Chitinase as well as preparation method and application thereof

Abstract

The invention belongs to the field of biology, and discloses chitinase, the amino acid sequence of which is as shown in SEQ ID NO.1. The chitinase can significantly destroy the throat structure of the nematode to cause nematode death, so that the death rate of the nematode is significantly increased; meanwhile, the invention further discloses a preparation method and application of the chitinase.

Description

Technical Field

[0001] The invention belongs to the biological field, and specifically relates to chitinase, a preparation method and application thereof. Background Art

[0002] Chitin, also known as chitosan, chitosan or chitosan, is a linear polysaccharide polymer composed of N-acetyl-D-glucosamine connected by β-1,4 glycosidic bonds. Chitin has a wide distribution and important biological functions in the biological world. It is an important component of the shell of crustaceans (such as shrimps, crabs, etc.), providing structural support and defense functions; the exoskeletons of insects and other arthropods also contain a large amount of chitin as an active ingredient. In order to develop an effective method to control parasites, we prepared chitinase by in vitro expression and studied its interaction with chitin.

[0003] Chitinase is a class of specific enzymes that can degrade chitin. It is divided into three families based on amino acid structure and sequence homology: GH-18, GH-19, and GH-20. GH-18 chitinase is ubiquitous and widely distributed in bacteria, fungi, viruses, animals, plants, etc., mainly including chitinase A and chitinase B. GH-19 chitinase mainly comes from plants and nematodes, and is rarely identified in microorganisms. The GH-20 family includes β-N-acetylhexosaminidase and chitosanase, which catalyze the decomposition of chitosan and acetylgalactosamine. Chitinases of the GH-18 and GH-19 families have completely different structures. The GH-18 catalytic domain is a TIM barrel composed of 8 α-helices and 8 parallel β-strands, with a conserved glutamate residue, and the hydrolysis reaction is carried out through a substrate-assisted mechanism; the chitinase GH-19 family has a bilobed structure composed of high α-helices, has structural similarities with lysozyme, and adopts a single displacement catalytic mechanism.

[0004] These enzymes break down chitin into oligosaccharides and N-acetylglucosamine monomers by hydrolyzing the β-1,4 glycosidic bonds in chitin. Chitinase has a destructive effect on the integrity of the insect exoskeleton, thereby weakening or killing the parasite.

[0005] Chitinase can be efficiently produced by genetic engineering technology in an in vitro expression system. These recombinant chitinases can maintain their enzyme activity under experimental conditions and effectively degrade chitin in insect exoskeletons. Studies have shown that treating mosquito larvae with chitinase can significantly reduce their survival rate, because chitinase destroys the integrity of the larval exoskeleton, causing it to lose its protective function and leading to the death of the parasite. Other studies have pointed out that chitinase is also effective in controlling other insect pests. For example, a study on the fungus Beauveria bassiana showed that after exposure to a solution containing chitinase, the survival rate of mosquitoes was significantly reduced, with only 14% of mosquitoes surviving after 48 hours. In addition, in terms of biological control, chitinase is used as a biological insecticide. By spraying a solution containing chitinase, pests in the field can be effectively killed. As an environmentally friendly alternative to chemical pesticides, it has significant anti-insect and anti-fungal biological control activity. These enzymes can specifically degrade chitin and destroy the defense barrier of pests and pathogens, thereby achieving insecticidal and antibacterial effects. A study in Beauveria bassiana found that the addition of serine significantly increased the bioinsecticide activity of chitinase from Beauveria bassiana through kinetic studies. This suggests that by optimizing the production and application conditions of chitinase, its effectiveness as a bioinsecticide can be enhanced.

[0006] Chitinase is a biological control agent. Although there are few reports on chitinase killing nematodes, its application prospects in this field are undoubtedly very important and worthy of further exploration. Nematodes are important pests in agricultural production, resulting in a significant reduction in global crop yields. Currently, chemical nematicides are the main means of control, but these chemical agents are often toxic and pose potential hazards to the environment and human health. Summary of the invention

[0007] In view of the shortcomings of the prior art, the object of the present invention is to provide a chitinase, which can significantly destroy the throat structure of nematodes, causing the death of nematodes, resulting in a significant increase in the mortality rate of nematodes;

[0008] Meanwhile, the invention also discloses a preparation method and application of the chitinase.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] A chitinase, the amino acid sequence of which is as shown in SEQ ID NO.1.

