Application of rhamnosyl lectin from Pinctada martensii and its acetylation modification in the preparation of products regulating antibacterial ability

Abstract

The present invention provides a rhamnosyl lectin of a pearl oyster and its acetylation modification in the preparation of a product for regulating antibacterial ability, belonging to the field of genetic engineering technology. The present invention provides a rhamnosyl lectin of a pearl oyster, and the amino acid sequence of the rhamnosyl lectin is shown in SEQ ID NO.2. The present invention clones the amino acid sequence of a rhamnosyl lectin (RBL) of a pearl oyster by RACE technology. The present invention can achieve the effect of regulating the antibacterial ability of rhamnosyl lectin by increasing acetylation or deacetylation modification of the amino acid sequence of the rhamnosyl lectin of the pearl oyster. Deacetylation modification of K at position 159 in the amino acid sequence of the rhamnosyl lectin protein of the pearl oyster provided by the present invention can improve the antibacterial ability against bacteria such as Escherichia coli and Aeromonas hydrophila.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an application of a rhamnosyl lectin of Pinctada martensii and an acetylation modification thereof in preparing a product for regulating antibacterial ability. Background Art

[0002] In recent years, due to high-density farming, eutrophication of farming waters, and deterioration of the ecological environment, shellfish farming species have frequently suffered from various infectious diseases caused by bacteria or viruses. The large-scale use of traditional drugs such as antibiotics in the prevention and treatment process has destroyed the microecological balance of the water environment, making some pathogens resistant to drugs, and unable to effectively control the occurrence of diseases, resulting in a large number of deaths of Pinctada martensii due to diseases, causing huge economic losses to my country's pearl industry.

[0003] Therefore, understanding the immune mechanism of Pinctada martensii and improving its stress resistance on this basis is an urgent problem to be solved. Rhamnosyl lectin (RBL) is a newly discovered lectin in Pinctada martensii in recent years. It can identify foreign invasive pathogens. Understanding how rhamnosyl lectin acts on pathogens has become an important key to the prevention and control of diseases in Pinctada martensii. In addition, lysine acetylation plays a very important role in the immune stress of marine microorganisms. By studying the effect of acetylation modification of rhamnosyl lectin on the immune benefit of rhamnosyl lectin, we can further improve our understanding of this gene.

[0004] Understanding the characteristics of rhamnosin is of great significance for the development of new biological antibiotics derived from rhamnosin from Pinctada martensii. However, there are currently few reports on rhamnosin from Pinctada martensii, and there is no report on the sequence of rhamnosin from Pinctada martensii in the database. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide a Pinctada martensii rhamnosus lectin, which has an antibacterial effect. At the same time, by acetylation or deacetylation modification of the rhamnosus lectin, the antibacterial ability of the rhamnosus lectin can be regulated, thereby contributing to the development of new biological antibiotics.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a rhamnosyl lectin of Pinctada martensii, and the amino acid sequence of the rhamnosyl lectin is shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the gene encoding the rhamnosyl lectin is shown in SEQ ID NO.1.

[0009] The present invention provides the use of the rhamnosyl lectin described in the above technical solution in the preparation of disease prevention and control and / or antibacterial drugs.

[0010] The present invention provides an application of the acetylation modification or deacetylation modification of rhamnosyl lectin described in the above technical solution in the preparation of a product for regulating the antibacterial ability of rhamnosyl lectin.

[0011] Preferably, the acetylation modification method comprises mutating K in the amino acid sequence of rhamnosyl lectin to Q; the deacetylation modification method comprises mutating K in the amino acid sequence of rhamnosyl lectin to R; and the K in the amino acid sequence refers to the K at position 159 of rhamnosyl lectin.

[0012] Preferably, the regulation includes increasing the antibacterial ability or decreasing the antibacterial ability.

[0013] Preferably, the K at position 159 in the amino acid sequence of the rhamnosyl lectin is deacetylated to improve the antibacterial ability of the rhamnosyl lectin.

[0014] Preferably, the K at position 159 in the amino acid sequence is deacetylated to improve the antibacterial ability against Escherichia coli and / or Aeromonas hydrophila.

[0015] Preferably, the K at position 159 in the amino acid sequence of the rhamnosyl lectin is acetylated to reduce the antibacterial ability of the rhamnosyl lectin.

[0016] Preferably, the acetylation modification of K at position 159 in the amino acid sequence reduces the antibacterial ability of rhamnosyl lectin against one or more of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus and Escherichia coli.

[0017] Beneficial effects of the present invention:

[0018] The present invention provides a Pinctada martensii rhamnosyl lectin (PmRBL), and the amino acid sequence of the PmRBL is shown in SEQ ID NO.2. The present invention clones the nucleotide sequence of PmRBL by RACE technology. The present invention can achieve the effect of regulating the antibacterial ability of PmRBL by performing acetylation modification or deacetylation modification on the amino acid sequence of PmRBL. The results of the examples show that deacetylation modification of K at position 159 in the amino acid sequence of PmRBL provided by the present invention can improve the antibacterial ability against Aeromonas hydrophila and show a significant antibacterial effect against Escherichia coli. Acetylation modification of K at position 159 in the amino acid sequence can reduce the antibacterial ability of PmRBL against bacteria such as Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus and Escherichia coli. Furthermore, the present invention observes the agglutination effect of PmRBL and its acetylated mutants and deacetylated mutants, and obtains that acetylation or deacetylation of PmRBL can regulate the agglutination effect of PmRBL on different bacteria, thereby affecting the antibacterial ability of PmRBL. The acetylation or deacetylation modification of the PmRBL provided by the present invention can achieve the effect of regulating the antibacterial ability, thereby contributing to the development of new biological antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 The nucleotide sequence of PmRBL and the amino acid sequence it encodes;

[0021] Figure 2 It is the hydrophobicity analysis diagram of PmRBL;

[0022] Figure 3 This is the analysis diagram of the transmembrane domain of PmRBL;

[0023] Figure 4 This is a diagram of the PmRBL domain analysis;

[0024] Figure 5 It is a homology alignment diagram of PmRBL;

[0025] Figure 6 Phylogenetic tree diagram constructed for the Neighbor-joining method;

[0026] Figure 7 This is the expression distribution map of PmRBL gene in various tissues of Pinctada martensii;

[0027] Figure 8This is the result diagram of the verification of the PmRBL prokaryotic expression recombinant plasmid;

[0028] Fig. 9 Figures are the results of rPmRBL purification analysis; A is a purification analysis (SDS-PAGE) diagram of the wild type rPmRBL, B is a purification analysis (SDS-PAGE) diagram of the rPmRBLK159R mutant, and C is a purification analysis (SDS-PAGE) diagram of the rPmRBLK159Q mutant;

[0029] Fig.10 The results of the refolding and concentration diagram of rPmRBL are shown in Figure 1; A is the refolding and concentration diagram of the wild type rPmRBL, B is the refolding and concentration diagram of the rPmRBLK159R mutant, and C is the refolding and concentration diagram of the rPmRBL K159Q mutant;

[0030] Fig.11 This is the antibacterial effect diagram of the wild type rPmRBL;

[0031] Fig.12 This is a diagram showing the antibacterial effect of the rPmRBLK159Q mutant;

[0032] Fig.13 This is a diagram showing the antibacterial effect of the rPmRBLK159R mutant;

[0033] Fig.14 This is a graph showing the detection of the agglutination effect of rPmRBL. DETAILED DESCRIPTION

[0034] The present invention provides a Pinctada martensii rhamnosus lectin (PmRBL), and the amino acid sequence of the rhamnosus lectin is shown in SEQ ID NO.2.

