Application of northeast forest frog lysozyme gene, mature polypeptide region gene, recombinant protein and recombinant lysozyme genetically engineered bacteria

Abstract

The present application belongs to the technical field of biological gene engineering, and relates to a Rana chensinensis lysozyme gene, a mature polypeptide region gene, a recombinant protein and a recombinant lysozyme genetically engineered bacterium, and application of the recombinant lysozyme genetically engineered bacterium. The present application provides a Rana chensinensis lysozyme gene with a sequence shown in SEQ ID NO. 1 and a Rana chensinensis lysozyme mature polypeptide region gene with a sequence shown in SEQ ID NO. 4. After the mature polypeptide region gene is connected to a vector and transformed into Escherichia coli BL21 (DE3), a recombinant lysozyme genetically engineered bacterium for expressing a recombinant protein is obtained, and the bacteriostatic ability of the recombinant lysozyme genetically engineered bacterium on Micrococcus luteus, Staphylococcus aureus, Escherichia coli and Aeromonas hydrophila from amphibians is detected. The Rana chensinensis lysozyme provided by the present application has bacteriostatic ability on Micrococcus luteus, Aeromonas hydrophila, Escherichia coli and Staphylococcus aureus, and can be used for developing antibacterial drugs and feed additives.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of a lysozyme gene, a mature polypeptide region gene, a recombinant protein, and a recombinant lysozyme genetically engineered bacterium from the Northeast Forest Frog. Background Technology

[0002] Lysozyme (LYZ), an N-acetylaminohydrolase, is widely found in bodily fluids such as saliva, tears, plasma, and breast milk, as well as tissues like the liver and kidneys in humans and animals. It is also abundant in avian egg whites. Lysozyme specifically acts on the β-1,4 glycosidic bonds between N-acetylglucosamine and N-acetycarbamic acid in the peptidoglycan backbone of bacterial cell walls, disrupting the bacterial cell wall and leading to bacterial lysis and death. It can also kill pathogens by indirectly stimulating macrophages. Based on differences in amino acid sequence characteristics, chemical properties, and biological activity, lysozymes can be classified into six types: type C, type G, type I, plant lysozyme, microbial lysozyme, and bacteriophage lysozyme. Studies have found that both C-type and G-type lysozymes are commonly found in humans and vertebrates. For example, donkeys contain two G-type lysozymes and six C-type lysozymes, and both C-type and G-type lysozymes have also been identified in the Chinese giant salamander, a species of amphibian with a caudal order. These findings are significant for understanding the application of lysozymes in amphibian immunity and disease defense. As a naturally occurring immune substance, lysozyme exhibits various biological functions, including antibacterial, anti-inflammatory, antiviral, immunomodulatory, and digestive effects. Lysozyme can also be molecularly modified through physicochemical and biological enhancement techniques to further expand and enhance its biological functions. As our understanding of the structure, function, and mechanism of action of lysozyme grows, its applications will continue to expand, attracting increasing attention from researchers.

[0003] The Northeast Forest Frog (Rana dybowskii) is a dominant amphibian species in Northeast China with high economic value. The oviducts of its females can be used to prepare the valuable traditional Chinese medicine, forest frog oil, which is widely used in my country's traditional medicine and health product industries. The industrial demand for the Northeast Forest Frog has been steadily increasing, playing an important role in the economic development of forest areas. However, with the development of artificial breeding of the Northeast Forest Frog, high stocking densities and water pollution have made the frogs susceptible to various infectious diseases. The overuse of antibiotics has severely impacted the survival of the Northeast Forest Frog population and caused environmental pollution. In the breeding process, the use of lysozyme has become an important antibacterial agent and feed additive in the context of "antibiotic reduction and replacement." Currently, lysozyme derived from poultry is commonly used as a feed additive to improve the production performance of the Northeast Forest Frog. However, due to the differences between poultry and amphibian Northeast Forest Frog species, their living environments, and the differences in lysozyme sequences, the inhibitory effects of lysozyme on pathogens vary significantly. Therefore, it is necessary to develop effective lysozyme-related gene products derived from the Northeast Forest Frog itself to meet its disease resistance needs. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides an application of a lysozyme gene from the Northeast Forest Frog, a mature polypeptide region gene, a recombinant protein, and a genetically engineered strain of the recombinant lysozyme. The lysozyme provided by this invention exhibits antibacterial activity against Aeromonas hydrophila, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus, particularly showing a very significant antibacterial effect against Aeromonas hydrophila derived from amphibians. Compared to commercially available avian egg white lysozyme with an activity of 20,000 U / mg, the lysozyme provided by this invention has a higher antibacterial effect against Aeromonas hydrophila.

[0005] The first aspect of the present invention provides a lysozyme gene from the Northeast Forest Frog, the gene sequence of which is shown in SEQ ID NO.1.

[0006] The second aspect of this invention provides a method for cloning the lysozyme gene of the Northeast Forest Frog described above, the specific steps of which are as follows:

[0007] RNA was extracted from tissues of the Northeast Forest Frog;

[0008] After the RNA is denatured, it is reverse transcribed to synthesize the first strand of cDNA;

[0009] The obtained cDNA was amplified by PCR using primers with the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification products;

[0010] The amplified product was ligated to a vector and introduced into the expression host cell. After screening and sequencing, the lysozyme gene of the Northeast Forest Frog was obtained.

[0011] Furthermore, the nucleotide sequences of SEQ ID NO.2 and SEQ ID NO.3 are as follows:

[0012] SEQ ID NO.2: 5'-CTCCATTACATTTGTTGCCATA-3',

[0013] SEQ ID NO. 3: 5'-ACTGTAACTGAATCCTTGCGAG-3'.

[0014] Further, the PCR amplification reaction system is as follows: 1-2 μL cDNA, 0.2-0.4 μL primers of the sequence shown in SEQ ID NO.2, 0.2-0.4 μL primers of the sequence shown in SEQ ID NO.3, 0.8 μL dNTP, 0.2 μL Taq enzyme, 1 μL 10×PCRBuffer, and ddH2O to a final volume of 10 μL.

