CN110590926B - A kind of compound crucian carp ITLN protein and its preparation method and application - Google Patents

Abstract

The invention discloses a crucian carp ITLN protein, the amino acid sequence of which is shown in SEQ ID NO: 2, or the amino acid sequence shown in SEQ ID NO: 2 is substituted, deleted and/or added by one or more amino acids and and/or a protein with equivalent or higher activity obtained by terminal modification. The discovery of the crucian carp ITLN protein not only enriches the gene pool of crucian carp, but also can be applied to the preparation of recombinant proteins, and further applied to inhibiting the growth of Aeromonas hydrophila and/or Streptococcus agalactiae, and preparing the immunity of aquatic animals. Preparations or feed additives provide a new practical basis for the research on physiological immunity of Hefang crucian carp. The invention also discloses a preparation method of the compound crucian carp ITLN protein, and the compound crucian carp ITLN protein is used in inhibiting the growth of Aeromonas hydrophila and/or Streptococcus agalactiae, preparing immune preparations or feed additives for aquatic animals Applications.

Description

A kind of compound crucian carp ITLN protein and its preparation method and application

technical field

The invention belongs to the technical field of genetic engineering research and development, and in particular relates to a hybrid crucian carp Intelectin (ITLN) protein and a preparation method thereof, as well as immune preparations for inhibiting the growth of Aeromonas hydrophila and/or Streptococcus agalactiae, preparing aquatic animals or Application in feed additives.

Background technique

Intestinal lectin (Intelectin, ITLN), belonging to the lectin family, is a new type of soluble β-galectin. Mammalian ITLN was first discovered in mouse small intestinal Paneth cells, and two subtypes of mouse and human ITLN have been found. Vertebrate ITLN mainly contains the N-terminal fibrinogenlike-domain (FReD) and the C-terminal intestinal lectin domain (intelectin domain). In general, the C-terminal β and γ chains of ITLN are conserved, while the N-terminus has various modifications. The trimeric structure formed by the aggregation of FReD domains is related to the ability of ficolin to bind acetylglucosamine, which is a typical fibrinogen protein, which can not only participate in the activation of the complement system, but also can resist bacterial infection. In addition, in most mammals, ITLN contains about 10 conserved cysteine residues, and its domain is composed of a random coil and 3 β-sheets surrounded by 12 short α-helices , but the quaternary structure of ITLN is different in different species. Ascidian ITLN exists in various multimeric forms such as dimer, trimer and hexamer, while mouse and human ITLN exist in monomer and trimer forms, respectively. More and more studies now point out that ITLN is a multifunctional lectin protein, which plays a key regulatory role in a variety of life activities, mainly focusing on polyspermia, bone density regulation, iron metabolism, pattern receptor recognition , immune defense mechanisms induced by bacterial infection and Th2 immune responses caused by parasites. In addition, ITLN is closely related to the pathological process of various diseases, such as heart disease, obesity, asthma, cancer, enteritis and emphysema.

Evidence suggests that the innate immune defense system of invertebrates contains multiple pathogen recognition systems, but this is only the basic immune system of teleost fish. Teleost fish contain mammalian-like immune defense systems and apoptotic signaling pathways, and members of the fish lectin superfamily play an important role in defense against bacterial invasion. As a newly discovered member of fish galectins, ITLN has certain differences compared with ITLN of higher animals, but given that its amino acid sequence contains CRD characteristic structure, successive reports speculate that it may be involved in fish galectins immune regulation. Therefore, the development and utilization of fish-derived ITLN will be more beneficial to participate in the mechanism of immune regulation and antibacterial and bacteriostasis of fish efficiently. Hefang crucian carp (WR, 2n=100) is a freshwater fish developed by the State Key Laboratory of Freshwater Fish Developmental Biology jointly established by Hunan Normal University and the Ministry of Education, and is also a new national aquatic species. Hefang crucian carp (WR, 2n=100) is the first generation of the hybrid of Japanese white crucian carp (Carassius auratus cuvieri, WCC, ♀, 2n=100) and red crucian carp (Carassius auratus red var, RCC, ♂, 2n=100). Hefang crucian carp looks like wild crucian carp, has strong fecundity, fast growth, high fertilization rate and hatching rate, and is suitable for large-scale production. Furthermore, the amino acids in the muscle of Hefang crucian carp were significantly higher than those of their parents, the meat was fresh and tender, and the nutritional value was high, which was very popular among farmers and consumers. However, due to drastic climate change and seriously polluted water quality problems, the health of fish farming has been severely hit, and it is also a difficult problem to solve. Hybrid breeding technology can not only cultivate high-quality aquaculture fish species, but also further develop high-quality fish-derived feed additive protein on this basis, which is conducive to the development and application of high-quality fish feed, which can reduce environmental pollution and reduce environmental pollution. Can increase efficiency.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology, and provide a compound crucian carp ITLN protein and a preparation method and application thereof.

In order to solve the above-mentioned technical problems, the technical scheme proposed by the present invention is:

A compound crucian carp ITLN protein, its amino acid sequence is as shown in SEQ ID NO:2, or the amino acid sequence shown in SEQ ID NO:2 is substituted, deleted and/or added by one or more amino acids and/or terminal modification The resulting protein has the same or higher activity.

