Application of carp egg lectin rCcFEL2 in preparation of carp breeding feed additive

Abstract

The invention discloses application of carp roe lectin rCcFEL2 in preparation of a carp culture feed additive. The carp roe lectin rCcFEL2 has dual effects in improving disease resistance and growth performance of carps. The antibacterial function and the growth regulation effect of the carp roe lectin rCcFEL2 are systematically clarified for the first time, and the research blank of the lectin in antibacterial and immunological enhancement application of aquatic products is filled. An in-vitro antibacterial experiment proves that the carp roe lectin rCcFEL2 shows remarkable antibacterial activity on various common gram-negative bacteria and gram-positive bacteria in aquatic products, and is wide in antibacterial spectrum and stable in action effect; in-vivo challenge tests further show that the carp roe lectin rCcFEL2 can significantly enhance the disease-resistant defense capability of carp bodies and ovaries, effectively reduce the bacterial load in immune tissues infected by pathogenic bacteria, actively regulate the growth performance of carps and promote healthy growth of the bodies.

Description

Technical Field

[0002] This invention belongs to the field of aquaculture feed additive technology, and relates to a new use of carp egg lectin in the preparation of aquaculture feed additives, specifically the application of carp egg lectin rCcFEL2 in the preparation of carp aquaculture feed additives. Background Technology

[0004] Carp (Cyprinus carpio) is an important aquaculture species worldwide, widely favored by consumers for its delicious meat and rich nutrition. However, with the promotion of intensive, high-density aquaculture, problems such as deteriorating aquaculture environments and accelerated pathogen transmission have become increasingly prominent, leading to frequent outbreaks of bacterial, fungal, and parasitic diseases. Pathogen infection not only significantly inhibits the growth and development rate of carp and reduces aquaculture yield, but also directly damages the structure and function of their reproductive system, interferes with gamete formation and fertilization, and disrupts the homeostasis of the fish's own reproductive-immune regulatory network. This, in turn, leads to a decline in carp breeding efficiency and degradation of germplasm quality, posing a serious threat to the breeding of superior carp varieties and the upgrading of the industry.

[0005] In current aquaculture practices, antibiotics are commonly used to control bacterial diseases. However, long-term improper use can lead to drug residues and drug resistance, which not only harms the health of carp but also exacerbates food safety risks. Therefore, developing safe, efficient, environmentally friendly, and antibiotic-alternative green prevention and control technologies to address disease problems by strengthening the carp's own disease resistance has become a key technological bottleneck in promoting the high-quality development of the carp aquaculture industry and is an urgent industry need.

[0006] The innate immune system is the primary defense system of aquatic animals, and its activation core is pathogen recognition. Lectins, as key members of pattern recognition receptors (PRRs), can specifically recognize pathogen-associated molecular patterns such as bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN), initiating an immune response by binding to microbial sugars through their sugar recognition domains. Furthermore, lectins also possess functions such as promoting microbial aggregation, enhancing hemocyte phagocytosis and encapsulation, antiviral and antibacterial properties, and opsonization. They also exhibit strong thermal stability, tolerating the high temperatures of feed pelleting, making them highly promising candidate materials for green aquatic feed additives. Fish egg lectins are maternally derived immune-active molecules unique to fish, possessing both antibacterial and immunomodulatory functions. They can be transmitted to offspring through maternal reproduction, constructing a passive immune barrier in the early developmental stages of larvae, effectively resisting pathogen invasion, and playing a crucial role in ensuring larval survival and improving early developmental quality.

