A C-type lectin derived from the exoskeleton of Macrobrachium nipponense and its encoding gene and application

Abstract

The present invention provides a C-type lectin derived from the exoskeleton of Macrobrachium nipponense, its encoding gene, and its application, belonging to the field of genetic engineering technology; the amino acid sequence of the C-type lectin is shown in SEQ ID NO.1. The present invention discovered a new C-type lectin and its encoding gene in the exoskeleton of Macrobrachium nipponense, and demonstrated its role in improving the immunity and survival rate of Macrobrachium nipponense. After the C-type lectin was injected into the abdomen of Macrobrachium nipponense, the survival rate after infection with Aeromonas hydrophila was significantly increased, and the survival rate was 60% 96 hours after the infection.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a C-type lectin derived from the exoskeleton of Macrobrachium nipponense, a coding gene thereof and an application thereof. Background Art

[0002] The hard exoskeleton of crustaceans is composed of the epidermis and an inner layer of epithelial cells. The main chemical components of the epidermis are chitin, protein, and calcium carbonate. The vast majority of proteins are structural cuticular proteins (CPs), which bind to chitin to form the organic scaffolding of the epidermis. Recent discoveries have revealed the presence of proteins with immune functions in the epidermis, such as antimicrobial peptides and prophenoloxidase. This makes the exoskeleton more than just a physical barrier; it may also possess antimicrobial activity. However, the presence of other immune molecules in the epidermis and their associated functions remain unknown.

[0003] The innate immune system is the primary defense system of crustaceans, and a key step in its activation is the recognition of pathogenic microorganisms. C-type lectins, members of the pattern recognition receptors (PRRs), are an important class of immune factors. In crustaceans, C-type lectins play a variety of immune functions. As members of the PRRs family, C-type lectins specifically recognize and bind to PAMPs. Furthermore, C-type lectins promote microbial aggregation, facilitate phagocytosis and encapsulation of bacteria by blood cells, and have antiviral, antibacterial, and opsonic effects.

[0004] However, to date, there have been few reports on the discovery of C-type lectins in crustacean exoskeletons. Summary of the Invention

[0005] The purpose of the present invention is to provide a C-type lectin derived from the exoskeleton of Macrobrachium nipponense, its encoding gene and application. The present invention discovers a new C-type lectin in the exoskeleton of crustaceans.

[0006] The present invention provides a C-type lectin derived from the exoskeleton of Macrobrachium nipponense, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides a gene encoding the C-type lectin described in the above scheme, and the nucleotide sequence is shown in SEQ ID NO.2.

[0008] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0009] Preferably, the original plasmid of the recombinant vector includes pET-30a; the coding gene is inserted between the Nde I and Xho I restriction sites on pET-30a.

[0010] The present invention also provides a recombinant bacterium comprising the recombinant vector described in the above scheme.

[0011] The present invention also provides the use of the C-type lectin, the encoding gene, the recombinant vector or the recombinant bacteria described in the above scheme in preparing a product that improves the survival rate of Macrobrachium nipponense.

[0012] Preferably, the improvement of the survival rate of Macrobrachium nipponense is achieved by improving the immunity of Macrobrachium nipponense.

[0013] Preferably, said improving the immunity of Macrobrachium nipponense includes improving the ability of Macrobrachium nipponense to resist bacterial infection.

[0014] Preferably, the bacteria include Aeromonas hydrophila.

[0015] The present invention also provides the use of a reagent for reducing the expression amount of the encoding gene described in the above scheme in constructing a Japanese swamp shrimp animal model with reduced immunity; the reagent for reducing the expression amount of the encoding gene described in the above scheme includes an interfering primer; the nucleotide sequence of the upstream primer of the interfering primer is shown in SEQ ID NO.5; the nucleotide sequence of the downstream primer of the interfering primer is shown in SEQ ID NO.6.

