CN111233993B - Prawn coupling antibacterial peptide and gene, acquisition method of prawn coupling antibacterial peptide, expression vector, recombinant bacterium and application - Google Patents

Abstract

The invention provides a prawn coupling antibacterial peptide and a gene thereof, an acquisition method of the prawn coupling antibacterial peptide, an expression vector, a recombinant bacterium and application, belonging to the technical field of genetic engineering, wherein the amino acid sequence of the prawn coupling antibacterial peptide is shown as SEQ ID No.1, and the prawn coupling antibacterial peptide replaces antibiotics used in the current aquatic feed so as to achieve the purposes of preventing and treating animal diseases and reducing adverse consequences caused by the antibiotics.

Description

Prawn coupling antibacterial peptide and gene, acquisition method of prawn coupling antibacterial peptide, expression vector, recombinant bacterium and application

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a prawn coupling antibacterial peptide, a gene, a prawn coupling antibacterial peptide acquisition method, an expression vector, a recombinant bacterium and application.

Background

Aquatic products such as fish, shrimp and the like are important sources of human daily food protein, so that aquaculture plays a very important role in global economy. However, aquaculture suffers from huge economic losses each year due to bacterial or viral insults. In order to prevent and control the occurrence of diseases, farmers widely use chemical drugs for site disinfection or antibiotics are added to feed for disease control. The use of a large amount of chemical drugs or antibiotic drugs can cause serious ecological damage and cause great worry on food safety. Under the situation of strengthening environmental protection and food safety supervision of China, the novel fishing medicament which is pollution-free, residue-free, safe and effective and can completely or partially replace chemical medicaments or antibiotic medicaments has important social significance.

Disclosure of Invention

In view of the above, the present invention provides a prawn coupling antibacterial peptide and a gene, a prawn coupling antibacterial peptide obtaining method, an expression vector, a recombinant bacterium and an application thereof, and the prawn coupling antibacterial peptide provided by the present invention replaces antibiotics used in current aquatic feeds, so as to achieve the purposes of preventing and treating animal diseases and reducing adverse consequences caused by abuse of the antibiotics.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention provides a prawn coupling antibacterial peptide, and the amino acid sequence of the prawn coupling antibacterial peptide is shown in SEQ ID No. 1.

The invention also provides a gene for coding the prawn coupling antibacterial peptide, and the nucleotide sequence of the gene is shown in SEQ ID No. 2.

The invention also provides an expression vector, and the gene in the technical scheme is connected between the EcoR1 site and the Not1 site of the yeast expression vector pPIC9K to obtain the expression vector.

The invention also provides a recombinant bacterium, wherein the expression vector of the technical scheme is transferred into a Top10 clone strain, plasmids are extracted, and the plasmids are electrically transformed into pichia pastoris to obtain the recombinant bacterium.

Preferably, the pichia pastoris includes pichia pastoris GS 115.

The invention also provides a method for obtaining the prawn coupling antibacterial peptide, which comprises the steps of carrying out liquid culture on the recombinant bacteria in the technical scheme, centrifuging the obtained culture solution to obtain a supernatant, and purifying the prawn coupling antibacterial peptide and the prawn coupling antibacterial peptide in the supernatant.

The invention also provides application of the recombinant bacteria in the technical scheme in preparation of aquatic animal antibacterial drugs, vaccines or feed additives.

Preferably, the aquatic animals include fish or shrimp.

The invention provides a prawn coupling antibacterial peptide and a gene thereof, an acquisition method of the prawn coupling antibacterial peptide, an expression vector, a recombinant bacterium and application, wherein the amino acid sequence of the prawn coupling antibacterial peptide is shown as SEQ ID No.1, and the prawn coupling antibacterial peptide replaces antibiotics used in the current aquatic feed so as to achieve the purposes of preventing and treating animal diseases and reducing adverse consequences caused by abuse of antibiotics.

Drawings

FIG. 1 is a plasmid map of pPIC9K vector;

FIG. 2 is a schematic diagram of the insertion of the WAP + Crustinlike + pvHM117 tandem expression sequence into the pPIC9K vector;

FIG. 3 is a diagram showing the results of agarose electrophoresis;

FIG. 4 is a 1% agarose gel electrophoresis image;

FIG. 5 is a graph showing the results of the growth of the yeast on the plate after the electric transformation;

FIG. 6 is a result diagram of PCR identification of a recombinant yeast positive clone strain;

FIG. 7 is a diagram showing the identification of recombinant proteins in the supernatant from WB (Western blot);

FIG. 8 shows the results of recombinant protein purification;

FIG. 9 is a Salmonella bacteriostatic profile;

FIG. 10 is a bacterial inhibition pattern of Vibrio alginolyticus;

FIG. 11 is a Staphylococcus bacteriostasis pattern.