[0015] Preferably, the PCR amplification reaction system is as follows: 1 μL cDNA, 0.2 μL primers of the sequence shown in SEQ ID NO.2, 0.2 μL primers of the sequence shown in SEQ ID NO.3, 0.8 μL dNTP, 0.2 μL Taq enzyme, 1 μL 10×PCRBuffer, and ddH2O to a final volume of 10 μL.

[0016] Further, the PCR amplification reaction conditions are: (1) 90-95℃ for 5 min; (2) 94℃ for 30 s, 52-54℃ for 30 s, 72℃ for 30 s, 30-35 cycles; (3) 72℃ for 10 min.

[0017] Preferably, the PCR amplification reaction conditions are: (1) 95℃ for 5 min; (2) 94℃ for 30 s, 52.3℃ for 30 s, 72℃ for 30 s, for 30 cycles; (3) 72℃ for 10 min.

[0018] The third aspect of the present invention provides a mature polypeptide region gene of lysozyme from the Northeast Forest Frog, wherein the mature polypeptide region gene of lysozyme from the Northeast Forest Frog is obtained by removing the signal peptide sequence from the lysozyme gene described above, and the sequence of the mature polypeptide region gene of lysozyme from the Northeast Forest Frog is shown in SEQ ID NO.4.

[0019] The fourth aspect of the present invention provides a recombinant protein encoded by the mature polypeptide region gene of the lysozyme of the Northeast Forest Frog described above, the amino acid sequence of which is shown in SEQ ID NO.6.

[0020] The fifth aspect of this invention provides a method for preparing the recombinant protein described above, the specific steps of which are as follows:

[0021] EcoR I and Xho I restriction sites were added to both ends of the mature polypeptide region gene of the Northeast Forest Frog lysozyme, and then linked to the vector pET28a to construct the recombinant plasmid LYZ-pET28a.

[0022] The recombinant plasmid LYZ-pET28a was introduced into expression host cells to induce protein expression, and the protein was purified to obtain the recombinant protein.

[0023] Furthermore, the expression host cell is Escherichia coli.

[0024] Furthermore, the expression host cell is Escherichia coli BL21(DE3) competent cells.

[0025] The sixth aspect of the present invention provides a recombinant lysozyme genetically engineered bacterium, wherein the recombinant lysozyme genetically engineered bacterium can express the recombinant protein described above.

[0026] The seventh aspect of this invention provides the application of the above-described Northeast Forest Frog lysozyme gene, Northeast Forest Frog lysozyme mature polypeptide region gene, recombinant protein, or recombinant lysozyme genetically engineered bacteria in the preparation of antibacterial products.

[0027] Furthermore, the antibacterial activity is the inhibition of any one or more of Aeromonas hydrophila, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus.

[0028] In summary, compared with the prior art, the present invention has the following advantages and effects:

[0029] (1) The lysozyme gene from the Northeast Forest Frog provided by this invention encodes a lysozyme from the Northeast Forest Frog. This lysozyme is a type C lysozyme with high antibacterial activity, exhibiting inhibitory effects against Micrococcus luteus, Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus, especially against Aeromonas hydrophila derived from amphibians. Compared with commercially available avian egg white lysozyme with an activity of 20,000 U / mg, the lysozyme provided by this invention has a higher antibacterial effect against Aeromonas hydrophila.

[0030] (2) This invention, starting from the perspective of the Northeast Forest Frog itself, explores and analyzes the types of amphibian lysozymes, obtaining a Northeast Forest Frog lysozyme gene, a mature polypeptide region gene of Northeast Forest Frog lysozyme, recombinant protein, and recombinant lysozyme genetically engineered bacteria. This provides a novel, efficient, non-toxic, pollution-free, and stable antibacterial product for Northeast Forest Frog farming, changing the current situation of antibiotic and preservative abuse. It also has significant value for the development and utilization of amphibian lysozymes and type C lysozymes, and provides a reference for lysozyme research in other species. Attached Figure Description

[0031] Figure 1Electrophoresis diagrams for RNA extraction from skin tissue of the Northeast Forest Frog; lane 1 shows the electrophoresis results of RNA from an abdominal skin sample of the Northeast Forest Frog, and lane 2 shows the electrophoresis results of RNA from a leg skin sample of the Northeast Forest Frog.

[0032] Figure 2 The image shows the PCR electrophoresis results after the amplification of the target gene; lane M is DNAMarker2000, and lane 1 is the location of the C-type lysozyme gene of the Northeast Forest Frog.

[0033] Figure 3 This is the plasmid map of the recombinant plasmid LYZ-pET28a.

[0034] Figure 4 This is a PCR electrophoresis result of recombinant plasmid LYZ-pET28a; the left lane is DNAMarker5000, and the red box on the right lane indicates the location of the PCR product of recombinant plasmid LYZ-pET28a.

[0035] Figure 5 This is an electrophoresis result of the target fragment digested with EcoRI / XhoI; the left lane shows the electrophoresis result of the digested product, and the red box indicates the position of the electrophoretic band of the digested product. The right lane is for DNA Marker5000.

[0036] Figure 6 The images show the nucleotide and amino acid sequencing peaks of the mature polypeptide region of lysozyme from the Northeast Forest Frog. Image B is a supplement to image A, and image C is a supplement to image B.

[0037] Figure 7 This figure shows the expression of recombinant lysozyme protein from the Northeast Forest Frog. Lane M represents the protein marker. Lanes 1-3 show the protein expression results after 2h, 3h, and 4h without IPTG induction, respectively. Lanes 4-6 show the protein expression results after 2h, 3h, and 4h with 0.1 mmol / L IPTG induction, respectively. Lanes 7-9 show the protein expression results after 2h, 3h, and 4h with 0.5 mmol / L IPTG induction, respectively. Lanes 10-12 show the protein expression results after 2h, 3h, and 4h with 1 mmol / L IPTG induction, respectively.