[0016] The present invention provides a C-type lectin derived from the exoskeleton of Macrobrachium nipponense. The amino acid sequence is shown in SEQ ID NO. 1. The present invention discovered a new C-type lectin from the exoskeleton of Macrobrachium nipponense and demonstrated its role in improving the immunity and survival rate of Macrobrachium nipponense. Injecting the C-type lectin into the abdomen of Macrobrachium nipponense significantly increased the survival rate after infection with Aeromonas hydrophila, reaching 60% 96 hours after challenge. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the gel electrophoresis result of the amplified product of the full-length coding region of MnLec3;

[0018] Figure 2 The expression analysis and purification results of rMnLec3 (Coomassie Brilliant Blue staining), where M is a protein molecular marker, 1 is the supernatant (uninduced), 2 is the precipitate (uninduced), 3 is the supernatant (IPTG induced), 4 is the precipitate (IPTG induced), and 5 is the rMnLec3 purified from inclusion bodies;

[0019] Figure 3 is the survival rate of Macrobrachium nipponense infected with Aeromonas hydrophila after injection of rMnLec3;

[0020] Figure 4 Double-stranded RNA gel electrophoresis diagram, where A: dsMnLec3 gel electrophoresis diagram; B: dsGFP gel electrophoresis diagram; M: DNA molecular weight marker;

[0021] Figure 5 is the expression of MnLec3 after interference;

[0022] Figure 6 The survival rate of Macrobrachium nipponense infected with Aeromonas hydrophila after MnLec3 interference. DETAILED DESCRIPTION

[0023] The present invention provides a C-type lectin MnLec3 derived from the exoskeleton of Macrobrachium nipponense, the amino acid sequence of which is shown in SEQ ID NO.1, specifically:

[0024] ETDVANTIGLGIGDISATLSDHEDRCSYTHVEGKLEEQLEHNERFLGFQESSVVAMKNNTEVLRNKTEEALERYYKTADKEVPDVCGEPFQRSAGSCFWAHKGPALSWG DAREFCLQEGGDLATPEDILRVVEFLNEELGSGWWYVWLGGKQDASGDWKWLESDKTMDAGDSFWEDEEVTEADPERMRCAAFRSESGYKIAKQDCGNTNWFLCEKKI.

[0025] The present invention has no particular limitation on the source and preparation method of the C-type lectin MnLec3. In the specific implementation of the present invention, the C-type lectin MnLec3 is prepared and purified by genetic engineering.

[0026] The present invention also provides the C-type lectin encoding gene MnLec3 described in the above scheme, the nucleotide sequence of which is shown in SEQ ID NO.2, specifically:

[0027] .

[0028] The present invention also provides a recombinant vector into which the coding gene described in the above scheme is inserted.

[0029] In the present invention, the original plasmid of the recombinant vector preferably includes pET-30a; the coding gene is preferably inserted between the Nde I and Xho I restriction sites on pET-30a.

[0030] The present invention has no particular limitation on the method for constructing the recombinant vector, and conventional construction methods in the art may be used.

[0031] The present invention also provides a recombinant bacterium comprising the recombinant vector described in the above scheme.

[0032] In the present invention, the original bacteria of the recombinant bacteria preferably include Escherichia coli, more preferably Escherichia coli competent cells BL21 (DE3).

[0033] The present invention has no particular limitation on the method for constructing the recombinant bacteria, and conventional construction methods in the art may be used.

[0034] The present invention also provides the use of the C-type lectin, the encoding gene, the recombinant vector or the recombinant bacteria described in the above scheme in preparing a product that improves the survival rate of Macrobrachium nipponense.

[0035] In the present invention, the product preferably comprises a medicine; the dosage form of the medicine is preferably an injection. The single injection dose of the medicine is preferably 1-3 μg / Macrobrachium nipponense, more preferably 2 μg / Macrobrachium nipponense, calculated as C-type lectin.

[0036] In the present invention, the improvement of the survival rate of Macrobrachium nipponense is preferably achieved by improving the immunity of Macrobrachium nipponense.

[0037] In the present invention, the method of improving the immunity of Macrobrachium nipponense preferably includes improving the ability of Macrobrachium nipponense to resist bacterial infection. In the present invention, the bacteria preferably include Aeromonas hydrophila.

[0038] The present invention also provides the use of a reagent for reducing the expression amount of the encoding gene described in the above scheme in constructing a Japanese prawn animal model with reduced immunity; the reagent for reducing the expression amount of the encoding gene described in the above scheme includes an interfering primer; the nucleotide sequence of the upstream primer of the interfering primer is shown in SEQ ID NO.5, specifically: 5'(tgaggaccgctgctcttac)3'; the nucleotide sequence of the downstream primer of the interfering primer is shown in SEQ ID NO.6, specifically: 5'(cagattccagccatttccag)3'.