[0035] In the present invention, the rhamnosus rhamnosus lectin contains 212 amino acids; the molecular weight of the rhamnosus lectin is 22.9KDa, the theoretical isoelectric point of the rhamnosus lectin is 8.99; the rhamnosus lectin is positively charged overall. In the present invention, the random coil in the secondary structure of the rhamnosus lectin accounts for 56.13% of the whole, the α-helical structure is 9.43%, and the extended chain is 29.25%. In the present invention, the rhamnosus lectin forms a transmembrane domain at positions 189-211; the amino acid sequence of the rhamnosus lectin forms two tandem galactoside-binding lectin domains, which are located at amino acids 11-89 and 107-188, respectively.

[0036] The nucleotide sequence of the rhamnosyl lectin gene of the present invention is preferably as shown in SEQ ID NO.1.

[0037] In the present invention, the full length of the sequence of the rhamnosyl lectin gene is 1045 bp. In the present invention, the 5'-UTR of the PmRBL gene is 375 bp, the 3'-UTR is 3 bp, the PolyA is 28 bp, and the ORF frame is 639 bp encoding 212 amino acids. Figure 1 The present invention performs homology analysis on the PmRBL gene, and concludes that the PmRBL gene is clustered with molluscs; the PmRBL gene is most similar to the Ciona indica and the long oyster at 45.74% and 42.93% respectively, the PmRBL gene is 35.38% similar to the zebrafish, the PmRBL gene is 41.63% similar to the giant scallop, the PmRBL gene is 39.57% similar to the calyx columnar coral, the PmRBL gene is 41.51% similar to the trumpet poisonous spine sea urchin, the PmRBL gene is 39.57% similar to the lancelet fish, and the PmRBL gene is 41.51% similar to the trumpet poisonous spine sea urchin, the PmRBL gene is 41.51% similar to the lancelet fish, and the PmRBL gene is 41.51% similar to the lancelet fish. The similarity with Acropora is 24.49%, the similarity with green sea urchin is 33.44%, the similarity with American shad is 40.08%, the similarity with mountain star coral is 35.89%, the similarity with lancelet is 34.03%, the similarity with Tibetan plateau loach is 26.76%, the similarity with scallop is 38.76%, the similarity with long oyster is 42.93%, and the similarity with milkfish is 41.67%. In the present invention, the expression of the PmRBL gene is tissue-specific; the expression level of the PmRBL gene is the highest in the gills, followed by the mantle, and is almost not expressed in other tissues, such as hemolymph, adductor muscle and hepatopancreas.

[0038] The present invention provides the use of the rhamnosyl lectin described in the above technical solution in the preparation of disease prevention and control and / or antibacterial drugs.

[0039] The present invention provides the use of the rhamnosyl lectin described in the above technical solution in the preparation of a drug for disease control. In the present invention, the disease preferably includes one or more bacteria caused by Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi and Vibrio alginolyticus. The present invention shows through the results of the examples that the rhamnosyl lectin has a significant antibacterial effect on Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi and Vibrio alginolyticus.

[0040] The present invention provides the use of the rhamnosyl lectin described in the above technical solution in the preparation of antibacterial drugs. In the present invention, the antibacterial preferably includes inhibiting one or more of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi and Vibrio alginolyticus.

[0041] The present invention provides an application of the acetylation modification or deacetylation modification of rhamnosyl lectin described in the above technical solution in the preparation of a product for regulating the antibacterial ability of rhamnosyl lectin.

[0042] In the present invention, the acetylation modification or deacetylation modification of the rhamnosyl lectin can play an effect of regulating the antibacterial ability of the rhamnosyl lectin.

[0043] In the present invention, the antibacterial activity preferably includes inhibiting any one or two or more of Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Escherichia coli and Staphylococcus aureus.

[0044] In the present invention, the acetylation modification method preferably includes mutating K in the amino acid sequence of rhamnosyl lectin to Q; the deacetylation modification method preferably includes mutating K in the amino acid sequence of rhamnosyl lectin to R. In the present invention, the regulation preferably includes increasing the antibacterial ability or decreasing the antibacterial ability. In the present invention, the K in the amino acid sequence preferably includes the K at position 159 of the rhamnosyl lectin described in the above technical solution.

[0045] In the present invention, the deacetylation modification of K at position 159 in the amino acid sequence of the rhamnosyl lectin can preferably improve the antibacterial ability of the rhamnosyl lectin. In the present invention, the deacetylation modification of only K at position 159 in the amino acid sequence of the rhamnosyl lectin can preferably improve the antibacterial ability of the rhamnosyl lectin. In the present invention, the deacetylation modification of K at position 159 in the amino acid sequence preferably improves the antibacterial ability of Escherichia coli and / or Aeromonas hydrophila. In the present invention, the deacetylation modification of K at position 159 in the amino acid sequence improves the antibacterial ability of Aeromonas hydrophila and has a significant antibacterial effect on Escherichia coli. In the present invention, the deacetylation modification of K at position 159 in the amino acid sequence preferably includes mutating K at position 159 to R.

[0046] In the present invention, the acetylation modification of K at position 159 in the amino acid sequence of the rhamnosyl lectin can preferably reduce the antibacterial ability of the rhamnosyl lectin. In the present invention, the acetylation modification preferably includes mutating the K to Q. In the present invention, the acetylation modification of the rhamnosyl lectin only at position 159 in the amino acid sequence can preferably reduce the antibacterial ability of the rhamnosyl lectin to one or more of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus and Escherichia coli.