Detailed Description

The invention provides a prawn coupling antibacterial peptide, which is characterized in that the amino acid sequence of the prawn coupling antibacterial peptide is shown as SEQ ID No.1, and specifically comprises the following steps:

EFVPTRHSRPRPQPKPRPGTCPDTSDIFSICVVTERNCFSDSECGPGQKCCPIGCGRECLAVVPPYGSGRGGGGSGGGGSGGGGSQDKDKAGTRLGGGFGVPGAGGVFPGAGGVPGVGGVFPGAGGVFPGAGGIGPGPGGLIPGGGFNCNYCRTPVGYVCCKPGRCPPVRDVCPSTRFGPPVCRQDLDCSGSDKCCYDVCLEDTVCKPIVAGSQGGGGGSGGGGSGGGGSGKFRGFGQPFGGLGGPGGGVGVGGGFPGGGLGVGGGLGVGGGLGVGGGLGVGGGLGTGTSDCRYWCKTPEGQAYCCESAHEPETPVGTKPLDCPQVRPTCPRFHGPPTTCSNDYKCAGLDKCCFDRCLGEHVCKPPSFFGSQVFGHHHHHH。

the invention also provides a gene for coding the prawn coupling antibacterial peptide, the nucleotide sequence of the gene is shown as SEQ ID No.2, and the gene is as follows:

gaattcgttccaactagacattctagaccaagaccacaaccaaaaccaagaccaggtacttgcccagatacttccgatattttctctatttgtgttgttactgagagaaactgtttttctgattccgaatgtggacctggtcaaaagtgttgcccaattggatgtggaagagagtgtttggctgttgttccaccatacggttctggtagaggtggtggtggttctggtggtggtggatctggtggtggaggatctcaggataaggataaggctggtactagattgggtggtggttttggtgttcctggtgctggtggtgtttttcctggtgctggaggtgttcctggagttggtggtgttttccctggtgctggtggagtttttcctggagctggtggtattggtcctggtcctggtggtttgattcctggtggtggtttcaattgtaattattgtagaactcctgtcggttatgtttgttgtaagccaggtagatgtcctcctgttagagatgtttgtccatctactagatttggtcctcctgtttgtagacaagatttggattgttctggttctgataagtgttgttatgatgtttgtttggaagatactgtttgtaagcctattgttgctggttctcaaggtggtggtggtggttccggtggtggtggttcaggtggtggtggttctggaaagtttagaggttttggtcagccttttggtggtttgggtggtcctggtggaggtgttggtgttggtggtggttttcctggtggtggattgggtgttggtggaggtttgggtgttggaggtggtttgggagttggtggaggattgggtgtcggtggtggtttgggtactggtacttctgattgtagatattggtgtaagactcctgagggtcaggcttattgttgtgagtctgctcatgagcctgagactcctgttggtactaagcctttggattgtcctcaagttagacctacttgtcctagatttcatggtccaccaactacatgttcaaatgattacaagtgtgctggtttggataagtgttgctttgatagatgtttgggtgagcatgtttgtaaaccaccatctttttttggttcccaagtctttggtcatcatcatcatcatcattaa。

in the present invention, the gene is preferably a WAP gene, which has NCBI accession number: AY464465.1, NCBI accession number of the Crustinlike gene: JQ824114.1, NCBI accession number of the pvHM117 gene: AY 488497.1.

The invention also provides an expression vector, and the gene in the technical scheme is connected between the EcoR1 site and the Not1 site of the yeast expression vector pPIC9K to obtain the expression vector. The method for connecting the gene into the yeast expression vector pPIC9K is not particularly limited, and the method for connecting the gene into the yeast expression vector can be realized by adopting the conventional method.