[0038] Figure 8 Electrophoresis image confirming inclusion bodies of recombinant lysozyme protein from the Northeast Forest Frog; lane M is the protein marker, lane 1 is the cultured bacterial solution, lane 2 is the supernatant of the bacterial solution after ultrasonic disruption and centrifugation, and lane 3 is the precipitate of the bacterial solution after ultrasonic disruption and centrifugation.

[0039] Figure 9The results show the gradient elution of the target protein; lane M is the protein marker, lane 1 is the elution result of the target protein eluted with Buffer C, and lanes 2-6 are the electrophoresis results of the target protein eluted with Buffer C containing 100 mmol / L, 200 mmol / L, 300 mmol / L, 400 mmol / L, and 500 mmol / L imidazole, respectively.

[0040] Figure 10 This is an electrophoresis image of recombinant protein after dialysis refolding; lane M is the protein marker, and lane 1 is the electrophoresis result of the recombinant protein sample after dialysis refolding.

[0041] Figure 11 The results of Western blotting for detecting the expression of His-tagged whole bacterial recombinant proteins are shown below. Lane 1 sample is whole bacterial cells diluted 30 times, lane 2 sample is whole bacterial cells diluted 10 times, lane 3 sample is whole bacterial cells diluted 3 times, and lane 4 sample is whole bacterial stock solution.

[0042] Figure 12 The results of Western blotting for the purification of recombinant protein containing the His tag were obtained; the samples in lanes 1-4 contained 10 ng, 30 ng, 100 ng and 300 ng of recombinant protein, respectively.

[0043] Figure 13 Figure A shows the electrophoresis results of bovine serum albumin (BSA) standards, where the concentrations of BSA standards in each lane from right to left are 0 μg / mL, 10 μg / mL, 30 μg / mL, 50 μg / mL, 70 μg / mL, 90 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL, 500 μg / mL, 600 μg / mL, 700 μg / mL, 800 μg / mL, 900 μg / mL, 1000 μg / mL, 1500 μg / mL, 2000 μg / mL, 3000 μg / mL, and 4000 μg / mL. Figure B shows the protein concentration standard curve plotted using the BCA method, with protein concentration on the x-axis and absorbance on the y-axis.

[0044] Figure 14 This is a mass spectrometry image of the partial amino acid sequence of the recombinant protein (DPNGMKAWVGWR).

[0045] Figure 15 This is a mass spectrometry image of the partial amino acid sequence (AWVGWR) of the recombinant protein.

[0046] Figure 16The graph shows the antibacterial activity of lysozyme from the Northeast Forest Frog. Figures A1, B1, C1, and D1 represent the negative control groups inoculated with Aeromonas hydrophila, Micrococcus luteus, Escherichia coli, and Staphylococcus aureus, respectively, and supplemented with PBS. Figures A2, B2, C2, and D2 represent the experimental groups inoculated with Aeromonas hydrophila, Micrococcus luteus, Escherichia coli, and Staphylococcus aureus, respectively, and supplemented with lysozyme from the Northeast Forest Frog. Figures A3, B3, C3, and D3 represent the positive control groups inoculated with Aeromonas hydrophila, Micrococcus luteus, Escherichia coli, and Staphylococcus aureus, respectively, and supplemented with avian egg white lysozyme.

[0047] Figure 17 Figure A shows the antibacterial effect of lysozyme; Figure B shows the antibacterial ability of different concentrations of lysozyme from the Northeast Forest Frog and Figure C shows the antibacterial ability of different concentrations of avian egg white lysozyme.

[0048] Figure 18 Electron micrographs of the morphological changes of Aeromonas hydrophila cells after treatment with lysozyme; Figure A is the negative control group (1 mL Aeromonas hydrophila culture + 100 μL phosphate buffer), Figure B is the positive control group (1 mL Aeromonas hydrophila culture + 100 μL 50 μg / mL avian egg white lysozyme), and Figures C-F are the experimental groups with 100 μL of 1000 μg / mL, 250 μg / mL, 500 μg / mL, and 5 μg / mL Rhizopus spp. 100 μL of 1000 μg / mL, 250 μg / mL, 500 μg / mL, and 5 μg / mL Rhizopus spp. The images on the same horizontal row, from left to right, show magnifications at 5 μm, 2 μm, 1 μm, and 500 nm scales; red arrows indicate bacterial morphological changes; yellow arrows indicate the action state of Rhizopus spp. ... Detailed Implementation

[0049] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0050] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0051] Artificially raised frog populations are susceptible to bacterial pathogens, and conventional treatment involves using antibiotics to suppress infection. However, the overuse of antibiotics has severely impacted frog populations' survival and caused environmental pollution. Therefore, a highly effective, safe, and environmentally friendly antimicrobial agent is needed. Lysozyme is an excellent natural antibacterial agent. Currently, egg white-derived lysozyme is used as a feed additive in animal production to improve animal performance. However, in frog farming, because egg white lysozyme originates from poultry, and poultry and the Northeast Forest Frog (a type of amphibian) are different species with different living environments, their lysozyme sequences differ, leading to different inhibitory effects on pathogens. The current practical application of lysozyme is still quite insufficient. In production practice, the Northeast Forest Frog requires effective lysozyme-related gene products derived from its own organism to meet its disease resistance needs.

[0052] This invention first studies the lysozyme gene of the Northeast Forest Frog (Frog melilotus), clones and identifies the species of the lysozyme gene, and obtains a lysozyme gene with the sequence shown in SEQ ID NO.1. This lysozyme gene is then modified to obtain the mature polypeptide region gene of the Northeast Forest Frog lysozyme. A recombinant lysozyme genetically engineered bacterium expressing recombinant lysozyme protein from the Northeast Forest Frog is then constructed using this mature polypeptide region gene. Finally, the inhibitory effects of the Northeast Forest Frog lysozyme on *Micrococcus luteus*, *Staphylococcus aureus*, *Aeromonas hydrophila* (amphibian-derived), and *Escherichia coli* are verified, and the destructive effect of the Northeast Forest Frog lysozyme on *Aeromonas hydrophila* is observed using electron microscopy.