[0011] The amino acid sequence of chitinase is as follows:

[0012] DSPKQSQKIVGYFPSWGVYGRNYQVADIDASKLTHLNYAFADICWNGKHGNPSTHPDNPNKQTWNCKESGVPLQNKEVPNGTLVLGEPWADVTKSYPGSGTTWEDCDKYARCGNFGELKRLKAKYPHLKTIISVGGWTWSNRFSDMAADEKTRKVFADST VAFLRAYGFDGVDLDWEYPGVETIPGGSYRPEDKQNFTLLLQDVRNALNKAGAEDGKQYLLTIASGASQRYADHTELKKISQILDWINIMTYDFHGGWEATSNHNAALYKDPNDPAANTNFYVDGAINVYTNEGVPVDKLVLGVPFYGRGWKSCGKENNGQ YQPCKPGSDGKLASKGTWDDYSTGDTGVYDYGDLAANYVNKNGFVRYWNDTAKVPYLYNATTGTFISYDDNESMKYKTDYIKTKGLSGAMFWELSGDCRTSPKYSCSGPKLLDTLVKELLGGPISQKDTEPPTNVKNIVVTNKNSNSVQLNWTASTDNVG VTEYEITAGEEKWSTTTNSITIKNLKPNTEYTFSVIAKDAAGNKSQPTAFTVKTDEANTTPPDGNGTATFSVTSNWGSGYNFSIIIKNNGTTPIKNWKLEFDYSGNLTQVWDSKISSKTNNHYVITNTGWNGEIPPGGSITIGGAGTGNPAELLNAVISEN

[0013] At the same time, the present invention also discloses a method for preparing the chitinase as described above, wherein the chitinase gene is connected to a vector to obtain a plasmid, and the plasmid is transferred into a cell for expression to obtain the chitinase;

[0014] The nucleotide sequence of the chitinase gene is as shown in SEQ ID NO.2.

[0015] The nucleotide sequence of chitinase is as follows:

[0016]

[0017] In the above preparation method, the vector is pET28a plasmid; the cells are E. coli DH5α and BL21 (DE3) competent cells.

[0018] Finally, the present invention also discloses the use of the chitinase as described above to prepare a medicament for killing nematodes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention clones chitinase genes from Bacillus licheniformis to explore the killing effect of chitinase genes on nematodes.

[0021] First, the present invention obtains a chitinase gene from Bacillus proteolyticus. The gene is amplified by PCR and cloned into an expression vector. In order to determine that the chitinase gene can be efficiently expressed in a host cell, the present invention truncates the chitinase, transforms the constructed recombinant plasmid into an Escherichia coli expression host strain, induces protein expression and purifies the chitinase, and the purified chitinase has biological activity.

[0022] Secondly, the present invention conducts an in vitro interaction experiment with nematodes. The purified chitinase protein is added to a culture system containing nematodes to observe its effect on the nematodes. The results show that the chitinase protein can significantly damage the throat structure of the nematodes, causing the nematodes to die, resulting in a significant increase in the mortality rate of the nematodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a photo of Bacillus proteolyticus on LB solid medium;

[0024] Figure 2 It is a picture of Bacillus proteolyticus;

[0025] Figure 3 This is a reference diagram of the genetic evolutionary tree of the 16S rRNA gene sequence of Bacillus proteolyticus

[0026] Figure 4 This is the result of using specific primers to detect chitinase genes of four tick-derived Bacillus sp.;

[0027] Figure 5A This is a graph showing the PCR test results of pET28a-RChiA;

[0028] Figure 5B This is the result diagram of optimized expression of pET28a-RChiA;

[0029] Figure 5CIt is the result of expression of recombinant chitinase protein;

[0030] Figure 6 It is the expression result of the recombinant protein pET28a-RchiA;

[0031] Figure 7 It is chitinase that breaks down colloidal chitin to produce a transparent circle diagram;

[0032] Figure 8 This is the result of the reaction between the product of chitinase decomposing colloidal chitin and the color developer;

[0033] Fig. 9 This is a graph showing the survival rate of nematodes co-cultured with chitinase solution in a 96-well plate compared with the control group and s-buffer group;

