Mannose-binding lectin ptmbl gene of Portunus trituratus and its encoded protein and application

The invention relates to a technology for encoding a protein of Portunus trituratus, which is applied in the field of the mannose-binding lectin PtMBL gene and its encoded protein of Portunus trituratus, and can solve the problem that the research on the Portunus trituratus is still lacking.

Active Publication Date: 2022-04-15
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are few studies on PtMBL of Portunus trituberculatus

Method used

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  • Mannose-binding lectin ptmbl gene of Portunus trituratus and its encoded protein and application
  • Mannose-binding lectin ptmbl gene of Portunus trituratus and its encoded protein and application
  • Mannose-binding lectin ptmbl gene of Portunus trituratus and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The base sequence of the mannose-binding lectin PtMBL gene of Portunus trituberculatus is shown in SEQ ID No.1.

[0038] The sequence listing SEQ ID No.1 is:

[0039]GTTCCTCAGGTTTGATGCTCGGGGACTTCTCCTCCGCAAGATGAAGGTCGCTGTCGTCCTGCTGTCGTGCCTCGCCTTCGCCGCCTCCTCCCGGCGTCCCTACCCGAGCCGCTACCCAAGCTCCGGCGGCGGCTTCCCTGGCAGAGGCTCAGTTATCGTCTTCCCCGACGAGGTCAAGGGCGGTGGAGGAAGCGGGGGCCACGTCCACCCTGGAGTTGGTGTTGGGAATATTTACCCCCCACCAGTCGGTCATGGTGGCGAGCCTCTGGTCACTAGCCACTGCCCACGCGCTATTTCCAAAGATGTCCACGGCACCTTCCTGGGACACAACTACCACTTCTCTTGGTGCGCTGATGGCGGCCAGCGCTACACTTGGGAGGCCGCCCGCGACTACTGCACCAGGCTGGGCCCCGGCTGGTACCCTGTGGCCATCGAAAGTAGGGATGAAGACAACTACATCATCGACATTGTTGGCAGCCACCAATCTCCCTGGATCTGGACTGGCGGGAACACTTTGAGCAACACTAACTACGTCTGGCAATGGCTGGATGGAAGTTCTCTCATCTACACTAACTGGGGACAGACTGGATCATTGGACAGACCACAGCCAGACAACGCCGAAAACAACAACGAACGGTGCCTGGCTATCCTCAACCAGTTCTACGCAGGAGACTTCATTACTTTCCACGACATCGGATGCCATCACACCAAACCAACCATCTGCGAGAACTCTAATGTCCAGGTTCCCGTCCCCGTGCCCCAGTATGGTTAAGAGCGATGGCTAAGGTATAATAGGTTCGGAGATAGTGACTGTCCAACG...

Embodiment 2

[0071] The base sequence of Portunus trituberculatus mannose-binding lectin PtMBL is described in SEQ ID No. 1 in the sequence listing, and the amino acid sequence is described in SEQ ID No. 2 in the sequence listing.

[0072] The sequence listing SEQ ID No.2 is:

[0073]MKVAVVLLSCLAFAASSRRPYPSRYPSSGGGFPGRGSVIVFPDEVKGGGGSGGHVHPGVGVGNIYPPPVGHGGEPLVTSHCPRAISKDVHGTFLGHNYHFSWCADGGQRYTWEAARDYCTRLGPGWYPVAIESRDEDNYIIDIVGSHQSPWIWTGGNTLSNTNYVWQWLDGSSLIYTNWGQTGSLDRPQPDNAENNNERCLAILNQFYAGDFITFHDIGCHHTKPTICENSNVQVPVPVPQYG

[0074] It has a complete coded protein containing 243 amino acids, 1-16 amino acids of the coding sequence are signal peptides, the mature peptide contains 227 amino acids, the predicted molecular weight is 24.87kDa, and the isoelectric point is 5.95. The mature peptide lacks the cysteine-rich, collagen-like, and neck regions reported in vertebrates, but has a typical CRD domain (92-229) containing four cysteine ​​residues capable of forming Two disulfide bonds (C 11...

Embodiment 3

[0078] 1. Antibacterial test in vitro of recombinant protein of Portunus trituberculatus mannose-binding lectin PtMBL:

[0079] Culture and preparation of microorganisms: culture Vibrio alginolyticus in TSB medium at 28°C, Pseudomonas aeruginosa in TSB medium at 37°C, Staphylococcus aureus in LB medium at 37°C, Micrococcus luteus in LB medium Cultivate at 37°C, Pichia pastoris was cultured with YPD medium at 28°C, and each of the above strains was cultured on a shaker at 220rpm / min to make the bacterial concentration reach the logarithmic growth phase, and the bacteria were diluted with 50mM Tris-HCl (pH=8.0) buffer respectively. body, so that the number of colonies per milliliter of bacterial liquid is about 1×10 3 indivual.

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Abstract

The invention belongs to the technical field of molecular biology, and specifically relates to a PtMBL gene of Portunus trituberculatus mannose-binding lectin, its encoded protein and its application. The present invention uses the unigene and RACE technology obtained by transcriptome sequencing to amplify the PtMBL gene cDNA from Portunus trituberculatus, and finds that the recombinant PtMBL protein has significant bacteriostasis, bacterium binding and bacterium agglutination activities. The recombinant protein PtMBL has obvious inhibitory effects on Gram-negative bacteria (Vibrio alginolyticus, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus, Micrococcus luteus), and the minimum inhibitory concentrations are 0.81~1.61μM, 0.81μM, 0.81~1.61μM, 3.22~6.44μM. The recombinant protein PtMBL has obvious binding activity to Vibrio alginolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus and Pichia pastoris. In addition, in Ca 2+ In the presence of the recombinant protein PtMBL, it has obvious agglutination effect on Vibrio alginolyticus, Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus and Pichia pastoris. The invention lays a foundation for the disease prevention and treatment of portunus trituberculatus, and has potential application value in the development of antibacterial drugs, bacterial agglutination preparations, new immune preparations, feed additive production and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a gene of Portunus trituberculatus mannose-binding lectin PtMBL, its encoded protein and its application. Background technique [0002] The complement system is an important part of the innate immune system and a link between innate immunity and acquired immunity. The activation of the vertebrate complement system is mainly through three pathways, namely the classical pathway, the lectin pathway and the alternative pathway. Invertebrates lack real antibodies and specific immune cells, and the body's defense response mainly relies on the innate immune system, in which the complement lectin pathway does not require the presence of antigen-antibody complexes in the classical pathway, nor does it lack the specificity of the alternative pathway. It is activated by the combination of pattern recognition receptors and microbial polysaccharides, which has very impor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435A23K20/147A23L3/3526A61K38/17A61P31/04A61P31/10
Inventor 刘媛崔朝霞张孟洁
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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