[0047] The present invention also provides a method for preparing rhamnosyl lectin and / or rhamnosyl lectin mutant, comprising the following steps:

[0048] Synthesizing the PmRBL gene or PmRBL mutant gene described in the above technical solution to obtain a target gene or a vector containing the target gene;

[0049] Connecting the target gene into a vector to obtain a recombinant plasmid;

[0050] Transforming the recombinant plasmid into competent Escherichia coli cells to obtain a recombinant strain;

[0051] Inducing the recombinant strain to express the fusion protein;

[0052] The fusion protein is separated and purified to obtain rhamnosyl lectin and / or rhamnosyl lectin mutant.

[0053] The present invention synthesizes the PmRBL gene or PmRBL mutant gene described in the above technical solution to obtain the target gene or a vector containing the target gene.

[0054] The present invention has no special limitation on the synthesis method, and any conventional synthesis method in the art can be used. The present invention can further prepare rhamnosyl lectin by synthesizing the PmRBL gene. The present invention synthesizes the target gene by mutating the K159 site in the PmRBL gene to R159, and further prepares the R159 rhamnosyl lectin mutant. The present invention synthesizes the target gene by mutating the K159 site in the PmRBL gene to Q159, and further prepares the Q159 rhamnosyl lectin mutant. In the present invention, the acetylation modification of rhamnosyl lectin is simulated by mutating K to Q; and the deacetylation modification is simulated by mutating K to R. In the process of synthesizing the above-mentioned target gene of the present invention, it is preferred to further include introducing restriction sites MluI and XhoI into the target gene.

[0055] After obtaining the target gene, the present invention connects the target gene into a vector to obtain a recombinant plasmid.

[0056] In the present invention, the expression vector preferably includes a pSmart1 plasmid. The present invention does not specifically limit the method for connecting the target gene to the expression vector, and any conventional method in the art can be used to express the target gene. The present invention preferably utilizes MluI and XhoI restriction endonucleases to digest the expression vector and the target gene, and then connects the target gene to the expression vector by homologous recombination. The present invention does not specifically limit the conditions for the digestion and the method for connection, and any conventional method in the art can be used. After obtaining the connection product, the present invention preferably transforms the connection product, screens positive clones, and sequences to determine that a recombinant vector containing the target gene is obtained. After determining that a recombinant vector containing the target gene is obtained, the present invention preferably extracts a plasmid from the recombinant vector to obtain a recombinant plasmid.

[0057] After obtaining the recombinant plasmid, the present invention transforms the recombinant plasmid into competent Escherichia coli cells to obtain a recombinant strain.

[0058] In the present invention, the competent E. coli cells preferably include competent cells BL21 (DE3). The present invention does not specifically limit the transformation method, and any conventional transformation method in the art can be used. In the present invention, the transformation method preferably includes mixing the recombinant plasmid and the competent E. coli cells, first incubating on ice for 30 minutes, heating at 42°C for 90 seconds, and placing on ice for 3 minutes to obtain a recombinant strain. After obtaining the recombinant strain, the present invention preferably incubates the recombinant strain in a culture medium at 37°C and 200rpm for 60 minutes; the culture medium is preferably LB culture medium. After the culture is completed, the present invention preferably spreads the obtained bacterial solution on an LB agar plate containing 50μg / mL kanamycin, inverts the plate and incubates overnight at 37°C to obtain positive colonies. After obtaining the positive colonies, the present invention preferably clones and sequences the positive colonies to determine that the correct recombinant strains are obtained.

[0059] After determining that the correct recombinant strain is obtained, the present invention induces the recombinant strain to express the fusion protein. In the present invention, in order to obtain a large amount of fusion protein, the recombinant strain is cultured at 37°C and 220 rpm until the bacterial OD reaches 600nm After the pH value is 0.6 to 0.8, induction is performed. The culture medium of the present invention is preferably an LB medium containing 50 μg / mL kanamycin. In the present invention, the induction is preferably induced by IPTG. In the present invention, the molar concentration of IPTG is preferably 0.5 to 1 mM, more preferably 1 mM; the induction time is preferably 4 hours; the induction temperature is preferably 37°C.

[0060] After induction, the present invention preferably separates and purifies the fusion protein to obtain rhamnosyl lectin and / or rhamnosyl lectin mutant. The present invention has no particular limitation on the separation and purification method, and any conventional method in the art can be used. In the present invention, the separation and purification method preferably includes nickel agarose affinity chromatography.

[0061] The present invention prepares rhamnosyl lectin and / or rhamnosyl lectin mutant with high purity by the above method. The three recombinant proteins prepared by the present invention all show a clear single band at about 37KDa. The concentrations of the three proteins are measured by Bradford protein assay, and the concentrations are all 1.00mg / mL.

[0062] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0063] Example 1

[0064] Obtaining the full-length sequence of PmRBL gene

[0065] 1. Design of primers related to PmRBL gene

[0066] The specific primers of PmRBL gene were designed using Primer Premier 5.0. The 5' and 3' specific primers were designed based on the RACE amplification principle, and the full-length cDNA of the gene was obtained after alignment and splicing. The relevant primer information is shown in Table 1.

[0067] Table 1 Primer information for obtaining full-length cDNA of genes

[0068] Primers Label Sequence (5'-3') use PmRBL-3'-inner SEQ ID NO.3 AGAGCAAGTAATGGTGTTTTCGGGG 3'-RACE PmRBL-3'-outer SEQ ID NO.4 ACTCAGAGCAAGTAATGGTGTTTTC 3'-RACE PmRBL-5'-inner SEQ ID NO.5 GCTTTGCAATTAGTGTCACGGT 5'-RACE PmRBL-5'-outer SEQ ID NO.6 GCAATTGTAAACCACAGTCAG 5'-RACE PmRBL-R SEQ ID NO.7 GGGTCCCCGAAAACACCATTA Middle segment PmRBL-F SEQ ID NO.8 GGAACTTGTCGGAGCAGCTTAG Middle segment

[0069] 2. Total RNA Extraction

[0070] (1) Whole tissue samples of Pinctada martensii were obtained, Trizol was added and each tissue was ground, and total RNA was extracted according to the Trizol method.

[0071] (2) 1% agarose gel electrophoresis was used to detect RNA integrity and quality.

[0072] (3) The concentration and purity of total RNA were determined by micro-nucleic acid quantification instrument.

[0073] (4) The obtained total RNA was stored in a -80℃ ultra-low temperature refrigerator for future use.

[0074] 3. Synthesis of the First Strand of cDNA

[0075] Prepare the required template according to the Reverse Transcriptase M-MLV kit. The specific procedure is as follows:

[0076] (1) RNA is combined with primers. The PCR program is 70°C for 10 min and placed on ice for 2 to 3 min. The sample loading system is shown in Table 2.