[0034] Fig.10 is the plasmid map of pET28a-RChiA;

[0035] Fig.11 It is a photo of a living nematode;

[0036] Fig.12 It is a picture of a dead nematode;

[0037] Fig.13 This is a diagram of the degradation of the pharynx of Caenorhabditis elegans by chitinase. DETAILED DESCRIPTION

[0038] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0039] Experiment 1: In vitro cloning and expression of chitinase

[0040] Using Bacillus proteolyticus preserved in our laboratory as a template and referring to the CDS sequence of chitinase, the chitinase gene (RChiA) of Bacillus proteolyticus was amplified, and a pair of specific primers were designed using the Novozymes CE Design tool.

[0041] The process of obtaining proteolytic Bacillus is as follows: crush the tick, separate and identify the Bacillus carried by the tick. The colonies of the isolated proteolytic Bacillus on LB solid medium are milky white (reference Figure 1 ), the colony surface is smooth and the edges are neat (reference Figure 2 ). Genetic phylogenetic tree reference of 16S rRNA gene sequence of Bacillus proteolyticus Figure 3 ;

[0042] The chitinase genes of four tick-derived Bacillus species were detected using specific primers. Figure 4 , Figure 4 Among them, 1 is Bacillus thuringiensis; 2 is Bacillus proteolyticus; 3 is Bacillus paramycoides; 4 is Bacillus cereus.

[0043] pass Figure 4 It can be seen that among the tick-borne Bacillus, only the proteolytic Bacillus has an obvious bright band.

[0044] The specific sequence is as follows:

[0045] Upstream primer (PF-RChiA): 5'-cagcaaatgggtcgcggatccATGGATTCACCAAAGCAAAGTCA-3'; (SEQ ID NO.3)

[0046] Downstream primer (PR-RChiA): 5'-gtggtggtggtggtgctcgagCTAGTTTTCGCTAATGACGGC-3'; (SEQ ID NO.4)

[0047] A BamHI restriction site sequence was introduced at the 5' end of the upstream primer, immediately following the start codon (ATG).

[0048] An XhoI restriction site sequence was inserted at the 5' end of the downstream primer, followed by a stop codon (TAA).

[0049] The estimated length of the amplified fragment is 1932 bp.

[0050] Chitinase was subjected to PCR using homologous recombination technology, and the PCR reaction conditions were set as follows: pre-denaturation at 94°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 1 minute, for a total of 30 cycles, and final extension at 72°C for 10 minutes.

[0051] The RChiA gene was amplified using the designed specific primers. After the PCR reaction, the amplified product was detected by agarose gel electrophoresis.

[0052] The 1932 bp DNA fragment (RChiA gene fragment, SEQ ID NO. 2) obtained by PCR amplification was purified by a gel recovery kit to obtain a purified DNA fragment. At the same time, the pET28a plasmid was also purified after being double-digested with BamHI and XhoI to remove small molecule fragments and enzymes.

[0053] The purified RChiA gene fragment and the pET28a plasmid treated with double enzymes were ready for ligation reaction. The ligation reaction was carried out using Exnase II ligase from Novozymes, and then the reaction product was transformed into E. coli DH5α competent cells, and the plasmid was promoted to enter the cells by heat shock method.

[0054] The transformed bacteria were spread on LB solid medium containing kanamycin and cultured overnight at 37°C. Single colonies were selected for PCR verification to confirm the successful construction of the recombinant plasmid.

[0055] The positive strains were sent to Sangon Biotech for sequencing to ensure the correctness of the inserted sequence. The successfully constructed recombinant plasmid was named pET28a-RChiA. The plasmid map is shown in Fig.10 shown.

[0056] like Figure 5A As shown, the target gene was found by PCR identification of pET28a-RChiA.

[0057] Experiment 2: Protein expression and purification

[0058] Optimize (time) expression:

[0059] The recombinant plasmid pET28a-RChiA with correct sequencing was transformed into E. coli BL21 (DE3) competent cells. Subsequently, a single clone was inoculated into a 100 mL conical flask containing LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 180 rpm until OD 600 When the p-value reaches 0.4–0.8, add IPTG with a final concentration of 1 mmol / L. Then, induce for 6 hours at 20°C and 150 rpm, and collect the 0–6 hour cells. After collecting the cells by centrifugation, perform denaturing polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the optimal expression time.

