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PtCrustin-1 gene of Portunus trituberculatus, encoding protein of PtCrustin-1 gene and application of PtCrustin-1 gene

A technology of portunus trituberculatus and gene coding, which is applied in the field of molecular biology, can solve the problem that the research on Portunus trituberculatus Crustin is still scarce, and achieve significant antibacterial activity

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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  • PtCrustin-1 gene of Portunus trituberculatus, encoding protein of PtCrustin-1 gene and application of PtCrustin-1 gene
  • PtCrustin-1 gene of Portunus trituberculatus, encoding protein of PtCrustin-1 gene and application of PtCrustin-1 gene
  • PtCrustin-1 gene of Portunus trituberculatus, encoding protein of PtCrustin-1 gene and application of PtCrustin-1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] The PtCrustin-1 gene of Portunus trituberculatus is the base sequence shown in SEQ ID No.1.

[0025] The sequence listing SEQ ID No.1 is:

[0026]

[0027]

[0028] (a) Sequential features

[0029] ●Length: 1105bp (effective length 86-382)

[0030] ●Type: base sequence

[0031] ●Chain type: single chain

[0032] ●Topological structure: linear

[0033] (b) Molecular type: double-stranded DNA

[0034] (c) Assumption: No

[0035] (d) Antisense: no

[0036] (e) Original source: Portunus trituberculatus

[0037] (f) Specific name: CDS

[0038] The specific operation of building is as follows:

[0039] 1. Extraction of total RNA from Portunus trituberculatus and purification of mRNA: use Trizol reagent from Invitrogen Company to extract total RNA from Portunus trituberculatus, and use Oligitex mRNA purification kit from QIAGENE Company to purify mRNA.

[0040] 2. Construction of the cDNA library of Portunus trituberculatus: the cDNA library was constructed usi...

Embodiment 2

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

[0060] The sequence listing SEQ ID No.2 is:

[0061]

[0062] It has a complete coded protein containing 98 amino acids, of which 1-21 amino acids of the coding sequence are signal peptides, and the mature peptide contains 77 amino acids, with a predicted molecular weight of 8.78kDa and an isoelectric point of 4.97. 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 four cysteine ​​residues in the cysteine-rich region are relatively conservatively arranged 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 bond cores (4-DSC).

[0063] To obtain the recombinant protein of Portun...

Embodiment 3

[0066] In vitro antibacterial test of PtCrustin-1 recombinant protein from Portunus trituberculatus:

[0067] 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 with YPD medium at 28°C, culture each of the above strains with a shaker at 220rpm / min to make the bacterial concentration reach the logarithmic growth phase, dilute the cells with 50mM Tris-HCl (pH=8.0) buffer respectively, Make the number of colonies per milliliter of bacteria liquid about 1×10 3 .

[0068] 2. Determination of antibacterial activity of recombinant protein PtCrustin-1: Utilize the recombinant protein PtCrustin-1 obtained in the above-mentioned examples to use Tris-HCl (50mM, pH=8.0) gradient dilution (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256), 50 μl was added to a sterile flat-bottom 96-well plate (Costa...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a PtCrustin-1 gene of Portunus trituberculatus, encoding protein of the PtCrustin-1 gene and application of the PtCrustin-1 gene. Complementary deoxyribonucleic acid (cDNA) of the PtCrustin-1 gene is amplified from the Portunus trituberculatus by using a cDNA library and rapid-amplification of cDNA ends (RACE); and the gene has an important effect in the aspect of immune defence of the Portunus trituberculatus. Recombinant PtCrustin-1 protein has a remarkable effect of inhibiting the growth of Gram-negative bacteria, namely Pseudomonas aeruginosa and Gram-positive bacteria, namely Staphylococcus aureus and Micrococcus luteus, and the minimum inhibitory concentrations are 1.50mu M, 24.03mu M, 24.03mu M and 48.06mu M respectively; and the recombinant PtCrustin-1 protein does not have a remarkable effect of inhibiting the Gram-negative bacteria, namely Vibrio alginolyticus and a fungus, namely Pichia pastoris. The invention lays a foundation for the disease control, gene-assisted breeding, and development of feed additives of the Portunus trituberculatus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a PtCrustin-1 gene of Portunus trituberculatus 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 a WAP (whey acidic protein) domain, which is composed of 8 Cys. Sulfur-bonded core (4-DSC) formation. 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 carcinin, which was found in the granule blood cells of Carcinus maenas, and has obvious inhib...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P31/04A61P37/04A23K1/16A23L3/3544
Inventor 崔朝霞刘媛宋呈文李希红
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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