[0077] Table 2 Sample addition system

[0078] Reagents dose Total RNA 1ng~1μg Oligo(dT)(50uM) 1μL RNase-free water Add to 8 μL

[0079] (2) Add the following reagents in Table 3 to the above RNA system:

[0080] Table 3 Reagents

[0081] Reagents dose 5×M-MLVBuffer 2.0μL dNTP Mixture (10 mM) 0.5μL RNaseInhibitor 0.25μL RTaseM-MLV(RNaseH-) 0.25~1μL RNase-free water Make up to 12 μL

[0082] (3) Centrifuge and mix for a few seconds, then perform PCR with the following procedures: 42°C, 60 min; 70°C, 15 min to obtain cDNA.

[0083] 4. Intermediate fragment cloning

[0084] (1) PrimeSTARHS high-fidelity enzyme amplification obtains the target fragment. The PCR reaction system is shown in Table 4:

[0085] Table 4 Reaction system

[0086] Reagents volume PrimeSTARHS 5μL template 0.4μL PmRBL-F 0.4μL PmRBL-R 0.4μL <![CDATA[ddH2O]]> 3.8μL Total 10μL

[0087] A three-step amplification method was used. The PCR reaction program was: 94°C, 5 min; 98°C, 15 s, 62°C, 30 s, 72°C, 2 min, 35 cycles; 72°C, 10 min.

[0088] (2) Gel detection and target fragment recovery

[0089] After obtaining the PCR product, 1 μL of the PCR product was aspirated and detected by 1% agarose electrophoresis to amplify the target fragment system, and the PCR product was recovered according to the instructions of the PCR product recovery kit (Gene JET Gel Extraction Kit).

[0090] (3) Target fragment connection

[0091] The purified PCR product was connected to the pMD-19T Vector (Dalian Bao Biotechnology Co., Ltd.). The system is shown in Table 5.

[0092] Table 5 System

[0093]

[0094]

[0095] The connection procedure is: 16°C for about 16 hours.

[0096] (4) Conversion

[0097] Take the DH5α competent cells out of the ultra-low temperature refrigerator, put them on ice until slightly dissolved, then add the ligation product to the tube containing 100μL of DH5α competent cells, gently blow and mix evenly; put it on ice for 30 minutes, heat shock at 42℃ for 60-90s, and put it on ice for 2-3 minutes; then add 890μL of LB liquid culture medium preheated at 37℃ in advance, shake and culture at 37℃ and 200rpm for 1h; then centrifuge at 4000rpm for 2min, discard the supernatant, leave about 100μL of bacterial solution, gently blow off the suspended cells, and apply it to LA (Amp + 50 μg / mL) solid plate; finally, culture at 37°C upright for half an hour and then inverted overnight.

[0098] (5) Colony PCR detection

[0099] Pick a single colony and inoculate it in LA (containing Amp + 50μg / mL) liquid culture medium, 37℃, 220rpm shaking culture for about 6h. M13 universal primers were used to identify positive clones. The nucleotide sequence of M13-F is shown in SEQ ID NO.9, specifically: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3'; the nucleotide sequence of M13-R is shown in SEQ ID NO.10, specifically: 5'-GAGCGGATAACAATTTCACACAGG-3'. The positive colonies detected by 1% agarose gel electrophoresis of colony PCR products were sent to Guangzhou Biotech for sequencing. The specific system is shown in Table 6.

[0100] Table 6 System

[0101] Reagents volume PremixTaq 5.0μL Primer M13-F (10 μM) 0.4μL Primer M13-R (10 μM) 0.4μL Bacterial DNA 0.4μL <![CDATA[ddH2O]]> 3.8μL Total 10μL

[0102] The colony PCR program was: 94°C, 5 min; 94°C, 30 s, 54°C, 30 s, 72°C, 2 min, 30 cycles; 72°C, 10 min.

[0103] 5.3′ and 5′ RACE amplification

[0104] The cDNA templates for 3′ and 5′ RACE were prepared according to the instructions of SMARTTM RACE cDNA Amptification kit. Nested PCR was performed with 3′ and 5′ specific primers combined with universal primers (NUP and UPM) for amplification.

[0105] (1) The first round of RACE reaction, the specific system is shown in Table 7.

[0106] Table 7 System

[0107] Reagents volume PrimeSTARHS 5μL 3′ / 5′ cDNA template 0.4μL UPM Primers 0.4μL Outer specific primer 0.4μL <![CDATA[ddH2O]]> 3.8μL Total 10μL

[0108] In the first round of RACE reaction, PCR reaction program: 94°C, 5 min; 98°C, 15 s, 58°C, 30 s, 72°C, 3 min, 30 cycles; 72°C, 10 min.

[0109] (2) The second round of RACE reaction, the specific system is shown in Table 8.

[0110] Table 8 System

[0111]

[0112]

[0113] PCR reaction program: 94°C, 5 min; 94°C, 30 s, 58°C, 30 s, 72°C, 2 min, 35 cycles; 72°C, 10 min.

[0114] (3) PCR gel recovery / product purification, ligation, transformation and colony PCR detection refer to 4(2)~(5).

[0115] 6. Bioinformatics Analysis

[0116] NCBI blastx was used for sequence homology and similarity analysis; DNAMAN 6.0 software was used for sequence search, splicing and alignment; ExPASy was used for online prediction of its theoretical molecular weight (MW) and isoelectric point (pI); SignalP 4.0Server was used to predict the signal peptide sequence; TMHMM Server v.2.0 was used to analyze the transmembrane domain of the sequence; MEGA 6 software was used to construct a phylogenetic tree using the NJ method; SoftBerry Psite was used to predict the functional sites of its deduced amino acid sequence; and SOPMA was used to predict the protein secondary structure.

[0117] 7. The full length of the PmRBL (Pinctada martensii rhamnosus lectin) gene cloned by RACE technology is 1045 bp, and the specific nucleotide sequence is shown in SEQ ID NO.1, specifically:

[0118]

[0119] Among them: the underline is the open reading frame of the Pinctada martensii rhamnosyl agglutinin (RBL) gene.

[0120] The 5'-UTR of the PmRBL gene is 375 bp, the 3'-UTR is 3 bp, the PolyA is 28 bp, and the ORF frame is 639 bp encoding 212 amino acids, such as Figure 1 As shown, Figure 1 The boxes in the figure represent the start codon and the stop codon respectively. The gray part represents the structural domain.