[0060] A lot of expression:

[0061] Resuscitate the positive receptor strain stored at -80℃, inoculate 200μL of bacterial suspension into 20mL LB liquid medium (Kanamycin), and shake and culture at 37℃, 180rpm for 12h. Inoculate into 1000mL LB liquid medium (Kanamycin) at a volume fraction of 1:100, shake and culture at 37℃, 180rpm. When the OD60nm value is higher than 0.6, add IPTG to a final working concentration of 1mmol / L, and shake and culture slowly at 25℃, 150rpm for 6h. Dispense the bacterial solution into 50mL centrifuge tubes, centrifuge at 4℃, 5000rpm for 15min, discard the supernatant, add 3mL of non-denaturing lysis buffer to the precipitated bacteria, and mix slowly by pipetting. Add 30μL lysozyme to a final working concentration of 1mg / mL, mix slowly by pipetting, and ice bath for 30min. Use an ultrasonic disruptor to break the bacteria until transparent. Use a refrigerated centrifuge to high-speed centrifuge at 4°C, 9000rpm for 30 minutes, collect the supernatant into a new centrifuge tube, add non-denaturing lysis buffer to the broken precipitate, blow and resuspend, and perform SDS-PAGE electrophoresis to identify whether the protein is expressed in a soluble form.

[0062] like Figure 5B As shown, by optimizing the expression of pET28a-RChiA, its expression level was highest in the 6th hour.

[0063] like Figure 5C As shown, the recombinant chitinase protein can be expressed in the supernatant;

[0064] The expression of the target protein can be observed through the SDS-PAGE electrophoresis pattern to determine its solubility and stability.

[0065] To purify the expressed His-tagged chitinase protein, BeyoGold TM His-tag Purification Resin was used for affinity chromatography purification. Take 6 mL of 50% BeyoGold TMHis-tag Purification Resin was added to a 50mL centrifuge tube. Centrifuge at 1000g for 10 seconds at 4°C, discard the storage solution, add 3mL of non-denaturing lysis buffer to the gel and mix well to balance the gel, centrifuge again at 1000g for 10 seconds at 4°C, and discard the liquid. Repeat the balancing step 1-2 times, discarding the liquid each time to ensure that the gel is fully balanced. Add the supernatant of the bacterial lysate to the balanced gel, place the centrifuge tube in ice, and shake it slowly on a horizontal shaker for 60 minutes to allow the protein to fully bind to the gel. Allow the liquid in the column to flow out under gravity, collect about 50μL of the flow-through liquid for use, wash the column 5 times, and add 6mL of non-denaturing washing solution each time. Collect about 50μL of the washing solution from the column for subsequent analysis and detection to ensure that the non-specifically bound impurity proteins are fully eluted. Elute with 12mL of non-denaturing elution solution (50mM imidazole), and collect the eluate into different centrifuge tubes each time. Repeat the elution 5 times, collect each elution to obtain a purified His-tagged chitinase protein sample. Then, the chitinase protein was replaced and concentrated. The purified chitinase (containing 50 mM imidazole) was added to Ultra 10K ultrafiltration tube. Centrifuge at 4500g for 15 minutes to concentrate the protein and discard the filtrate. Add 10mL PBS (0.01M) solution and centrifuge again at 4500g for 15 minutes. Repeat the above liquid replacement steps three times to fully remove impurities and imidazole and ensure that the protein is in pure PBS buffer. Add 5mL PBS solution and mix well to ensure that the protein is fully dissolved. Dispense the recombinant chitinase pET28a-RChiA solution with a concentration of 0.584mg / ml into 1.5mL centrifuge tubes, with an appropriate amount in each tube for subsequent use.

[0066] like Figure 6 As shown, an obvious band of the size of the recombinant protein pET28a-RchiA can be seen from Figures E1, E2, and E3, proving that it has been purified; Figure 6 The meanings of the symbols are as follows: M: marker; CL: cell lysate; FT: flow through; W1-W3: wash 1-3; E1-E4: elution 1-4.

