Northeast wood frog lysozyme gene, mature polypeptide region gene, recombinant protein and application of recombinant lysozyme gene engineering bacteria

Abstract

The invention belongs to the technical field of biological gene engineering, and relates to application of a northeast wood frog lysozyme gene, a mature polypeptide region gene, a recombinant protein and a recombinant lysozyme gene engineering bacterium. The invention provides a northeast wood frog lysozyme gene with a sequence as shown in SEQ ID NO.1 and a northeast wood frog lysozyme mature polypeptide region gene with a sequence as shown in SEQ ID NO.4. The mature polypeptide region gene is connected with a carrier and then is transformed into escherichia coli BL21 (DE3) to obtain a recombinant lysozyme genetically engineered bacterium for expressing recombinant protein. And the bacteriostatic ability of the bacillus subtilis to micrococcus luteus, staphylococcus aureus, escherichia coli and aeromonas hydrophila from amphibians is detected. The northeast wood frog lysozyme provided by the invention has bacteriostatic ability to micrococcus luteus, aeromonas hydrophila, escherichia coli and staphylococcus aureus, and can be used for developing antibacterial drugs and feed additives.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and specifically relates to the application of a Northeast forest frog lysozyme gene, a mature polypeptide region gene, a recombinant protein and a recombinant lysozyme gene engineering bacterium. Background Art

[0002] Lysozyme (LYZ), as an N-acetylaminohydrolase, is widely present in human and animal body fluids such as saliva, tears, plasma, and milk, as well as in tissues such as the liver and kidney. It is also very rich in poultry egg white. Lysozyme can specifically act on the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylaminoformic acid in the peptidoglycan polysaccharide skeleton of bacterial cell walls, destroying bacterial cell walls and causing bacterial lysis and death. It can also kill pathogens by indirectly stimulating macrophages. Lysozyme can be divided into 6 types based on the differences in amino acid sequence characteristics, chemical properties, and biological activities, including C-type, G-type, I-type, plant lysozyme, microbial lysozyme, and phage lysozyme. Studies have found that C-type and G-type lysozymes are commonly found in humans and vertebrates. For example, donkeys contain 2 G-type lysozymes and 6 C-type lysozymes. C-type and G-type lysozymes have also been identified in the Chinese giant salamander of the Caudata order of the Amphibian class. These findings are of great significance for understanding the application of lysozymes in the immunity and disease defense of amphibians. Lysozymes, as natural immune substances existing in organisms themselves, exhibit a variety of biological effects in antibacterial and anti-inflammatory, antiviral, immune regulation and digestion. Lysozymes can also be molecularly modified through physical and chemical as well as biological improvement technologies to further expand and enhance their biological functions. As people understand the structure, function and mechanism of action of lysozymes, their scope of application will continue to expand and receive increasing attention from researchers.

[0003] The Northeast Forest Frog (Rana dybowskii) is a dominant amphibian species in Northeast China with high economic value. The oviduct of its female forest frog can be used to prepare the precious Chinese medicinal material forest frog oil, which is widely used in traditional Chinese medicine and health care products in my country. The industrial demand for the Northeast Forest Frog has been on the rise, and it plays an important role in the economic development of forest areas. With the development of the artificial breeding industry of the Northeast Forest Frog, the high stocking density and water pollution have made the Northeast Forest Frog susceptible to a variety of infectious diseases. The abuse of antibiotics has seriously affected the survival of the Northeast Forest Frog population and brought environmental pollution. In the breeding process, the use of lysozyme has become an important antibacterial agent and feed additive in the context of "reducing antibiotics and replacing antibiotics". At present, lysozyme from poultry is often used as a feed additive in the breeding of Northeast Forest Frog to improve the production performance of the Northeast Forest Frog. However, due to the different species of Northeast Forest Frogs of poultry and amphibians, different living environments, and different lysozyme sequences, the inhibition of lysozyme on pathogenic bacteria is also quite different. Therefore, it is necessary to develop effective lysozyme-related gene products from the Northeast Forest Frog itself to meet its disease resistance needs. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an application of a lysozyme gene of a northeastern forest frog, a mature polypeptide region gene, a recombinant protein and a recombinant lysozyme genetic engineering bacterium. The lysozyme provided by the present invention can inhibit Aeromonas hydrophila, Escherichia coli, Micrococcus luteus and Staphylococcus aureus, especially Aeromonas hydrophila from amphibians. Compared with the commercially available avian egg white lysozyme with an activity of 20,000 U / mg, the lysozyme provided by the present invention has a higher antibacterial effect on Aeromonas hydrophila.

[0005] The first aspect of the present invention provides a Northeastern forest frog lysozyme gene, the gene sequence of which is shown in SEQ ID NO.1.

