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Polypeptide separated from hexagrammos otakii

A gene and microbial technology, applied in the field of antibacterial polypeptide screening, can solve the problems of reduced growth rate of farmed animals

Active Publication Date: 2018-03-23
MARINE BIOLOGY INST OF SHANDONG PROVINCE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, exogenous antimicrobial peptides often cause stress reactions in fed animals due to bioincompatibility; cause problems such as reduced growth rates in farmed animals

Method used

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  • Polypeptide separated from hexagrammos otakii
  • Polypeptide separated from hexagrammos otakii
  • Polypeptide separated from hexagrammos otakii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Embodiment 1: Screening of antibacterial polypeptide

[0017] By in vitro injection of bacterial immunization to the reared Hexa ostaki, the livers of the experimental group and the control group were extracted, and then differential display PCR (DDRT-PCR) was performed to obtain differentially expressed genes.

[0018] Specific steps are as follows:

[0019] 1) Prepare fish for experiment

[0020] Put the Otaki hexaline fish into the net cage for breeding, select fish with a weight of about 150-200g, and feed them fresh miscellaneous fish such as jade tendon fish, once in the morning and once in the evening, and the feeding amount is 3% of the fish's body weight. Prepare in vitro injection of strains for infection experiments in about a week;

[0021] 2) Inoculate Aeromonas hydrophila, Vibrio parahaemolyticus, Edwardsiella lentus, and Staphylococcus aureus into peptone medium for culture, shake culture until the logarithmic growth phase of bacteria, and mix various b...

Embodiment 2

[0030] Embodiment 2: Antibacterial detection of HO-AP-1 polypeptide

[0031] The antibacterial properties of HO-AP-1 polypeptide against Aeromonas hydrophila, Vibrio parahaemolyticus, Edwardsiella tarda and Staphylococcus aureus were detected.

[0032] Specific steps are as follows:

[0033] 1. Activation treatment of bacterial strains: Obtain purified single colonies of Aeromonas hydrophila, Vibrio parahaemolyticus, Edwardsiella tarda, and Staphylococcus aureus by streaking on a solid medium. Select and expand the culture in 25ml liquid LB medium respectively, and dilute the cultured bacteria solution to a concentration of 5×10 5 CFU / mL, take 60 μl in turn and add to each well of a 96-well plate to prepare for the experiment.

[0034] 2. After the recombinantly expressed HO-AP-1 polypeptide is quantified, it is sequentially diluted in liquid medium. 40 μl of the diluted antimicrobial peptides were sequentially added to each well of the 96-well plate, and the reaction syste...

Embodiment 3

[0038] Embodiment 3: Recombinant expression of HO-AP-1 polypeptide

[0039] 1. Linking the nucleotide sequence of the HO-AP-1 polypeptide (SEQ ID NO: 1) into the expression vector of Pichia pastoris. Both the vector containing the antimicrobial peptide gene and the yeast expression vector were digested with XhoI and XbaI, and the digested products were recovered and ligated for PCR identification and sequencing.

[0040] 2. After the positive plasmid was linearized by SacI single enzyme digestion, it was added to the competent cell suspension of Pichia pastoris. After electroporation, spread evenly on YPDS selection plate containing 100 μg / mL Zeocin, and incubate at 30°C for 3-5 days. When the positive transformants on the YPDS plate grow larger, each transformant is inoculated onto the YPDS selection plate containing Zeocin 200 μg / mL, 500 μg / mL, and 1000 μg / mL in turn, and the colonies that grow normally on the high-concentration Zeocin plate are Possibly high copy recombin...

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Abstract

The invention provides a polypeptide separated from hexagrammos otakii. Amino acid sequences of the polypeptide are shown as SEQ ID NO. 2. The polypeptide can be used for preparing disease-resistant microbial products and feed additives. The polypeptide separated from the hexagrammos otakii jordan and starks has the advantages that the polypeptide has antibacterial activity, is obvious in resistance to aquatic disease germs such as aeromonas hydrophila, vibrio parahaemolyticus, edwardsiella tarda and staphylococcus aureus, is free of strong stress response on the hexagrammos otakii jordan andstarks and can be used as a feed additive for the hexagrammos otakii jordan and starks.

Description

technical field [0001] The invention belongs to the technical field of antibacterial polypeptide screening, and in particular relates to a polypeptide isolated from six-line fish. Background technique [0002] Hexagrammos otakii, also known as Hexagrammos otakii, also known as Hexagrammos otakii, commonly known as yellow croaker, belongs to the order Scorpaeniformes, the family Hexagrammidae, and the genus Hexagrammos. Sexual offshore demersal reef fish. It is mainly distributed along the coasts of the Yellow Sea and the Bohai Sea, and is also found in the seas of North Korea, Japan and the Far East of Russia. This fish is resistant to low temperature and its survival temperature is 2-26°C. In China, it is mainly produced in coastal rocky sea areas such as Shandong and Liaoning. Its tender meat and delicious taste are known as "Northern Grouper". It is deeply loved by consumers and has high economic value. Dataki hexaline is an ideal species for cage culture in northern C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/46C12N15/12A61K38/17A61P31/04A23K20/147A23K50/80
CPCA23K20/147A23K50/80A61K38/00C07K14/461
Inventor 胡发文李莉王雪刘元文菅玉霞潘雷高凤祥郭文
Owner MARINE BIOLOGY INST OF SHANDONG PROVINCE
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