pretreated by Binding Buffer solution (Binding Buffer), 2mL of target protein dissolved in the Binding Buffer solution (Binding Buffer) is added into a pretreated centrifugal tube containing the magnetic beads, the centrifugal tube is vigorously shaken for several times, and the target protein and the magnetic beads are combined by gently reversing and uniformly mixing the mixture for 20 minutes at room temperature. After carefully discarding the supernatant (flow-through solution), 2mL of a Washing solution (Washing buffer) is added into a centrifuge tube containing only magnetic beads, the centrifuge tube is turned over for several times, resuspended, magnetically separated, collected and washed again, and the two Washing solutions are collected and combined, so that the Washing solution after protein purification by magnetic beads is called as the Washing solution after protein purification. Adding 1mL of eluent (Elution Buffer) with different imidazole concentrations into a tube which only contains magnetic beads and is removed from a cleaning solution to elute SWP12 bound on the magnetic beads, and collecting an eluted SWP12 protein sample (wherein the 'eluent working solution with different imidazole concentrations' is a working solution formed by mixing a Binding Buffer solution (Binding Buffer) and an eluent (Elution Buffer) according to a certain ratio, starting cleaning from 1mL of the Binding Buffer solution as the eluent working solution, collecting the eluted liquid each time, sequentially increasing 0.1mL of the eluent and correspondingly reducing 0.1mL of the Binding Buffer solution, and keeping the volume of the eluent working solution to be 1mL constant until the eluent working solution is completely the eluent). Eluting three times by using complete imidazole eluent without Binding Buffer, and respectively collecting the three eluents. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) verification is carried out on the supernatant (shown in a lane 1) after the ultrasonication of the recombinant engineering bacteria, the washing liquid (shown in a lane 2 of a lane 1) after the protein purification by magnetic beads, the supernatant (shown in a lane 3 of a lane 1) after the dissolution of the inclusion bodies, SWP12 protein samples (shown in lanes 4-14 of a lane 1) eluted by 11 eluent with different imidazole concentrations and SWP12 protein samples (shown in lanes 15-17 of a lane 1) eluted by three complete imidazole eluents, and the obtained target protein band is single, and the result is shown in a lane 1.

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

Wherein, Binding Buffer: 8M Urea (Urea), 50mM NaH₂PO₄5-50 mM Imidazole (Imidazole), 100mM Tris-HCl. And the pH was adjusted to 8.0.

Elution Buffer: 8M Urea (Urea), 50mM NaH₂PO₄，500mM Imidazol (imidazole), 100mM Tris-HCl. And the pH was adjusted to 8.0.

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

3.1 preparation and potency assay of SWP12 polyclonal antibodies

Taking 2mL of ear vein blood of a new Zealand rabbit which has been raised for one week to prepare negative serum, standing for 1 hour at 37 ℃, subpackaging the serum into different centrifuge tubes after the serum is separated out, 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 equal volume of Freund's complete adjuvant, first immunization was performed, and serum was collected from the marginal vein of the ear and titer was measured. Two weeks later, the booster immunization was carried out, the antigen amount was 500. mu.g/mL, blood was collected from the heart of New Zealand rabbits, polyclonal antibody serum was collected, and the titer reached 1: 51200 and packaging in refrigerator at-80 deg.C.

3.2 specificity test of SWP12 polyclonal antibody

Transferring the recombinant expressed SWP12 protein to a PDVF membrane after SDS-PAGE, drying, using a rabbit antibody (marked as SWP12 polyclonal antibody) resisting the recombinant SWP12 protein as a primary antibody, and testing the specificity of the polyclonal antibody by Western blotting, wherein the specificity is as follows: SWP12 polyclonal antibody was raised in TBST solution at 1: diluting with 5000 volume ratio, incubating with membrane at 37 deg.C for 2 hr, washing membrane with TBST solution for 5 min for 4 times, and rinsing with TBS solution for 1 time and 5 min; using TBST solution according to the weight ratio of 1: diluting the goat anti-rabbit antibody IgG-HRP at 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; when the color development band is clear, the film is washed with deionized water for 3 times to stop color development, and the film is photographed and stored. As shown in FIG. 2, a clear band was found at about 29kDa, which is consistent with the size of the target band, indicating that the polyclonal antibody specifically binds to the target protein.