[0006] The second aspect of the present invention provides a method for cloning the above-mentioned Northeast Forest Rana lysozyme gene, the specific steps are as follows:

[0007] Extract RNA from Rana edulis tissue;

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

[0009] The obtained cDNA was amplified by PCR using primers of sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplified product;

[0010] The amplified product was connected to a vector and introduced into an expression host cell, and the Northeast Forest Rana lysozyme gene was obtained through screening and sequencing.

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

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

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

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

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

[0019] In a fourth aspect, the present invention provides a recombinant protein encoded by the above-mentioned gene of the mature polypeptide region of Rana chensinensis lysozyme, wherein the amino acid sequence of the recombinant protein is shown in SEQ ID NO.6.

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

[0021] EcoR I and Xho I restriction sites were added to both ends of the gene of the mature polypeptide region of the Northeast Forest Rana lysozyme, and connected with the vector pET28a to construct a recombinant plasmid LYZ-pET28a;

[0022] The recombinant plasmid LYZ-pET28a was introduced into the expression host cells, protein expression was induced, and the recombinant protein was obtained by purification.

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

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

[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] A seventh aspect of the present invention provides a use of the above-mentioned Rana chensinensis lysozyme gene, Rana chensinensis lysozyme mature polypeptide region gene, recombinant protein or recombinant lysozyme genetically engineered bacteria in the preparation of an antibacterial product.

[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 Northeast Forest Rana lysozyme gene provided by the present invention can encode a Northeast Forest Rana lysozyme, which is a C-type lysozyme with high antibacterial activity. It has an antibacterial effect on Micrococcus luteus, Aeromonas hydrophila, Escherichia coli and Staphylococcus aureus, especially on Aeromonas hydrophila from amphibians. Compared with the commercially available avian egg white lysozyme with an activity of 20,000 U / mg, the lysozyme provided by the present invention has a higher antibacterial effect on Aeromonas hydrophila.

[0030] (2) The present invention starts from the perspective of the Northeast Forest Rana, explores and analyzes the types of amphibian lysozymes, obtains a Northeast Forest Rana lysozyme gene, a Northeast Forest Rana lysozyme mature polypeptide region gene, a recombinant protein and a recombinant lysozyme genetic engineering bacterium, and can provide a new, efficient, non-toxic, pollution-free and stable antibacterial product for Northeast Forest Rana breeding, changing the current abuse of antibiotics and preservatives. At the same time, it has important value for the development and utilization of amphibian lysozymes and C-type lysozymes, and also provides a reference for the study of lysozymes of other species. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The figure is the electrophoresis diagram of RNA extracted from the skin tissue of the Northeast Forest Rana; Lane 1 is the electrophoresis result of RNA from the abdominal skin sample of the Northeast Forest Rana, and Lane 2 is the electrophoresis result of RNA from the leg skin sample of the Northeast Forest Rana.

[0032] Figure 2 The figure is the result of PCR electrophoresis after amplification of the target gene; Lane M is DNAMarker2000, and Lane 1 is the location of the Northeast Forest Rana C-type lysozyme gene.

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

[0034] Figure 4 The figure shows the PCR electrophoresis result of the recombinant plasmid LYZ-pET28a; the left lane is DNAMarker5000, and the red frame in the right lane indicates the location of the PCR product of the recombinant plasmid LYZ-pET28a.

[0035] Figure 5 The figure is the electrophoresis result of the target fragment double digested with EcoR I / Xho I; the left lane is the electrophoresis result of the double digestion product, the red frame indicates the position of the electrophoresis band of the digestion product, and the right lane is DNA Marker5000.

[0036] Figure 6 Figure 1 is the nucleotide and amino acid sequencing peak diagram of the mature polypeptide region of the Northeast Forest Rana lysozyme. Figure B is a subsequent supplement to Figure A, and Figure C is a subsequent supplement to Figure B.

[0037] Figure 7 It is an expression diagram of the recombinant lysozyme protein of Rana edulis; wherein lane M is a protein marker, lanes 1 to 3 are the protein expression results without IPTG induction for 2h, 3h, and 4h, respectively, lanes 4 to 6 are the protein expression results after adding 0.1mmoL / L IPTG for induction for 2h, 3h, and 4h, respectively, lanes 7 to 9 are the protein expression results after adding 0.5mmoL / L IPTG for induction for 2h, 3h, and 4h, respectively, and lanes 10 to 12 are the protein expression results after adding 1mmoL / L IPTG for induction for 2h, 3h, and 4h.

[0038] Figure 8 This is the electrophoresis diagram for the confirmation of the inclusion bodies of the recombinant lysozyme protein of the Northeast Forest Rana; wherein lane M is the protein marker, lane 1 sample is the cultured bacterial solution, lane 2 sample is the supernatant of the bacterial solution after ultrasonic disruption and centrifugation, and lane 3 sample is the precipitate of the bacterial solution after ultrasonic disruption and centrifugation.