The above-mentioned crucian carp ITLN protein, preferably, the ITLN protein of crucian carp Hefang is encoded by the ITLN gene of crucian carp Hefang, and the nucleotide sequence of the ITLN gene of crucian carp is shown in SEQ ID NO: 1.

Based on a general technical concept, the present invention also provides a method for preparing the ITLN protein of Hefang crucian carp, comprising the steps of: amplifying the ITLN gene of Hefang crucian carp by PCR method and then cloning it into the pMD19-T vector, and then using the obtained The cloning vector is cloned into the expression vector pET32a, and then the expression vector pET32a containing the ITLN gene of Hefang crucian carp is transferred into Escherichia coli BL21 (E.coliBL21) for expression by the CaCl ₂ transformation method, and finally the described Hefang crucian carp is obtained. ITLN protein.

The above-mentioned preparation method, preferably, the specific operation of cloning the Hefang crucian carp ITLN gene into the pMD19-T vector after amplification by PCR method includes the following steps: using the Hefang crucian carp liver cDNA obtained by reverse transcription as a template , Amplify the ITLN gene of crucian carp by PCR method, and then ligated with pMD19-T vector at 4°C, the ligation product was transformed into Escherichia coli DH5α (Escherichia coli), and screened out by Amp ⁺ resistance and/or blue-white spot method Positive clone means that the ITLN gene sequence of Hefang crucian carp was successfully cloned into the pMD19-T vector; the nucleotide sequence of the ITLN gene of Hefang crucian carp is shown in SEQ ID NO: 1.

Preferably, the PCR amplification reaction conditions are: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30s, annealing at 72°C for 60s, for a total of 5 cycles; denaturation at 94°C for 30s, annealing at 65°C for 30s, extension at 72°C for 60s, A total of 5 cycles; denaturation at 94°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 60s, a total of 35 cycles; the final extension was continued at 72°C for 10min; the primers used in the PCR amplification were upstream primer ITLN-F1 and downstream primer ITLN-R1, the nucleotide sequence of the upstream primer ITLN-F1 is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer ITLN-R1 is shown in SEQ ID NO: 4.

Preferably, the PCR amplification reaction conditions are as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 60s, for a total of 40 cycles; and a final extension at 72°C for 10 min; the PCR The primers used in the amplification method are upstream primer ITLN-F2 and downstream primer ITLN-R2, the nucleotide sequence of the upstream primer ITLN-F2 is shown in SEQ ID NO: 5, the nucleoside of the downstream primer ITLN-R2 The acid sequence is shown in SEQ ID NO:6.

Preferably, the specific operation of cloning the obtained cloning vector into the expression vector pET32a includes the following steps: after extracting the obtained cloning vector with a plasmid mini-kit, use restriction endonuclease EcoRI and positive clone XhoI Carry out double-enzyme digestion and agarose gel electrophoresis to recover the product of the ITLN gene sequence of crucian carp. The ITLN gene sequences were mixed and ligated with T4 ligase at 16°C for 15h, that is, the cloning vector was successfully cloned into the expression vector pET32a.

Based on a general technical concept, the present invention also provides a corresponding application of a crucian carp ITLN protein in inhibiting the growth of Aeromonas hydrophila and/or Streptococcus agalactiae.

Above-mentioned application, preferably, its application method comprises the following steps: mix described compound crucian carp ITLN protein and the solution containing Ca ²⁺ , contact with Aeromonas hydrophila and/or Streptococcus agalactiae, after the reaction is terminated That is, the inhibition of exposure to Aeromonas hydrophila and/or Streptococcus agalactiae is accomplished.

Based on a general technical concept, the present invention also provides a corresponding application of a compound crucian carp ITLN protein in the preparation of an immune preparation or a feed additive for aquatic animals.

Compared with the prior art, the beneficial effects of the present invention are:

1. The compound crucian carp ITLN protein of the present invention has been proved by experiments to inhibit the growth activity of Aeromonas hydrophila and Streptococcus agalactiae, and can be applied to inhibit the growth of Aeromonas hydrophila and/or Streptococcus agalactiae, The preparation of immune preparations or feed additives for aquatic animals has broad application potential.

2. The preparation method of the present invention expresses the ITLN nucleotide sequence of Hefang crucian carp through a recombinant strain, so that the recombinant protein of Hefang crucian carp ITLN can be produced in large quantities, which greatly reduces the production cost and has high economic value.