The invention also provides a recombinant bacterium, wherein the expression vector of the technical scheme is transferred into a Top10 clone strain, plasmids are extracted, and the plasmids are electrically transformed into pichia pastoris to obtain the recombinant bacterium. In the present invention, the pichia pastoris preferably includes pichia pastoris GS 115. The method for transferring the expression vector into the Top10 clone strain is not specially limited, and a conventional method is adopted, and the method for extracting the plasmid and performing the electrotransformation is not specially limited, and the conventional method is adopted.

The invention also provides a method for obtaining the prawn coupling antibacterial peptide, which comprises the steps of carrying out liquid culture on the recombinant bacteria in the technical scheme, centrifuging the obtained culture solution to obtain a supernatant, and purifying the prawn coupling antibacterial peptide in the supernatant to obtain the prawn coupling antibacterial peptide. The invention preferably uses a Ni-NTA chromatographic column to purify the prawn coupling antibacterial peptide in the supernatant.

The invention also provides application of the recombinant bacteria in the technical scheme in preparation of aquatic animal antibacterial drugs, vaccines or feed additives. In the present invention, the aquatic animals preferably include fish or shrimp.

In the invention, the stable recombinant protein expression strain can be obtained by transforming the gene into pichia pastoris GS 115. The recombinant expression method of the invention enables the target recombinant protein to obtain post-translational modification processing, thus being closer to the protein expressed in the prawn body; the gene is integrated on a pichia pastoris chromosome, is replicated along with the replication of the chromosome and is not easy to lose; the recombinant coupling peptide has high expression level, can be expressed in cells and secreted expression, can be secreted into fermentation liquor, and is favorable for separation and purification. The yeast for expressing the recombinant coupling peptide contains various nutrient substances necessary for the growth and development of animals, and can be used as a high-quality protein source to be added into feeds such as chickens, pigs, ruminants, aquatic products and the like. The recombinant bacteria prepared by the invention and the recombinant antibacterial peptide protein expressed by the recombinant bacteria have good antibacterial effect, and can be applied to producing antibacterial drugs, vaccines or feed additives for fishes and shrimps.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual,3rd edition,2001, NY, Cold Spring Harbor). The primers and the DNA sequences are synthesized by Shenzhen technology Limited.

The pPIC9K vector and the red yeast GS115 adopted by the invention are purchased from Nanjing Belding biotechnology limited, and the plasmid maps of the pPIC9K vector are respectively shown in figure 1; restriction enzyme and pfu DNA polymerase were purchased from TaKaRa, T4 DNA ligase was purchased from NEB, gel recovery kit and plasmid small quantity extraction kit were purchased from OMEGA, and plasmid large quantity extraction kit was purchased from Nucleo Bond; the inverted microscope is an Olympus product, and the fluorescent inverted microscope is a Nikon product. Protein Marker beijing holo-type gold biotechnology limited), PVDF membrane (available from Millipore corporation, usa), X-ray film (available from kodak corporation, usa), ECL color developing solution (available from prilley corporation, china), mouse anti-His mab (beijing holo-type gold biotechnology limited), rabbit anti-mouse HRP secondary antibody (beijing holo-type gold biotechnology limited), Acr, Bis, Tris, etc. (available from Sigma corporation), SDS (available from Amresco corporation), Tyrptone, Yeast Extract (available from OXOID corporation), PCR reaction tube (available from Fisher corporation), 0.22 μm sterile filter and dialysis bag (available from Millipore corporation), Ni2+ IDA affinity chromatography gel (zoonbio corporation) Agarose (available from shanghai gene corporation), DNA gel purification kit (available from ygen corporation), SACI (available from baobao biosciences), and conventional reagent is national analysis pure. Other reagents used in the present invention are commercially available unless otherwise specified.

Example 1

(1) And synthesizing a WAP + Crustinlike + pvHM117 serial expression sequence according to the sequence information of the WAP, Crustinlike and pvHM117 genes. As shown in FIG. 2, 3 protein signal peptide regions were truncated and then concatenated with a linker (green region), and the sequence was constructed between EcoRI and NotI cleavage sites (purple region) of ppic9K vector after codon optimization according to the Pichia expression system. The N-terminal double-chain-line sequence is an a-factor section signal sequence on ppic9K vector, and the C-terminal double-chain-line sequence is a His tag sequence.