[0039] Fig. 9The graph shows the gradient elution results of the target protein; lane M is the protein marker, lane 1 is the elution result of the target protein using Buffer C, and lanes 2 to 6 are the electrophoresis results of the target protein eluted using Buffer C containing 100mmoL / L, 200mmoL / L, 300mmoL / L, 400mmoL / L, and 500mmoL / L imidazole, respectively.

[0040] Fig.10 It is the protein electrophoresis diagram of the dialysis renaturation of the recombinant protein; wherein lane M is the protein marker, and lane 1 is the electrophoresis result of the dialysis renaturation recombinant protein sample.

[0041] Fig.11 The results of Western blotting for detecting the expression of whole-cell recombinant protein containing His tag; the sample in lane 1 is the whole-cell diluted 30 times, the sample in lane 2 is the whole-cell diluted 10 times, the sample in lane 3 is the whole-cell diluted 3 times, and the sample in lane 4 is the whole-cell stock solution.

[0042] Fig.12 This is the result of Western blotting detection of purified recombinant protein containing His tag; the samples in lanes 1 to 4 are 10 ng, 30 ng, 100 ng and 300 ng of recombinant protein, respectively.

[0043] Fig.13 Figure 2 is a graph showing the results of bovine serum albumin concentration determination; Figure A is a graph showing the electrophoresis results of bovine serum albumin standards, wherein the concentrations of bovine serum albumin standards in each lane from right to left are 0μg / mL, 10μg / mL, 30μg / mL, 50μg / mL, 70μg / mL, 90μg / mL, 100μg / mL, 200μg / mL, 300μg / mL, 400μg / mL, 500μg / mL, 600μg / mL, 700μg / mL, 800μg / mL, 900μg / mL, 1000μg / mL, 1500μg / mL, 2000μg / mL, 3000μg / mL, and 4000μg / mL; Figure B is a protein concentration standard curve drawn by the BCA method, with the abscissa representing protein concentration and the ordinate representing absorbance value.

[0044] Fig.14 This is the mass spectrometry detection diagram of the partial amino acid sequence of the recombinant protein (DPNGMKAWVGWR).

[0045] Fig.15 This is the mass spectrometry detection diagram of the partial amino acid sequence (AWVGWR) of the recombinant protein.

[0046] Fig.16Figure 1 is a graph showing the antibacterial ability of the Northeast Forest Rana lysozyme; A1, B1, C1 and D1 are negative control groups inoculated with Aeromonas hydrophila, Micrococcus luteus, Escherichia coli and Staphylococcus aureus and added with PBS; A2, B2, C2 and D2 are experimental groups inoculated with Aeromonas hydrophila, Micrococcus luteus, Escherichia coli and Staphylococcus aureus and added with Northeast Forest Rana lysozyme; A3, B3, C3 and D3 are positive control groups inoculated with Aeromonas hydrophila, Micrococcus luteus, Escherichia coli and Staphylococcus aureus and added with avian egg white lysozyme.

[0047] Fig.17 Figure 2 is a graph showing the antibacterial effect of lysozyme; Figure A shows the antibacterial ability of different concentrations of Northeast Forest Rana lysozyme, and Figure B shows the antibacterial ability of different concentrations of avian egg white lysozyme.

[0048] Fig.18 These are electron micrographs of the morphological changes of Aeromonas hydrophila cells after lysozyme treatment; A is the negative control group (1 mL of Aeromonas hydrophila bacterial solution + 100 μL of phosphate buffer), B is the positive control group (1 mL of Aeromonas hydrophila bacterial solution + 100 μL of 50 μg / mL avian egg white lysozyme), and C to F are the experimental groups in which 100 μL of 1000 μg / mL, 250 μg / mL, 500 μg / mL, and 5 μg / mL of Rana edulis lysozyme were added to 1 mL of Aeromonas hydrophila bacterial solution, respectively; the same horizontal row shows magnification pictures of 5 μm scale, 2 μm scale, 1 μm scale, and 500 nm scale from left to right, respectively; red arrows indicate bacterial morphological changes; yellow arrows indicate the action status of Rana edulis lysozyme. DETAILED DESCRIPTION

[0049] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0050] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0051] Artificially bred frogs are susceptible to infection by bacterial pathogens, and conventional treatment methods use antibiotics to inhibit the infection of pathogens. Since the abuse of antibiotics has seriously affected the survival of frogs and caused environmental pollution, there is a need for an efficient, safe, and environmentally friendly preparation against harmful microorganisms. Lysozyme is an excellent natural antibacterial agent. Currently, egg white-derived lysozyme is used as a feed additive in animal production practices to improve animal production performance. However, in frog breeding, since egg white lysozyme comes from poultry, poultry and amphibian Northeast Forest Rana belong to different species, and the two have different living environments, there are differences in the sequence of lysozyme, resulting in different inhibitory effects on pathogenic bacteria. The current practical application of lysozyme is still quite insufficient. In production practice, Northeast Forest Rana needs more effective lysozyme-related gene products derived from itself to meet its disease resistance needs.