3. The present invention obtains the ITLN gene sequence of Hefang crucian carp for the first time, which not only enriches the gene pool of Hefang crucian carp, but also can be applied to the preparation of recombinant proteins, and further applied to inhibit Aeromonas hydrophila and/or Streptococcus agalactiae. The growth and preparation of immune preparations or feed additives for aquatic animals provides a new practical basis for the study of physiological immunity of Hefang crucian carp.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are For some embodiments of the present invention, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Fig. 1 is the electrophoresis identification diagram of the PCR amplification product of the ITLN gene ORF of Hefang crucian carp in the embodiment (M: DNA molecular weight standard; 1, 2: PCR product of the full-length sequence of the ITLN gene ORF of Hefang crucian carp);

Fig. 2 is the electrophoresis identification diagram of the PCR amplification product of the ORF ligase ligase linker (EcoRI, XhoI) of the crucian carp ITLN gene in the embodiment (M: DNA molecular weight standard; 1, 2: the hefang crucian carp with the restriction enzyme cleavage linker) ITLN gene ORF full-length sequence PCR product);

Figure 3 is the electrophoresis identification diagram of the cloning vector pMD19-T-ITLN double-enzyme digestion (EcoRI, XhoI) in the Example (M: DNA molecular weight standard; 1: double-enzyme-digested pMD19-T-ITLN plasmid; 2: pMD19-T- ITLN plasmid);

Figure 4 is an electrophoresis identification diagram of the expression vector pET32a-ITLN double-enzyme digestion (EcoRI, XhoI) in the Example (M: DNA molecular weight standard; 1: pET32a-ITLN plasmid; 2: double-enzyme digestion pET32a-ITLN plasmid);

Figure 5 is the PAGE electrophoresis analysis chart and the Western blot identification chart of the recombinant strain pET32a-ITLN-BL21 expression product ITLN recombinant protein in the Example (M: protein molecular weight standard; 1: pET32a-BL21 induced product; 2: pET32a-WR-ITLN -BL21-induced product; 3: supernatant of pET32a-WR-ITLN-BL21-induced product after disruption; 4: precipitation of pET32a-WR-ITLN-BL21-induced product after disruption; 5: purified recombinant protein WR-ITLN; 6: Western blot verification of the purified recombinant protein WR-ITLN);

Fig. 6 is the construction schematic diagram of pET32a-ITLN expression vector in the embodiment;

Fig. 7 is the comparison diagram of the amino acid sequence of compound crucian carp ITLN and the amino acid sequence of human ITLN in the embodiment;

Fig. 8 is the predicted analysis result of the secondary structure of the crucian carp ITLN protein in the embodiment;

Fig. 9 is the ELISA binding experiment of different concentrations of crucian carp ITLN recombinant protein and Aeromonas hydrophila in the embodiment (different letters indicate significant differences, P<0.05);

Fig. 10 is the ELISA binding experiment of different concentrations of Hefang crucian carp ITLN recombinant protein and Streptococcus agalactiae in the embodiment (different letters indicate significant differences, P<0.05);

Figure 11 is the effect of the treatment of the recombinant crucian carp ITLN protein in the embodiment on the activity of Aeromonas hydrophila (different letters indicate significant differences, P<0.05);

Figure 12 is the effect of the treatment of the recombinant crucian carp ITLN protein in the embodiment on the activity of Streptococcus agalactiae (different letters indicate significant differences, P<0.05);

Figure 13 is the effect on the colony formation of Aeromonas hydrophila after the treatment with the crucian carp ITLN recombinant protein in the embodiment;

Figure 14 is the effect on the colony formation of Streptococcus agalactiae after the treatment with the Recombinant Crucian carp ITLN recombinant protein in the embodiment;

Figure 15 shows the expression changes of Aeromonas hydrophila hemolysin hlyA gene in the liver, kidney and spleen of fish treated with Trx and ILTN after Aeromonas hydrophila infection in the Example (different letters indicate the same as the control group) There is a significant difference in the ratio, P<0.05);

Figure 16 shows the expression changes of Streptococcus agalactiae CAMP factor gene in the liver, kidney and spleen of fish treated with Trx and ILTN after Streptococcus agalactiae infection in the examples (different letters indicate significant differences compared with the control group) , P<0.05);

Figure 17 shows the changes of TNFα-1 gene levels in the liver, kidney and spleen of fish treated with Trx and ILTN after Aeromonas hydrophila infection in the example (different letters indicate significant differences compared with the control group, P< 0.05);

Figure 18 shows the changes of TNFα-2 gene levels in the liver, kidney and spleen of fish treated with Trx and ILTN after Aeromonas hydrophila infection in the example (different letters indicate significant differences compared with the control group, P< 0.05);

Figure 19 shows the changes of TNFα-1 gene levels in the liver, kidney and spleen of fish treated with Trx and ILTN after Streptococcus agalactiae infection in the example (different letters indicate significant differences compared with the control group, P<0.05) ;

Figure 20 shows the changes of TNFα-2 gene levels in the liver, kidney and spleen of fish treated with Trx and ILTN after Streptococcus agalactiae infection in the example (different letters indicate significant differences compared with the control group, P<0.05) .

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and in detail below with reference to the accompanying drawings and preferred embodiments of the specification, but the protection scope of the present invention is not limited to the following specific embodiments.

Unless otherwise defined, all technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments, and are not intended to limit the protection scope of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or can be prepared by existing methods.

