C-type lectin gene MytiCTL17A and application of coding protein thereof

Abstract

The invention relates to the technical field of molecular biology, in particular to a C-type lectin gene MytiCTL17A and application of coding protein of the C-type lectin gene MytiCTL17A. The invention discovers and verifies that the C-type lectin protein MytiCTL17A from mytilus coruscus has calcium ion dependence, remarkable and broad-spectrum microalgae agglutination activity for the first time. Experiments prove that the protein can effectively agglutinate multiple different microalgae (including chlorella and two kinds of isochrysis) in taxonomy, and an obvious agglutination block mass is formed in a short time. Compared with lectin reported in early literatures, the protein provided by the invention shows excellent broad spectrum, which means that a single preparation can cope with various microalgae problems, and has wider application potential. The protein provides a brand new core material basis for developing a new generation of efficient, environment-friendly and broad-spectrum microalgae treating agent.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and in particular to the application of the C-type lectin gene MytiCTL17A and its encoded protein. Background Technology

[0002] Microalgae are a type of tiny, simple aquatic planktonic organisms. Their pollution of water resources mainly stems from a series of chain reactions triggered by excessive proliferation. Currently known aspects of microalgae pollution include: 1. Causing algal blooms and red tides: the proliferation of large numbers of microalgae consumes dissolved oxygen in the water, leading to oxygen depletion and death of aquatic organisms such as fish; 2. Some microalgae (such as cyanobacteria) produce algal toxins, polluting drinking water sources. Long-term exposure may harm the human liver and nervous system; 3. Microalgae accumulation blocks sunlight from reaching the water body, inhibiting photosynthesis in aquatic plants, disrupting the ecological balance of the water body, and leading to a decline in biodiversity; 4. Affecting water resource utilization: polluted water bodies cannot meet the needs of irrigation, aquaculture, and landscaping, increasing water treatment costs.

[0003] Currently, common methods for treating microalgae in water bodies (such as controlling red tides and harvesting economically valuable microalgae) include centrifugation, filtration, and chemical flocculation. Centrifugation and filtration are energy-intensive and have low throughput; chemical flocculation may introduce secondary pollution and is environmentally unfriendly. Although there are reports of isolating lectins from other marine organisms, there are no reports on lectins specific to multiple microalgae. Therefore, developing efficient, environmentally friendly, and broad-spectrum bio-flocculating agents has become a research hotspot in this field. Summary of the Invention

[0004] The purpose of this invention is to provide applications of the C-type lectin gene MytiCTL17A and its encoded protein to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides the application of the C-type lectin gene MytiCTL17A, the C-type lectin protein rMytiCTL17A, or biomaterials containing the C-type lectin gene MytiCTL17A in promoting microalgal flocculation. The nucleotide sequence of the C-type lectin gene MytiCTL17A is shown in SEQ ID NO.1; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.2.

[0007] Preferably, the microalgae include one or more of Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis.

[0008] This invention provides the application of the C-type lectin gene MytiCTL17A, the C-type lectin protein rMytiCTL17A, or biomaterials containing the C-type lectin gene MytiCTL17A in the preparation of formulations that promote microalgal aggregation. The nucleotide sequence of the C-type lectin gene MytiCTL17A is shown in SEQ ID NO.1; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.2.

[0009] Preferably, the microalgae include one or more of Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis.

[0010] The present invention provides a formulation for promoting microalgal aggregation, the formulation comprising a C-type lectin protein rMytiCTL17A and a calcium ion donor; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.2.

[0011] Preferably, the calcium ion provider includes calcium chloride.

[0012] Preferably, the microalgae include one or more of Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis.

[0013] This invention provides the application of the C-type lectin gene MytiCTL17A, the C-type lectin protein rMytiCTL17A, or biological materials containing the C-type lectin gene MytiCTL17A in the preparation of microalgae scavengers. The nucleotide sequence of the C-type lectin gene MytiCTL17A is shown in SEQ ID NO.1; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.2.

[0014] The present invention provides a formulation of a microalgae scavenger, the microalgae scavenger comprising a C-type lectin protein rMytiCTL17A and a calcium ion donor; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.2.

[0015] Preferably, the calcium ion provider includes calcium chloride.