[0052] The present invention first studies the Northeast Forest Rana lysozyme gene, clones and identifies the type of the Northeast Forest Rana lysozyme gene, obtains a Northeast Forest Rana lysozyme gene with a gene sequence as shown in SEQ ID NO.1, transforms the lysozyme gene to obtain the Northeast Forest Rana lysozyme mature polypeptide region gene, and then uses the Northeast Forest Rana lysozyme mature polypeptide region gene to construct a Northeast Forest Rana recombinant lysozyme gene engineering bacterium expressing recombinant lysozyme protein. Finally, the antibacterial ability of the Northeast Forest Rana lysozyme on Micrococcus luteus, Staphylococcus aureus, Aeromonas hydrophila from amphibians and Escherichia coli is verified, and the destructive effect of the Northeast Forest Rana lysozyme on Aeromonas hydrophila is observed by electron microscopy.

[0053] Example 1: Cloning and sequencing of Rana edulis lysozyme

[0054] 1. Extraction and quality detection of total RNA from the skin of Rana edulis

[0055] Based on the instructions of the total RNA Trizol extraction kit (purchased from Invitrogen), the skin RNA of male Northeast Forest Rana (purchased from Xingwang Breeding Farm, Songbei District, Harbin City, Heilongjiang Province) was extracted. The EP tubes, tips, mortars, tweezers, etc. required in the experiment were soaked in 1‰ DEPC water in advance, sterilized and dried for use.

[0056] (1) Place an appropriate amount of liquid nitrogen in a mortar and pestle, grind about 0.1 g of abdominal and leg skin tissues in liquid nitrogen, transfer to an EP tube containing 750 μL Trizol, mix well, and centrifuge at 12,000 rpm for 5 min at 4°C;

[0057] (2) The supernatant was transferred to a new EP tube containing 150 μL of chloroform. After sufficient shaking, the tube was placed in an ice bath for 10 min, centrifuged at 12,000 rpm at 4°C for 15 min, and the upper aqueous phase was transferred to a new EP tube.

[0058] (3) Add an equal volume of isopropanol to the upper water layer into the EP tube, mix thoroughly by inversion, and place on ice for 10 min. Centrifuge at 12,000 rpm at 4°C for 10 min, discard the supernatant, and keep the precipitate.

[0059] (4) Add an appropriate amount of 75% ethanol to the EP tube with the white RNA precipitate at the bottom, aspirate repeatedly until the precipitate is dissolved, centrifuge at 12,000 rpm at 4°C for 5 min, and discard the supernatant;

[0060] (5) Leave the EP tube containing the RNA precipitate open at room temperature for several minutes to allow the ethanol to evaporate completely, then add 20 μL of RNase-free ddH2O to dissolve the RNA sample;

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

[0062] (7) The quality of the extracted total RNA was qualitatively tested by horizontal agarose gel electrophoresis (1%), and the concentration of the total RNA was quantitatively determined by ultraviolet spectrophotometer. The RNA samples with qualified quality were frozen in a -80°C refrigerator for future use.

[0063] The electrophoresis results are as follows Figure 1 As shown, there are three complete and clear bands of 28S, 18S and 5S in the lane, which meets the requirements of the next step of reverse transcription.

[0064] 2. RT-PCR amplification of the lysozyme gene of Rana edulis

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

[0066] use II 1st Strand cDNA Synthesis Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was used for RNA reverse transcription reaction, and the reaction system was 20 μL.

[0067] First, the reaction system shown in Table 1 was added to a pre-cooled centrifuge tube, heated at 65°C for 5 minutes, and then placed on ice for 2 minutes to complete the denaturation of the RNA template.

[0068] Table 1 Denaturation reaction system of RNA template

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

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

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

[0072]

[0073]

[0074] (2) PCR amplification of target gene

[0075] Primer design: By comparing the sequence with the C-type lysozyme sequences of human, mammal, fish, African clawed frog and other species published on NCBI, the conserved region was selected, and a pair of primers were designed in the conserved region using PrimerPremier5.0 software. The sequences of the upstream primer and the downstream primer are shown in SEQ ID NO.2 and SEQ ID NO.3:

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

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

[0078] The PCR amplification reactions of the lysozyme gene were all 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 Target gene PCR amplification reaction system

[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 Target gene PCR amplification reaction conditions

[0082]

[0083]

[0084] Note: “-” means the corresponding step will not be cycled.

[0085] The PCR amplified product was detected by 1% agarose gel electrophoresis, and then the size of the target band was observed and the image was collected in a UV gel imaging system.

