Recombined bursicon protein for facilitating increase of caridina antimicrobial peptide and application thereof

An antibacterial peptide, rice shrimp technology, applied in the biological field, can solve the problems of shrimp and crab anorexia, muscle white turbidity, body surface ulcer, etc., and achieve the effect of reducing mortality and improving antibacterial ability.

Inactive Publication Date: 2019-03-01

TIANJIN NORMAL UNIVERSITY

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Problems solved by technology

In the process of shrimp and crab breeding, diseases often occur, including bacterial diseases and viral diseases, which cause an...

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Method used

(1) mortar, scissors and tweezers vessel are placed in oven in advance and bake 4h at 180 DEG C, for preventing RNase pollution, the mortar that has baked is poured appropriate amount after cooling at room tem...

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Abstract

The invention discloses a recombined bursicon protein for facilitating increase of a caridina antimicrobial peptide and application thereof. A Bursalpha protein has an amino acid sequence shown as SEQUENCE LISTING NO.2, and a Bursabeta protein has an amino acid sequence shown as SEQUENCE LISTING NO.4. Both the Bursalpha protein and the Bursbeta have the beneficial effect of improving the gene expression amount of the caridina antimicrobial peptide. By adopting the recombined bursicon protein, the antibacterial performance of aquaculture shrimps and crabs is improved, the death rate in aquaculture production is lowered remarkably, and the shrimps and crabs can grow healthily, so that the culturing yield and profit are improved greatly.

Application Domain

Peptide preparation methodsAnimals/human peptides

Technology Topic

Gene expressionShrimp +7

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Examples

Experimental program(1)
Effect test(1)

Example Embodiment

[0032] Zhejiang rice prawns are purchased from the Zhejiang aquatic product market. They are about 1.5-2.5cm in length and weigh about 50-100mg each. They are healthy and vigorous. The rice prawns are purchased and kept in the laboratory. Water aeration, the temperature is about 26-28°C. Shrimp food was fed twice a day, and fresh aeration water was changed every two days.

[0033] Short distance: time is 3s, 1000rpm.

[0034] Bursicon-α has the DNA sequence described in SEQUENCE LISTING NO.1.

[0035] The Bursa protein has the amino acid sequence described in SEQUENCE LISTING NO.2.

[0036] Bursicon-β has the DNA sequence described in SEQUENCE LISTING NO.3.

[0037] The Bursβ protein has the amino acid sequence described in SEQUENCE LISTING NO.4.

[0038] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0039] Step 1, Trizol method to extract total RNA

[0040] Take 1.5ml enzyme-free EP tube and place it in liquid nitrogen to pre-cool, select 3-4 rice shrimps and quickly put it into the pre-cooled EP tube, press Reagent (Thermo Fisher Scientific) reagent instructions to extract total RNA from rice shrimp, the specific steps are as follows:

[0041] (1) Put the mortar, scissors, tweezers and utensils in an oven in advance and bake at 180°C for 4 hours to prevent RNase contamination. Cool the baked mortar at room temperature 20~25°C and pour an appropriate amount of liquid nitrogen again Cool down to obtain a pre-cooled mortar. At this time, put the frozen rice shrimp in the pre-cooled mortar, and quickly grind it into powder with a grinding rod;

[0042] (2) Transfer 50 mg of powder to an enzyme-free EP tube that has added 1 mL of TRIzol (Chinese), mix thoroughly with a shaker, and let stand at room temperature 20~25℃ for 10 min, centrifuge at 4℃ for 10 min, the centrifugal force of centrifugation is 12000g;

[0043] (3) After centrifugation, transfer the supernatant to a new 1.5mL enzyme-free EP tube, let it stand at room temperature 20-25℃ for 5 minutes, add 200μL chloroform, shake with a shaker for 15s, and let it stand at room temperature 20-25℃ for 10 minutes;

[0044] (4) Centrifuge the static liquid at 4°C for 15 minutes, the centrifugal force of the centrifugation is 12000g, the liquid is divided into three layers in the EP tube: the upper layer is colorless and contains RNA, the middle layer is white flocculent containing DNA, and the lower layer is pink containing protein;

[0045] (5) Pipette the colorless upper layer solution into a new EP tube. When pipetting, be careful to prevent DNA from being absorbed. After pipetting, add 500 μL of isopropanol to the pipetted solution and mix it upside down by hand. Let it stand at room temperature 20~25℃ for 10min, and put it in the refrigerator at -20℃ for 1h (because the RNA content is less).