[0007] Patent document CN200910066648.8 discloses the bullfrog lectin gene and its encoded lectin and applications. The bullfrog lectin gene was screened from a constructed bullfrog skin cDNA library. The obtained bullfrog lectin can be used as an antibacterial feed additive, in drug development, and as a guiding functional molecule in drug delivery vectors. Patent document CN200610035461.8 discloses the sea bass lectin gene sequence. Degenerative primers were designed using conserved regions of CDS sequences of lectin homologous genes from closely related species. Total RNA was extracted from the spleen of sea bass, amplified by PCR, and cloned using RACE technology to obtain the full expression sequence of the sea bass lectin gene. This gene not only lays the foundation for studying the role of fish lectins in inflammatory responses and further exploring the mechanisms of inflammatory responses in fish, but also allows for the acquisition of bioactive purified proteins through gene sequencing, providing a theoretical basis for the research of novel disease-fighting immune adjuvants. Patent document CN201510423083.X discloses the application of lectin-modified anchovy antimicrobial peptide liposomes in inhibiting Listeria monocytogenes and its biofilm. Based on the specific affinity of plant lectins for glycounits in the extracellular polysaccharide matrix of the biofilm, lectin-modified anchovy antimicrobial peptide liposomes were prepared. A Listeria monocytogenes biofilm model was established, and the inhibitory effect of lectin-modified anchovy antimicrobial peptide liposomes on Listeria monocytogenes and its biofilm was evaluated by bactericidal curves, colony counts, and crystal violet staining. The results show that lectin-modified anchovy antimicrobial peptide liposomes have a bactericidal effect on Listeria monocytogenes, can interfere with its biofilm formation, and can remove existing biofilms. Patent document CN202110559923.0 discloses the amphioxus C-type lectin BjCTL4 gene and its applications. It is the first discovery that the protein encoded by the C-type lectin BjCTL4 gene possesses the basic characteristics of traditional CTLs, including two or three pairs of conserved disulfide bonds, a motif similar to the characteristic WIGL motif, and a similarity to Ca²⁺. 2+Similar motifs to the related sugar-binding motifs QPD / EPN and WND have been preliminarily verified to bind to polysaccharides on bacterial surfaces relatively broadly, exhibiting significant bacterial binding and agglutination effects. These motifs could be developed into natural antibacterial active substances and used to prepare drugs for treating infectious diseases. Patent document CN201810089975.4 discloses a method for preparing and applying a fish-derived galactosyl lectin, CaGal, recombinant protein. The prepared CaGal recombinant protein retains its natural biological activity and can effectively inhibit pathogens such as Aeromonas hydrophila, demonstrating significant antibacterial effects. It can be used as a novel fish immune additive or antibacterial drug. Patent document CN200610035329.7 discloses the isolation and application of the AmphiGAL13 gene from the digestive tract of amphioxus. Recombinant amphioxus GST-PGAL13 protein is expressed through GST fusion and digested with EK enzyme, yielding a protein with general lectin functions. This protein can be developed and applied as a natural small-molecule bioadhesive microparticle formulation. However, none of the aforementioned patent documents mention the dual application of carp egg lectin rCcFEL2 in improving the disease resistance and growth performance of carp.

[0008] Currently, there are no publicly available or authorized patent records for carp egg lectins, and non-patent research literature only sporadically mentions that some fish egg lectins possess basic antibacterial activity. Related research is significantly insufficient: First, there is a lack of systematic analysis of the antibacterial spectrum and bactericidal mechanism of fish egg lectins; second, the impact on the overall immune function and specific immune regulation of the reproductive system in fish has not been explored in depth; third, research on its application in improving the growth performance and enhancing the disease resistance of farmed fish is extremely scarce; fourth, no research has yet combined fish egg lectins with the protection of aquatic germplasm resources, and its application potential in improving carp germplasm breeding efficiency and enhancing germplasm quality has not been explored. In summary, there are many gaps in the basic research and application exploration of carp egg lectins, and targeted research is urgently needed to fill these gaps and provide new technical pathways for the green development and breeding of superior varieties in the carp aquaculture industry. Summary of the Invention

