Odontobutis potamophila chitinase gene and application thereof
By cloning and expressing the chitinase gene of the river Shatang ferment, recombinant plasmids were constructed and expressed in yeast, the problem of insufficient digestibility was solved, the growth performance and immunity of fish were significantly improved, and efficient Breeding effect.
Patent Information
- Application Number
- CN202411356215.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2024-12-27
AI Technical Summary
Due to the lack of suitable feed in the breeding of river sand pond birds, the digestive system lacks the digestive capacity of chitin, which affects protein utilization and growth performance. The existing technology lacks cloning of its own endogenous chitinase and Applied research.
Screen and clone the chitinase gene of the fermented bird in Shatang, Hechuan, construct the recombinant plasmid pPICZαA, and transform it into yeast to express it to obtain chitinase expression protein. The digestive ability and immunity of fish are enhanced by adding different concentrations of chitinase. improves growth performance.
By expressing and adding chitinase, the weight gain and specific growth rate of river sand pond ferns are significantly improved, the mortality rate is reduced, intestinal health and immunity is enhanced, digestive enzyme activity and antioxidant ability are improved, and it has green Efficient application prospects.
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Figure CN119193633A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of genetic engineering, in particular to a river Channa tangna chitinase gene and application thereof. Background Art
[0002] The river snakehead (Odontobutispotamophila) is a carnivorous fish endemic to my country. It is widely distributed in rivers and lakes in the middle and lower reaches of the Yangtze River. It mainly feeds on live animals, including shrimps and zooplankton. Its meat is delicious, with more edible parts and fewer bones. It is an excellent economic aquatic product. However, due to the lack of suitable feed, the breeding of river snakehead depends on arthropods and small fish in pond water. The chitin contained in its feed needs to be digested and decomposed by its own digestive system, which affects the utilization and digestibility of protein, and thus affects its growth performance. Based on its feeding habits, the activity of chitinase in the digestive system of river snakehead is crucial to its growth.
[0003] At present, many studies have focused on the cloning of enzyme genes produced by exogenous environmental microorganisms, the study of enzymatic properties, and the construction and expression of engineered bacteria, while there are few reports on the cloning, heterologous expression, and application of chitinases synthesized and secreted endogenously by river Channa tang in aquaculture. Therefore, screening endogenous chitinases in river Channa tang, analyzing its chitinase-related genes, cloning and expressing related genes using molecular biological methods, displaying proteins on the surface of yeast cells, expressing chitinase genes, and applying them in the culture process of river Channa tang is of great significance for improving digestion ability, maintaining intestinal health, enhancing immunity, and improving growth performance. Summary of the invention
[0004] The purpose of the present invention is to provide a river Channa sand tang chitinase gene and its application. The purpose of the present invention is to provide an expression protein of the river Channa sand tang chitinase gene, and its application in improving the growth performance of fish. The gene and its expression protein can improve the digestion ability of river Channa sand tang to chitin, maintain intestinal health, enhance immunity, and improve growth performance.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The invention provides a river Channa tangna chitinase gene, the nucleotide sequence of which is shown as SEQ ID NO.1.
[0007] The present invention also provides a river Channa tangna chitinase encoded by the river Channa tangna chitinase gene. The amino acid sequence of the river Channa tangna chitinase is shown as SEQ ID NO.2.
[0008] The invention also provides an expression vector, comprising an initial vector and the river Channa tangna chitinase gene.
[0009] Preferably, the initial vector is pPICZαA.
[0010] The present invention also provides a host transformed or transfected with the expression vector; the host is a microorganism.
[0011] Preferably, the microorganism is yeast.
[0012] The present invention also provides the use of the riverine sand channan chitinase gene, the riverine sand channan chitinase, the expression vector and the host in preparing feed for improving fish growth performance or improving intestinal health.
[0013] The present invention also provides the use of the riverine sand channan chitinase gene, the riverine sand channan chitinase, the expression vector and the host in the preparation of a feed additive for improving fish growth performance or improving intestinal health.