[0016] The present invention discloses the following technical effects:

[0017] This invention is the first to discover and confirm that the C-type lectin protein rMytiCTL17A derived from the thick-shelled mussel possesses calcium-dependent, significant, and broad-spectrum microalgal agglutination activity. Experiments demonstrate that this protein can effectively agglutinate various taxonomically different microalgae (including Chlorella and two species of Isochrysis), forming distinct aggregates within a short time. Compared to lectins reported in earlier literature, the protein provided by this invention exhibits superior broad-spectrum activity, meaning that a single formulation can address multiple microalgal problems, demonstrating broader application potential. This protein provides a novel core material basis for developing a new generation of highly efficient, environmentally friendly, and broad-spectrum microalgal treatment agents. Furthermore, due to the inherent high calcium ion content in seawater, this characteristic makes the protein naturally suitable for microalgal treatment in real aquatic environments such as oceans and estuaries, requiring no additional cofactors, thus offering significant practical convenience and promising prospects. Therefore, this invention provides a novel technical solution to address the problems of high energy consumption and environmental unfriendliness in existing microalgal treatment technologies. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 SDS-PAGE electrophoresis images of recombinant expression and purification of rMytiCTL17A protein; lane 1 is whole bacterial protein before induction, lane 2 is whole bacterial protein after induction, lane 3 is purified MytiCTL17A protein; lane M is protein molecular weight standard.

[0020] Figure 2 Microscopic images showing the agglutination effect of rMytiCTL17A protein on Chlorella; PBS is the control group, rMytiCTL17A is the MytiCTL17A protein group, and rMytiCTL17A (Ca) is the protein group containing MytiCTL17A. 2+ () is the MytiCTL17A protein + CaCl2 group; the scale bar is 50 μm. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] The nucleotide sequence of the C-type lectin gene MytiCTL17A from the thick-shelled mussel is shown in SEQ ID NO.1, and is as follows:

[0027] ATGCTCTTCTTACTTGAGAGAGGAAACAGACGTGTTATTGCGTTCCAGTCATGACAATGATCCGTTTAGGAGCGCTCTTTTTTTGTTGTCTGTGTGTGTCATCAACACGAGCTGACTGTGGAAATG GTTGGAAAAGTTTCAGGGATTCTTGCTATTTGTTTGTTGATAACTTTATGGACTGGTATGACGCACAGgcAGATTGTGTCAGAAGAGGAGGACACTTAGTAGATATAGTAGACGCTGGGGAAAACGAA TTTATACATTCAAATCTTCACGATTACTGGAATTGGCACCATATTGGGCTAACTGACCGTATGGATGAAGGTACATTTAAATGGGTTACTGGAACCGCCATGGAATATGACAACTTCTGGCCAGGAGAACCTAACAACTCCAGAGGAAAGGAAGACTGTGCAGAAATGAGATATTCTGGACTTTGGAACGATTTTCATGTAATAAAAATCAGTACTACATATGTGAAAAGGAAGCGTCTGACTCTGGAAATATAG;

[0028] The amino acid sequence of the C-type lectin protein rMytiCTL17A from the thick-shelled mussel, encoded by the gene MytiCTL17A, is shown in SEQ ID NO.2, as follows:

[0029] MLFLLERGNRRVIAVPVMTMIRLGALFFCCLCVSSTRADCGNGWKSFRDSCYLFVDNFMDWYDAQADCVRRGGHLVDIVDAGENEFIHSNLHDYWNWHHIGLTDRMDEGTFKWVTGTAMEYDNFWPGEPNNSRGKEDCAEMRYSGLWNDFHVIKISTTYVKRKRLTLEI.