The molecular biology experimental techniques used in the following examples include PCR amplification, plasmid extraction, plasmid transformation, DNA fragment ligation, enzyme digestion, gel electrophoresis, etc. Unless otherwise specified, they are usually operated according to conventional methods. Molecular Cloning Experiment Guide (Third Edition) (translated by Sambrook J, Russell DW, Janssen K, Argentine J. Huang Peitang et al., 2002, Beijing: Science Press), or according to the conditions suggested by the manufacturer.

Example:

1. Obtain the ORF of the ITLN gene of the congenital crucian carp by designing degenerate primers:

Degenerate primers were designed according to the conservation of ITLN amino acid sequence in various species. The first primer was ITLN-F1: 5'-ATGCTGCGCTTCATTTTCTTCCT (see SEQ ID NO: 3), the second primer was ITLN-R1: 5'-TTAACGGTAGAAGAGCAGCACGG (see SEQ ID NO: 4), reverse transcribed with M-MLV Enzyme kit (Promega, Madison, WI, USA) reverse-transcribed the liver cDNA of Hefang crucian carp as the template. Through touchdown PCR (touchdown PCR) technology, the PCR amplification product was obtained, sequenced and compared, and it was determined as Hefang crucian carp ITLN gene, its length is 945bp (see SEQ ID NO: 1), and its amino acid sequence is deduced (see SEQ ID NO: 2). PCR reaction conditions were: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30s, annealing at 72°C for 60s, a total of 5 cycles; denaturation at 94°C for 30s, annealing at 65°C for 30s, extension at 72°C, a total of 5 cycles; denaturation at 94°C for 30s , annealing at 55 °C for 30 s, and extending at 72 °C for 60 s, for a total of 35 cycles; the final extension was continued at 72 °C for 10 min. The electropherogram of PCR amplification products is shown in Figure 1.

Using the two sequence analysis software ClustalX and GeneDoc, the amino acid sequence of ITLN of Hefang crucian carp (WR-ITLN) and the amino acid sequence of human ITLN (Hs ITLN) were compared, and it was found that the amino acid sequence of ITLN of Hefang crucian carp was similar to that of human ITLN. High homology (Fig. 7), and the secondary structure of ITLN protein of Hefang crucian carp was analyzed by software (Fig. 8).

2. Synthesis of the hybrid crucian carp ITLN gene sequence containing the enzyme cleavage site:

Based on the analyzed cDNA sequence of the ITLN gene of Hefang crucian carp, the primers at the upstream and downstream ends were designed and synthesized. The upstream primer was ITLN-F2: 5'-CCGGAATTCATGCTGCGCTTCATTTTCTTC (see SEQ ID NO: 5); the downstream primer was ITLN-R2: 5'-CCGCTCGAGTTAATGATGATGATGATGATGACGGTAGAAGAGCAGCAC GGC (see SEQ ID NO: 6). Using the reverse-transcribed cDNA of the liver of Hefang crucian carp as a template, the ITLN gene of Hefang crucian carp was amplified by PCR. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 60s , a total of 40 cycles; the final extension was continued at 72 °C for 10 min. The electrophoresis identification map of PCR amplification products is shown in Figure 2.

3. The construction of the cloning vector pMD19-T-ITLN of the hybrid crucian carp ITLN gene containing the restriction site:

The gene fragments obtained above were separated by agarose gel electrophoresis, and then the DNA fragments were purified by the Universal DNA purification kit (Tiangen Biotech), and then combined with the pMD19-T (TAKARA) vector according to the system provided by the kit. Ligation overnight at 4°C, the ligation product was transformed into Escherichia coli DH5α (Escherichia coli), the gene sequence was amplified with the growth of the bacteria, and positive clones were screened by Amp ⁺ resistance and blue-white spot method; (Omega, USA) extracted the plasmid. The plasmid was verified by double enzyme digestion and sequencing, which proved that the ITLN gene sequence of Hefang crucian carp was successfully cloned into the pMD19-T vector. The vector containing the ITLN gene sequence of the Hefang crucian carp was named pMD19-T- ITLN, the recombinant strain obtained by transforming the cloning vector into Escherichia coli DH5α was named pMD19-T-ITLN-DH5α. The cloning vector pMD19-T-ITLN was double-enzyme digested using EcoRI and XhoI, and the restriction map is shown in Figure 3.

4. Construction of the expression vector pET32a-ITLN containing the ITLN gene sequence of Hefang crucian carp:

GelExtraction Kit回收，分离纯化得到约945bp的合方鲫ITLN基因序列；The cloning vector pMD19-T-ITLN was extracted with a plasmid mini-extraction kit (Omega, USA), and then double-enzyme digested with restriction endonuclease EcoRI and positive clone XhoI, and subjected to agarose gel electrophoresis, and the product was EZNA

The GelExtraction Kit was recovered, and the ITLN gene sequence of about 945bp was obtained by separation and purification;