4. Laser confocal localization of sporoderm protein SWP12 distribution in shrimp liver enterocytozoon

10 μ L of purified shrimp liver enterozoon spores were dropped on an anti-shedding slide, uniformly coated, and dried at room temperature for 10 minutes. 300. mu.L of 5% paraformaldehyde was added dropwise to cover the shrimp liver enterocytozoon spores and fixed for 15 minutes. Carefully abandoning the supernatant, sucking 0.1M PBS buffer solution by a pipette to slowly wash the surface of the slide for three times, slowly dripping 0.5 percent Triton-100 to cover and permeabilize for 15 minutes, and changing the permeability of cell membranes; carefully abandoning the liquid on the surface of the slide, dripping 1% BSA (bovine serum albumin) and sealing at room temperature for 1 hour; carefully blotting the blocking solution with absorbent paper, adding 500-fold dilution of SWP12 polyclonal antibody and negative control serum diluted with 1% BSA, and incubating overnight at 4 deg.C; discarding the liquid on the surface of the slide, sucking the PBST solution to wash the slide slowly for three times; adding FITC fluorescent labeled goat anti-rabbit secondary antibody diluted by 500 times with 1% BSA in dark, and incubating for one hour in a wet box in dark; removing liquid on the surface of the slide, taking PBST solution to slowly wash the slide for three times; add 10. mu.M DiI (cell membrane red fluorescent dye) and incubate for 15 minutes at room temperature; discarding the supernatant, and slowly washing the slide three times with PBST solution; adding 5 mu g/mL DAPI, incubating for 15 minutes at normal temperature, removing supernatant, and taking PBST solution to wash the slide slowly for three times; after the slide is slightly dried, two drops of the anti-fluorescence quenching sealing piece are dripped into the center of the sample area, a cover glass is covered, and the edge of the sample area is coated with a nail polish sealing piece; and observing under a laser confocal scanning microscope. The results are shown in FIG. 3, and blue, green and red fluorescence signals respectively appear in the experimental group, and the green and red fluorescence respectively form a circle, when the green and red fluorescence overlap, the obvious yellow fluorescence appears, which indicates that the SWP12 protein is distributed on the wall of the shrimp liver enterocytozoon spore. While there was no green fluorescence in the negative control group and no yellow fluorescence after the overlap.

5. Prawn anti-shrimp enterocytozoon infection effect by feeding sporoderm protein SWP12

5.1 Sporosporin protein SWP12 was fed

The healthy litopenaeus vannamei boone is divided into a blank control group (feeding common feed), an irrelevant protein group (feeding feed added with BSA with the content of 65mg/kg feed), a low-concentration protein group (feeding feed added with SWP12 with the content of 15mg/kg feed), a medium-concentration protein group (feeding feed added with SWP12 with the content of 40mg/kg feed) and a high-concentration protein group (feeding feed added with SWP12 with the content of 65mg/kg feed), and three parallel controls are arranged in each group. The feeding amount is about 10 percent of the weight of the shrimps every day, the feeding is carried out three times every day for 14 days continuously, and the feeding of the recombinant SWP12 protein is stopped immediately.

The common feed is a compound feed for penaeus monodon produced by Oncorhynchus feed limited company in Jiangmen city, and the main raw materials comprise: imported fish meal, cuttlefish powder, shrimp powder, high gluten flour, yeast powder, mineral substances, trace elements, multiple vitamins and a mildew preventive.

5.2EHP challenge experiment

After 14 days of feeding, all groups are changed to be fed with EHP infected shrimps, namely hepatopancreas, and the feeding is continuously carried out for 7 days.