[0086] from Figure 2 It can be seen that the target gene has an obvious 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 recovery

[0089] The target band was recovered according to the instructions of the gel recovery kit DC301-01 (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) to obtain a DNA sample, which was frozen at -20°C.

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

[0091] The ligation system shown in Table 5 was added to the PCR tube, mixed and connected in a 16°C low-temperature water bath for 16 h to obtain a ligation product between the target fragment and the pMD18-T vector (purchased from Nanjing Novozyme 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 plasmid

[0095] The obtained ligation product was transformed into Escherichia coli DH5α competent cells (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) with the following steps:

[0096] ①Mix the ligation product with competent Escherichia coli DH5α cells at a ratio of 1:10, mix gently and place on ice for 30 minutes; ②Heat shock in a 42℃ water bath for 90 seconds and then quickly place on ice for 5 minutes; ③Add 0.5mL of LB liquid culture medium and shake and culture at 180rpm on a constant temperature shaker at 37℃ for 1 hour. ④Centrifuge the bacterial solution at 2000rpm for 2 minutes, discard 400μL of the supernatant, and resuspend the bacterial solution at the bottom of the tube; ⑤Evenly and gently spread the resuspended bacterial solution on the surface of the LB solid plate containing 100μg / mL Amp, and culture at 37℃ for 12 hours.

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

[0098] A single white colony was randomly picked and placed in 20 μL of LB liquid culture medium containing 100 μg / mL Amp, and cultured in a constant temperature shaker at 37°C and 180 rpm for 4 h. The cultured bacterial solution was used as a template for PCR amplification, and the 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 of recombinant plasmid

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

[0101] The reaction products were detected by 1% agarose gel electrophoresis, and the cloned bacterial solution that was initially determined to be positive was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequence determination.

[0102] 4. Sequence determination and analysis of Northeast Forest Rana lysozyme

[0103] After sequence determination, the nucleotide sequence of the lysozyme gene coding region of the Northeast Forest Rana is shown in SEQ ID NO.1, the full length of the sequence is 459 bp, and the amino acid sequence is shown in SEQ ID NO.5. By comparing the obtained nucleotide sequence and amino acid sequence with the nucleotide and amino acid sequences of other reference species such as the European forest frog, the alpine pygmy frog, and humans disclosed on NCBI, and using MEGA software to construct a phylogenetic tree of amino acids, it was found that the Northeast Forest Rana lysozyme gene and the C-type lysozyme gene of the reference species were clustered in the same group. Therefore, the Northeast Forest Rana lysozyme provided by the present invention belongs to the C-type lysozyme.

[0104] SEQ ID NO.1:

[0105] ATGAGGTCTCTGATGCAAATCATGGGAGTCCTTTTCCTAACAATTGCATTAGTCGATGCAAAACAGTATGAGAGATGTGAACTTGCAAAAGCCATGAAAATTCTGGGTCTGGATGGATATCAAGGATACAGCCTGCCAAACTGGGTTTGCACAGCTTTCTACGAGAGTTCCTTTAACACATACAGCATAAACTTCAACCCTGGAGACAGCAGCACTGATTACGGTATTCT TCAAATAAACAGCCATTGGTGGTGTAATGATTACAAAACTCCGAGAGCCCACAATGCTTGCAACATTGATTGCTCAGACCTCCTATCCAATGACATCTCATTATCTGTGGAATGCGCAAAACGTGTTGTGAGGGATCCCAATGGTATGAAGGCATGGGTTGGATGGAGAAACCATTGCAAAGGAAAAGATTTGTCTGCATGGATTCAAGGCTGCGGGCTTGACTTTTAA;

[0106] SEQ ID NO.5:

[0107] MRSLMQIMGVLFLTIALVDAKQYERCELAKAMKILGLDGYQGYSLPNWVCTAFYESSFNTYSINFNPGDSSTDYGILQINSHWWCNDYKTPRAHNACNIDCSDLLSNDISLSVECAKRVVRDPNGMKAWVGWRNHCKGKDLSAWIQGCGLDF, *, indicates stop codon.