The vector plasmid pET32a (purchased from Novagen) was double digested with restriction endonuclease EcoRI and positive clone XhoI, and then the digested vector fragment was separated by agarose gel electrophoresis, and then the Universal DNA purification kit was used. (Tiangen Biotech) to purify the DNA fragment, and then mix it with the above-obtained hybrid crucian carp ITLN gene sequence containing the restriction site in a volume ratio of 1:3, and ligate with T4 ligase at 16°C overnight (about 15h). Then, the plasmid obtained after ligation was transformed into E. coli DH5a by the CaCl ₂ method, and the transformants with Amp ⁺ resistance were screened on the LB plate. The plasmid was extracted by standard methods, sent to Invitrogen for sequencing, the sequencing results were compared, and it was confirmed that it was the gene sequence of Hefang crucian carp ITLN, and was correctly inserted into the expression vector pET32a, containing the recombinant plasmid of the Hefang crucian carp ITLN gene sequence Named pET32a-ITLN. The expression vector pET32a-ITLN was double-enzyme digested using EcoRI and XhoI, and the digestion analysis diagram is shown in Figure 4. The plasmid construction process is shown in Figure 6.

5. Construction of Escherichia coli recombinant strain pET32a-ITLN-BL21 that can efficiently express the ITLN protein of Hefang crucian carp:

According to the calcium chloride transformation method, the above-mentioned recombinant plasmid pET32a-ITLN obtained by ligating T4 ligase was transferred into Escherichia coli BL21 (E.coliBL21) which can efficiently express and clone into the pET series expression vector containing the phage T7 promoter. The transformants with Amp ⁺ resistance were screened by plate, and the recombinant strain was pET32a-ITLN-BL21.

6. Using Escherichia coli recombinant bacteria pET32a-ITLN-BL21 to produce recombinant crucian carp ITLN protein:

Pick the recombinant BL21 monoclonal engineering bacteria, inoculate them in LB medium containing Amp ⁺ resistance, cultivate at 37°C, 200 rpm, expand the strain and change to fresh LB medium containing Amp ⁺ resistance, when the OD600 reaches 0.8 Stop, and add about 1 mM IPTG to induce 5 hours to express the ITLN gene of Hefang crucian carp. After induction, collect the cells at 7000 rpm and 4 °C; then add TEBbuffer to suspend the cells, add an appropriate amount of lysozyme, overnight at 4 °C, and then resuspend the cells. Sonicate in an ice bath until clear. Resuspend in Buffer A containing 30% Trition X-100, and then stand on ice for 30 min, and add buffer B containing urea to dissolve the expression product Hefang crucian carp ITLN protein at room temperature. The solubilized protein was purified according to the method of His-tag bind resin (Millipore). The results are shown in Figure 5.

7. Hefang crucian carp ITLN protein antigen activity identification experiment:

The protein of recombinant crucian carp ITLN was identified by western blot. Among them, the primary antibody was mouse-derived His monoclonal antibody (Novagen), and the secondary antibody was horse anti-mouse IgG-AP (Dingguo Biological Company). The results are shown in Figure 5, showing that the mouse-derived His monoclonal antibody can recognize the recombinant crucian carp ITLN protein expressed by Escherichia coli, which proves that the obtained protein is the recombinant crucian carp ITLN protein.

8. Aeromonas hydrophila and Streptococcus agalactiae ELISA experiment:

Aeromonas hydrophila was inoculated in LB medium, Streptococcus agalactiae was inoculated in brain heart infusion broth (BHI) medium, cultured continuously at 30°C, 200rpm to OD600=0.8, and then centrifuged with PBS for bacterial analysis. The seeds were resuspended, and the bacterial concentration was adjusted to 1×10 ⁷ CFU ml ^-1 , and the above bacteria were inoculated into 96-well ELISA plates; after overnight at 4°C, the ELISA plates were blocked with 5% skim milk for 2 hours, and then 0.5 After washing with %Tween-20/PBS, add 1.25μg, 6.25μg, 12.50μg, 25.00μg and 50.00μg of Trx protein/PBS, ITLN protein/PBS or LITN protein/PBS+2mM Ca ²⁺ respectively, and incubate at room temperature 2h; then after washing with 0.5% Tween-20/PBS, the ELISA plate was blocked with mouse His-tag primary antibody and horse anti-mouse HRP secondary antibody respectively; finally, 200 μl TMB solution was added in the dark environment, and then 2M sulfuric acid was added The indicated reaction was terminated and the readings were analyzed at OD450. Calculation formula: OD450 reading of experimental group/OD450 reading of negative control group, the results are shown in Figure 9 and Figure 10.

The above-mentioned formula of adopting brain heart infusion broth (BHI) medium is: peptone 10.0g/L, dehydrated calf brain infusion powder 12.5g/L, dehydrated bovine heart infusion powder 5.0g/L, sodium chloride 5.0g/ L, glucose 2.0g/L, disodium hydrogen phosphate 2.5g/L, pH=7.4±0.2, temperature 25℃.

The results showed that compared with the Trx control group, when the protein concentration of ITLN was gradually increased and 2 mM Ca ²⁺ was present, the binding ratio of it to Aeromonas hydrophila and Streptococcus agalactiae was gradually increased, indicating that the ITLN of Hefang crucian carp was gradually increased. The protein binds Aeromonas hydrophila and Streptococcus agalactiae in a Ca ²⁺ environment in a dose-dependent manner.