[0039] To further illustrate the present invention, a C-type lectin derived from the exoskeleton of Macrobrachium nipponense, its encoding gene, and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] 1. RNA extraction and reverse transcription:

[0042] Five Macrobrachium nipponense were selected, and the cephalothorax was carefully removed using forceps. Because the inner surface of the cephalothorax is difficult to separate from other tissues, such as connective tissue, the freed tissue was quickly dissected under a stereomicroscope using an RNase-free scalpel. The freed tissue was then transferred to a liquid nitrogen-cooled centrifuge tube and stored at −80°C. To verify the accuracy of the sample, a randomly selected cephalothorax tissue piece was cut into 6–8 mm pieces. The pieces were fixed in Davidson's fixative (DF) for 24 hours, rinsed in tap water for 1 hour, dehydrated at various concentrations of alcohol, and then transparentized overnight in n-butanol. The pieces were then paraffin-embedded and cross-sectioned at 6 μm thickness. Following hematoxylin and eosin staining, the exoskeleton structure was observed under a microscope and photographed. Total RNA was extracted using a column-based Total RNA Miniprep kit. After quality testing, cDNA was synthesized using reverse transcription procedures according to the manufacturer's instructions.

[0043] 2. Amplification of the full-length coding region of MnLec3:

[0044] Design specific primers for sequence amplification:

[0045] Upstream primer MnLec3-Nde I (SEQ ID NO. 3):

[0046] 5'(ggaattccatatggagaccgacgttgcaaatacg)3',

[0047] Downstream primer MnLec3-Xho I (SEQ ID NO.4):

[0048] 5'(ccgctcgaggatcttcttttcgcataagaaccag)3';

[0049] The amplified fragment was 651 bp in length. PCR amplification was performed using the reverse-transcribed cDNA as a template. A 50 μL PCR reaction system was established with the following reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 35 s, and 35 cycles followed by extension at 72°C for 10 min. The amplified product was detected by 1.0% agarose gel electrophoresis. The results are shown in the table. Figure 1 After electrophoresis detection, a 651 bp PCR product was recovered using a gel recovery kit; the PCR amplification product was recovered and purified by gel recovery to obtain the target fragment.

[0050] 3. Construction of pET-30a-MnLec3 expression vector:

[0051] The MnLec3 PCR product / pET-30a empty vector was double-digested with Nde I and Xho I, and the enzyme-digested fragments and plasmids were recovered by gel recovery and ligation. 10 μL of the ligation product was taken and added to 10 μL of the engineered bacteria (DH5α, BL21) and incubated on ice for 30 minutes. The mixture was placed in a 42°C water bath for heat shock for 90 seconds and then immediately placed on ice for a second incubation for 3 minutes. The mixture was added to 500 μL of liquid LB culture medium (without antibiotics) and cultured at 37°C for 1 hour. The supernatant was discarded at room temperature at 3500r for 5 minutes, and the retained precipitate was spread on LB solid medium containing Kana and cultured in a 37°C incubator overnight. A single colony was picked for bacterial liquid PCR using primer-MnLec3 primers, and the positive clone was sent for sequencing.

[0052] 4. Inducible expression of pET-30a-MnLec3 fusion protein:

[0053] Positive clones that were sequenced correctly were expanded and the recombinant plasmids were extracted. The recombinant plasmids were transformed into competent E. coli BL21(DE3) cells using the same method, and positive clones were identified by PCR. Correctly identified positive clones were cultured in LB liquid medium containing Kana at 37°C until the OD600 reached 0.6. IPTG was added at a final concentration of 0.5 mmol / L and expression was induced at 37°C for 6 h. The cells were then harvested and resuspended in PBS and ultrasonically disrupted at 4°C for 30 min. After ultrasonic disruption, the culture solution was centrifuged at 12,000 rpm for 10 min at 4°C to separate the supernatant and precipitate. 10 μL of each supernatant and precipitate after ultrasonic treatment were collected and analyzed by SDS-PAGE electrophoresis.

[0054] 5. Purification of recombinant protein (rMnLec3):

[0055] The expression of rMnLec3 was induced under the optimal induction conditions, and the supernatant of the sonicated cells was collected and chromatographed according to the instructions of Ni-IDA agarose gel electrophoresis. Figure 2 .