[0046] Take out the EP tube containing RNA from the refrigerator, centrifuge at 4°C for 10 minutes, with a centrifugal force of 12000g, discard the supernatant, and suck up the remaining liquid in the EP tube with a gun. Add 1 mL of the prepared 75 to the liquid sucked out by the gun. ﹪(Volume concentration) ethanol aqueous solution, repeatedly blow RNA with a gun to wash away impurities;

[0047] (6) Centrifuge the liquid obtained in step (5) at 4°C for 5 minutes in an EP tube with a centrifugal force of 7500g. Discard the supernatant liquid. Use a gun to suck the remaining liquid in the EP tube and place it on the filter paper and let it dry for 5-10 minutes. Allow ethanol to evaporate completely. Among them, the air-drying time is generally 5-10min, too long time will cause RNA degradation;

[0048] (7) Add 30 μL of enzyme-free water to dissolve the RNA according to the amount of RNA extracted to obtain an RNA-enzyme-free aqueous solution. If the RNA is not completely dissolved, use a water bath at 55℃ for 5 minutes to promote its dissolution;

[0049] (8) Use an ultra-micro spectrophotometer (Thermo Fisher Scientific) to detect the concentration of RNA in the RNA-enzyme-free aqueous solution: take 1 μL of the RNA-enzyme-free aqueous solution and use 1% agarose gel electrophoresis to detect the RNA extraction quality. The RNA-enzyme-free aqueous solution is frozen in Reserve in the refrigerator at -80℃.

[0050] Step 2. Synthesize the first strand of cDNA

[0051] Reference Reverse Transcription Kit ( One-step gDNA Remover and cDNA SynthesisSuperMix) instruction manual, reverse transcription to obtain the first strand of cDNA, the specific steps are as follows:

[0052] (1) Processing complex RNA

[0053] RNA enzyme-free water component: deionized water without RNase.

[0054]

[0055] Aspirate and mix the RNA-enzyme-free aqueous solution, then shortly separate, incubate at 65°C for 5 minutes, and then ice bath for 2 minutes, the temperature of the ice bath is 0°C.

[0056] (2) First strand cDNA synthesis and gDNA removal

[0057]

[0058] Continue to add the above reagents and short-circuit, and incubate at 42°C for 30 min.

[0059] (3) Heating at 85°C for 5s to make RT/RI and gDNA Remover are inactivated.

[0060] Step 3. Clone bursicon-α and bursicon-β genes

[0061] (1) According to the transcriptome prompts, design specific primers for amplifying bursicon-α and bursicon-β ORF regions-(bursicon-αF, bursicon-αR, and bursicon-βF and bursicon-βR, see Table 1.

[0062] (2) Using the cDNA obtained by reverse transcription as a template, a PCR machine is used to amplify the target gene fragment, and the reaction system is as follows:

[0063]

[0064] Add the corresponding reagents to the PCR tube according to the above requirements, and the reaction system is 25μL.

[0065] The PCR reaction program is as follows (the annealing temperature of bursicon-α and bursicon-β are 55°C and 60°C, respectively):

[0066]

[0067] (3) Connection: After detecting the PCR products by 1% agarose gel electrophoresis, expand the system, refer to the instruction manual of the agarose gel DNA recovery kit, cut and purify the target fragment, and detect with an ultra-micro spectrophotometer After cutting the gel and recovering the DNA concentration, ligate the target fragment to pMDTM-18-T Vector, and ligate overnight (12 hours) at 16°C to obtain the ligation products pMD18-T-α and pMD18-T-β. The system is as follows:

[0068]

[0069] (4) Transformation: using heat shock method to transfer the above-mentioned ligation products pMD18-T-α and pMD18-T-β into DH5α competent cells respectively, and the specific steps for transferring each linker into DH5α competent cells are as follows:

[0070] ①Inoculate the DH5α strain in 6ml of LB medium and reshake it to an OD value of 0.6. Take 1ml of the LB medium containing the DH5α strain in a 1.5ml EP tube and add 100μl of 0.1M Cacl 2 Aqueous solution treatment: Put the EP tube on ice for 5 hours, so that the temperature of the solution in the EP tube drops to 0 degrees Celsius.

[0071] ②Add the ligation product to DH5α competent cells, gently pipette to mix, and ice bath for 30 minutes to obtain a mixture.

[0072] ③Heat the mixture in a water bath at 42°C for 90s and then quickly transfer it to ice for 3 minutes to reduce the temperature of the mixture to 0°C.