[0053] Example 1: Cloning and Sequencing of Lysozyme from the Northeast Forest Frog

[0054] 1. Extraction and quality testing of total RNA from the skin of the Northeast Forest Frog.

[0055] RNA was extracted from the skin of male Northeast Forest Frog (purchased from Xingwang Breeding Farm, Songbei District, Harbin City, Heilongjiang Province) according to the instructions of the Trizol Total RNA Extraction Kit (purchased from Invitrogen). All EP tubes, tips, mortars, tweezers and other materials used in the experiment were soaked in 1‰ DEPC water beforehand, sterilized and dried for later use.

[0056] (1) Put an appropriate amount of liquid nitrogen into the mortar, place about 0.1g of abdominal and leg skin tissue into the liquid nitrogen and grind it quickly and thoroughly. Then transfer it to an EP tube containing 750μL Trizol and mix well. Centrifuge at 12000rpm for 5min at 4℃.

[0057] (2) The supernatant was aspirated into a new EP tube containing 150 μL of chloroform, shaken thoroughly, placed in an ice bath for 10 min, centrifuged at 12000 rpm for 15 min at 4 °C, and then the upper aqueous phase was transferred to a new EP tube.

[0058] (3) Add an equal volume of isopropanol to the EP tube, mix by inverting, let stand on ice for 10 min, centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant and keep the precipitate.

[0059] (4) Add an appropriate amount of 75% ethanol to the EP tube with white RNA precipitate at the bottom, aspirate repeatedly until the precipitate dissolves, centrifuge at 12000 rpm for 5 min at 4℃, and discard the supernatant.

[0060] (5) Open the EP tube containing RNA precipitate and let it stand at room temperature for a few minutes until the ethanol has fully evaporated. Then add 20 μL of RNase-free ddH2O to dissolve the RNA sample.

[0061] (6) Take 4 μL of the obtained RNA sample for mass and concentration detection;

[0062] (7) The total RNA extracted was qualitatively detected by horizontal agarose gel electrophoresis (1%), and the concentration of total RNA was quantitatively determined by ultraviolet spectrophotometer. The qualified RNA samples were frozen and stored at -80℃ for later use.

[0063] Electrophoresis results as follows Figure 1 As shown, the lane contains complete and clear 28S, 18S and 5S bands, which meets the requirements for the next step of reverse transcription.

[0064] 2. RT-PCR amplification of the lysozyme gene from the Northeast Forest Frog

[0065] (1) Reverse transcription to synthesize the first strand of cDNA

[0066] use The RNA reverse transcription reaction was performed using the 1st Strand cDNA Synthesis Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.), with a reaction volume of 20 μL.

[0067] First, add the reaction system shown in Table 1 to a pre-cooled centrifuge tube, heat at 65°C for 5 min, and then let stand on ice for 2 min to complete the denaturation of the RNA template.

[0068] Table 1. Denaturation reaction system for RNA template

[0069] Element content Oligo(dT)23VN(50μM) 1μL RNA 1μg <![CDATA[RNase-freeddH2O]]> 6μL

[0070] Synthesis of cDNA first strand: Add the reaction system shown in Table 2 to a centrifuge tube, heat at 50°C for 5 min, then heat at 85°C for 2 min. After the reaction is complete, freeze the product at -20°C.

[0071] Table 2. Reaction system for cDNA first-strand synthesis

[0072]

[0073]

[0074] (2) PCR amplification of the target gene

[0075] Primer design: Sequence alignment with publicly available C-type lysozyme sequences from humans, mammals, fish, and African clawed frogs on NCBI was performed to select conserved regions. Primer pairs were designed within these conserved regions using PrimerPremier 5.0 software. The sequences of the upstream and downstream primers are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0076] SEQ ID NO.2: 5'-CTCCATTACATTTGTTGCCATA-3',

[0077] SEQ ID NO. 3: 5'-ACTGTAACTGAATCCTTGCGAG-3'.

[0078] PCR amplification of the lysozyme gene was carried out in a 10 μL system. The specific reaction system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0079] Table 3 PCR amplification reaction system for the target gene

[0080] Element content <![CDATA[ddH2O]]> 6.6μL 10×PCRBuffer 1μL dNTP 0.8μL upstream primer 0.2μL Downstream primer 0.2μL cDNA 1μL Taq enzyme 0.2μL

[0081] Table 4. PCR amplification reaction conditions for the target gene

[0082]

[0083]

[0084] Note: "-" indicates that the corresponding step is not looped.

[0085] The PCR amplification products were detected by 1% agarose gel electrophoresis, and then the size of the target band was observed and images were acquired using a UV gel imaging system.

[0086] from Figure 2 As can be seen, the target gene has a clear band at 459bp, which is consistent with the expected result.

[0087] 3. Recovery, ligation, transformation, and identification of PCR amplified fragments

[0088] (1) Target fragment retrieval

[0089] The target band was recovered according to the instructions of the gel recovery kit DC301-01 (purchased from Nanjing Novizan Biotechnology Co., Ltd.), and the DNA sample was obtained and stored at -20℃.

[0090] (2) Ligation of the target fragment with the pMD18-T vector

[0091] Add the ligation system shown in Table 5 to the PCR tube, mix well, and incubate at 16℃ for 16 hours to obtain the ligation product of the target fragment and the pMD18-T vector (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0092] Table 5 Connection System

[0093] Element content PCR product of lysozyme gene 4μL pMD18-T 1μL solutionI 5μL

[0094] (3) Transformation of recombinant plasmids

[0095] The obtained ligation product was transformed into Escherichia coli DH5α competent cells (purchased from Nanjing Novizan Biotechnology Co., Ltd.), and the operation steps are as follows:

[0096] ① Mix the ligation product with Escherichia coli DH5α competent cells at a ratio of 1:10, gently mix, and incubate on ice for 30 min; ② Heat shock at 42℃ for 90 s, then immediately incubate on ice for 5 min; ③ Add 0.5 mL of LB liquid medium and incubate at 180 rpm on a shaker at 37℃ for 1 h; ④ Centrifuge the bacterial suspension at 2000 rpm for 2 min, discard 400 μL of supernatant, and resuspend the bacterial cells at the bottom of the tube; ⑤ Spread the resuspended bacterial suspension evenly and gently on the surface of an LB agar plate containing 100 μg / mL Amp and incubate at 37℃ for 12 h.