[0056] The steps of chromatography are:

[0057] ① Take 2 mL of Ni-IDA agarose stored in 20% alcohol and add it to an empty affinity chromatography column. Slowly drain the alcohol at room temperature. After the Ni-IDA agarose gradually settles, add 5 mL of deionized water and 10 mL of binding buffer in sequence.

[0058] ② Add the supernatant from the sonicated bacterial cells to the column dropwise, 1 mL at a time. After all the supernatant has passed through, rinse with 5 mL of binding buffer.

[0059] ③ According to the imidazole concentration from low to high, add 5 mL (1 mL each time) of elution buffer with different imidazole concentrations in sequence and collect the outflowing liquid (eluate) respectively.

[0060] ④SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining were used to analyze the protein bands in the eluates with different imidazole concentrations.

[0061] ⑤ Add the accurately sized and impurity-free eluate to the dialysis bag and dialyze it in 1 L of dialysate at 4°C for 8–10 hours. Repeat this step once. After dialysis, collect the liquid in the dialysis bag and centrifuge at 12,000 rpm at 4°C for 10 minutes. Concentrate the recombinant protein using an ultrafiltration tube, determine the concentration, and store at -80°C.

[0062] 6. Effects of rMnLec3 on the survival rate of Macrobrachium nipponense

[0063] Healthy Macrobrachium nipponense (body length 1.0-1.5 cm) were randomly divided into two groups, with 60 in each group.

[0064] Control group: Each shrimp was injected with 2 μg of bovine serum albumin into the abdomen using a microsyringe;

[0065] Experimental group: 2 μg rMnLec3 was injected into the abdomen of each shrimp;

[0066] 20 min after injection, each shrimp in the control and experimental groups was injected with 2 μl of Aeromonas hydrophila suspension (7.5×10 5 CFU / mL), and the mortality of each group of shrimp was recorded at 0, 12, 24, 48, 72, and 96 hours after injection. The results are shown in Tables 1 and Figure 3 .

[0067] Table 1 Effects of rMnLec3 on the survival rate of Macrobrachium nipponense

[0068]

[0069] From Table 1 and Figure 3 It can be seen that compared with the control group, the survival rate of Macrobrachium nipponense in the experimental group after infection with Aeromonas hydrophila increased significantly. 96 hours after infection, the survival rate of the experimental group was 60%, while the survival rate of the control group was only 42.22%.

[0070] 7. Using RNA interference (RNAi) technology, interference primers were designed based on the MnLec3 gene sequence:

[0071] Upstream primer dsMnLec3 (SEQ ID NO. 5): 5' (tgaggaccgctgctcttac) 3',

[0072] Downstream primer dsMnLec3 (SEQ ID NO. 6): 5′(cagattccagccatttccag)3′;

[0073] The amplified fragment was 425 bp long. Using cDNA from the cephalothorax exoskeleton of Macrobrachium nipponense as a template, the cDNA template for dsMnLec3 was amplified and synthesized. The target fragment, containing the T7 promoter, was recovered by gel extraction, ligated with the pMD19-T vector, and transformed into Escherichia coli DH5α. The fragment was grown on LB solid medium containing Amp (overnight at 37°C). Single colonies were selected for PCR using the primer-dsMnLec3 primer, and positive clones were sent for sequencing. The strains that were sequenced correctly were expanded, and the plasmid was extracted and its concentration determined. The extracted plasmid was then digested with EcoRI and HindIII, and double-stranded RNA (dsMnLec3) was synthesized in vitro using the plasmid DNA as a template according to the instructions of the Invitro Transcription T7 Kit (for siRNA Synthesis). The nucleotide sequence of the double-stranded RNA sequence of MnLec3 is shown in SEQ ID NO. 7 and is specifically:

[0074] tgaggaccgctgctcttacacccatgtcgaaggaaagctggaagagcagttagagcataacgagagattcttgggattccaagagtcatcagtcgttgcaatgaag aataacactgaggtactgcggaacaagacagaggaggctttggaacgctactataagactgccgataaagaagttcccgacgtctgtggagagccgttccagaggtc tgcaggggagctgcttctgggctcacaagggccctgccctctcgtggggcgacgcccgtgagttctgcctacaagaaggaggagatctggcgactccagaggatatc cttcgggtggttgagttccttaacgaggaactcggctcaggatggtggtacgtgtggcttggaggaaagcaggacgctagcggagactggaaatggctggaatctg.