[0121] The amino acid sequence of the protein encoded by the PmRBL gene is shown in SEQ ID NO.2, specifically: MVICEHKSSSVSCPAGHLIKITSANYGRTSKGTCRSSLDRDTNCKASKSLSIVKSICDGNNVCSLEATNSVFGDPCVGTYKYLTVVYNCYKSDEKSLVICEHKSGSLSCPIGKKIEIVYANYGRTAPSSKGTCPTSLDKNTNCRASKSLSIVQSQCEGKSTCSLRASNGVFGDPCVGTYKYLRILYKCSSGGRYIIIDIFAVFFLYCHMGMS.

[0122] 8. Analysis of physical and chemical properties of PmRBL

[0123] Depend on Figure 2 It can be obtained that the molecular weight of PmRBL is 22.9KDa, the theoretical isoelectric point is 8.99, and its hydrophilicity is predicted to be -0.107, indicating that it is a hydrophilic protein. It is also predicted that it has no signal peptide. The total number of negatively charged residues is: (Asp+Glu): 14; the total number of positively charged residues is (Arg+Lys): 25, so it is positively charged overall. TMHMM server 2.0 predicted that a transmembrane domain is formed at positions 189-211 (such as Figure 3 The secondary structure predicted by SOPMA software showed that the random coil accounted for 56.13% of the whole, the α-helix structure accounted for 9.43%, and the extended chain accounted for 29.25%. The results of SMART analysis showed that the amino acid sequence of PmRBL formed two tandem galactoside-binding lectin domains, located at amino acids 11-89 and 107-188 (e.g. Figure 4 ).

[0124] 9. PmRBL Homology Analysis

[0125] Analysis of PmRBL compared with other aquatic RBL results Figure 5 and Figure 6 shown.

[0126] Its highest similarity with Ciona indica and Crassostrea gigas is 45.74% and 42.93% respectively. Others are zebrafish: 35.38%, giant scallop: 41.63%, pistillate coral: 39.57%, trumpet-spined sea urchin: 41.51%, lancelet fish: 39.57%, antler coral: 24.49%, green sea urchin: 33.44%, American shad: 40.08%, mountain star coral: 35.89%, lancelet: 34.03%, Tibetan plateau loach: 26.76%, Yesso scallop: 38.76%, Crassostrea gigas: 42.93%, milkfish: 41.67% ( Figure 5).

[0127] Then, MEGA 6 was used to construct a phylogenetic tree, in which mollusks were clustered into one branch ( Figure 6 ).

[0128] Example 2

[0129] Tissue expression of PmRBL gene

[0130] 1. Pinctada martensii processing and sample collection

[0131] Tissue quantification: 10 clams of uniform size and good vitality were selected, and the tissues collected included gills, mantle, hemolymph, adductor muscle and hepatopancreas.

[0132] 2. Primer Design

[0133] The primers used in the fluorescence quantitative PCR reaction are PmRBL-RT-F and PmRBL-RT-R. The nucleotide sequence of PmRBL-RT-F is shown in SEQ ID NO.11, specifically: 5'-TCGGAGCAGCTTAGACCGTGACACT-3'; the nucleotide sequence of PmRBL-RT-R is shown in SEQ ID NO.12, specifically: 5'-GCAATTGTAAACCACAGTCAGG-3'. GAPDH was used as the internal reference gene in the fluorescence quantitative PCR, and the primers were GAPDH-F and GAPDH-R; the nucleotide sequence of GAPDH-F is shown in SEQ ID NO.13, specifically: 5'-GCAGATGGTGCCGAGTATGT-3'; the nucleotide sequence of GAPDH-R is shown in SEQ ID NO.14, specifically: 5'-CGTTGATTATCTTGGCGAGTG-3'.

[0134] 3. Extraction of Total RNA

[0135] The method for extracting total RNA is the same as steps (1) to (4) in 2 of Example 1.

[0136] 4. Preparation of cDNA Template for Fluorescence Quantification

[0137] The procedure was performed according to the instruction manual of Reverse Transcriptase M-MLV (RNase H).

[0138] (1) RNA is combined with primers. The PCR procedure is 70°C for 10 min and placed on ice for 2-3 min. The sample loading system is shown in Table 9.

[0139] Table 9 Sample addition system

[0140] Reagents volume Total RNA 1ng~1μg Random Primers (25 μM) 1μL RNase-free water Add to 8 μL

[0141] (2) Add the reagents in Table 10 to the above RNA system:

[0142] Table 10 Reagents

[0143] Reagents dose 5×M-MLVBuffer 2.0μL dNTP Mixture (10 mM) 0.5μL RNaseInhibitor 0.25μL RTaseM-MLV(RNaseH-) 0.25~1μL RNase-free water Make up to 12 μL

[0144] (3) Mix by centrifugation for a few seconds and perform PC with the following program: 42°C, 60 min; 70°C, 15 min. The obtained first-chain cDNA was stored at -20°C.

[0145] 5. Fluorescence quantitative PCR

[0146] The 4 reverse transcribed cDNAs were used as templates, and GAPDH was selected as the internal reference gene. Sample wells were set according to 3 experimental replicates. Real-time fluorescence quantitative PCR was performed according to the reaction system in Table 11 and the following reaction conditions:

[0147] Table 11 Reaction system

[0148]

[0149]

[0150] Reaction procedure:

[0151]

[0152] 6. Data statistics and analysis

[0153] Fluorescence quantitative data were obtained using 2 -△△ct The results were expressed as mean ± standard deviation, SPSS17.0 software was used for one-way ANOVA, and Duncan's multiple comparison was used to analyze the differences in the means.

[0154] 7. The results of tissue quantitative analysis of PmRBL gene mRNA are as follows Figure 7 As shown, Figure 7 M is mantle, B is blood cell, Gi is gill, A is adductor muscle, and He is hepatopancreas.

[0155] Real-time PCR was used to detect the expression pattern of PmRBL rhamnosyl lectin gene mRNA in five tissues of Pinctada martensii, including hemocytes. The results showed that the expression level of PmRBL rhamnosyl lectin gene was highest in gills (p<0.01), followed by mantle, while there was almost no expression in other tissues (such as Figure 7 ).

[0156] Example 3

[0157] Preparation of pSmartl-PmRBL recombinant plasmid and its mutant recombinant plasmid

[0158] 1. Gene Synthesis

[0159] Gene synthesis was performed according to the existing target gene sequence, and the existing K159 site was mutated to R159 and Q159, simulating the acetylation modification (K mutated to Q) and the deacetylation modification (K mutated to R) mutants of rhamnosyl lectin. This was used as a template for subsequent subcloning, and restriction sites MluI and XhoI were introduced.