[0014] The invention also provides a method for preparing chitinase, which comprises culturing the host to obtain the chitinase.
[0015] Preferably, after the cultivation is completed, the method further comprises the step of purifying chitinase: adding the supernatant containing chitinase to a chromatography column containing nickel ion filler, balancing the column with 5-7 mL Lysis buffer, and repeating 3-4 times; passing 50-60 mL of protein solution through the column twice; adding 10-15 mL Wash buffer for washing, and repeating 5-7 times; and adding 5-7 mL Elution buffer for elution.
[0016] Compared with the prior art, the present invention has the following beneficial effects:
[0017] The invention successfully constructs a recombinant plasmid pPICZαA-OpCht with a chitinase gene, and then transforms the recombinant plasmid containing the chitinase gene into Pichia pastoris X-33 to obtain a yeast recombinant expression vector; the yeast recombinant expression vector is purified by large-scale shaking to obtain a chitinase expression product, which is fed to river Channa sphaeroides for 56 days. It is found that adding three different concentrations of chitinase can increase the weight gain rate and specific growth rate of river Channa sphaeroides, reduce mortality, and improve growth performance; it is beneficial to improve digestive enzyme activity, increase the width, height and thickness of intestinal villi, increase antioxidant capacity, and enhance fish nonspecific immunity. It can be specifically applied to river Channa sphaeroides breeding, which is green and efficient and has a promising future. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.
[0019] Figure 1 This is the PCR identification of the recombinant plasmid pPICZαA-OpCht in Example 1 of the present invention, 1-5: recombinant plasmid PCR product; 6: original plasmid PCR product.
[0020] Figure 2 This is the protein expression detection in Example 2 of the present invention, 1-7: target protein expression, 8: positive control (eGFP protein-28kDa).
[0021] Figure 3 This is the purification status of the target protein for staining and detection in Example 2 of the present invention, 1: purified protein, 2: effluent, 3: washing solution, 4: positive control (eGFP-28kDa).
[0022] Figure 4 This is the WB detection of the target protein purification in Example 2 of the present invention, 1: purified protein, 2: effluent, 3: washing solution, 4: positive control (eGFP-28kDa).
[0023] Figure 5 This is the protein standard curve in Example 2 of the present invention.
[0024] Figure 6 This is the 4MU standard curve in Example 3 of the present invention.
[0025] Figure 7 The effect of adding recombinant chitinase on digestive enzyme activity in Example 6 of the present invention. Digestive enzyme activity indicators include protease (A), butyrate (B) and chitinase (C). One-way ANOVA was used to detect significant differences between groups, and then Duncan's multiple comparison test was used (*P<0.05, **P<0.01, ***P<0.001). Data are expressed as mean ± standard deviation (n=9).
[0026] Figure 8 The effect of adding fish chitinase in Example 6 of the present invention on liver antioxidant activity and immunity. Liver biochemical indicators include SOD (A), CAT (B), AKP (C), LZM (D), MDA (E). One-way analysis of variance was used to detect significant differences between the groups, and then Duncan's multiple comparison test was used (*P<0.05, **P<0.01, ***P<0.001). Data are expressed as mean ± standard deviation (n=9). DETAILED DESCRIPTION
[0027] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0028] Example 1: Construction of chitinase recombinant plasmid
[0029] (1) Synthesis of target gene: Based on the chitinase sequence of Channa sphaerocephala, Primer Premier 5.0 software was used for analysis. At the same time, the codon preference data of Pichia pastoris was used to optimize the codons by taking advantage of the degeneracy of codons. The chitinase gene sequence without signal peptide was synthesized without changing the encoded amino acid sequence. EcoRI and SalI restriction sites were introduced upstream and downstream, respectively. The target gene was commissioned to Qingke Biotechnology Co., Ltd. for synthesis.