[0010] The purpose of this invention is to provide the application of carp egg lectin rCcFEL2 in the preparation of carp aquaculture feed additives. This invention clones an egg lectin named rCcFEL2 from carp ovaries and obtains a large amount of carp egg lectin through prokaryotic expression. This carp egg lectin has good antibacterial properties and can improve the disease resistance of carp and enhance the growth performance of carp.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] The first aspect of this invention relates to the application of carp egg lectin rCcFEL2 in the preparation of carp aquaculture feed additives, wherein the gene sequence of carp egg lectin rCcFEL2 is shown in SEQ ID NO.1 of the sequence listing. A large amount of carp egg lectin rCcFEL2 is obtained through prokaryotic expression. Plasmid construction: The CcFEL2 coding sequence (shown in SEQ ID NO.1) is inserted into the EcoRI / XhoI restriction site of the pET30a-CcFEL2 vector to construct the recombinant plasmid pET30a-CcFEL2, which is then expressed in prokaryotic cells and transformed into E. coli BL21(DE3) and cultured at 37°C until OD200. 600 =0.6, add 0.5 mM IPTG, and induce at 37℃ for 4 h; Protein purification: after sonication, the supernatant was collected, passed through a Ni-NTA column (equilibrated with 20 mM imidazole, eluted with 250 mM imidazole), and the purity was detected by SDS-PAGE.

[0013] Furthermore, the amino acid sequence of the carp egg lectin rCcFEL2 is shown in SEQ ID NO.2.

[0014] Furthermore, the carp egg lectin rCcFEL2 is added to carp farming feed at a dosage of 40-80 mg / kg.

[0015] In a second aspect, the application of carp egg lectin rCcFEL2 in the preparation of antibacterial feed additives for carp farming, wherein rCcFEL2 exhibits inhibitory effects against both Gram-negative and Gram-positive bacteria. The antibacterial activity of rCcFEL2 against common fish-derived Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus) and Gram-negative bacteria (Aeromonas hydrophila, Aeromonas velutipes, Aeromonas salmonidae, and Pseudomonas aeruginosa) was determined using solid and liquid antibacterial assays.

[0016] The third aspect of this invention relates to the application of carp egg lectin rCcFEL2 in the preparation of a carp aquaculture feed additive that inhibits Aeromonas hydrophila biofilm. This carp egg lectin rCcFEL2 exhibits an inhibitory effect on Aeromonas hydrophila biofilm. Free-living bacteria have limited tolerance to environmental stresses, while biofilm formation enhances the bacterial community's ability to resist adverse environmental pressures, thereby helping bacteria escape the killing effect of antibacterial substances. Therefore, biofilm detection has become a core indicator for evaluating the effectiveness of sterilization processes. Combined observation using ELISA, optical microscopy, and laser confocal microscopy confirmed that carp egg lectin rCcFEL2 significantly inhibits both the formation and maturation of Aeromonas hydrophila biofilms.

[0017] In a fourth aspect, the invention relates to the application of carp ovum lectin rCcFEL2 in the preparation of carp aquaculture feed additives that enhance disease resistance in carp. Dietary supplementation with rCcFEL2 can improve the disease resistance of carp. Three feeds were prepared, with 40 mg / kg and 80 mg / kg of rCcFEL2 added to a basal commercial feed, respectively. A control diet was prepared using a basal commercial feed (without rCcFEL2), and carp were subsequently fed this diet for 60 consecutive days. Artificial infection experiments were conducted on carp using Aeromonas hydrophila. The results showed that compared to the control group fed a basal diet, the treatment group with 80 mg / kg rCcFEL2 in their diet exhibited a significant reduction in Aeromonas hydrophila load in the intestines, blood, and ovarian tissues. Furthermore, it effectively alleviated the increase in antioxidant enzyme activity caused by Aeromonas hydrophila attack in carp.

[0018] The fifth aspect of this invention relates to the application of carp ovum lectin rCcFEL2 in the preparation of a carp aquaculture feed additive that enhances the reproductive endocrine function of carp. Dietary supplementation with carp ovum lectin rCcFEL2 can enhance the reproductive endocrine function of carp. Ovarian health status was assessed using H&E section analysis, and changes in carp reproductive hormones were detected, revealing the interfering effect of carp ovum lectin rCcFEL2 on the carp reproductive endocrine system. Results showed that supplementation with carp ovum lectin rCcFEL2 can effectively alleviate ovarian damage caused by Aeromonas hydrophila and maintain healthy ovarian morphology and estradiol and testosterone levels.