[0030] Example 1: Obtaining the recombinant C-type lectin protein rMytiCTL17A from thick-shelled mussels

[0031] Based on the DNA sequence corresponding to SEQ ID NO.1, an NcoI restriction endonuclease cleavage site (CCATGG) and a protective base (GC) were introduced at the 5' end, and a nucleotide sequence encoding a 6×His tag (CATCATCATCATCATCAC, SEQ ID NO.3) was added upstream of it; an XhoI restriction endonuclease cleavage site (CTCGAG) was introduced at the 3' end, so that the final encoded amino acid sequence only has a short peptide "MGHHHHHH" (SEQ ID NO.4) added at the N-terminus. The target fragment was amplified by PCR, and after double digestion with NcoI and XhoI, the digested fragment was recovered and purified. The digested target fragment was ligated with the pET-28a(+) expression vector, which had also been double-digested, using T4 DNA ligase to construct a recombinant plasmid. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing kanamycin (50 μg / mL), and incubated overnight at 37°C. Single colonies were picked, and positive clones were verified by bacterial PCR and sequencing (T7 universal primers (T7-F: 5'-TAATACGACTCACTATAGGG-3', SEQ ID NO.5; T7-R: 5'-GCTAGTTATTGCTCAGCGG-3', SEQ ID NO.6)). The correctly sequenced bacterial cultures were expanded and plasmids were extracted to obtain the recombinant expression plasmid pET-28a(+) / rMytiCTL17A.

[0032] The validated recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. Positive clones were screened on LB agar plates containing kanamycin (50 μg / mL), and the correctness of the expression cassette was further confirmed by sequencing. The successfully validated frozen bacterial culture was inoculated at a 1:100 volume ratio into LB liquid medium containing kanamycin (50 μg / mL) and cultured at 37°C with shaking at 200 rpm until OD500. 600 nm ≈ 0.6. IPTG was added to a final concentration of 0.6 mM, and expression was induced for 12 h. After induction, the cells were collected by centrifugation at 5000 rpm for 5 min. The cells were washed twice with pre-chilled PBS buffer and resuspended (resuspended volume: original culture medium volume = 1:200), and PMSF protease inhibitor was added to a final concentration of 1 mM. The cells were disrupted using an ultrasonic cell disruptor (300 W, cycle: 10 s sonication, 5 s interval, total duration 30 min) under ice bath conditions. The disrupted bacterial solution was centrifuged at 12000 rpm at 4℃ for 20 min, and the precipitate was collected as coarse inclusion bodies. The coarse inclusion bodies were washed twice with pre-chilled PBS buffer to obtain fine inclusion bodies, which were stored at -20℃ for later use.

[0033] Recombinant proteins were purified using Ni-NTA affinity chromatography. Fine inclusion bodies were resuspended in lysis equilibration buffer (100 mM NaH₂PO₄, 10 mM Tris-HCl, 8 M urea, pH 8.0) and magnetically stirred at room temperature for 4–6 h until the solution was clear. The supernatant was collected after centrifugation at 12000 rpm for 30 min and filtered through a 0.45 μm filter. Protein concentration was determined using a BCA protein quantification kit. Based on the binding efficiency of 5 mg histidine-tagged protein per mL of Ni-NTA resin, an appropriate amount of resin was packed into a column and washed and equilibrated sequentially with pure water, 0.1 M nickel sulfate solution, pure water, and lysis equilibration buffer. The filtered inclusion body solution was loaded onto the column at a flow rate of 0.333 mL / min. The contaminating proteins were washed thoroughly with lysis equilibration buffer and washing buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, 20 mM imidazole, pH 8.0) in sequence. Finally, the target protein was eluted with elution buffer (100 mM NaH2PO4, 10 mM Tris-HCl, 8 M urea, 300 mM imidazole, pH 8.0), and the eluent was collected stepwise.

[0034] The collected eluent was subjected to gradient dialysis refolding. At 4°C, dialysis was performed sequentially using refolding solutions containing 6 M, 4 M, 2 M, 1 M, and 0 M urea (100 mM NaCl, 50 mM Tris-HCl, 5% glycerol, 2 mM reduced glutathione, 0.2 mM oxidized glutathione, pH 8.0), with each concentration for at least 4 hours. After refolding, the protein solution was transferred to ultrapure water and dialyzed for 2 hours to completely remove other components. Then, it was freeze-dried overnight to obtain purified rMytiCTL17A recombinant C-lectin protein powder from thick-shelled mussels. SDS-PAGE analysis was then performed. The SDS-PAGE electrophoresis images of the whole bacterial protein before induction, the whole bacterial protein after induction, and the purified rMytiCTL17A recombinant C-lectin protein from thick-shelled mussels are shown below. Figure 1 As shown in the figure. The results showed that the size of the purified C-type lectin recombinant protein rMytiCTL17A from the thick-shelled mussel was 20.5 kDa.