(2) The constructed expression vector pPIC9K-WAP + Crustinlike + pvHM117 is transformed into the Top10 clone strain. The specific method comprises the following steps: taking out corresponding Top10 competent cells from a refrigerator at-80 deg.C, thawing on an ice box at room temperature, and labeling; adding 2 μ l of recombinant vector into competent cells, placing in a refrigerator at 4 deg.C, and ice-cooling for 30 min; thermally shocking in 42 deg.C water bath for 1min, and immediately placing on ice box for 2 min; adding 900 mul LB culture medium into each tube, shaking by a shaker at 37 ℃, and rotating at 150rpm for 45 min; adding 100 mul of the transformation liquid into a plate with ampicillin resistance, and uniformly coating the liquid; the plate was placed upside down in a 37 ℃ incubator overnight.

(3) Extracting and identifying recombinant plasmids: and (3) selecting the positive clone colony in the step (2), inoculating the colony to an LB culture medium containing ampicillin, and placing the colony on a constant temperature shaking table to culture for 20 hours at 37 ℃. Plasmid extraction is carried out by using a plasmid extraction kit, and the specific method refers to the kit description. The extracted plasmid is identified by EcoRI-NotI alkaline double enzyme digestion, and the enzyme digestion system is as follows: mu.l of plasmid, 0.25. mu.l of EcoRI, 0.25. mu.l of NotI, 1.0. mu.l of 10 XBuffer, 5.5. mu.l of ultrapure water, and the reaction tube was placed in a 37 ℃ water bath for 3 hours, and then 5. mu.l of the digested reaction solution was subjected to agarose electrophoresis. The results are shown in FIG. 3: m is DNA ladder 1000,2000,3000,4000,5000,6000,7000,8000,10000bp, 1 is plasmid before enzyme cutting, 2 is plasmid after enzyme cutting. The cleavage product contained two DNA bands: one corresponds to the molecular weight of the plasmid pPIC9K, and the other corresponds to the molecular weight of the WAP + Crustinlike + pvHM117 coupling sequence. The enzyme digestion experiment result shows that the extracted plasmid contains WAP + Crustinlike + pvHM117 target fragments, and the next experiment can be carried out.

(4) pPIC9K-WAP + Crustinlike + pvHM117 plasmid linearization: about 20. mu.g of plasmid DNA was subjected to SacI linearization, reaction system: 10 XBuffer 40. mu.L, SacI 20. mu.L, plasmid 20. mu.g, ultrapure water was added to 320. mu.L. Digest for 3h at 37 ℃. mu.L of the sample was subjected to 1% agarose gel electrophoresis, and the results are shown in FIG. 4. In the figure, M is a DNA standard (from bottom to 2000,3000,4000,5000,6000,8000,10000bp), 1 is a SacI digestion product, and 2 is a recovered target fragment.

(5) Transgenic yeast cell GS 115: 10uL of the linearized plasmid pPIC9K-WAP + Crustinlike + pvHM117 was added to a 1.5mLEP tube containing 80uL of Pichia competent yeast cells, mixed well and added to an electrotransformation cuvette of 0.2cm in diameter. The electric shock conditions are as follows: voltage 1700V, time 8mS, shock 2 times. 50uL, 100uL and 200uL of linearized plasmid mixed solution are respectively sucked and coated on a YPD plate, and cultured at constant temperature of 30 ℃ for 48 hours, when a colony grows out of the plate (the result is shown in figure 5), a single bacterium growing on the plate is picked by using an inoculating loop, and the single bacterium is inoculated into a test tube filled with 10ml of YPD liquid culture medium and cultured at 30 ℃ and 180rpm overnight.

(6) PCR identification of positive clone strains: 10 positive clones were selected, genomic DNAs (No. 12345678910) were extracted, and the desired gene was identified by PCR using 5 'pGAP prime and 3' AOX1 prime primers (SEQ ID No. 3: 5'-gaattcgttccaactagaca-3'; SEQ ID No. 4: 5'-gcggccgcttaatgatgatgat-3'), and all the results were positive. Positive clones were identified by PCR, with an expected band size of about 1.0K. As shown in fig. 6, in the figure: m is DNAmarker, and each band is 100,250,500,750,1000,2000bp from bottom to top; 1 is an idle plasmid amplification band, 2-11 is a positive clone bacterium PCR band, and 12 is a negative control.