[0067] Experiment 3: Kit for detecting exogenously expressed chitinase activity

[0068] Chitinase activity was determined using a commercial kit (Solarbio, BC0820) according to the manufacturer's instructions.

[0069] Reagents 1-4 in the test can refer to the instructions of the kit (Solarbio, BC0820).

[0070] 1. Preparation of standard solution: dilute the standard with reagent 1 to different concentrations of standard solution (125, 62.5, 31.25, 15.625, 7.8125, 3.9, 1.953, 0.976, 0.488 μg / mL) and mix well;

[0071] 2. Sample preparation:

[0072] 80 μL of the recombinant chitinase pET28a-RChiA sample with a concentration of 0.584 mg / ml was added to the control tube and the assay tube, and no sample was added to the blank tube and the standard tube.

[0073] 3. Add reagents:

[0074] Add 40 μL of reagent to each control tube and assay tube.

[0075] 80 μL of Reagent 2 was added to each of the control tube and the assay tube, and no Reagent 2 was added to the blank tube and the standard tube.

[0076] 4. Enzymatic reaction:

[0077] The centrifuge tube containing the above liquid was placed at 37°C for 1 hour for enzymatic reaction, and then placed in a boiling water bath for 5 minutes (to terminate the enzymatic reaction).

[0078] 5. Centrifugation:

[0079] The control tube and the assay tube after the reaction were centrifuged at 10,000 rpm for 5 minutes at room temperature, and the supernatant was collected.

[0080] 6. Supernatant treatment:

[0081] 100 μL of supernatant was added to each of the new control tube and the assay tube, but not added to the blank tube and the standard tube.

[0082] 7. Color development:

[0083] Add 100μL of reagent 1 to each blank tube, and do not add to other tubes. Add 100μL of standard solution to the blank tube. Add 20μL of reagent 3 to all tubes and mix well. Let the control tube and the test tube stand at room temperature for 5 minutes. Then put the control tube and the test tube in a boiling water bath for 5 minutes, and then cool to room temperature with running water. Add 300μL of reagent 4 to all tubes.

[0084] 8. Preheat the spectrophotometer / ELISA reader for 30 min, adjust the wavelength to 585 nm, and zero it with distilled water.

[0085] 9. Incubate all centrifuge tubes at 37℃ for 20min, draw 200μL into a 96-well plate and measure the absorbance at 585nm, record them as A control, A test, A blank, A standard respectively. Calculate ΔA=A test-A control, ΔA standard=A standard-A blank. (The standard curve and blank tube only need to be measured 1-2 times).

[0086] Chitinase activity calculation:

[0087] 1. Draw a standard curve and establish a standard curve based on the concentration and absorbance of the standard tube.

[0088] 2. Calculate chitinase activity.

[0089] Definition of enzyme activity: At 37°C, the amount of enzyme that decomposes chitin to produce 1 μg N-acetylglucosamine per mL of liquid per minute is one unit of enzyme activity.

[0090] Chitinase activity (U / mL) = x × V enzymatic ÷ V sample ÷ T = 2.5 × x ÷ 60

[0091] Venzymatic: total volume of enzymatic reaction, 0.2 mL; Vsample: sample volume in the reaction system, 0.08 mL; Vsampletotal: volume of added extract, 1 mL; W: sample mass, g; Cpr: sample protein concentration, mg / mL; T: reaction time, 1 h.

[0092] Chitinase decomposes colloidal chitin to produce a transparent circle Figure 7 ;

[0093] The product of chitinase decomposing colloidal chitin reacts with the color developer to produce a purple color with a characteristic absorption peak at 585nm. Figure 8 .

[0094] After testing, the enzyme activity was 7.529±0.859U / ml and the enzyme specific activity was 12.526±1.471U / mg.

[0095] Experiment 4: Evaluation of the effect of chitinase in killing nematodes

[0096] First, for the synchronization and culture of C. elegans, 2 to 3 dishes were selected from the culture dishes with a large number of L4 nematodes or a large number of eggs for synchronization. These culture dishes ensure that the nematodes are at the same developmental stage to ensure consistency in subsequent experiments.