[0030] The nucleotide sequence of the chitinase from Channa tanglii is as follows (1458 bp):
[0031]
[0032] The amino acid sequence of the chitinase from Channa sphaeroides is as follows (SEQ ID No. 2):
[0033] MTKLIVLAGLCLVLCQLVTASQMVCYFTNWSQYRPGTGKYMPQNVDPFLCTHLIYAFSIINPKNELVTYEWNDETLYKTFNGLKSKNPGLKTLLAVGGWNFGSTQFSIMVSTPANRQTFIQ SSIKFLRTHGFDGLDLDWEYPGARGSPPIDKQRFTLLCRELVEAYAAEAKASGRPQLMLTAAVSAGKGTIDAGYEINEIAKYLDFINIMTYDFHGAWERFTGHNSPLYRGSHDSGDLIYFN TDYAMKYWRDQGTPAHKLRMGFASYGRTFRLSSSNTGVGAPASGAASAGPYTREAGFWSYYEICTFLKGTTIQWIADQKVPYASKNSEWVGFDTRESYETKVQYMKDNKFGGAFVWALDLD DFNGEFCGQGKHPLLSHLRNLINGDMPPPPATTTNKPGVTTTAPATKTTAKPATTTPRPIGTTTTPVPGPGFCKDLPDGIYAHPTDNTLFYECTGGRTYLKKCGGGTVFEQSCKCCVWPKT
[0034] (2) Enzyme digestion: The synthesized target gene and vector pPICZαA were double-digested with EcoRI and SalI, respectively, and the target gene fragment and pPICZαA vector fragment were recovered using a kit, and then connected with T4 DNA ligase at 37°C for 3h to construct the expression vector pPICZaA-OpCht. Subsequently, the plasmid was transformed into E. coli Top10, and the plasmid was extracted and identified by nucleic acid sequencing and double enzyme digestion.
[0035] (3) Transformation and positive single clone screening: Linearization by Sac I restriction enzyme digestion, electrotransformation into Pichia pastoris X33 expression strain, and transformation of plasmid pPICZaA-OpCht into Pichia pastoris X-33 competent cells. After transformation, evenly spread on YPD plates and culture at 30°C for 3 days. Pick several single clone yeasts for PCR verification, and sequence the bacterial solution of positive clones. Upstream primer F: ATGACTAAGCTCATAGTTCTAG CAGGC (SEQ ID No.3), downstream primer R: GACTGTTTTGGGCCACACACA (SEQID No.4). The results showed that the positive clone strain amplified a fragment consistent with the size of the target gene in agarose gel electrophoresis ( Figure 1 ).
[0036] The PCR reaction program was as follows: pre-denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 94°C for 15 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and finally extension at 72°C for 10 min. Reaction system (Table 1).
[0037] Table 1 PCR reaction system
[0038]
[0039] Example 2: Induced expression and purification
[0040] (1) Induced expression: Pick a round and plump single colony and inoculate it into 50mL YPG liquid medium, culture at 30℃ and 220rpm for 1d until the bacterial liquid is saturated, transfer it to a 50mL centrifuge tube, centrifuge it at 4000rpm for 5min, and discard the supernatant; resuspend the bacteria in 50mL BMMY medium, transfer it to a new sterile 250mL conical flask, add methanol to a final concentration of 0.75%, and culture it at 28℃ and 220rpm for 6d; add methanol every 24h; collect the bacterial liquid on the morning of the 6th day, centrifuge it at 4000rpm for 5min, and collect the supernatant; aspirate 20μL of the supernatant, add 5μL of 5× loading buffer, mix well, boil it at 100℃ for 10min, and aspirate 20μL for Western Blot identification. The results showed that a protein with a molecular weight of about 52kDa was detected in the supernatant ( Figure 2 ).