[0075] Double-stranded RNA of the green fluorescent protein gene (GFP) (dsGFP) was used as a control.

[0076] For double-stranded RNA gel electrophoresis, see Figure 4, where A: dsMnLec3 gel electrophoresis; B: dsGFP gel electrophoresis. M: DNA molecular weight marker.

[0077] (1) Healthy Macrobrachium zebulii (body length 1.0-1.5 cm) were randomly divided into two groups, with 30 in each group.

[0078] Control group: Each shrimp was injected with dsGFP into the abdomen using a microsyringe. The injection amount was calculated based on the shrimp weight (5 μg dsGFP / g body weight).

[0079] Interference group: Each shrimp was injected with dsMnLec3 into the abdomen, with an injection dose of 5 μg dsMnLec3 / g body weight.

[0080] Samples were taken at 0, 24, 48, 72, and 96 hours after injection. Three Macrobrachium nipponense were randomly selected from each group at each time point. Total RNA was extracted from their cephalothorax, and the first-strand cDNA was synthesized. The expression of the MnLec3 gene was detected by qRT-PCR. Figure 5 The results showed that 12 hours after injection, the expression of MnLec3 in the interference group was reduced by 49.59% compared with the corresponding control group (P<0.01). At 48 hours, the reduction in MnLec3 expression reached 91.83% (P<0.01). At 96 hours, the expression of MnLec3 was still 56.61% lower than that in the control group (P<0.01).

[0081] (2) Healthy Macrobrachium zebulii (body length 1.0-1.5 cm) were randomly divided into two groups, with 60 shrimp in each group.

[0082] Control group: Each shrimp was injected with dsGFP into the abdomen using a microsyringe. The injection amount was calculated based on the shrimp weight (5 μg dsGFP / g body weight).

[0083] Interference group: Each shrimp was injected with dsMnLec3 in the abdomen, with an injection dose of 5 μg dsMnLec3 / g body weight.

[0084] 48 h after double-stranded RNA injection, each shrimp was injected with 2 μl of Aeromonas hydrophila suspension (7.5×10 5 CFU / mL), and the mortality of each group of shrimp was recorded at 0, 12, 24, 48, 72, and 96 hours after injection. The results are shown in Tables 2 and Figure 6 .

[0085] Table 2 Survival rate of Macrobrachium nipponense infected with Aeromonas hydrophila after Mnlec3 interference

[0086]

[0087] From Table 2 and Figure 6It can be seen that silencing of the MnLec3 gene resulted in a significant decrease in the survival ability of Macrobrachium nipponense after infection with Aeromonas hydrophila. 96 hours after infection, the survival rate of the dsMnLec3 group dropped to 10.56%, while the survival rate of the dsGFP group was 46.67%.

[0088] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A C-type lectin derived from the exoskeleton of Macrobrachium nipponense, characterized in that The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the C-type lectin according to claim 1, characterized in that The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant vector, characterized in that The coding gene according to claim 2 is inserted.

4. The recombinant vector according to claim 3, characterized in that The original plasmid of the recombinant vector includes pET-30a; the coding gene is inserted between the Nde I and Xho I restriction enzyme cutting sites on pET-30a.

5. A recombinant bacterium, characterized in that Comprising the recombinant vector according to claim 3 or 4.

6. Use of the C-type lectin according to claim 1, the encoding gene according to claim 2, the recombinant vector according to claim 3 or 4, or the recombinant bacterium according to claim 5 in the preparation of a product for improving the survival rate of Macrobrachium nipponense in the presence of Aeromonas hydrophila.

7. The use according to claim 6, characterized in that The improvement of the survival rate of Macrobrachium nipponense is achieved by improving the ability of Macrobrachium nipponense to resist infection by Aeromonas hydrophila.

8. Use of a reagent for reducing the expression level of the encoding gene described in claim 2 in constructing a Japanese prawn animal model with reduced immunity; the reagent for reducing the expression level of the encoding gene described in claim 2 comprises an interfering primer; the nucleotide sequence of the upstream primer of the interfering primer is shown in SEQ ID NO.5; the nucleotide sequence of the downstream primer of the interfering primer is shown in SEQ ID NO.6.

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