[0019] In a sixth aspect of this invention, the application of carp roe lectin rCcFEL2 in the preparation of carp aquaculture feed additives to improve carp growth performance is described. Dietary supplementation with carp roe lectin rCcFEL2 enhances carp growth performance. A 60-day feeding trial was conducted with diets supplemented with 40 mg / kg and 80 mg / kg of carp roe lectin rCcFEL2. Condition factor (CF), weight gain (WGR), hepatic-to-body ratio (HIS), visceral-to-body ratio (VSI), gonadal index (GSI), and specific growth rate (SGR) were measured. The results showed that dietary supplementation with 40 mg / kg and 80 mg / kg of carp roe lectin rCcFEL2 significantly improved carp weight gain, specific growth rate, and gonadal index.

[0020] Compared with existing technologies, this invention has the following advantages and beneficial effects: The carp egg lectin rCcFEL2 in this invention has a dual effect in improving the disease resistance and growth performance of carp. This invention is the first to systematically elucidate the antibacterial function and growth regulation of carp egg lectin rCcFEL2, filling the research gap in the application of this type of lectin in aquatic antibacterial and immune-enhancing applications. In vitro antibacterial experiments confirmed that carp egg lectin rCcFEL2 exhibits significant antibacterial activity against a variety of common Gram-negative and Gram-positive bacteria in aquaculture, with a broad antibacterial spectrum and stable effects. In vivo challenge experiments further demonstrate that this carp egg lectin rCcFEL2 can significantly enhance the disease resistance and defense capabilities of carp bodies and ovaries, effectively reduce the bacterial load in immune tissues after pathogen infection, and actively regulate carp growth performance, promoting healthy growth. This invention provides a novel natural active lectin that can be developed into a highly efficient natural fish immune enhancer or antibacterial agent. It offers a new strategy for the green control of bacterial diseases in economic fish such as carp, avoiding the problems of drug resistance and environmental residues caused by the overuse of chemical antibacterial agents. It has important industrial application prospects and market value in the aquaculture industry. Attached Figure Description

[0022] Figure 1 This is a diagram showing the expression of carp egg lectin protein in Example 1.

[0023] Figure 2 The following graph shows the antibacterial effect of carp egg lectin in Example 2: A is a representative plate image from the antibacterial experiment; B is a statistical graph of the antibacterial rate of solid antibacterial agents; and C is a growth curve of liquid antibacterial agents.

[0024] Figure 3 In Example 3, carp egg lectin inhibits the formation of Aeromonas hydrophila biofilm. A represents the inhibition of the formation process of Aeromonas hydrophila biofilm, and B represents the inhibition of the mature biofilm of Aeromonas hydrophila.

[0025] Figure 4 In Example 5, the carp egg lectin reduced the bacterial load and anti-infection ability of carp tissues after pathogen infection. A represents the tissue bacterial load, B represents the MDA level, C represents the CAT level, and D represents the SOD level.

[0026] Figure 5 In Example 6, carp egg lectin rCcFEL2 was added to maintain ovarian function. A is an H&E section of the ovary, B is the testosterone content, and C is the estradiol content.

[0027] Figure 6 Adding carp egg lectin rCcFEL2 to Example 6 improved the growth performance of carp. Detailed Implementation

[0029] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0030] Example 1

[0031] cDNA cloning of carp egg lectin CcFEL2 and construction of recombinant rCcFEL2 strain

[0032] 1. RNA Extraction and cDNA Synthesis: Total RNA was extracted from carp ovarian tissue using Trizol reagent, and RNA concentration was assessed using NanoDrop 2000. First-strand cDNA was then synthesized using the PrimeScript First-Strand cDNA Synthesis Kit.