[0035] Example 2: Application of rMytiCTL17A protein

[0036] Chlorella vulgaris (C. vulgaris), Isochrysis galbana (I. galbana), and Isochrysis zhanjiangensis (I. zhanjiangensis) were purchased from the Ganxie Aquaculture Area in Shengsi County, Zhoushan City.

[0037] 1. Microalgae preparation: Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis were cultured to the logarithmic growth phase. The algal cells were collected by centrifugation at 5000 rpm for 10 minutes, washed three times with 1×PBS buffer to remove the culture medium, and finally resuspended in PBS.

[0038] 2. Agglutination experiment

[0039] The experiment was divided into three groups: a control group (PBS), a negative control group (rMytiCTL17A), and an experimental group (rMytiCTL17A (Ca)). 2+ )).

[0040] Control group: Mix 20 μL of algal solution with 50 μL of 1×PBS buffer.

[0041] Negative control group: Take 20 μL of algal solution and mix it with 50 μL of the purified thick-shelled mussel C-type lectin recombinant protein rMytiCTL17A solution (the concentration of C-type lectin recombinant protein rMytiCTL17A is 1 mg / mL).

[0042] Experimental group: Take 20 μL of algal solution and 50 μL of the purified thick-shelled mussel C-type lectin recombinant protein rMytiCTL17A solution (the concentration of C-type lectin recombinant protein rMytiCTL17A is 1 mg / mL) from Example 1 and mix well. At the same time, add CaCl2 solution with a final concentration of 10 mM.

[0043] 3. Incubation and observation: Incubate all samples at 4°C for 30 minutes. After incubation, gently pipette a small amount of sample onto a glass slide and observe and photograph it under an Olympus BX53 biological microscope.

[0044] 4. Results: such as Figure 2 As shown, compared with the control group, all experimental groups treated with the recombinant C-type lectin protein MytiCTL17A and CaCl2 from thick-shelled mussels showed significant microalgal cell aggregation, forming visible aggregates, demonstrating that the protein has strong calcium ion-dependent aggregation activity on the microalgae.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the C-type lectin gene MytiCTL17A, the C-type lectin protein rMytiCTL17A, or biomaterials containing the C-type lectin gene MytiCTL17A in promoting microalgal agglutination, characterized in that, The nucleotide sequence of the C-type lectin gene MytiCTL17A is shown in SEQ ID NO.1; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The microalgae include one or more of Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis.

3. The application of the C-type lectin gene MytiCTL17A, the C-type lectin protein rMytiCTL17A, or biomaterials containing the C-type lectin gene MytiCTL17A in the preparation of formulations that promote microalgal aggregation, characterized in that, The nucleotide sequence of the C-type lectin gene MytiCTL17A is shown in SEQ ID NO.1; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.

2.

4. The application according to claim 3, characterized in that, The microalgae include one or more of Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis.

5. A formulation for promoting microalgae aggregation, characterized in that, The formulation comprises a C-type lectin protein rMytiCTL17A and a calcium ion donor; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.

2.

6. The formulation according to claim 5, characterized in that, The calcium ion provider includes calcium chloride.

7. The formulation according to claim 6, characterized in that, The microalgae include one or more of Chlorella vulgaris, Isochrysis galbana, and Isochrysis zhanjiangensis.

8. The application of the C-type lectin gene MytiCTL17A, the C-type lectin protein rMytiCTL17A, or biomaterials containing the C-type lectin gene MytiCTL17A in the preparation of microalgae scavengers, characterized in that, The nucleotide sequence of the C-type lectin gene MytiCTL17A is shown in SEQ ID NO.1; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.

2.

9. A microalgae remover, characterized in that, The microalgae scavenger includes a C-type lectin protein rMytiCTL17A and a calcium ion donor; the amino acid sequence of the C-type lectin protein rMytiCTL17A is shown in SEQ ID NO.

2.

10. The microalgae remover according to claim 9, characterized in that, The calcium ion provider includes calcium chloride.

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