C-type lectin PtCLec1 gene of portunus trituberculatus and encoded protein and application thereof

A technology of Portunus trituberculatus and gene encoding, which is applied in the field of molecular biology and can solve problems such as unclear influence of gene function

Active Publication Date: 2019-10-18
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the C-type lectins of Portunus trituberculatus, and the

Method used

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  • C-type lectin PtCLec1 gene of portunus trituberculatus and encoded protein and application thereof
  • C-type lectin PtCLec1 gene of portunus trituberculatus and encoded protein and application thereof
  • C-type lectin PtCLec1 gene of portunus trituberculatus and encoded protein and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0029] The C-type lectin PtCLec1 gene of Portunus trituberculatus has the base sequence shown in SEQ ID No.1.

[0030] Depend on figure 1 It can be seen that the sequence table SEQ ID No.1 is:

[0031] GTAAGAATGACTGTTGGGTGTTTCTCCGTACAGCCACTCTTATGGGAAAGCCGGTCTGTTCGTAGTACAATGCAGAGATAGGGGTTCGGCTTGCGCAAGACGAGAGCACCAGCAAAACAGTCGTGCATATATGTAAGGTGGATCGCGGCACAGAACAGCACACATCCTCCTTCAGCTGCACACTTTGCCTCCACCTGCTCCGCCGCCAACACTTGCATCATGAGTCGCTACTTGCTCCTCGCATTCTTCGGAGCCTTGACTGTGGAGGCGACGACACATGTGAACTTATCTGACAGTATCTCCGGTGATTCTTCGGGGGGCTGCAGCTCCCCGTACACTATGGTTGCTGGCCGCTGCTTATACATTAACTTCGCTACCTCTGGCACTTGGGACCGCTTCCACTCCATGTACTGCAAAGACACAGGAGGGAATGACCTGGTGACAGTGGACGACGCTAACTTCCTTGCCGACCTTGTTCAGTACATCACAAGTATTGGATGGCAAGACGGTTTGTTCTGGATCGGTGCCTCGGACAAGAGTCACGAGGGCCACTGGATATGGCCTAACGGCTCCCCCGTCAAGATGGGTACACCCTTCTGGGGCACGTACGGCTGCTACAACCAGCAGTATCCCGATGGAGGAACCAATGAAAACTGCGCAATACTAGATGGAAATGCAAATCTATATTTCAACGACTTTAAATGTGACACAGCATTAGCTGTCTTCGGTATCTGCGAGAAGAATATCTAGGAAAATTATTGAGCTTATAATTTAGTAATAATGTATGTCA...

Embodiment 2

[0063] The base sequence of the C-type lectin of Portunus trituberculatus 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.

[0064] The sequence listing SEQ ID No.2 is:

[0065] MSRYLLLAFFGALTVEATTHVNLSDSISGDSSGGCSSPYTMVAGRCLYINFATSGTWDRFHSMYCKDTGGNDLVTVDDANFLADLVQYITSIGWQDGLFWIGASDKSHEGHWIWPNGSPVKMGTPFWGTYGCYNQQYPDGGTNENCAILDGNANLYFNDFKCDTALAVFGICEKNI

[0066] It has a complete coded protein containing 176 amino acids, the coded sequence has a signal peptide (1-17), the predicted molecular weight is 19.285kDa, and the isoelectric point is 4.45. The mature peptide contains 159 amino acids with a typical CRD domain (35-173), which contains 4 cysteine ​​residues capable of forming two disulfide bonds. The CRD domain lacks the typical vertebrate and invertebrate mannose-recognizing motif EPN (Glu-Pro-Asn), but contains an unusual YPD motif (Tyr-Pro-Asp), which may It is mutated from the QPD (Gln-...

Embodiment 3

[0070] 1. In vitro antibacterial test of the C-type lectin PtCLec1 recombinant protein of Portunus trituberculatus:

[0071] Culture and preparation of microorganisms: Vibrio alginolyticus was cultured in TSB medium at 28°C, Vibrio parahaemolyticus was cultured in TSB medium at 28°C, Pseudomonas aeruginosa was cultured in TSB medium at 37°C, Staphylococcus aureus was cultured in LB Base cultured at 37°C, Micrococcus luteus cultured at 37°C with LB medium, Pichia pastoris with YPD medium at 28°C, and the above-mentioned strains were cultured on a shaker at 220rpm / min to make the bacterial concentration reach the logarithmic growth phase, respectively. 50mM Tris-HCl (pH=8.0) buffer solution dilutes the bacterium so that the number of colonies per milliliter of the bacterium solution is about 1×10 3 indivual.

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a C-type lectin PtCLec1 gene of portunus trituberculatus and an encoded protein and application thereof.Through unigene obtained by transcriptome sequencing and the RAC technology, amplification is conducted in the portunus trituberculatus to obtain PtCLec1 gene cDNA, and the recombinant protein PtCLec1has obvious bacteriostasis, bacterium binding and bacterium agglutination activity. The recombinant protein PtCLec1 has an obvious inhibition effect on gram negative bacteria and gram-positive bacteria. The recombinant protein PtCLec1 has obvious binding activity to vibrio alginolyticus, vibrio parahaemolyticus, pseudomonas aeruginosa, micrococcus luteus and pichia pastoris. In the presence of Ca<2+>, the recombinant protein PtCLec1 has an obvious agglutination effect on vibrio alginolyticus, vibrio parahaemolyticus, pseudomonas aeruginosa, staphylococcus aureus, micrococcus luteus and pichiapastoris.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a C-type lectin PtCLec1 gene of Portunus trituberculatus and its encoded protein and application. Background technique [0002] Invertebrates lack an adaptive immune system, which recognizes "non-self" substances mainly by recognizing pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Invertebrate pattern recognition receptors mainly include: peptidoglycan recognition protein, Gram-negative bacteria binding protein, sulfur ester bond-containing protein, scavenger receptor, sulfur-dependent lectin, hemoglobin, Toll-like receptor and C type lectins. C-type lectins are a class of calcium ion-dependent proteins, all of which have a carbohydrate recognition domain (CRD) that can recognize and bind carbohydrates. The CRD domain generally contains four very conserved cysteines, through which two pairs of disulfide bonds are for...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P31/04A61P37/04A23K20/147A23L3/3526
CPCC07K14/43509A61P31/04A61P37/04A23K20/147A23L3/3526A61K38/00A23V2002/00A23V2250/54Y02A50/30
Inventor 刘媛苏越崔朝霞
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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