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

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

[0110] SEQ ID NO.4:

[0111] AAACAGTATGAGAGATGTGAACTTGCAAAAGCCATGAAAATTCTGGGTCTGGATGGATATCAAGGATACAGCCTGCCAAACTGGGTTTGCACAGCTTTCTACGAGAGTTCCTTTAACACATACAGCATAAACTTCAACCCTGGAGACAGCAGCACTGATTACGGTATTCTTCAAATAAACAGCCATTGGTGGTGTAATGA TTACAAAACTCCGAGAGCCCACAATGCTTGCAACATTGATTGCTCAGACCTCCTATCCAATGACATCTCATTATCTGTGGAATGCGCAAAACGTGTTGTGAGGGATCCCAATGGTATGAAGGCATGGGTTGGATGGAGAAACCATTGCAAAGGAAAAGATTTGTCTGCATGGATTCAAGGCTGCGGGCTTGACTTTTAA;

[0112] The protein molecular weight of the mature polypeptide region of Rana sylvaticum lysozyme is 15KDa, and the amino acid sequence is shown in SEQ ID NO.6. SEQ ID NO.6:

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

[0114] Example 2: Construction of prokaryotic expression vector of Northeastern Rana lysozyme gene

[0115] 1. Construction of recombinant plasmid

[0116] For recombinant expression, the present invention carries a stretch of amino acids at the N-terminus of the constructed recombinant plasmid, which carries a 6-histidine tag, and the amino acid sequence is shown in SEQ ID NO.7. The protein molecular weight of the recombinant expression vector finally constructed is about 17KDa.

[0117] SEQ ID NO.7: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF. EcoR I and Xho I restriction sites were added to both ends of the gene shown in SEQ ID NO.4, and connected with the vector pET28a (Nanjing Novogene Biotechnology Co., Ltd.) to construct a recombinant plasmid LYZ-pET28a. The plasmid map is shown in Figure 3 shown.

[0118] 2. Target fragment identification

[0119] The recombinant plasmid LYZ-pET28a was tested 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). In principle, the size of the PCR product is 690 bp, which is consistent with the position of the band in the electrophoresis result ( Figure 4 ), which 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 target fragment

[0123] The obtained PCR product solution was recovered and double-digested with EcoR I and Xho I. Figure 5 A band of about 690 bp can be clearly seen in the electrophoresis diagram, and the size of the enzyme-digested product fragment is consistent with the expected result.

[0124] 4. Sequencing results: The recombinant plasmid LYZ-pET28a was sent for testing. The sequencing results were as follows Figure 6 As shown, the sequencing map showed that the nucleotide sequence was identical to the nucleotide sequence SEQ ID NO.4 of the provided Northeast Forest Rana lysozyme mature polypeptide region gene, the amino acid sequence was identical to the sequence SEQ ID NO.6, and the recombinant sequence was consistent with expectations, indicating that the recombinant plasmid LYZ-pET28a was successfully constructed.

[0125] 5. Inducible expression of recombinant C-type lysozyme in Rana edulis

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

[0127] (2) Transfer the activated bacterial solution to LB culture medium with a kanamycin concentration of 50 μg / mL and culture at 37°C and 220 r / min until the bacterial solution OD 600 When the value was 0.6, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to make the final concentration of the solution 0mmoL / L, 0.1mmoL / L, 0.5mmoL / L and 1mmoL / L, respectively. The culture was induced at 37°C and 220rpm for 2h, 3h and 4h, respectively. The bacterial solution was collected and centrifuged to remove the supernatant. The bacterial precipitate obtained was the recombinant lysozyme genetically engineered bacteria, and samples were taken for electrophoresis analysis.

[0128] from Figure 7 It can be clearly seen from the electrophoresis results that lanes 1 to 12 can express the recombinant lysozyme protein of the Northeast Forest Rana. The recombinant lysozyme protein of the Northeast Forest Rana is marked as a recombinant protein. The protein molecular weight is about 17 kDa, and the recombinant protein can be expressed without adding IPTG.

[0129] (3) Resuspend the cells without IPTG induction in 20 mL of inclusion body cell lysis buffer (purchased from Bio-Tech Biotechnology Co., Ltd.), lyse the cells by ultrasonication, centrifuge at 10,000 rpm and 4°C for 15 min, separate the supernatant and precipitate, and perform protein electrophoresis. Figure 8 The results show that there is no recombinant protein in the supernatant. The recombinant protein mainly exists in the inclusion bodies and needs to be released after the bacteria are broken.

[0130] (4) Add 10 mL of inclusion body cell lysis buffer to resuspend the precipitate in step (3), let it stand at room temperature overnight, 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 by nickel column affinity purification using a His tag protein purification kit (purchased from Bio-Tech Biotechnology Co., Ltd.) according to the instructions. The specific steps are as follows:

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

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

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

[0137] (4) Elution of recombinant protein: Column 1 was used to elute the recombinant protein with Buffer C, and column 2 was used to elute the recombinant protein with different concentrations of imidazole Buffer C. 20 μL of 2× loading buffer was added to 20 μL of the eluate containing the recombinant protein to obtain a liquid sample of the recombinant protein with a gradient concentration, and an electrophoresis experiment was performed.

[0138] Protein electrophoresis results Fig. 9 As shown, the loading samples in lanes 1, 3, and 4 contained purified recombinant proteins.