[0097] (4) Colony PCR identification of recombinant plasmids

[0098] White single colonies were randomly selected and placed in 20 μL of LB liquid medium containing 100 μg / mL Amp. The medium was then placed in a constant temperature shaker at 37℃ and 180 rpm for 4 h. The cultured bacterial solution was used as a template for PCR amplification. Positive clones were identified according to the PCR reaction system shown in Table 6 and the PCR reaction conditions shown in Table 4.

[0099] Table 6. Colony PCR amplification reaction system for recombinant plasmids

[0100] Element content <![CDATA[ddH2O]]> 7.5μL 10×PCRBuffer 1μL dNTPMixture 0.8μL upstream primer 0.2μL Downstream primer 0.2μL bacterial solution 0.1μL Taq enzyme 0.2μL

[0101] The reaction products were detected by 1% agarose gel electrophoresis, and the cloned bacterial solutions that were initially identified as positive were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing.

[0102] 4. Sequence determination and analysis of lysozyme from the Northeast Forest Frog

[0103] Sequencing revealed that the nucleotide sequence of the coding region of the lysozyme gene from the Northeast Forest Frog is shown in SEQ ID NO.1, with a full length of 459 bp. The amino acid sequence is shown in SEQ ID NO.5. By comparing the obtained nucleotide and amino acid sequences with those of other reference species such as the European Forest Frog, the Alpine Frog, and humans published on NCBI, and constructing a phylogenetic tree of amino acids using MEGA software, it was found that the lysozyme gene from the Northeast Forest Frog clustered with the C-type lysozyme genes of the reference species within the same group. Therefore, the lysozyme from the Northeast Forest Frog provided in this invention belongs to the C-type lysozyme group.

[0104] SEQ ID NO.1:

[0105] ATGAGGTCTCTGATGCAAATCATGGGAGTCCTTTTCCTAACAATTGCATTAGTCGATGCAAAACAGTATGAGAGATGTGAACTTGCAAAAGCCATGAAAATTCTGGGTCTGGATGGATATCAAGGATACAGCCTGCCAAACTGGGTTTGCACAGCTTTCTACGAGAGTTCCTTTAACACATACAGCATAAACTTCAACCCTGGAGACAGCAGCACTGATTACGGTATTCT TCAAATAAACAGCCATTGGTGGTGTAATGATTACAAAACTCCGAGAGCCCACAATGCTTGCAACATTGATTGCTCAGACCTCCTATCCAATGACATCTCATTATCTGTGGAATGCGCAAAACGTGTTGTGAGGGATCCCAATGGTATGAAGGCATGGGTTGGATGGAGAAACCATTGCAAAGGAAAAGATTTGTCTGCATGGATTCAAGGCTGCGGGCTTGACTTTTAA;

[0106] SEQ ID NO.5:

[0107] MRSLMQIMGVLFLTIALVDAKQYERCELAKAMKILGLDGYQGYSLPNWVCTAFYESSFNTYSINFNPGDSSTDYGILQINSHWWCNDYKTPRAHNACNIDCSDLLSNDISLSVECAKRVVRDPNGMKAWVGWRNHCKGKDLSAWIQGCGLDF, * indicates the stop codon.

[0108] The amino acid sequence of the obtained lysozyme from the Northeast Forest Frog was analyzed. The signal peptide was removed, and the mature polypeptide region was retained. Gene synthesis of the mature polypeptide region was performed without codon optimization.

[0109] The nucleotide sequence of the mature polypeptide region of the Northeast Forest Frog lysozyme is shown in SEQ ID NO.4. The full-length gene sequence is 399 bp (stop codon retained), encoding 132 amino acids (*stop codon not included).

[0110] SEQ ID NO.4:

[0111] AAACAGTATGAGAGATGTGAACTTGCAAAAGCCATGAAAATTCTGGGTCTGGATGGATATCAAGGATACAGCCTGCCAAACTGGGTTTGCACAGCTTTCTACGAGAGTTCCTTTAACACATACAGCATAAACTTCAACCCTGGAGACAGCAGCACTGATTACGGTATTCTTCAAATAAACAGCCATTGGTGGTGTAATGA TTACAAAACTCCGAGAGCCCACAATGCTTGCAACATTGATTGCTCAGACCTCCTATCCAATGACATCTCATTATCTGTGGAATGCGCAAAACGTGTTGTGAGGGATCCCAATGGTATGAAGGCATGGGTTGGATGGAGAAACCATTGCAAAGGAAAAGATTTGTCTGCATGGATTCAAGGCTGCGGGCTTGACTTTTAA;

[0112] The mature polypeptide region of the lysozyme from the Northeast Forest Frog has a molecular weight of 15 kDa, and its amino acid sequence is shown in SEQ ID NO. 6. SEQ ID NO. 6:

[0113] KQYERCELAKAMKILGLDGYQGYSLPNWVCTAFYESSFNTYSINFNPG DSSTDYGILQINSHWWCNDYKTPRAHNACNIDCSDLLSNDISLSVECAKRVV RDPNGMKAWVGWRNHCKGKDLSAWIQGCGLDF, * indicates the stop codon.

[0114] Example 2: Construction of a prokaryotic expression vector for the lysozyme gene of the Northeast Forest Frog

[0115] 1. Construction of recombinant plasmids

[0116] For recombinant expression, the present invention carries an amino acid segment at the N-terminus of the constructed recombinant plasmid. This amino acid segment carries a 6-histidine tag, and the amino acid sequence is shown in SEQ ID NO.7. The final recombinant expression vector has a protein molecular weight of approximately 17 kDa.