5.3 feeding ordinary fodder and sampling and sample processing

After the challenge period is finished, all groups are changed to be fed with common feed, 5 time points (respectively 10 th, 14 th, 21 st, 28 th and 31 th days after the feeding of recombinant SWP12 protein is stopped) are sampled at 5 time points of all groups which are changed to be fed with common feed, 5 shrimps are respectively taken from each parallel time point, the shrimps are dissected, the hepatopancreas are taken out, the shrimps are placed in a freezing storage tube, and the shrimps are frozen by liquid nitrogen and then placed in a refrigerator at minus 80 ℃ for storage.

5.4 protective effect of prawn against shrimp liver enterocytozoon infection by feeding recombinant SWP12 protein

The artificial infection of EHP for 7 days was carried out on the prawns fed with the recombinant SWP12 protein for 14 days, and as a result, as shown in fig. 4, it was found that 100% of the prawns in the high concentration group (65mg/kg feed) were not infected with EHP within 10 days after the termination of the recombinant SWP12 protein, whereas EHP infection was detected in each group after the 14 days (i.e., 7 days after the termination of challenge) after the termination of the recombinant SWP12 protein, but the infection rate in the high concentration group was 6.67%, which was significantly lower than that in each other group (33.33% to 53.33%) (p <0.05), and the infection rate in each group showed an increasing trend with time, and reached the highest on the 31 day (24 days after the termination of 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 EHP infection resistance of the litopenaeus vannamei can be enhanced by feeding the litopenaeus vannamei with the high-concentration protein (65mg/kg) mixed with the feed, and 100 percent of shrimp enterocytozoon infection can be prevented within at least 10 days after the feeding of the recombinant SWP12 protein is stopped; when the recombinant SWP12 protein is stopped to be fed for 21 days, the infection rate of the shrimp liver enterocytozoon is obviously lower than that of the prawns which are not fed or fed with the low (medium) concentration SWP12 protein, which shows that the shrimp liver enterocytozoon sporoderm protein SWP12 with high concentration can obviously enhance the capability of the prawns to resist the shrimp liver enterocytozoon infection.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such modifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

Application of <120> sporoderm protein in prevention and control of shrimp liver enterocytozoon infection in shrimp mixing feeding

<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 sporoderm 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 sporoderm protein SWP12 in preparing the product for preventing and controlling shrimp liver enterocytozoon infection is characterized in that: the amino acid sequence of the spore wall protein SWP12 is shown in SEQ ID NO. 1.

2. Use according to claim 1, characterized in that:

the spore wall protein SWP12 is applied to the preparation of products for preventing and controlling shrimp enterocytozoon infection of mixed feeding and feeding shrimps.

3. Use according to claim 2, characterized in that:

the shrimp is litopenaeus vannamei.

4. Use according to any one of claims 1 to 3, wherein:

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

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

the addition amount of the spore wall protein SWP12 in the product is above 60 mg/kg.

6. Use according to claim 5, characterized in that:

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

7. A sporoderm protein polyclonal antibody, characterized in that: the polyclonal antibody is prepared by immunizing an animal with the sporoderm protein SWP12 described in claim 1.

8. The sporoderm protein polyclonal antibody according to claim 7, characterized in that:

the specific steps of the polyclonal antibody comprise:

(2) collecting serum of the immunized animal: after 2 times of animal immunization, blood collection and detection are carried out, and the titer of antiserum to the spore wall protein SWP12 is determined until the titer is more than or equal to 1: 51200 collecting antiserum by final blood collection;

(3) and (5) purifying the antibody.

9. Use of a sporoderm protein polyclonal antibody of any one of claims 7 to 8 in the preparation of a reagent or kit for diagnosing or detecting shrimp liver enterocytozoon.

10. A kit for diagnosing or detecting shrimp liver enterocytozoon is characterized in that: a polyclonal antibody against a spore wall protein, comprising the polyclonal antibody against a spore wall protein according to any one of claims 7 to 8.