[0139] 2. Recombinant protein dialysis refolding

[0140] 0.01% skl (sodium lauryl sarcosine) was added to 1×PBS solution and the pH value was adjusted to 7.4 to obtain a refolding buffer solution.

[0141] Will Fig. 9 The eluted proteins loaded in lanes 1, 3, and 4 were dialyzed in a refolding buffer, and the resulting solution was the active recombinant protein, and protein electrophoresis was performed.

[0142] The electrophoresis results are as follows Fig.10 As shown, the molecular weight of the recombinant protein after dialysis renaturation was approximately 17 kDa, which was consistent with the expectation.

[0143] 3. Recombinant protein electrophoresis detection

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

[0145] First, WB detection of whole bacterial recombinant protein was performed. The original bacterial solution and the bacterial solution diluted 3, 10, and 30 times were subjected to 10% SDS-PAGE electrophoresis, and the bands were transferred to a nitrocellulose membrane. Then, it was blocked with 5% skim milk powder and incubated at room temperature for 2 hours. Then, a 500-fold diluted rabbit anti-His tag antibody was added and incubated at 4°C overnight. Then, the membrane was incubated with a 1000-fold diluted mouse anti-rabbit IgG at room temperature for 1 hour, and then the membrane was washed and the results were observed after color development with ECL chemiluminescent solution and photographed.

[0146] The results are as follows Fig.11 As shown, clear bands can be seen at about 17 kDa in all four lanes. The recombinant protein may also form polymers or undergo post-translational modification, so some bands may also appear at other positions.

[0147] Then, the recombinant protein solution after renaturation and purification was identified by WB, and the content of the recombinant protein was calculated by ImageJ software. The expression level was calculated according to the formula: protein relative content = target protein optical density value / color scale optical density value.

[0148] like Fig.12 As shown, it can be seen that the recombinant protein after renaturation and purification can bind to the rabbit anti-His tag antibody, indicating that a certain amount of recombinant protein has been obtained.

[0149] 4. Determination of recombinant protein concentration

[0150] The BCA method was established using bovine serum albumin (BSA) standard. Fig.13 The protein concentration standard curve shown is Y = 2E-05X 2 +0.1715X+1.7615, 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 Image J software. According to the protein concentration standard curve, the concentration of the 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 Fig.14 and Fig.15 As shown, the partial amino acid sequences of the captured recombinant proteins DPNGMKAWVGWR (SEQ ID NO.10) and AWVGWR (SEQ ID NO.11) are consistent with the amino acid sequences of Rana chensinensis lysozyme, indicating that the recombinant protein expressed by the prokaryotic expression vector is Rana chensinensis lysozyme.

[0152] Example 4: Application of Northeast Forest Rana Lysozyme

[0153] In this embodiment, the information of each strain is as follows:

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

[0155] For information on Aeromonas hydrophila, please refer to “Shao Jie, Chai Longhui, Wang Boju, et al. Identification and pathogenicity of a strain of Aeromonas hydrophila from Rana mandshurica[J]. Journal of Wildlife Science, 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. with the product number CMCC(B)44102.

[0158] 1. Plate antibacterial experiment

[0159] In order to detect the antibacterial ability of the Northeast Forest Rana lysozyme, the present invention specifically detects the antibacterial effect of the Northeast Forest Rana lysozyme on Micrococcus luteus, Aeromonas hydrophila, Staphylococcus aureus and Escherichia coli. Micrococcus luteus and Staphylococcus aureus are Gram-positive bacteria, and the culture temperature is 28°C. Aeromonas hydrophila and Escherichia coli are Gram-negative bacteria, and the culture temperature is 37°C. The specific steps of the experiment are as follows:

[0160] (1) The above four bacteria were first inoculated on LB solid plate medium for streak activation culture, and then a single colony was picked and inoculated into liquid LB medium for overnight culture. Then 100 μL of the bacterial solution was transferred to 100 mL of liquid LB medium and cultured until OD 600 The value of 0.6 is reserved.

[0161] (2) The Northeast Forest Rana recombinant lysozyme protein was sterilized by filtration through a 0.22 μm microporous filter. 1 mL of the above four bacterial cultures was added with 10 μL of the recombinant protein. After culturing for 30 min, 100 μL was taken and coated on a plate. 10 μL of sterilized PBS buffer was added to the negative control group. The experimental results are as follows: Fig.16 shown.

[0162] (3) From Fig.16It can be seen that A1, B1, C1, and D1 are negative control groups, and no lysozyme is added, and the bacteria can grow normally. A2, B2, C2, and D2 are experimental groups to which the Northeast Forest Rana lysozyme of the present invention is added, and 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 a certain extent. A3, B3, C3, and D3 are positive control groups to which commercialized avian egg white lysozyme (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was added. It can be found that Aeromonas hydrophila can still grow in A3, indicating that the antibacterial effect of avian egg white lysozyme is lower than that of the recombinant lysozyme of Rana simiana on Aeromonas hydrophila. The inhibitory effect of avian egg white lysozyme on Micrococcus luteus in the B3 dish is also low, the inhibitory effect of avian egg white lysozyme on Escherichia coli in the C3 dish is also low, and the antibacterial effect of avian egg white lysozyme on Staphylococcus aureus in the D3 dish is also low.