(7) Small test expression: the size of the WAP + Crustinlike + pvHM117 protein is 36.11KDa, and the translated amino acid sequence is shown in SEQ ID No. 1. Taking 50 mu L of the identified positive strains 2#, 3#, 8#, 11#, inoculating into a conical flask filled with 10mL of BMGY, culturing overnight at 30 ℃ and 220r/min, and shaking until OD600 is 2-6 (logarithmic growth, about 16-18 h); centrifuging at room temperature for 5min at 5000r/min, collecting cells, removing supernatant, resuspending the cells with 10mL of BMMY, and performing induced expression; sampling 1mL of the culture medium every 24h, and adding methanol to a final concentration of 0.5% to continue induction; samples of 0, 24, 48, 72 and 96h were centrifuged at 10000r/min for 2min and subjected to SDS-PAGE.

(8) WB (Western blot) identification: and (3) after the SDS-PAGE electrophoresis in the step (11) is finished, cutting the gel to be in a proper size, and balancing the gel with a transfer membrane buffer solution for 20 min. Cutting several pieces of filter paper and one piece of PVDF membrane according to the size of gel, and soaking in membrane transfer buffer for 10 min. In the film transfer device, 24 layers of filter paper, PVDF film, gel and 24 layers of filter paper are stacked in sequence from the positive electrode to the negative electrode and are accurately aligned, and the glass rod carefully excludes air bubbles. And after the placement is finished, switching on a power supply, and performing constant-current transfer for 1-2h at a constant voltage of 20V or a constant current of 30 mA. After the transfer was completed, the PVDF membrane was taken out, and it was observed whether the pre-dyed marker transferred well. The PVDF membrane was removed with forceps and rinsed 3 times for 5min each time by soaking in PBS-T solution. The PVDF membrane is taken out and placed in the sealing liquid, and the PVDF membrane is stably shaken for 2 hours at room temperature. The PVDF membrane was then washed three times with PBST, 5min each. Putting the membrane into a plastic bag, marking the front surface of the membrane, then dropwise adding the diluted primary antibody into the bag, adding the 6-Histag monoclonal antibody, and incubating for 1h at 37 ℃. The membranes were removed and washed three times with PBS-T for 5min each. PVDF membrane was incubated with goat anti-mouse IgG-HRP antibody for 1-2h in the same manner. After the secondary antibody was applied, the membranes were removed and washed three times with PBS-T, 3-5min each. Then the membrane is put into a plastic bag, a proper amount of DAB color developing solution is dripped to be shaded and developed until a clear target protein band appears, ddH2O is used for stopping the reaction, the membrane is placed on filter paper, and after the membrane is dried completely, the membrane is photographed or sealed for storage.

The results are shown in FIG. 7, where Lane M: protein marker; 1: the GS115 strain is cultured for 72 hours to secrete supernatant; 2: culturing the 2# positive strain for 72 hours to secrete supernatant; 3: culturing the 2# positive strain for 96 hours to secrete supernatant; 4: culturing the 3# positive strain for 72 hours to secrete supernatant; 5: culturing the 3# positive strain for 96 hours to secrete supernatant; 6: 8# positive strain is cultured for 72 hours to secrete supernatant; 7: culturing the 8# positive strain for 96 hours to secrete supernatant; 8: culturing the 11# positive strain for 72 hours to secrete supernatant; 9: the 11# positive strain was cultured for 96 hours to secrete the supernatant. The analysis result shows that the supernatants of the strains No.2, No.3, No. 8 and No. 11 have target proteins, wherein the 11# strain has a better expression effect, and the supernatants of the cultures at 72 hours and 96 hours have recombinant protein expression.

(9) And (3) purifying the WAP + Crustinlike + pvHM117 recombinant protein: and (3) selecting the 11# strain to carry out strain amplification culture, culturing for 96 hours according to the method in the step (7), centrifuging the bacterial liquid at 5000rpm multiplied by 10min, and taking the supernatant to carry out protein purification. The specific method comprises the following steps: fixing the chromatographic column on the bracket, and closing the cap of the chromatographic column; slightly mixing Ni-NTA resin filler (the filler contains 30% ethanol with the same volume), loading 4-5ml into a chromatographic column, standing until the filler is completely naturally settled to the bottom, loosening a cap to allow 30% ethanol to flow out, and balancing the column once with buffer B with the volume of 8 times of the column volume; adding protein solution with about 8 times of column volume into the well-balanced column, adjusting a cap to control the flow rate, and naturally passing through the column under the action of gravity; the column was washed 2 times with 8 column volumes of buffer C and the effluent collected. Eluting the column with 1 column volume of buffer D for 4 times, and collecting effluent; eluting the column with 1 column volume of buffer E for 4 times, and collecting effluent; and taking the penetrating fluid and the effluent of each gradient to perform SDS-PAGE electrophoresis and WB (Western blot) identification, and analyzing the distribution condition of the recombinant protein in each tube. Protein concentration was determined using a protein quantification kit. The protein purification results are shown in fig. 8, 1: GS115 control strain was cultured for 96 hours supernatant; 2: the 11# positive strain was cultured for 96 hours in supernatant.