[0117] SEQ ID NO.7: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF. EcoRI and XhoI restriction sites were added to both ends of the gene shown in SEQ ID NO.4, and then ligated to the vector pET28a (Nanjing Novizan Biotechnology Co., Ltd.) to construct the recombinant plasmid LYZ-pET28a. The plasmid map is shown below. Figure 3 As shown.

[0118] 2. Identification of the target fragment

[0119] The recombinant plasmid LYZ-pET28a was detected by PCR using universal T7 primers (the upstream primer nucleotide sequence is shown in SEQ ID NO. 8, and the downstream primer nucleotide sequence is shown in SEQ ID NO. 9). The PCR product size should ideally be 690 bp, consistent with the band position in the electrophoresis results. Figure 4 This is in line with expectations;

[0120] SEQ ID NO.8: 5'-TAATACGACTCACTATAGGG-3',

[0121] SEQ ID NO. 9: 5'-TGCTAGTTATTGCTCAGCGG-3'.

[0122] 3. Double digestion of the target fragment

[0123] The obtained PCR product solution was recovered and digested with EcoRI and XhoI. Figure 5 The electrophoresis image clearly shows a band of approximately 690 bp in size, and the size of the enzyme digestion product fragment is consistent with the expected result.

[0124] 4. Sequencing Results: The recombinant plasmid LYZ-pET28a was sent for sequencing, and the results are as follows: Figure 6 As shown in the sequencing map, the nucleotide sequence is identical to that of the mature polypeptide region gene of the Northeast Forest Frog lysozyme (SEQ ID NO.4), and the amino acid sequence is identical to that of SEQ ID NO.6. The recombinant sequence is as expected, indicating that the recombinant plasmid LYZ-pET28a was successfully constructed.

[0125] 5. Induced expression of recombinant C-type lysozyme from the Northeast Forest Frog

[0126] (1) The recombinant plasmid LYZ-pET28a was transformed into competent Escherichia coli BL21(DE3) cells (purchased from Nanjing Novizan Biotechnology Co., Ltd.) and inoculated into LB medium with a kanamycin concentration of 50 μg / mL. The cells were cultured overnight at 37℃ and 220 r / min with shaking to obtain activated bacterial solution.

[0127] (2) The activated bacterial culture was transferred to LB medium with a kanamycin concentration of 50 μg / mL and cultured at 37℃ and 220 r / min until the bacterial culture reached OD. 600 When the value was 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to make the final concentrations of the solution 0 mmol / L, 0.1 mmol / L, 0.5 mmol / L and 1 mmol / L, respectively. The solutions were induced and cultured at 37℃ and 220 rpm for 2 h, 3 h and 4 h, respectively. The bacterial solutions were collected, and after centrifugation and removal of the supernatant, the bacterial precipitates obtained were the recombinant lysozyme genetically engineered bacteria. Samples were taken for electrophoretic analysis.

[0128] from Figure 7 The electrophoresis results clearly show that lanes 1 to 12 can express the recombinant lysozyme protein of the Northeast Forest Frog. The recombinant lysozyme protein of the Northeast Forest Frog is labeled as a recombinant protein with a molecular weight of approximately 17 kDa. The recombinant protein can also be expressed without the addition of IPTG.

[0129] (3) Resuspend the uninduced IPTG-treated bacterial cells in 20 mL of inclusion body cell lysis buffer (purchased from Beyotime Biotechnology Co., Ltd.), lyse the cells by sonication, centrifuge at 10,000 rpm, 4°C for 15 min, separate the supernatant and precipitate, and perform protein electrophoresis. Figure 8 The results show that there are no recombinant proteins in the supernatant. The recombinant proteins are mainly found in inclusion bodies and need to be released after the bacterial cells are broken down.

[0130] (4) Add 10 mL of inclusion body cell lysis buffer to resuspend the precipitate in step (3), let stand overnight at room temperature, centrifuge at 17000 rpm for 15 min, collect the supernatant, and then perform preliminary purification of the recombinant protein.

[0131] Example 3: Purification and refolding of recombinant protein

[0132] 1. Recombinant protein purification

[0133] The recombinant protein was purified using a nickel column affinity purification method and a His-tagged protein purification kit (purchased from Beyotime Biotechnology Co., Ltd.). The specific steps are as follows:

[0134] (1) Column equilibration: Equilibrate the column with 20 mL of Buffer A at an equilibration flow rate of 1.5 mL / min;

[0135] (2) Sample loading: The sample loading flow rate is 0.5 mL / min;

[0136] (3) Washing off contaminants: Wash off contaminants with 40 mL of Buffer B at a flow rate of 2 mL / min;

[0137] (4) Elution of recombinant protein: Column 1 was eluted with Buffer C, and column 2 was eluted with different concentrations of imidazole Buffer C. 20 μL of 2× loading buffer was added to 20 μL of elution buffer containing recombinant protein to obtain liquid samples of recombinant protein with gradient concentrations, and electrophoresis experiments were performed.

[0138] Protein electrophoresis results as follows Figure 9 As shown, the samples loaded into lanes 1, 3, and 4 contained purified recombinant protein.

[0139] 2. Recombinant protein dialysis refolding

[0140] Add 0.01% SKL (sodium dodecyl sarcosinate) to 1×PBS solution and adjust the pH to 7.4 to obtain the refolding buffer.

[0141] Will Figure 9 The eluted proteins loaded into lanes 1, 3, and 4 were dialyzed in refolding buffer, and the resulting solution contained the active recombinant protein, which was then subjected to protein electrophoresis.

[0142] Electrophoresis results as follows Figure 10 As shown, the molecular weight of the recombinant protein after dialysis refolding is approximately 17 kDa, which is consistent with expectations.