[0160] 2. Construction of prokaryotic expression vector

[0161] (1) Recombinant PmRBLORF amplification, PCR system: 10 μL Premix ExTaq, 0.5 μL synthetic template, 0.4 μL each of Forward Primer and Reverse Primer, ddH2O to make up the system to 20 μL. The program is: 94°C denaturation for 5 min; 98°C for 10 s, 55°C for 30 s, 72°C for 60 s, 30 cycles. The reaction product was detected by gel electrophoresis. Among them, the nucleotide sequence of Forward Primer is shown in SEQ ID NO.15, specifically: 5'-AAGCAGTTCAGTATCATGTCCAGC-3': The nucleotide sequence of Reverse Primer is shown in SEQ ID NO.16, specifically: 5'-ACCTCCAGAGGAACATTTGTAT-3'. After PCR amplification, the PmRBL gene fragment was obtained.

[0162] (2) The cloning and sequencing of PmRBL are shown in 4 of Example 1.

[0163] (3) Plasmid extraction was performed according to the instructions of the Easy Pure Plasmid Mini Prep Kit silica gel membrane centrifugal column plasmid DNA small-scale extraction kit.

[0164] (4) Double digest the empty plasmid pSmartl (pSmartl plasmid purchased from Hangzhou Huaan Biotechnology Co., Ltd.) and the extracted recombinant plasmid with MluI and XhoI endonucleases. The digestion system is 5.0 μL of 10×FD Buffer, 3.0 μL of MluI endonuclease, 3.0 μL of XhoI endonuclease, 20 μL of plasmid, and ddH2O to make up the system to 50 μL. Mix gently and keep warm at 37°C for 2 h.

[0165] (5) Gel excision purification was performed according to step (2) in 4 of Example 1.

[0166] (6) The purified product was connected to the pSmartl vector by T4 ligase to construct the recombinant expression plasmid pSmartl-PmRBL. The ligation system is: 6 μL of the purified product, 2 μL of pSmartl (+), 1 μL of 10×T4 buffer, 1 μL of T4 ligase, and the total system is 10 μL. Mix gently and incubate at 16°C overnight.

[0167] (7) The transformation, positive clone screening and sequencing of the ligation product refer to steps (4) to (5) in 4 of Example 1. After digestion with MluI-XhoI restriction endonucleases, the digested PmRBL gene fragment was detected to be approximately 1500 bp.

[0168] (8) Plasmid extraction Refer to the above step (3) to obtain the pSmartl-PmRBL recombinant plasmid, which is stored at -20°C for future use. For the positive clone results, plasmid extraction is performed, and the extracted plasmid is the pSmartI-PmRBL recombinant plasmid, and the estimated molecular weight of the recombinant protein is 37.32KDa. Colony PCR identification, MluI-XhoI digestion identification (identification results are as follows Figure 8 ) and sequencing results verified that the pSmartI-PmRBL vector was successfully constructed, and the sequencing results further proved that the PmRBL wild-type prokaryotic expression vector was successfully constructed.

[0169] The preparation of the other two mutants (mutating the K159 site in the existing target gene into R159 and Q159) was the same as above.

[0170] Example 4

[0171] Inducible expression of PmRBL recombinant fusion protein in prokaryotic expression vector of PmRBL wild type / K159Q / K159R mutant

[0172] 1. Transformation and screening of positive clones

[0173] Take out competent cells BL21(DE3) from the ultra-low temperature refrigerator and thaw them first; add 100ng of plasmid DNA (i.e., pSmartl-PmRBL recombinant plasmid) to the BL21(DE3) strain and mix gently, incubate the test tube on ice for 30min, heat the shaking tube at 42℃ for 90s without shaking, place the test tube on ice for 3min, add the transformed strain to 100μL of room temperature LB medium. Shake and incubate at 37℃ at 200rpm for 60min, spread on LB agar plates containing 50μg / mL kanamycin, and incubate the plates upside down at 37℃ overnight. Screen positive clones and sequence.

[0174] 2. Choose the best induction conditions

[0175] Three well-separated single colonies were selected from each positive clone obtained after transformation with the recombinant prokaryotic expression vector and inoculated into 4 mL LB medium containing 50 μg / mL kanamycin. The cells were shaken and incubated at 200 rpm on a shaker at 37°C. When the OD 600nm When the value reached 0.6-0.8, IPTG was added to two of the three tubes to make the final concentration of 1 mM IPTG and induced at 37°C for 4 h.

[0176] 3. Recombinant protein preparation and detection

[0177] ① Harvest the cell pellet from 450 μL of the induced culture in step (2) above and lyse it using an ultrasonic device for 1 min.

[0178] ② Whole cell lysate: Mix 50 μL of 5× loading buffer with 100 μL of cell lysate as the sample of whole cell lysate, heat the sample at 100°C for 10 min, and then centrifuge at 15,000 rpm for 5 min.

[0179] ③Supernatant and fragments of cell lysate: Centrifuge the remaining 200 μL of cell lysate at 15,000 rpm for 10 min, collect the supernatant and cell fragments of the cell lysate, and mix 90 μL of 5× loading buffer with 180 μL of supernatant as samples. Resuspend all precipitates with 150 μL of 5× loading buffer as samples of cell lysate fragments. Heat the sample at 100°C for 10 min and centrifuge at 15,000 rpm for 5 min before loading.

[0180] ④ Use SDS-PAGE to detect protein expression and solubility, and finally induce the expression of fusion protein in large quantities.

[0181] 4. Isolation and Purification of Fusion Protein

[0182] (1) Ultrasonic disruption of bacteria

[0183] ① Dissolve the collected bacterial cells with Buffer (50 mM Tris, 150 mM NaCl, 5% glycerol pH 8.0), and disrupt the cells by ultrasonication in an ice bath at a power of 350 W for 30 min (ultrasonication for 4 s, pause for 6 s as one cycle).

[0184] ② After ultrasonication, centrifuge at 12,000 rpm and 4°C for 20 min, discard the supernatant, dissolve the precipitate with crushing buffer (50 mM Tris, 150 mM NaCl, 5% glycerol pH 8.0), and ultrasonically crush in an ice bath at a power of 350 W for 30 min (ultrasonication for 4 s, pause for 6 s as one cycle).

[0185] ③After ultrasonication, centrifuge at 12,000 rpm and 4°C for 20 min and collect the supernatant for the next purification step.

[0186] (2) Nickel agarose affinity chromatography

[0187] ① Take 5mL Ni-NTA and wash and equilibrate the column with 5 times the column bed volume of Binding buffer at a flow rate of 5mL / min.

[0188] ②After incubating the filler and sample for 1 hour, load them onto the column and collect the penetration liquid.

[0189] ③ Wash the column with 5 times the volume of column bed Binding buffer at a flow rate of 5 mL / min.

[0190] ④Wash impurities with Wash buffer at a flow rate of 5 mL / min and collect the eluate.