(10) WAP + Crustinlike + pvHM117 recombinant protein bacteriostasis experiment: adding 30uL of bacteria in logarithmic phase into LB culture medium, uniformly mixing and coating on a flat plate; the mixture was thoroughly soaked in a sterilized paper sheet in a WAP + Crustinlike + pvHM117 protein solution at a concentration of 0.03mg/ml, and then removed and applied to a plate for 20h of culture. PBS (phosphate buffer solution) is set as a negative control, the operation method is the same as the WAP + Crustinlike + pvHM117 protein experimental group, ampicillin and streptomycin drug sensitive paper sheets are set as positive controls, and the drug sensitive paper sheets are directly taken and pasted on a flat plate coated with bacteria.

The bacteriostatic effect was identified by zone of inhibition experiments using salmonella, vibrio alginolyticus and staphylococcus as experimental strains, respectively, and the results are shown in fig. 9, 10 and 11, respectively. In fig. 9, 1: cephalothin; 2: a recombinant protein; 3: PBS negative control; 4: ampicillin; 5: streptomycin; 6: bacitracin. In fig. 10, 1: cephalothin; 2: a recombinant protein; 3: PBS negative control; 4: ampicillin; 5: streptomycin; 6: bacitracin. In fig. 11, 1: streptomycin; 2: kanamycin; 3: florfenicol; 4: a recombinant protein; 5: PBS negative control. The experimental result shows that the recombinant protein has obvious bacteriostatic effect on staphylococcus and has certain bacteriostatic action on vibrio alginolyticus and salmonella.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Guangxi Zhuang nationality autonomous region aquatic science institute

<120> prawn coupling antibacterial peptide, gene, acquisition method of prawn coupling antibacterial peptide, expression vector, recombinant bacterium and application

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gaattcgttc caactagaca ttctagacca agaccacaac caaaaccaag accaggtact 60

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gattccgaat gtggacctgg tcaaaagtgt tgcccaattg gatgtggaag agagtgtttg 180

gctgttgttc caccatacgg ttctggtaga ggtggtggtg gttctggtgg tggtggatct 240

ggtggtggag gatctcagga taaggataag gctggtacta gattgggtgg tggttttggt 300

gttcctggtg ctggtggtgt ttttcctggt gctggaggtg ttcctggagt tggtggtgtt 360

ttccctggtg ctggtggagt ttttcctgga gctggtggta ttggtcctgg tcctggtggt 420

ttgattcctg gtggtggttt caattgtaat tattgtagaa ctcctgtcgg ttatgtttgt 480

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cctgtttgta gacaagattt ggattgttct ggttctgata agtgttgtta tgatgtttgt 600

ttggaagata ctgtttgtaa gcctattgtt gctggttctc aaggtggtgg tggtggttcc 660

ggtggtggtg gttcaggtgg tggtggttct ggaaagttta gaggttttgg tcagcctttt 720

ggtggtttgg gtggtcctgg tggaggtgtt ggtgttggtg gtggttttcc tggtggtgga 780

ttgggtgttg gtggaggttt gggtgttgga ggtggtttgg gagttggtgg aggattgggt 840

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Claims (1)

1. The application of the recombinant bacteria in preparing aquatic animal antibacterial drugs or feed additives;

transferring the expression vector into a Top10 clone strain, extracting a plasmid, and electrically converting the plasmid into pichia pastoris to obtain a recombinant strain;

connecting the gene between EcoRI site and NotI site of yeast expression vector pPIC9K to obtain expression vector;

the nucleotide sequence of the gene is shown as SEQ ID No. 2;

the pichia pastoris comprises pichia pastoris GS 115;

the aquatic animals include fish or shrimp;

the recombinant bacteria inhibit staphylococcus, vibrio fusiformis and salmonella.

Images