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Portunus trituberculatus C-type lectin PtCLec2 gene and encoded protein thereof and application of encoded protein

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: 2019-10-11
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 effect of the variation of the Ca2+ binding site 2 motif on the gene function is still unclear

Method used

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  • Portunus trituberculatus C-type lectin PtCLec2 gene and encoded protein thereof and application of encoded protein
  • Portunus trituberculatus C-type lectin PtCLec2 gene and encoded protein thereof and application of encoded protein
  • Portunus trituberculatus C-type lectin PtCLec2 gene and encoded protein thereof and application of encoded protein

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Experimental program
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Effect test

Embodiment 1

[0029] The C-type lectin PtCLec2 gene of Portunus trituratus is the base sequence shown in SEQ ID No.1.

[0030] see figure 1 , the sequence listing SEQ ID No.1 is:

[0031]AAATAATAGTGGTTGCGGAACATTCCCTCTTGTCTCCCCGAGAATGCGAATCTACTTGCTTCTGGCGGTGGCGCTGGCGGTGATCTGCTCCGTAGCAGCCCAAGGCCGTGTGTTGGCGTTGCCGGAAATAGAACTATGTGATAATCGCCCAAAGCAGTGGAAGTTCCGCAACCACTATTATTTCTTCTCGTGGGACCAGGATGGCCCAGACTTCAAGGAAGTAAATCCTAAAACAGGACAACTGGAAGGTAGCAAGGTTGACTGGCTGAAAGCTCGCAACTTGTGCCGTCAGCGATGCATGGACGCTGTCGGCATGGAGAGCGAAGAGGAGAACAACATGATTTTCGACTTTATCAAAAGACGCAACATCACGTACATCTGGACGTCTGGCCGCCTCTGTGACTTCAAGGGGTGCGATGAGCGCGAGGACCTGAAGCCCATCAGTGTCAAGGGATGGTTCTTCTCCAACACCAACACTAAGATGGCCCCGACCAACGCATCGCCACCAGGCTGGAAGTACCAGCCATGGAGCGACAAGGGCCACACCGGTGGACCGCAGCCAGACAACGCTGAGTTCGATATCAACCAGACATCGGAGTCTTGTCTCGGCGTACTCAACAATCTGTACAATGACGGCATCAAATGGCACGACATTGCCTGCTACCACAAGAAGCCCTTCATTTGTGAGGACAGTGATGAACTACTTCGGTACATCGAGGGACAAAAGCAGCAACTGCAACAACAAAACCGCCCAGGGAACCAGGGACAGGGAAACCGTGGACAAGGAAACCGTGGACAAGGGAATAACGACAACGCCAA...

Embodiment 2

[0064] The base sequence of the C-type lectin of Portunus trituratus is described in SEQ ID No. 1 of the sequence listing, and the amino acid sequence is described in SEQ ID No. 2 of the sequence listing.

[0065] Sequence Listing SEQ ID No.2 is:

[0066] MRIYLLLAVALAVICSVAAQGRVLALPEIELCDNRPKQWKFRNHYYFFSWDQDGPDFKEVNPKTGQLEGSKVDWLKARNLCRQRCMDAVGMESEEENNMIFDFIKRRNITYIWTSGRLCDFKGCDEREDLKPISVKGWFFSNTNTKMAPTNASPPGWKYQPWSDKGHTGGPQPDNAEFDINQTSESCLGVLNNLYNDGIKWHDIACYGRGHKKPFICEDSDQELLYARANHRPGQFGRGRG

[0067] It has a complete coding protein containing 267 amino acids, the coding sequence has a signal peptide (1-19), the predicted molecular weight is 30.65kDa, and the isoelectric point is 7.53. The mature peptide contains 248 amino acids with a typical CRD domain (32-215), 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 galactose-recognizi...

Embodiment 3

[0071] 1. In vitro antibacterial test of C-type lectin PtCLec2 recombinant protein of Portunus trituratus

[0072] Culture and preparation of microorganisms: Vibrio alginolyticus was cultured with TSB medium at 28°C, Pseudomonas aeruginosa was cultured with TSB medium at 37°C, Staphylococcus aureus was cultured with LB medium at 37°C, and Micrococcus luteus was cultured with LB. The base was cultured at 37°C, Pichia pastoris was cultured with YPD medium at 28°C, and the above strains were cultured with a shaker at 220 rpm / min so that the bacterial concentration reached the logarithmic growth phase, and were diluted with 50 mM Tris-HCl (pH=8.0) buffer solution. Bacteria, so that the number of colonies per milliliter of bacterial solution is about 1 × 10 3 indivual.

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Abstract

The invention belongs to the technical field of molecular biology, and particularly discloses a portunus trituberculatus C-type lectin PtCLec2 gene and an encoded protein thereof and application of the encoded protein. A RACE technology and a unigene obtained by adopting transcriptome sequencing are adopted to acquire cDNA of the PtCLec2 gene from portunus trituberculatus through amplification, and it is found that a recombinant PtCLec2 gene has significant activity of bacterium inhibition, somatic agglutination and bacterium removal. The recombinant PtCLec2 gene has significant inhibiting effects on Gram-negative bacteria (vibrio alginolyticus and pseudomonas aeruginosa) and Gram-positive bacteria (staphylococcus aureus and micrococcus luteus). In the presence of Ca<2+>, the recombinant PtCLec2 gene has significant agglutination effects on vibrio alginolyticus, pseudomonas aeruginosa, staphylococcus aureus and micrococcus luteus. Meanwhile, the recombinant PtCLec2 gene has a removal effect on vibrio alginolyticus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to a C-type lectin PtCLec2 gene of Portunus trituratus and its encoded protein and applications. 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 bacterial binding protein, thioester bond-containing protein, scavenger receptor, sulfur-dependent lectin, hematin, Toll-like receptor and C type lectins. C-type lectins are a class of calcium-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 formed to maintain the conf...

Claims

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Application Information

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