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Portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof

A technology of swimming crab and gene encoding, which is applied in the field of molecular biology and can solve the problems that the research on the crustin of the swimming crab is less and other problems.

Inactive Publication Date: 2012-07-18
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there are few studies on Portunus trituberculatus Crustin

Method used

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  • Portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof
  • Portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof
  • Portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The PtCrustin-2 gene of Portunus trituratus is the base sequence shown in SEQ ID No.1.

[0026] Sequence Listing SEQ ID No.1 is:

[0027]

[0028]

[0029] (a) Sequence features

[0030] ●Length: 629bp (effective length 58-405)

[0031] ●Type: base sequence

[0032] ●Chain type: single chain

[0033] ●Topology: Linear

[0034] (b) Molecular type: double-stranded DNA

[0035] (c) Assumption: No

[0036] (d) Antonym: No

[0037] (e) Original source: Portunus trituberculatus

[0038] (f) Distinctive name: CDS

[0039] The specific operation of the construction is as follows:

[0040] 1. Extraction of total RNA and purification of mRNA from Pool crab: Trizol reagent from Invitrogen company was used to extract total RNA from pool crab, and mRNA was purified by Oligitex mRNA purification kit from QIAGENE company.

[0041] 2. Construction of the cDNA library of Portunus trituratus: The cDNA library was constructed using the instructions of the Creator Smart cDN...

Embodiment 2

[0059] The base sequence of the PtCrustin-2 sequence table of PtCrustin-2 is described in SEQ ID No. 1 of the sequence table, and the amino acid sequence of the amino acid sequence is described in the SEQ ID No. 2 of the sequence table.

[0060] Sequence Listing SEQ ID No.2 is:

[0061]

[0062]

[0063] It has a complete encoded protein containing 98 amino acids, of which 1-20 amino acids of the coding sequence are signal peptides, and the mature peptide contains 95 amino acids, the predicted molecular weight is 10.58kDa, and the isoelectric point is 6.74. The mature peptide has a typical type I Crustin pattern structure, including a cysteine-rich region (Cys-rich region) and a WAP domain (WAP domain). Among them, the arrangement of 4 cysteine ​​residues in the cysteine-rich region is relatively conservative, in a C-(X3)-C-(X8-12)-C-C pattern; the WAP domain contains 8 cysteine ​​residues acid residues, which are capable of forming four disulfide cores (4-DSC).

[006...

Embodiment 3

[0067] In vitro antibacterial test of PtCrustin-2 recombinant protein from Portunus trituratus:

[0068] 1. Culture and preparation of microorganisms: TSB medium for Vibrio alginolyticus at 28°C, TSB medium for Pseudomonas aeruginosa at 37°C, LB medium for Staphylococcus aureus at 37°C, and LB medium for Micrococcus luteus 37°C, Pichia pastoris was incubated with YPD medium at 28°C, and each of the above strains was incubated with a shaker at 220 rpm / min so that the bacterial concentration reached the logarithmic growth phase, the cells were diluted with 50 mM Tris-HCl (pH=8.0) buffer solution, The number of colonies per milliliter of bacterial solution is about 1 × 10, respectively. 3 .

[0069] 2. Determination of the antibacterial activity of the recombinant protein PtCrustin-2: The recombinant protein PtCrustin-2 obtained in the above example was diluted with Tris-HCl (50mM, pH=8.0) in a gradient (1, 1 / 2, 1 / 4, 1 / 8, After 1 / 16, 1 / 32, 1 / 64, 1 / 128), 50 μl was added to a ste...

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Abstract

The invention belongs to the technical field of molecular biology and particularly relates to a portunus trituberculatus PtCrustin-2 gene, and coded protein and application thereof. The PtCrustin-2 gene DNA is amplified from portunus trituberculatus by utilizing a cDNA library and an RACE technology and the gene exerts important effect in the aspect of immunologic defence of the portunus trituberculatus. The recombinant PtCrustin-2 protein has obvious bacteriostatic activity on Gram negative bacteria and Gram positive bacteria. The minimum inhibitory concentrations of the gene on vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus and micrococcus luteus are 24.75 MuM, 12.37 MuM, 49.51 MuM and 49.51 MuM respectively. The gene does not have obvious bacteriostatic effect on fungi pichia pastoris. Basis is laid for diseases prevention and treatment on the portunus trituberculatus, gene auxiliary breeding and development of feed additives.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a PtCrustin-2 gene of Portunus trituratus and its encoded protein and application. Background technique [0002] Crustin is a cationic antimicrobial peptide rich in cysteine ​​residues found in crustaceans. Its molecular weight is 7-14kDa, the isoelectric point is between 7.0-8.7, the N-terminal contains a signal peptide, and the C-terminal contains the WAP (whey acidic protein) domain, which is composed of 8 Cys. Sulfur bond core (4-DSC) composition. According to the structure of the intermediate sequence region between the signal peptide and the WAP domain, Crustin can be divided into three types I-III. Type I Crustins are mainly found in crabs, lobsters and crayfish; Types II and III Crustins are mainly found in prawns. [0003] Crustin was originally named as carcinin, which was found in the granular blood cells of Carcinus maenas, and has obvious inh...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P31/04A61P37/04A23K1/17A23L3/3526A23K20/195
Inventor 崔朝霞刘媛宋呈文李希红
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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