[0033] 2. cDNA cloning: This invention designed a pair of specific primers to amplify the CcFEL2 gene: rCcFEL2-F (5'-TACTCAGAATTCATGAAGGTTTATCAGGG-3') and rCcFEL2-R (5'-TACTCACTCGAGCGTAAATGTACTTTATAA-3');

[0034] 3. Perform the following PCR amplification: 94℃ for 5 min; 94℃ for 30 s, 48℃ for 30 s, 72℃ for 30 s, 34 cycles; 72℃ for 10 min.

[0035] 4. After PCR amplification, the product was recovered by 1 wt% agarose gel electrophoresis at 120 V for 20 min. The gel showing the correct band was cut off, and the agarose gel was recovered using a small-dose gel recovery kit according to the manufacturer's instructions to obtain the purified CcFEL2 fragment.

[0036] 5. Expression vector construction: The CcFEL2 fragment and pET30a plasmid were double-digested using EcoRI and XhoI enzymes, respectively. The digestion system was: DNA fragment digestion (20 μL system).

[0037] DNA 2μL <![CDATA[dd H2O]]> 14μL 10× Qustone buffer (colorless) 2μL Enzymes (1 μL each) 2μL

[0038] Enzyme digestion of plasmid (20 μL system)

[0039] plasmid 2μL <![CDATA[dd H2O]]> 16μL 10× Qustone buffer (colored) 2μL Enzymes (1 μL each) 2μL

[0040] Samples were added on ice, and the enzyme digestion was performed entirely on ice. Three tubes were used for each gene / plasmid digestion. PCR was performed at 37°C for 45 minutes.

[0041] 6. After enzyme digestion, the digested pET30a vector was detected and recovered using a gel microparticle kit, and the CcFEL2 fragment was recovered as PCR product. The recovered CcFEL2 fragment was ligated into the pET30a vector to construct the expression vector pET30a-CcFEL2.

[0042] 7. Transform the ligation product from the above steps into competent Escherichia coli DH5α, screen for positive strains, inoculate them into LB medium containing ampicillin sodium, and culture them at 37°C and 180 rpm for 10 h. Then, extract the expression plasmid using a kit.

[0043] 8. Construction of CcFEL2 recombinant strain: The above expression plasmid was transformed into Escherichia coli BL21 competent cells, inoculated into LB solid medium containing kanamycin sulfate, cultured at 37°C until colonies grew, and positive strains were screened.

[0044] 9. High-volume protein expression: The rCcFEL2 recombinant strain was grown in LB medium at 37°C and 180 rpm. 3 mL of the bacterial culture was transferred to 300 mL of LB medium at a volume ratio of 1:100 and cultured with shaking at 37°C and 180 rpm for 3 h. Then, 0.5 mM IPTG inducer was added for another 4 h of induction. The culture was then centrifuged at 4°C and 6000 rpm for 10 min. The supernatant was discarded, and the precipitate was collected. The precipitate was resuspended in 20 mL of denaturing lysis buffer and sonicated on ice. The precipitate was centrifuged at 4°C and 12000 rpm for 10 min, and the supernatant was collected. After purification, the precipitate was dialyzed (dialysis buffer was 50 times the volume of the supernatant) at 4°C for at least 24 h. Finally, the precipitate was centrifuged at 4°C and 10000 rpm for 10 min. The supernatant containing the target protein was stored at -80°C until use.

[0045] Figure 1 SDS-PAGE was used to detect the induced expression of carp egg lectin rCcFEL2; lane M: standard protein molecular marker; lane 1: uninduced protein; lane 2: recombinant protein induced by 0.5 mM IPTG; lane 3: purified carp egg lectin rCcFEL2.