[0191] ⑤Elute with Elution buffer at a flow rate of 2 mL / min and collect the eluate.

[0192] ⑥ Collect samples for SDS-PAGE detection

[0193] The buffer solution was prepared as follows:

[0194] Binding buffer: 20 mM sodium phosphate, 0.5 mM sodium chloride, 8 M urea, pH 7.4

[0195] Wash buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 10 mM imidazole, pH 7.4

[0196] Elution buffer: 20 mM sodium phosphate, 0.5 M sodium chloride, 100 mM imidazole, 8 M urea, pH 7.4

[0197] (3) SDS-PAGE detection of purified protein

[0198] Prepare 12% SDS-PAGE, Tris-Gly electrophoresis buffer, concentrate gel at 80V, 20min, separate gel at 120V, 60min, stain with Coomassie brilliant blue for 20min after gel electrophoresis, and decolorize overnight.

[0199] (4) Inclusion body protein renaturation

[0200] The purified target protein was dialyzed and renatured, and finally replaced into a soluble buffer (PBS, 10% glycerol, 1mL arginine, pH 7.4). SDS-PAGE was used to compare the purity of the purified protein before (precipitation) and after (dissolution) renaturation.

[0201] In order to obtain a large amount of fusion protein in the induced expression of the three recombinant proteins, the expression bacteria containing pSmartI-PmRBL were cultured at 37°C and 220 rpm until the bacterial OD 600nm The results showed that the five recombinant proteins all had a clear single band at about 37KDa, indicating that the recombinant proteins with high purity (such as Fig. 9 As shown, Fig. 9 The middle lane M is the marker; lane 1 is the lysis solution; lane 2 is the flow-through solution; lanes 3-4 are the elution samples; the results of SDS-PAGE detection of the three recombinant proteins after renaturation are as follows Fig.10 The five protein concentrations were determined by Bradford protein assay, and the concentrations were all 1.00 mg / mL.

[0202] Example 5

[0203] Antibacterial activity detection of purified protein

[0204] The purified protein showed antimicrobial activity against Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Staphylococcus aureus and Micrococcus luteus.

[0205] First, each bacterium was cultured in 2216E liquid medium to the logarithmic growth phase. The bacteria were then centrifuged (3,000×g, 10 min), washed three times with phosphate-buffered saline (1×PBS), and then resuspended in PBS. Next, 50 μL of purified protein (1 mg / mL) was mixed with 10 μL of each bacterial suspension using a 96-well ELISA plate and incubated at room temperature for 2 h. An equal amount of PBS was mixed with 10 μL of each bacterial suspension for use as a negative control. Then 140 μL of culture medium was added to each mixed well, incubated in a 37°C constant temperature incubator, and an ELISA reader (EnSpire, PerkinElmer) was used to measure the OD at 0, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, and 12h after adding purified protein and PBS. 600nm Three experiments were performed in parallel for each sample.

[0206] The results of the antibacterial activity assay of the wild type and two mutants of rPmRBL are shown in Figure 11 to Figure 13 As shown. Fig.11 A in the table is the antibacterial activity assay of 1 mg / mL rPmRBL (Vibrio parahaemolyticus), Fig.11 B is the antibacterial activity test of 1 mg / mL rPmRBL (Aeromonas hydrophila), Fig.11C is the antibacterial activity assay of 1 mg / mL rPmRBL (Pseudomonas aeruginosa), Fig.11 D is the antibacterial activity assay of 1 mg / mL rPmRBL (Vibrio harveyi), Fig.11 E is the antibacterial activity assay of 1 mg / mL rPmRBL (Vibrio alginolyticus), Fig.11 F is the antibacterial activity assay of 1 mg / mL rPmRBL (Staphylococcus aureus), Fig.11 G is the antibacterial activity assay of 1 mg / mL rPmRBL (Escherichia coli), Fig.11 Antibacterial activity assay of rPmRBL (Micrococcus luteus) at 1 mg / mL H. Fig.12 A is the antibacterial activity assay of 1 mg / mL rPmRBLK159Q (Vibrio parahaemolyticus), Fig.12 B is the antibacterial activity test of 1 mg / mL rPmRBLK159Q (Aeromonas hydrophila), Fig.12 C is the antibacterial activity assay of 1 mg / mL rPmRBLK159Q (Pseudomonas aeruginosa), Fig.12 D is the antibacterial activity assay of 1 mg / mL rPmRBLK159Q (Vibrio harveyi), Fig.12 E is the antibacterial activity assay of 1 mg / mL rPmRBLK159Q (Vibrio alginolyticus), Fig.12 F in the figure is the antibacterial activity assay of 1 mg / mL rPmRBLK159Q (Staphylococcus aureus), Fig.12 G is the antibacterial activity assay of 1 mg / mL rPmRBLK159Q (Escherichia coli). Fig.12 P<0.05 indicated that K159Q was significantly different from the control group. Fig.13 A is the antibacterial activity assay of 1 mg / mL rPmRBL K159R (Vibrio parahaemolyticus), Fig.13 B is the antibacterial activity assay of 1 mg / mL rPmRBLK159R (Aeromonas hydrophila), Fig.13 C is the antibacterial activity assay of 1 mg / mL rPmRBLK159R (Pseudomonas aeruginosa), Fig.13 D is the antibacterial activity assay of 1 mg / mL rPmRBLK159R (Escherichia coli), Fig.13 E in the figure is the antibacterial activity assay of 1 mg / mL rPmRBLK159R (Staphylococcus aureus). Fig.13 F is the antibacterial activity assay of 1 mg / mL rPmRBLK159R (Vibrio harveyi), Fig.13 G is the antibacterial activity test of 0.75 mg / mL rPmRBLK159R (Aeromonas hydrophila), Fig.13The antibacterial activity of rPmRBLK159R at 0.75 mg / mL was determined (Pseudomonas aeruginosa). Fig.13 P<0.05 indicates that K159R is significantly different from the control group. Fig.13 D in the middle indicates that the data corresponding to K159R are significantly different from those of the control group, while the data corresponding to the wild type are not significantly different from those of the control group.

[0207] The antibacterial effects of rPmRBL wild type and K159R mutant on Escherichia coli are shown in Table 12.

[0208] Table 12 Antibacterial effect of rPmRBL wild type and K159R mutant on Escherichia coli, corresponding Fig.13 D

[0209] Group Control group wild type K159R 0h 0.0445 0.0380 0.0380 1h 0.0435 0.0510 0.0565 2h 0.0440 0.0490 0.0695 3h 0.0455 0.0510 0.0740 4h 0.0595 0.520 0.0760 5h 0.1025 0.0640 0.0830 6h 0.1585 0.1010 0.0955 7h 0.2030 0.1710 0.1410 8h 0.2300 0.2130 0.2025 9h 0.2770 0.2410 0.2315 10h 0.3205 0.2730 0.2585 11h 0.3560 0.3090 0.2840 12h 0.3830 0.3540 0.3050

[0210] The antibacterial effects of rPmRBL wild type and K159R mutant on Aeromonas hydrophila are shown in Table 13.