9. Growth inhibition experiments of Aeromonas hydrophila and Streptococcus agalactiae:

Aeromonas hydrophila was inoculated in LB medium, Streptococcus agalactiae was inoculated in BHI medium, cultured continuously at 30°C, 200rpm to OD600=0.8, then centrifuged and resuspended with PBS solution, and adjusted the bacteria. The concentration was 1×10 ⁷ CFU ml ⁻¹ . 1.25 μg, 6.25 μg, 12.50 μg, 25.00 μg and 50.00 μg of Trx protein/PBS, ITLN protein/PBS or ITLN protein/PBS+2mM Ca ²⁺ were mixed with Aeromonas hydrophila in a volume ratio of 3:1, respectively. It was mixed with Streptococcus agalactiae, added to a 96-well culture plate, and mixed with 10 μl of the bacterial activity detection reagent CCK-8 (BestBio, Shanghai). After culturing for 5 h at 30°C, reading analysis was performed at OD450, and the Trx-treated group was used as the experimental control group. Calculation formula: (OD450 reading of experimental group/OD450 reading of control group)×100%, the results are shown in Figure 11 and Figure 12.

The results showed that when there was 2mM Ca ²⁺ in the environment, with the addition of ITLN protein concentration gradually increased, the viability of co-cultured Aeromonas hydrophila and Streptococcus agalactiae gradually decreased, and reached the lowest viability at 50.00μg value. This indicated that the recombinant protein of crucian carp ITLN had the activity of inhibiting the growth of Aeromonas hydrophila and Streptococcus agalactiae in the Ca ²⁺ environment.

10. Colony formation inhibition test of Aeromonas hydrophila and Streptococcus agalactiae:

Aeromonas hydrophila was inoculated in LB medium, Streptococcus agalactiae was inoculated in BHI medium, continuously cultivated at 30°C, 200rpm to OD600=0.8, then centrifuged and resuspended with PBS solution, and adjusted the bacterial concentration It is 1×10 ⁵ CFUml ^-1 . 50.00 μg Trx protein/PBS, ITLN protein/PBS or ITLN protein/PBS+2mM Ca ²⁺ were incubated with Aeromonas hydrophila and Streptococcus agalactiae at room temperature for 2h at a ratio of 49:1, respectively, and then coated On LB plate or BHI plate, cultured for 10h overnight, the results are shown in Figures 13 and 14.

The results showed that compared with the Trx control group, in the environment of 2mM Ca ²⁺ , the number of colonies of Aeromonas hydrophila and Streptococcus agalactiae treated with 50.00μg ITLN protein was significantly lower than that of the Trx control group and ITLN. group, indicating that ITLN protein can effectively inhibit the growth of Aeromonas hydrophila and Streptococcus agalactiae in the environment of 2 mM Ca ²⁺ .

11. Animal experiments:

1) Handling of animals and isolation of tissues

Select the same batch of Hefang crucian carp (about 28.50±0.79g) with healthy physique and similar specifications and temporarily raise them in a storage box of 1.0m×0.65m×0.65m. Each storage box is filled with about 40L. Pre-aerated fresh water, water temperature 25 ℃. Activated Aeromonas hydrophila and Streptococcus agalactiae were resuspended in PBS solution and adjusted to 1×10 ⁷ CFU ml ⁻¹ , respectively. About 30 minutes after intraperitoneal injection of 50 μl of the above resuspended bacterial solution, Trx protein and ITLN protein dissolved in 2mM Ca ²⁺ /PBS were intraperitoneally injected into the fish at a dose of 5.0 μg/g fish weight. Twenty-four hours after injection, the liver, kidney and spleen tissues of the fish were separated, quick-frozen in liquid nitrogen, and stored at -80°C.

2) Proliferation test of bacteria in Hefang crucian carp

Tissue DNAkit (Omega) was used to isolate DNA from the above-mentioned sample tissue, and the concentration of the isolated DNA was adjusted to 100 ng/μl. The total volume of the real-time quantitative qPCR system is 20 μl: each 0.8 μl diluted upstream and downstream primers (hlyA, CAMP, GAPDH), 5.0 μl ultrapure water, 3 μl DNA solution, 0.4 μl ROX reference dye and 10 μl SYBR premix ExTaq ^TM II (Perfect Real Time) (TaKaRa, Dalian, China). The reaction program included: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 30 seconds, annealing at 60°C for 25 seconds, a total of 40 cycles, and the amplified products were subjected to melting curve analysis. Each qRT-PCR experiment was repeated 3 times. The experimental results were analyzed by 7500SDS software (Applied Biosystems, USA), see Figures 15 and 16.