[0046] Example 2

[0047] Antibacterial activity assay of carp egg lectin rCcFEL2

[0048] 1. Antibacterial Activity Test: Six commonly used fish-derived bacterial species were selected: Gram-negative bacteria: *Aeromonas hydrophila*, *Aeromonas veronii*, *Pseudomonas aeruginosa*, and *Aeromonas salmonicida*; and Gram-positive bacteria: *Staphylococcus aureus* and *Micrococcus luteus*. The six bacterial strains were inoculated onto LB medium and cultured on a shaker at 37 / 28℃ and 180 rpm for 12 h. The six bacterial suspensions were then centrifuged at 6000 rpm for 5 min, the supernatant was discarded, and the suspensions were resuspended and diluted with sterile PB medium. 10 μL of the bacterial suspension (1×10⁻⁶) was collected. 5 50 μL of CFU and 50 μL of recombinant protein (50 μg / mL) were mixed with 140 μL of sterile PB and added to a sterile 96-well plate. The control group was prepared by mixing sterile PBS with bacterial suspension and PB medium. Finally, the 96-well plate was incubated at 37 / 28℃ and 110 rpm with slow shaking. The bacterial density was detected by measuring the absorbance at 600 nm using a microplate reader at multiple time points from 0 to 40 h. The experimental data were repeated three times.

[0049] 2. Antibacterial test: Dilute 50 μL of bacterial culture to 1×10⁻⁶. 5 CFU / mL was incubated with 50 μL of protein (0.5 mg / mL) at 37°C for 4 h. The mixture was then diluted 10-fold, and 100 μL of the diluted solution was plated on LB agar plates. After 12 h of incubation, colonies were counted and the average value was calculated.

[0050] Test results as follows Figure 2 As shown, carp egg lectin rCcFEL2 has significant antibacterial activity against the tested bacteria: Aeromonas hydrophila, Aeromonas vesiculosus, Pseudomonas aeruginosa, and Aeromonas salmonida; and Gram-positive bacteria: Staphylococcus aureus and Micrococcus luteus, especially against Aeromonas hydrophila.

[0051] Example 3

[0052] The ability of carp egg lectin rCcFEL2 to inhibit biofilm

[0053] 1. Since carp egg lectin rCcFEL2 has the strongest antibacterial activity against Aeromonas hydrophila, Aeromonas hydrophila was selected as the infecting strain for subsequent experiments. The activated Aeromonas hydrophila strain was inoculated into 24-well plates at a ratio of 1 wt%, and co-cultured with carp egg lectin rCcFEL2 for 72 h. OD values ​​were measured using a microplate reader at 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. 595nm , to represent the biomass in the biofilm during the biofilm formation process.

[0054] 2. The activated Aeromonas hydrophila strain was inoculated into 24-well plates at a ratio of 1 wt% and cultured at 30°C for 48 h, with half of the culture medium replaced every 12 h to allow for the formation of a mature biofilm. The biofilm was treated with carp egg agglutinin rCcFEL2. The dissipation effect of rCcFEL2 on the biofilm was observed using an optical microscope with crystal violet staining. The thickness and density of the mature Aeromonas hydrophila biofilm were observed using a laser confocal microscope with FITC-labeled bacteria.

[0055] Test results as follows Figure 3 As shown, the formation and maturation of biofilms by Aeromonas hydrophila are both inhibited by the carp egg lectin rCcFEL2, resulting in an increase in dead cells, a decrease in biofilm density and thickness, and biofilm dissipation.

[0056] Example 4

[0057] Carp egg lectin rCcFEL2, as a feed additive, reduces bacterial load in carp tissues after pathogen infection and maintains antioxidant enzyme activity.

[0058] 1. Three experimental diets were set up. Two experimental diets were prepared by adding 40 mg / kg and 80 mg / kg of carp lectin rCcFEL2 to a basic commercial feed, respectively. The control group was a standard commercial feed without added carp lectin rCcFEL2. The feed preparation process was as follows: First, the basic commercial carp feed was thoroughly pulverized into powder using an electric grinder. Then, different doses of carp lectin rCcFEL2 were added to the feed powder, and distilled water was added and stirred thoroughly to achieve a homogeneous state. The homogenized material was then granulated into pellets using a granulator. Finally, the pellets were sealed and dried at 25°C for 12 h. After preparation, they were stored at 4°C for later use.