[0097] In a 12mL centrifuge tube, mix 3mL of sodium hypochlorite, 4mL of 1M NaOH, and 5mL of water for subsequent nematode lysis. Use 1-2mL of S-buffer to gently rinse the surface of the culture dish to avoid rinsing too many bacteria. Transfer the rinsed nematodes to a 1.5mL centrifuge tube with a pipette to ensure that a sufficient number of adult worms are collected for subsequent processing. Subsequently, centrifuge the collected nematodes at 3000rpm for 2 minutes and discard the supernatant. Add the prepared lysis solution and mix it upside down until the nematode body curls up, stiffens, and releases eggs. Observe the color change of the liquid during the lysis process, from turbid to yellow and then to clear. After confirming that the lysis is complete, centrifuge again at 3000rpm for 2 minutes, discard the supernatant, rinse the eggs repeatedly for at least 3 times, and then collect the eggs enriched at the bottom.

[0098] The collected eggs were inoculated onto NGM medium containing E. coli without OP50 using a pipette. After natural drying in a sterile environment, the eggs were incubated overnight at 20°C to L1 larvae. The hatched L1 larvae were inoculated onto NGM medium containing E. coli with OP50 and continued to be cultured at 20°C until the nematodes reached the L4 stage.

[0099] Chitinase acts on Caenorhabditis elegans:

[0100] Grouping treatment: Add the resuspended nematodes into a 96-well plate at a volume of 20 μL (about 50 nematodes) per well. The experiment was set up in three groups, with three wells in each group:

[0101] Experimental group: 200 μL of recombinant chitinase pET28a-RChiA with a concentration of 0.584 mg / ml was added to each well.

[0102] Inactivated control group: 200 μL of high temperature inactivated RChiA was added to each well (incubated in a 100°C water bath for 10 minutes).

[0103] Blank control group: 200 μL S-buffer was added to each well.

[0104] The treated 96-well plates were incubated at 37°C for 6 hours. The mortality and morphological changes of nematodes were observed with an inverted microscope, and the mortality and morphological changes of nematodes in each group were recorded to evaluate the effect of RChiA. Control analysis: The killing effect of RChiA on nematodes was analyzed by comparing the mortality rates of the experimental group, the inactivated control group, and the blank control group. High-temperature inactivated RChiA and S-buffer were used as controls to ensure the reliability and accuracy of the experimental results.

[0105] refer to Fig. 9 The results of experiments showed that the survival rates of nematodes co-cultured with chitinase solution in 96-well plates decreased by 50.4% and 55.2% compared with the control group and s-buffer group, respectively.

[0106] Photos of live nematodes Fig.11 The photo of dead nematodes is shown in Fig.12 As shown in the figure, the living nematode is slender and cylindrical, transparent or translucent, highly flexible and continuously mobile, moves freely through S-shaped peristalsis, and shows sensitive responses to external stimuli. The dead nematode is stiff, its transparency decreases, and it loses its ability to move.

[0107] Experiment 5: Degradation of the Pharynx of Caenorhabditis elegans by Chitinase

[0108] The three groups of Caenorhabditis elegans in Experiment 3 were washed and suspended in 500 μl of pH 6 citrate-phosphate buffer (0.2 M NaH2PO4 and 0.1 M K citric acid), and then 10 μl of eosin Y stock (5 mg / ml in 70% ethanol) was added to the test tube and incubated in the dark for 10 min, followed by extensive washing in citrate-phosphate buffer and observation under a fluorescence microscope.

[0109] like Fig.13 As shown, eosin Y staining is used to highlight chitosan in the pharynx before exposure. The large white arrow points to the posterior pharyngeal bulb. The small white arrow points to the anterior pharyngeal bulb. A and B are nematodes treated with S-Buffer and 0.01M PBS. C is a nematode treated with chitinase. The pharynx of C is significantly thinner than that of A and B, proving that the pharynx of the nematode is decomposed by chitinase.

[0110] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, and it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A chitinase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. A method for preparing chitinase according to claim 1, characterized in that: Connecting the chitinase gene to a vector to obtain a plasmid, and transferring the plasmid into cells for expression to obtain chitinase; The nucleotide sequence of the chitinase gene is as shown in SEQ ID NO.

2.

3. The preparation method according to claim 2, characterized in that: The vector is pET28a plasmid; the cell is E. coli DH5α (DE3) competent cell.

4. Use of the chitinase as claimed in claim 1 in preparing a medicament for killing nematodes.

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