[0163] Fig.17 Figure A is a graph showing the antibacterial activity of different concentrations of Northeast Forest Rana lysozyme, and Figure B is a graph showing the antibacterial activity of different concentrations of avian egg white lysozyme. It can be seen from the figure that the Northeast Forest Rana lysozyme provided by the present invention has a high antibacterial activity and can effectively inhibit Aeromonas hydrophila at a concentration of 10 μg / mL. Compared with avian egg white lysozyme, the Northeast Forest Rana lysozyme provided by the present invention has a higher inhibitory effect on amphibian Aeromonas hydrophila than the avian egg white lysozyme.

[0164] 2. Scanning electron microscope observation

[0165] The present invention further uses electron microscopy scanning to observe the antibacterial effect of Northeast Forest Rana lysozyme on Aeromonas hydrophila, and the specific steps are as follows:

[0166] (1) Aeromonas hydrophila was streaked on LB solid plate medium and cultured at 28°C. After a single colony grew, a single colony was picked and added to LB liquid medium for overnight culture. Then, it was transferred to 100 mL LB liquid medium at a 1% inoculum dose and cultured until OD 600 The value is 0.5.

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

[0168] (3) The control group and the experimental group were incubated at 28°C for 8 h, and then centrifuged again at 4°C and 3000 rpm for 10 min. The bacteria were washed twice with PBS and then added with 2.5% glutaraldehyde solution. After fixation at 4°C overnight, the antibacterial effect of the Northeast Forest Rana lysozyme protein on Aeromonas hydrophila was observed using a G300 thermal field emission scanning electron microscope (TFE-SEM) from Zeiss, Germany.

[0169] like Fig.18 As shown, it can be seen that the bacterial morphology in A4 is normal and not destroyed; the bacterial morphology in B4 is basically normal, indicating that the egg white lysozyme has a small destructive effect; the bacterial rupture in C4 is obvious, indicating that the Northeast Forest Rana lysozyme at a concentration of 1000μg / mL can inhibit Aeromonas hydrophila; D4 is lysed under the action of 250μg / mL lysozyme; E4: under the action of 50μg / mL lysozyme, small holes appear on the surface of the bacterial body and the contents seep out; F4: under the action of 5μg / mL lysozyme, the bacterial body swells. It shows that the Northeast Forest Rana lysozyme provided by the present invention has a high antibacterial activity against Aeromonas hydrophila of amphibian origin, and has a stronger antibacterial effect on Aeromonas hydrophila than the commercially available avian egg white lysozyme.

Claims

1. A Northeast Forest Rana lysozyme gene, characterized in that: The gene sequence is shown in SEQ ID NO.

1.

2. A method for cloning the Northeast Forest Rana lysozyme gene according to claim 1, characterized in that: The specific steps are as follows: Extract RNA from Rana edulis tissue; RNA is reverse transcribed to synthesize the first strand of cDNA; The obtained cDNA was amplified by PCR using primers of sequences shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain an amplified product; The amplified product was connected to a vector and introduced into an expression host cell, and the Northeast Forest Rana lysozyme gene was obtained after screening and sequencing; SEQ ID NO.2: 5'-CTCCATTACATTTGTTGCCATA-3', SEQ ID NO. 3: 5'-ACTGTAACTGAATCCTTGCGAG-3'.

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

4. A gene of the mature polypeptide region of the Northeast Forest Rana lysozyme obtained by genetic modification of the Northeast Forest Rana lysozyme 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 Rana simiana 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 a recombinant protein according to claim 5, characterized in that: The specific steps are as follows: EcoR I and Xho I restriction sites were added to both ends of the gene of the mature polypeptide region of the Northeast Forest Rana lysozyme, and connected with the vector pET28a to construct a recombinant plasmid LYZ-pET28a; The recombinant plasmid LYZ-pET28a is introduced into the expression host cell, the protein is induced, and the protein is purified to obtain the recombinant protein.

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

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

9. Use of the Northeast Forest Rana lysozyme gene of claim 1, the Northeast Forest Rana lysozyme mature polypeptide region gene of claim 4, the recombinant protein of claim 5 or the recombinant lysozyme genetically engineered bacteria of claim 8 in the preparation of an antibacterial product.

10. The use according to claim 9, characterized in that: The antibacterial agent is any one or more of Aeromonas hydrophila, Escherichia coli, Micrococcus luteus and Staphylococcus aureus.

Images