[0041] (2) Purification: The supernatant was added to a chromatography column containing nickel ion filler, and the column was equilibrated with 5 mL of Lysis buffer (Lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH = 8.0, filtered and sterilized), and the process was repeated 3 times; 50 mL of the protein solution was passed through the column twice; 10 mL of Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH = 8.0, filtered and sterilized) was added for washing, and the process was repeated 5 times; 5 mL of Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH = 8.0, filtered and sterilized) was added for elution to obtain the purified protein; after purification, SDS-PAGE and Western Blot were used for detection, and the results showed that a clear protein band appeared at approximately 52 kDa ( Figure 3 and Figure 4 ), proving that the target protein was successfully expressed in yeast X-33, and the protein concentration was determined by BCA method ( Figure 5 ).
[0042] Example 3: Chitinase activity assay
[0043] The enzyme activity was determined using 4-methylumbelliferone (4-MU) fluorescent substrate (obtained in Example 2). The enzyme activity is defined as the amount of enzyme required to release 1 μmol 4-MU per minute at 37°C, expressed as U. The specific activity is expressed as the number of enzyme activity units (U / g) contained per gram of protein. A 4-MU standard sample with a gradient concentration of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1 nmol was prepared using PBS buffer at pH 7.4, and the sample to be tested was 1 μg of the purified product and 2.5 nmol of 4MU-(GlcNAc) 1 / 2 / 3 The final reaction volume of the standard sample and the sample to be tested was 100 μL, and the reaction was carried out at 37°C for 30 min. Finally, 100 μL of 0.5 mol / L Na2CO3 solution was added to each well to terminate the reaction. The fluorescence intensity was measured with an ELISA reader at an excitation wavelength of 370 nm and an emission wavelength of 448 nm ( Figure 6 ). The results showed that the protein concentration of the recombinant enzyme was 71ug / mL, the enzyme activity for degrading 4MU-GlcNAc was 0U / g, the enzyme activity for degrading 4MU-(GlcNAc)2 was 42.69U / g, and the enzyme activity for degrading 4MU-(GlcNAc)3 was 6.88U / g, according to the protein and 4MU standard curves.
[0044] Example 4: Effect of adding recombinant chitinase on the growth performance of river Channa tang
[0045] A total of 240 river Channa were randomly assigned to four treatment groups, three replicates in each group, and each replicate contained 20 fish. Using a 200-microliter micropipette, 0, 1, 2, and 4 μg / g (protein concentration / fish body weight) of recombinant fish-derived chitinase (obtained in Example 2) were injected into the stomach every 48 hours, and they were marked as the control group (CK), S1, S2, and S3 groups, respectively. They were fed with Chinese sawtooth shrimp twice a day (8:00 and 17:00) for 56 days until they were fully fed. The feeding status and death status of river Channa were observed and recorded. Weigh once a week and fast for 24 hours before weighing. After the experiment, 10 fish were randomly caught from each tank to collect intestinal and liver tissues.
[0046] After the 56-day breeding experiment, the final body weight FW, weight gain rate WGR and specific growth rate SGR were significantly increased (P < 0.05), among which the S3 group had the highest FW, WGR and SGR, which were 20.84g, 97.02% and 1.21% respectively; the survival rate SR of the S2 and S3 groups was statistically significant compared with the control group CK, which was 86.67% and 88.33% respectively, and SR had a trend of gradual increase with the increase of concentration. (Table 2)
[0047] Table 2 Effect of adding recombinant chitinase on the growth performance of river Channa
[0048]
[0049]
[0050] The results are expressed as mean ± standard deviation of three groups (n = 9). Data values with different superscript letters in the same column are significantly different (P < 0.05).
[0051] Example 5: Effect of adding recombinant chitinase on the improvement of intestinal structure of farmed fish
[0052] Fresh intestinal tissue was fixed with 4% paraformaldehyde before preparing paraffin sections. After fixation for at least 24 hours, the tissue samples were appropriately trimmed in a fume hood and then gradually dehydrated in ethanol with increasing concentrations from 75% to 100%. Subsequently, the intestinal samples were embedded in paraffin and cut into 4-micron sections using a microtome. They used hematoxylin and eosin (H&E) staining and acquired images under a microscope to observe tissue morphology. Image J software was used to measure mucosal thickness (MT), villus length (VL), and villus width (VW). The experimental results are shown in Table 2. The three treatment groups with the addition of recombinant fish-derived chitinase significantly increased the length and width of the intestinal villi and the thickness of the muscular layer of river sand tang Channa (P<0.05), and were concentration-dependent.