3) Detection of inflammatory cytokine expression changes in Hefang crucian carp caused by bacterial infection

Total RNA was extracted from the above samples using Trizol reagent (Invitrogen), and the quality and purity of the extracted RNA were determined by the values at 260 nm and 260/280 nm. Reverse transcription experiments were performed using 1000 ng of mRNA according to the method of Revert Aid ^™ M-MuLV Reverse Transcriptase Kit (MBI Fermentas, USA). The above reversed cDNA was subjected to real-time fluorescence quantitative qRT-PCR experiments, and the total system was 20 μl: 0.8 μl of each diluted upstream and downstream primers (TNFα-1, TNFα-1, β-actin), 6.0 μl ultrapure water, 2 μl DNA solution, 0.4 μl of ROX reference dye and 10 μl of SYBR premix ExTaq ^TM II (Perfect Real Time) (TaKaRa, Dalian, China). The reaction program includes: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 30 seconds, denaturation at 60°C Annealed for 35 seconds for a total of 40 cycles, and the amplified product was subjected to melting curve analysis. Each qRT-PCR experiment was repeated 3 times. The experimental results were analyzed by 7500SDS software (Applied Biosystems, USA), see Figures 17, 18, 19, 20.

The above experiments showed that the ITLN protein dissolved in 2mM Ca ²⁺ /PBS at a dose of 5.0μg/g fish weight could effectively inhibit Aeromonas hydrophila after 30min infection with Aeromonas hydrophila and Streptococcus agalactiae and Streptococcus agalactiae in various organs of the crucian carp, and reduced the inflammatory response caused by Aeromonas hydrophila and Streptococcus agalactiae infection. It can be seen that the ITLN recombinant protein of Hefang crucian carp has development potential in antibacterial and bacteriostasis, and can also be used to prepare aquatic animal immune preparations or feed additives.

sequence listing

<110> Hunan Normal University

<120> A kind of compound crucian carp ITLN protein and its preparation method and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 945

<212> DNA

<213> Hefang crucian carp (WR)

<400> 1

atgctgcgct tcattttctt cctgatgtgt cttttgcagt taaggcacac aacgctgggt 60

gttgtgggag ctgtcacccc aaagccaatt atcataaatg gcacctacat aaacccagaa 120

ttaggcaagt atcatgccag gttgagggtc ctcgctcgca gctgcaggga tatcaaagag 180

atacacggcg cttctcagga tggcatgtac atcctgacaa cagagagcgg tacatactac 240

cagactttct gtgacatgac cacagccggc ggtggctgga cgctggtggc cagtgtccac 300

gagaacaaca tcaaagggaa gtgctctctt ggtgatcgct ggtcgagcca gcagggaaac 360

gacccggacc tgcccgaagg agatggtaca tgggcaaaca ctgtgacatt cggcagcgcc 420

gaggcctcta ctagtgacga ctacaagaat cctgggtact acgacatctc tgcacaagac 480

gtgtcagtgt ggcacgttcc caataatgag cagctgacga gctggacgtc ttctgctgtc 540

ctgcgctacc atacagacag ccagttcctc acagagcatg gtggaaacct ttaccaccta 600

ttcaagaaat accccgtgaa gtttggagca ggacagtgta agagtgacat gggacccagc 660

agtccggtgg tgtacgatac tggagacaaa gattcaactg caaacttata tggaccacct 720

gttggaaagg agtttgagtc cggcttcatc accttcagag tgtttaacgc cgatcaagcc 780

gccatggcca tgtgctctgg agttaaacct actgaatgca atcctcaaca ctattgcatc 840

ggtggaggat atttctcggg ccgtcagcag tgtggagact tcaccgctct ggagcaagcg 900

tctaaaaatc tgactcaatc cgccgtgctg ctcttctacc gttaa 945

<210> 2

<211> 314

<212> PRT

<213> Hefang crucian carp (WR)