[0143] 3. Recombinant protein electrophoresis detection

[0144] The recombinant protein was identified using Western blotting (WB). Since the expressed recombinant protein contains a 6-histidine tag (6×His-tag), a rabbit anti-His-tag antibody (purchased from Beijing Solarbio Science & Technology Co., Ltd.) was used to detect the recombinant protein. The specific detection method is as follows:

[0145] First, Western blot analysis of whole-cell recombinant proteins was performed. The original bacterial solution and bacterial solutions diluted 3, 10, and 30 times were subjected to 10% SDS-PAGE electrophoresis. The bands were then transferred to a nitrocellulose membrane, blocked with 5% skim milk powder at room temperature for 2 hours, followed by incubation with rabbit anti-His tag antibody diluted 500 times at 4°C overnight. The membrane was then washed with mouse anti-rabbit IgG diluted 1000 times at room temperature for 1 hour, and the results were observed after development with ECL chemiluminescence solution and photographed.

[0146] The results are as follows Figure 11 As shown, clear bands can be seen at approximately 17 kDa in all four lanes. This recombinant protein may also form multimers or undergo post-translational modifications, so some bands may also appear in other locations.

[0147] The recombinant protein solution after refolding and purification was then identified by Western blotting. After identification, the content of recombinant protein was calculated using ImageJ software. The expression level was calculated according to the formula: relative protein content = target protein optical density value / color standard optical density value.

[0148] like Figure 12 As shown, the refolded and purified recombinant protein can bind to the rabbit anti-His tag antibody, indicating that a certain amount of recombinant protein was obtained.

[0149] 4. Determination of recombinant protein concentration

[0150] Establishment of a method using bovine serum albumin (BSA) standard via the BCA method, as follows: Figure 13 The protein concentration standard curve shown is expressed as Y = 2E-05X. 2 +0.1715X+1.7615, where X represents the absorbance value and Y represents the BSA concentration. Then, 1 μL of BSA and 1 μL of purified recombinant protein solution were subjected to protein electrophoresis, and grayscale analysis was performed using ImageJ software. Based on the protein concentration standard curve, the concentration of recombinant protein was calculated to be 1 mg / mL.

[0151] 5. Mass spectrometry detection of recombinant proteins: Randomly capture two amino acid sequences, such as... Figure 14 and Figure 15 As shown, the partial amino acid sequences of the captured recombinant protein, DPNGMKAWVGWR (SEQ ID NO.10) and AWVGWR (SEQ ID NO.11), are consistent with the amino acid sequence of the lysozyme of the Northeast Forest Frog, indicating that the recombinant protein expressed by the prokaryotic expression vector is the lysozyme of the Northeast Forest Frog.

[0152] Example 4: Application of lysozyme from Northeast Forest Frog

[0153] In this embodiment, the information for each bacterial species is as follows:

[0154] Micrococcus luteus was purchased from Beijing Biotechnology Co., Ltd., product number CMCC(B)28001;

[0155] For information on Aeromonas hydrophila strains, please refer to “Shao Jie, Chai Longhui, Wang Boju, et al. Identification and pathogenicity of an Aeromonas hydrophila strain from Northeast Forest Frog [J]. Acta Wildlife Sinica, 2018, 39(2):390-394.”;

[0156] Staphylococcus aureus was purchased from Shanghai Yaji Biotechnology Co., Ltd., product number CMCC(B)26003;

[0157] Escherichia coli was purchased from Shanghai Beinuo Biotechnology Co., Ltd., product number CMCC(B)44102.

[0158] 1. Plate antibacterial test

[0159] To detect the antibacterial activity of lysozyme from the Northeast Forest Frog, this invention specifically tested its antibacterial effects against *Micrococcus luteus*, *Aeromonas hydrophila*, *Staphylococcus aureus*, and *Escherichia coli*. *Micrococcus luteus* and *Staphylococcus aureus* are Gram-positive bacteria, and the culture temperature was 28℃. *Aeromonas hydrophila* and *Escherichia coli* are Gram-negative bacteria, and the culture temperature was 37℃. The specific experimental steps are as follows:

[0160] (1) The above four types of bacteria were first inoculated onto LB solid agar plates for streaking and activation culture. Then, a single colony was picked and inoculated into liquid LB medium for overnight culture. Then, 100 μL of bacterial culture was transferred into 100 mL of liquid LB medium and cultured until OD500. 600 The value is 0.6 for backup.

[0161] (2) The recombinant lysozyme protein from the Northeast Forest Frog was sterilized by filtration through a 0.22 μm microporous membrane. 10 μL of the recombinant protein was added to 1 mL of the above four bacterial cultures, and after culturing for 30 min, 100 μL was plated. The negative control group was treated with 10 μL of sterile PBS buffer. The experimental results are as follows: Figure 16 As shown.

[0162] (3) From Figure 16As can be seen, A1, B1, C1, and D1 are negative control groups, without the addition of lysozyme, and bacteria can grow normally. A2, B2, C2, and D2 are experimental groups with the addition of the lysozyme from the Northeast Forest Frog of this invention. It can be found that no bacteria grow in the A2 plate, a small amount of Micrococcus luteus grows in the B2 plate, and Escherichia coli in the C2 plate and Staphylococcus aureus in the D2 plate are also inhibited to some extent. A3, B3, C3, and D3 are positive control groups with commercially available avian egg white lysozyme (purchased from Nanjing Novizan Biotechnology Co., Ltd.). It was observed that Aeromonas hydrophila could still grow in A3, indicating that the antibacterial effect of avian egg white lysozyme was lower than that of recombinant lysozyme from the Northeast Forest Frog against Aeromonas hydrophila. The avian egg white lysozyme in plate B3 also showed a low inhibitory effect on Micrococcus luteus, while the avian egg white lysozyme in plate C3 showed a low inhibitory effect on Escherichia coli, and the avian egg white lysozyme in plate D3 also showed a low inhibitory effect on Staphylococcus aureus.