[0053] Table 3 Improvement of intestinal morphology of river Channa tangna by adding recombinant chitinase
[0054]
[0055] The results are expressed as mean ± standard deviation of three groups (n = 9). Data values with different superscript letters in the same column are significantly different (P < 0.05).
[0056] Example 6: Effect of adding recombinant chitinase on the digestion and immune-related enzyme activities of river Channa tang
[0057] The commercial detection kits of Nanjing Jiancheng Bioengineering Institute were used to determine the contents of protease and amylase in the intestinal tissue of river Channa tangchus, lysozyme, superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (AKP) and malondialdehyde (MDA) in the liver tissue. The chitinase in the gastric tissue was determined by 4MU method.
[0058] The experimental results are as follows Figure 7-8 As shown in the results, after 56 days of culture, the activities of protease, amylase (AMS) and chitinase in groups S2 and S3 were significantly increased compared with those in the control group (P<0.05). Compared with the control group, the CAT, AKP and LZM in the three groups treated with recombinant fish chitinase were significantly increased (P<0.05). The liver SOD in groups S2 and S3 increased significantly by 10.29% and 21.38%, respectively (P<0.05). The liver MDA in the three treatment groups with OpCht addition was significantly lower than that in the control group (P<0.05), and decreased with the decrease of the added concentration.
[0059] In summary, the recombinant chitinase expressed by the eukaryotic expression system of the present invention can be added to the stomach of fish in a certain amount to improve growth performance, improve digestive enzyme activity, increase the width and height of intestinal villi and the thickness of the muscular layer; enhance antioxidant capacity and non-specific immunity of fish. It can be specifically applied to river Channa tangensis breeding, which is green and efficient and has a promising future.
[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A chitinase gene of Channa sphaeroides, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.
1.
2. The river Channa sand mullet chitinase encoded by the river Channa sand mullet chitinase gene according to claim 1, wherein the amino acid sequence of the river Channa sand mullet chitinase is shown as SEQ ID NO.
2.
3. An expression vector, characterized in that: The invention comprises an initial vector and the chitinase gene of Channa tangli as claimed in claim 1.
4. The expression vector according to claim 3, characterized in that The initial vector is pPICZαA.
5. A host, characterized in that The host is transformed or transfected with the expression vector as claimed in claim 3; the host is a microorganism.
6. The host according to claim 5, characterized in that The microorganism is yeast.
7. Use of the chitinase gene of Channa tanglii as claimed in claim 1, the chitinase of Channa tanglii as claimed in claim 2, the expression vector as claimed in claim 3 or 4, and the host as claimed in claim 5 or 6 in preparing feed for improving fish growth performance or improving intestinal health.
8. Use of the chitinase gene of Channa tanglii as claimed in claim 1, the chitinase of Channa tanglii as claimed in claim 2, the expression vector as claimed in claim 3 or 4, and the host as claimed in claim 5 or 6 in the preparation of a feed additive for improving fish growth performance or improving intestinal health.
9. A method for preparing chitinase, characterized in that: Cultivate the host according to claim 5 or 6 to obtain chitinase.
10. The method according to claim 9, characterized in that After the cultivation is completed, the method also includes the steps of purifying chitinase: adding the supernatant containing chitinase to a chromatography column containing nickel ion filler, balancing the column with 5-7 mL Lysis buffer, repeating 3-4 times; passing 50-60 mL of protein solution through the column twice; adding 10-15 mL Wash buffer for washing, repeating 5-7 times; and adding 5-7 mL Elution buffer for elution.