[0073] ④ Add 1 mL of LB liquid medium to the mixture and incubate at 37°C and 150r/min for 45min.

[0074] ⑤ After centrifugation at 3000 rpm for 5 min, aspirate 900 μl of supernatant after centrifugation, and spread the remaining bacterial solution evenly on LB solid medium (medium with ampicillin), and incubate at 37°C for 12 hours (overnight).

[0075] (5) Pick a single colony: Use a sterilized toothpick to pick multiple single colonies of DH5α bacteria that have been transferred to the ligation product and place them in 50μl of ampicillin-resistant LB liquid medium at 37°C and 220r/min. After 3 hours of culture, the bacteria solution was obtained, and the bacteria solution was used as a template for PCR detection. The positive cloned bacteria solution was extracted and the plasmid was sent to Jinweizhi Biotechnology Co., Ltd. for sequencing.

[0076] Table 1 Specific primers used to amplify bursicon-α and bursicon-β ORF regions

[0077]

[0078] Step 4. Bioinformatics analysis of sequence

[0079] Use DNAStar (version7.1) software to analyze bursicon-α and bursicon-β gene sequence and amino acid sequence, use proteomics online website ExPASy (http://web.expasy.org/protparam/) to analyze protein physical and chemical properties, use SMART (http://smart.embl-heidelberg.de/) predict protein domain, SignalP 4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) detects whether the protein has signal peptide.

[0080] Prokaryotic expression protein of bursicon two subunit homodimer

[0081] Construction of bursicon-α and bursicon-β gene expression vectors

[0082] (1) According to the results of cloning, design specific primers to amplify the nucleotide sequence of the two subunit genes after removing the signal peptide-bursicon-αF', bursicon-αR', bursicon-βF', bursicon-βR', See Table 2, where a BamH I restriction site was added to the 5'end of the upstream primer of the specific primer, and Hind III restriction site was added to the 3'end of the downstream primer of the upstream primer of the two pairs of specific primers.

[0083] (2) Take the plasmid extracted from the single clone in step 3 (5) as a template, and use PCR to amplify the target gene fragment. The reaction system is as follows:

[0084]

[0085] Add the corresponding reagents to the PCR tube according to the above requirements, and the reaction system is 25μL.

[0086] The PCR reaction procedure is as follows (wherein, the annealing temperature of the primers of bursicon-α and bursicon-β fragments is 61°C and 64°C respectively):

[0087]

[0088] (3) According to the instructions of the plasmid extraction kit, extract the pET-28a-c(+) plasmid (the plasmid is stored in DH5α bacteria).

[0089] (4) Double enzyme digestion: After the PCR product is detected by electrophoresis, the PCR system is expanded, the target fragment is cut and purified, and the recovered DNA concentration is determined. The target gene fragments of bursicon-α and bursicon-β and pET-28a-c(+) plasmid were cut with restriction enzymes BamH I and Hind III.

[0090] The digestion system is as follows:

[0091]

[0092] After the digestion is completed, the target fragment and the large fragment of the vector are recovered and purified respectively. After measuring its concentration, electrophoresis detects the recovery quality of the target gene and plasmid fragment.

[0093] (5) Ligation: Use T4 DNA ligase to ligase the cut target fragment and vector, and the reaction condition is 16°C to obtain the ligated products pET-28a-α and pET-28a-β, respectively. The connection system is as follows:

[0094] Reagent

[0095] Step 5. Expression of Bursα and Bursβ

[0096] (1) Prepare Rosetta competent cells, extract expression plasmids pET-28a-α or pET-28a-β, and use heat shock method to transfer expression plasmids pET-28a-α and pET-28a-β into Rosetta competent cells. The specific steps are the same as step 3 (4) transformation.

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PUM

Property	Measurement	Unit
Relative molecular weight	13.15
Relative molecular weight	16.7
Relative molecular weight	12.7
tensile		MPa
Particle size		Pa
strength	10

Description & Claims & Application Information

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Recombined bursicon protein for facilitating increase of caridina antimicrobial peptide and application thereof

An antibacterial peptide, rice shrimp technology, applied in the biological field, can solve the problems of shrimp and crab anorexia, muscle white turbidity, body surface ulcer, etc., and achieve the effect of reducing mortality and improving antibacterial ability.

AI-Extracted Technical Summary

Problems solved by technology

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Abstract

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PUM

Description & Claims & Application Information

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