[0163] Figure 17 Figure A shows the antibacterial activity of different concentrations of *Rhizoctonia solani* lysozyme, and Figure B shows the antibacterial activity of different concentrations of avian egg white lysozyme. The figures demonstrate that the *Rhizoctonia solani* lysozyme provided by this invention exhibits high antibacterial activity, effectively inhibiting *Aeromonas hydrophila* at a concentration of 10 μg / mL. Furthermore, compared to avian egg white lysozyme, the *Rhizoctonia solani* lysozyme provided by this invention shows a higher inhibitory effect on *Aeromonas hydrophila* in amphibians.

[0164] 2. Scanning electron microscopy observation

[0165] This invention further utilizes scanning electron microscopy to observe the antibacterial effect of lysozyme from the Northeast Forest Frog on Aeromonas hydrophila. The specific steps are as follows:

[0166] (1) Streak Aeromonas hydrophila on LB solid agar plates and incubate at 28°C. After single colonies grow, pick a single colony and add it to LB liquid medium for overnight incubation. Then, transfer the colony to 100 mL of LB liquid medium at a 1% inoculation dose and incubate until OD. 600 The value is 0.5.

[0167] (2) Collect Aeromonas hydrophila cells by centrifuging 1 mL of bacterial solution at 4℃ and 3000 rpm for 10 min. Wash the cells three times with PBS buffer (pH 7.2) and then prepare a bacterial suspension with PBS buffer for later use. Then set up a negative control group, a positive control group and an experimental group. Group A was the negative control group (1 mL Aeromonas hydrophila bacterial solution + 100 μL phosphate buffer), Group B was the positive control group (1 mL Aeromonas hydrophila bacterial solution + 100 μL 50 μg / mL avian egg white lysozyme), and Groups C to F were the experimental groups in which 100 μL of 1000 μg / mL, 250 μg / mL, 500 μg / mL and 5 μg / mL Northeast Forest Frog lysozyme were added to 1 mL Aeromonas hydrophila bacterial solution, respectively.

[0168] (3) After incubating the control group and the experimental group at 28℃ for 8 hours, they were centrifuged again at 4℃ and 3000rpm for 10 minutes. The bacterial cells were washed twice with PBS, and then 2.5% glutaraldehyde solution was added. After fixation at 4℃ overnight, the inhibitory effect of the lysozyme protein of the Northeast Forest Frog on Aeromonas hydrophila was observed using a ZEISS G300 thermal field emission scanning electron microscope.

[0169] like Figure 18 As shown in the figures, in A4, the bacterial cells are morphologically normal and undamaged; in B4, the bacterial cells are basically normal, indicating that the destructive effect of egg white lysozyme is minimal; in C4, the bacterial cells are clearly ruptured, indicating that 1000 μg / mL of Northeast forest frog lysozyme can inhibit Aeromonas hydrophila; in D4, the bacterial cells lyse under the action of 250 μg / mL lysozyme; in E4, small pores appear on the bacterial surface and contents leak out under the action of 50 μg / mL lysozyme; and in F4, the bacterial cells swell under the action of 5 μg / mL lysozyme. This indicates that the Northeast forest frog lysozyme provided by this invention has high antibacterial activity against Aeromonas hydrophila from amphibians, and its antibacterial effect is stronger than that of commercially available avian egg white lysozyme against Aeromonas hydrophila.

Claims

1. A lysozyme gene from the Northeast Forest Frog, characterized in that, The gene sequence is shown in SEQ ID NO.

1.

2. A method for cloning the lysozyme gene of the Northeast Forest Frog as described in claim 1, characterized in that, The specific steps are as follows: RNA was extracted from tissues of the Northeast Forest Frog; RNA reverse transcription to synthesize the first strand of cDNA; The obtained cDNA was amplified by PCR using primers with the sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the amplification products; The amplified product was ligated to a vector and introduced into expression host cells. After screening and sequencing, the lysozyme gene of the Northeast Forest Frog was obtained. SEQ ID NO.2: 5'-CTCCATTACATTTGTTGCCATA-3', SEQ ID NO. 3: 5'-ACTGTAACTGAATCCTTGCGAG-3'.

3. The cloning method for the lysozyme gene of the Northeast Forest Frog according to claim 2, characterized in that, The PCR amplification reaction system is as follows: 1-2 µL cDNA, 0.2-0.4 µL primers of the sequence shown in SEQ ID NO.2, 0.2-0.4 µL primers of the sequence shown in SEQ ID NO.3, 0.8 µL dNTP, 0.2 µL Taq enzyme, 1 µL 10×PCR Buffer, and ddH2O to a final volume of 10 µL.

4. A mature polypeptide region gene of *Rhizophora stylosa* lysozyme obtained by gene modification according to claim 1, characterized in that, The gene sequence of the mature polypeptide region is shown in SEQ ID NO.

4.

5. A recombinant protein encoded by the mature polypeptide region gene of the Northeast Forest Frog lysozyme according to claim 4, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.

6.

6. A method for preparing recombinant protein according to claim 5, characterized in that, The specific steps are as follows: Add to both ends of the mature polypeptide region gene of the lysozyme of the Northeast Forest Frog. EcoR I and Xho The I restriction site was linked to the vector pET28a to construct the recombinant plasmid LYZ-pET28a; The recombinant plasmid LYZ-pET28a was introduced into host cells to induce protein production, and the protein was purified to obtain the recombinant protein.

7. The preparation method according to claim 6, characterized in that, The host cell for expression is Escherichia coli.

8. A recombinant lysozyme-producing genetically engineered bacterium, characterized in that, The recombinant lysozyme-producing engineered bacteria can express the recombinant protein as described in claim 5.

9. The application of the lysozyme gene of the Northeast Forest Frog as described in claim 1, the mature polypeptide region gene of the lysozyme of the Northeast Forest Frog as described in claim 4, the recombinant protein as described in claim 5, or the recombinant lysozyme genetically engineered bacteria as described in claim 8 in the preparation of antibacterial products, characterized in that, The antibacterial effect refers to the inhibition of any one or more of Aeromonas hydrophila, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus.

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