<400> 2

Met Leu Arg Phe Ile Phe Phe Leu Met Cys Leu Leu Gln Leu Arg His

1 5 10 15

Thr Thr Leu Gly Val Val Gly Ala Val Thr Pro Lys Pro Ile Ile Ile

20 25 30

Asn Gly Thr Tyr Ile Asn Pro Glu Leu Gly Lys Tyr His Ala Arg Leu

35 40 45

Arg Val Leu Ala Arg Ser Cys Arg Asp Ile Lys Glu Ile His Gly Ala

50 55 60

Ser Gln Asp Gly Met Tyr Ile Leu Thr Thr Glu Ser Gly Thr Tyr Tyr

65 70 75 80

Gln Thr Phe Cys Asp Met Thr Thr Ala Gly Gly Gly Trp Thr Leu Val

85 90 95

Ala Ser Val His Glu Asn Asn Ile Lys Gly Lys Cys Ser Leu Gly Asp

100 105 110

Arg Trp Ser Ser Gln Gln Gly Asn Asp Pro Asp Leu Pro Glu Gly Asp

115 120 125

Gly Thr Trp Ala Asn Thr Val Thr Phe Gly Ser Ala Glu Ala Ser Thr

130 135 140

Ser Asp Asp Tyr Lys Asn Pro Gly Tyr Tyr Asp Ile Ser Ala Gln Asp

145 150 155 160

Val Ser Val Trp His Val Pro Asn Asn Glu Gln Leu Thr Ser Trp Thr

165 170 175

Ser Ser Ala Val Leu Arg Tyr His Thr Asp Ser Gln Phe Leu Thr Glu

180 185 190

His Gly Gly Asn Leu Tyr His Leu Phe Lys Lys Tyr Pro Val Lys Phe

195 200 205

Gly Ala Gly Gln Cys Lys Ser Asp Met Gly Pro Ser Ser Pro Val Val

210 215 220

Tyr Asp Thr Gly Asp Lys Asp Ser Thr Ala Asn Leu Tyr Gly Pro Pro

225 230 235 240

Val Gly Lys Glu Phe Glu Ser Gly Phe Ile Thr Phe Arg Val Phe Asn

245 250 255

Ala Asp Gln Ala Ala Met Ala Met Cys Ser Gly Val Lys Pro Thr Glu

260 265 270

Cys Asn Pro Gln His Tyr Cys Ile Gly Gly Gly Tyr Phe Ser Gly Arg

275 280 285

Gln Gln Cys Gly Asp Phe Thr Ala Leu Glu Gln Ala Ser Lys Asn Leu

290 295 300

Thr Gln Ser Ala Val Leu Leu Phe Tyr Arg

305 310

<210> 3

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 3

atgctgcgct tcattttctt cct 23

<210> 4

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 4

ttaacggtag aagagcagca cgg 23

<210> 5

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 5

ccggaattca tgctgcgctt cattttcttc 30

<210> 6

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 6

ccgctcgagt taatgatgat gatgatgatg acggtagaag agcagcacgg c 51

Claims (8)

1. An ITLN protein of crucian carp, which has an amino acid sequence shown in SEQ ID NO: 2, respectively.

2. The crucian carp ITLN protein of claim 1 encoded by a crucian carp ITLN gene whose nucleotide sequence is set forth in SEQ ID NO: 1 is shown.

3. A preparation method of ITLN protein of synthetic crucian carp is characterized by comprising the following steps: the synthetic crucian carp ITLN gene is amplified by a PCR method and then cloned into a pMD19-T vector, then the obtained cloning vector is cloned into an expression vector pET32a, and then CaCl is added₂The transformation method transfers an expression vector pET32a containing the synthetic crucian carp ITLN gene into escherichia coli BL21（E. coli BL21）The ITLN protein of the synthetic crucian is finally obtained by expression; the nucleotide sequence of the synthetic crucian carp ITLN gene is shown in SEQ ID NO: 1 is shown.

4. The preparation method of claim 3, wherein the specific operation of cloning the ITLN gene of the crucian carp from the cross into the pMD19-T vector after amplification by the PCR method comprises the following steps: using the liver cDNA of the crucian carp obtained by reverse transcription as a template, amplifying the ITLN gene of the crucian carp by a PCR method, then connecting with a pMD19-T vector at 4 ℃, transforming the connecting product into escherichia coli DH5 alpha, and transforming the escherichia coli DH5 alpha by Amp⁺Screening positive clones by a resistance and/or blue-white spot method, namely successfully cloning the ITLN gene of the crucian carp of the cross into a pMD19-T vector; the nucleotide sequence of the synthetic crucian carp ITLN gene is shown in SEQ ID NO: 1 is shown.

5. The method according to claim 3 or 4, wherein the PCR amplification is carried out under the following conditions: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 72 ℃ for 60s, for 5 cycles; denaturation at 94 ℃ for 30s, annealing at 65 ℃ for 30s, and extension at 72 ℃ for 60s for 5 cycles; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s for 35 cycles; finally, continuing to extend for 10min at 72 ℃; the primers adopted by the PCR amplification method are an upstream primer ITLN-F1 and a downstream primer ITLN-R1, and the nucleotide sequence of the upstream primer ITLN-F1 is shown as SEQ ID NO: 3, the nucleotide sequence of the downstream primer ITLN-R1 is shown as SEQ ID NO: 4, respectively.

6. The method according to claim 3 or 4, wherein the PCR amplification is carried out under the following conditions: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 60s for 40 cycles; finally, continuing to extend for 10min at 72 ℃; the primers adopted by the PCR amplification method are an upstream primer ITLN-F2 and a downstream primer ITLN-R2, and the nucleotide sequence of the upstream primer ITLN-F2 is shown as SEQ ID NO: 5, the nucleotide sequence of the downstream primer ITLN-R2 is shown as SEQ ID NO: and 6.

7. The method for preparation according to any one of claims 3 to 6, characterized in that said specific operation of cloning the obtained cloning vector into the expression vector pET32a comprises the following steps: extracting the obtained cloning vector by a plasmid miniextraction kit, performing double enzyme digestion by using restriction enzyme EcoRI and positive clone XhoI, performing agarose gel electrophoresis, and recovering a product, namely a synthetic crucian ITLN gene; taking an expression vector pET32a, carrying out double enzyme digestion by using restriction enzyme EcoRI and positive clone XhoI, mixing the obtained large fragment of the vector with the recovery product, namely, the synthetic crucian ITLN gene, and carrying out connection for 15h at 16 ℃ by using T4 ligase, namely successfully cloning the cloning vector into the expression vector pET32 a.

8. The use of the synthetic crucian ITLN protein of claim 1 or prepared by the method of any one of claims 3-7 in the preparation of an immune preparation or feed additive for aquatic animals.

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