[0211] Table 13 Antibacterial effect of rPmRBL wild type and K159R mutant on Aeromonas hydrophila, corresponding Fig.13 G in

[0212] Group Control group wild type K159R 0h 0.0380 0.0640 0.0640 1h 0.0380 0.0740 0.0930 2h 0.0380 0.0730 0.0810 3h 0.0390 0.0740 0.0840 4h 0.0410 0.0750 0.0860 5h 0.0510 0.0770 0.0880 6h 0.0840 0.0770 0.0920 7h 0.1370 0.0850 0.0940 8h 0.2050 0.1050 0.1000 9h 0.2470 0.1570 0.1170 10h 0.2630 0.2080 0.1350 11h 0.2900 0.2510 0.1820 12h 0.3270 0.3120 0.2450

[0213] In the bacterial growth curve inhibition experiment, a total of 8 bacteria were measured, including 2 Gram-positive bacteria and 6 Gram-negative bacteria. Wild-type rPmRBL had significant antibacterial effects on Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi, and Vibrio alginolyticus. It had a partial antibacterial effect on Escherichia coli but it was not significant. It had no antibacterial effect on Staphylococcus aureus and Micrococcus luteus ( Fig.11 ).

[0214] In order to explore the differences between the two mutants and the wild-type RBL protein, namely the acetylation modification (K mutation to Q) and the simulated deacetylation modification (K mutation to R) mutants of rhamnosyl lectin, the bacteria that interacted with the wild-type protein were also selected for antibacterial experiments.

[0215] The results showed that the acetylated K159Q mutant protein had significant antibacterial effects on Vibrio parahaemolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa, but the effect was weaker than that of the wild-type protein; it had no antibacterial effect on Escherichia coli and Staphylococcus aureus, which was consistent with the effect of the wild-type protein; in addition, the antibacterial effects on Vibrio harveyi and Vibrio alginolyticus that appeared in the wild-type disappeared in the K159Q protein ( Fig.12 ).

[0216] The deacetylated K159R mutant protein had significant antibacterial effects on Pseudomonas aeruginosa and Aeromonas hydrophila. However, compared with the wild-type RBL protein, the K159R protein lost its antibacterial effects on Vibrio parahaemolyticus and Vibrio harveyi and showed significant antibacterial effects on Escherichia coli. It still had no antibacterial effect on Staphylococcus aureus. To further study whether the K159R mutant has an enhanced antibacterial effect, a lower protein concentration was used to select Aeromonas hydrophila and Pseudomonas aeruginosa, which had a common antibacterial effect with the wild-type protein, for further antibacterial experiments. The wild-type protein at a protein concentration of 0.75 mg / mL and the K159R mutant protein at a protein concentration of 0.75 mg / mL were used to act on Aeromonas hydrophila and Pseudomonas aeruginosa. The results showed that at a concentration of 0.75 mg / mL, there was a significant difference in the antibacterial curves of the K159R mutant protein and the wild-type protein after 9 hours, suggesting that K159R enhanced the antibacterial effect against Aeromonas hydrophila, while the antibacterial effect against Pseudomonas aeruginosa remained the same ( Fig.13 ).

[0217] In general, the antibacterial effect of the acetylated K159Q mutant protein was weakened compared with the wild-type protein; the deacetylated K159R mutant protein enhanced the antibacterial activity against Aeromonas hydrophila compared with the wild-type protein, and showed significant antibacterial activity against Escherichia coli.

[0218] Example 6

[0219] Recombinant protein lectin activity detection

[0220] (1) Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Escherichia coli, which have antibacterial effects on both wild type and mutant types in the above antibacterial experiment, were selected and overnight cultured bacteria were collected by centrifugation, resuspended and washed three times with PBS, and 10 mM FITC was added. The mixture was stained in the dark for 2 h, and the excess FITC dye was washed away with PBS.

[0221] (2) Adjust the concentration of FITC-labeled bacteria to 2.5 x 10 9 CFU / mL -1 , 10 μL of bacterial suspension was mixed with 25 μL of recombinant protein (wild type, K159R type, K159Q type) in a 96-well plate and incubated at room temperature for 2 h.

[0222] (3) Place the 96-well plate under an inverted fluorescence microscope for observation.

[0223] The results of agglutination activity observation were as follows Fig.14 shown. Fig.14AD in the figure are the effect diagrams of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli and wild-type protein, respectively. EH are the effect diagrams of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli and K159Q mutant protein, respectively. IL are the effect diagrams of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli and K159R mutant protein, respectively. UX are the control groups of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Escherichia coli, respectively.

[0224] FITC fluorescent dye was used to label Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Escherichia coli, and the agglutination activity of the five proteins on them was detected. The results showed that: first, in the control group, Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, and Escherichia coli were all in a uniformly distributed colony state ( Fig.14 MP in the wild-type protein), all four colonies showed agglutination under the action of wild-type protein, and agglutinated in clusters against Vibrio parahaemolyticus and Aeromonas hydrophila ( Fig.14 AB), flocculent agglutination of Pseudomonas aeruginosa and Escherichia coli ( Fig.14 Medium CD).

[0225] The K159Q mutation caused agglutination of Vibrio parahaemolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa ( Fig.14 There was no agglutination of Escherichia coli.

[0226] The agglutination of the K159R mutant was observed and it was found that it agglutinated all four bacteria ( Fig.14 However, compared with the wild type, its agglutination morphology against Pseudomonas aeruginosa and Escherichia coli was different.

[0227] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A rhamnosyl lectin from Pinctada martensii, characterized in that: The amino acid sequence of the rhamnosyl lectin is shown in SEQ ID NO.

2.

2. Use of the rhamnosyl lectin according to claim 1 in the preparation of a drug for inhibiting bacteria; the bacteria are any one or more of Vibrio parahaemolyticus, Aeromonas hydrophila, Pseudomonas aeruginosa, Vibrio harveyi and Vibrio alginolyticus.

3. Use of the deacetylated modification of rhamnosyl lectin according to claim 1 in the preparation of a product for improving the antibacterial ability of rhamnosyl lectin; The deacetylation modification method is to mutate K in the amino acid sequence of rhamnosyl lectin to R; The K in the amino acid sequence is the 159th K of rhamnosyl lectin; The bacterium is Aeromonas hydrophila.

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