[0059] 2. Carp (approximately 3-5 cm in length each) were randomly divided into three groups. The control group was fed a diet without carp egg lectin rCcFEL2, while the experimental groups were fed diets containing 40 mg / kg and 80 mg / kg of rCcFEL2, respectively. The feeding was repeated for 60 days. The carp were fed twice daily (8:00 AM and 5:00 PM) at 2 wt% of their body weight. After the feeding experiment, a pathogen infection experiment was conducted.

[0060] Test results as follows Figure 4 As shown, the addition of carp egg lectin rCcFEL2 can effectively reduce the colonization of Aeromonas hydrophila in the intestines, ovaries, and blood of carp, and alleviate oxidative stress caused by Aeromonas hydrophila attack.

[0061] Example 5

[0062] Adding carp egg lectin rCcFEL2 improves carp reproductive ability

[0063] After being challenged with Aeromonas hydrophila, the morphology of carp tissues and the changes in hormone levels of estradiol and testosterone in carp were detected by H&E staining.

[0064] Test results as follows Figure 5 As shown: the condition of carp ovaries can be directly observed after H&E staining, and the addition of carp egg lectin rCcFEL2 can maintain the level of reproductive endocrine hormones in carp.

[0065] Example 6

[0066] Effects of carp egg lectin rCcFEL2 addition on carp growth performance

[0067] Before and after the experiment, the body length, weight, liver weight, and visceral weight of the carp in each aquaculture tank were counted and weighed. The weight gain rate (WGR), visceral weight ratio (VSI), liver weight ratio (HPI), specific growth rate (SGR), condition factor (CF), and gonad index (GSI) of the carp were measured.

[0068] Test results as follows Figure 6 As shown, supplementation with carp egg lectin rCcFEL2 can significantly improve specific growth rate, weight gain rate, and gonad index in carp.

[0069] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

[0070] SEQUENCE LISTING

[0071] <110> Henan Normal University

[0072] <120> Application of carp egg lectin rCcFEL2 in the preparation of carp aquaculture feed additives

[0073] <130> 2026

[0074] <160> 4

[0075] <170> Patentin version 3.3

[0076] <210> 1

[0077] <211> 771

[0078] <212> DNA

[0079] <213> Artificial sequence

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[0090] <212> DNA

[0091] <213> Artificial sequence[[ID=1३]]

[0092] <400> 3

[0093] tactcagaattcatgaaggtttatcaggg 29

[0094] <210> 4

[0095] <211> 30

[0096] <212> DNA

[0097] <213> Artificial sequence

[0098] <400> 4

[0099] tactcactcgagcgtaaatgtactttataa 30

Claims

1. Application of carp egg lectin rCcFEL2 in the preparation of carp aquaculture feed additive, wherein the gene sequence of carp egg lectin rCcFEL2 is shown in the sequence listing SEQ ID NO.

1.

2. The application according to claim 1, characterized in that: The amino acid sequence of the carp egg lectin rCcFEL2 is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: The carp egg lectin rCcFEL2 is added to carp aquaculture feed at a rate of 40-80 mg / kg.

4. The application of the carp egg lectin rCcFEL2 according to claim 1 in the preparation of antibacterial feed additives for carp farming, wherein the carp egg lectin rCcFEL2 has an inhibitory effect on both Gram-negative and Gram-positive bacteria.

5. The application of the carp egg lectin rCcFEL2 according to claim 1 in the preparation of a carp aquaculture feed additive that inhibits Aeromonas hydrophila biofilm.

6. The application of the carp egg lectin rCcFEL2 as described in claim 1 in the preparation of a carp aquaculture feed additive that enhances the disease resistance of carp.

7. The application of the carp egg lectin rCcFEL2 according to claim 1 in the preparation of a carp aquaculture feed additive that enhances the reproductive endocrine function of carp.

8. The application of the carp egg lectin rCcFEL2 according to claim 1 in the preparation of carp aquaculture feed additives that improve the growth performance of carp.

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