Portunus trituberculatus mannose-binding lectin PtMBL gene, encoding protein thereof and application

A technology of Portunus trituberculatus and its encoded protein, which is applied in the field of Portunus trituberculatus mannose-binding lectin PtMBL gene and its encoded protein, and can solve the problems that Portunus trituberculatus has little research

Active Publication Date: 2019-05-24
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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  • Portunus trituberculatus mannose-binding lectin PtMBL gene, encoding protein thereof and application
  • Portunus trituberculatus mannose-binding lectin PtMBL gene, encoding protein thereof and application
  • Portunus trituberculatus mannose-binding lectin PtMBL gene, encoding protein thereof 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 particularly discloses a portunus trituberculatus mannose-binding lectin PtMBL gene, an encoding protein thereof and application.The portunus trituberculatus mannose-binding lectin PtMBL gene, the encoding protein and the application have the advantages that PtMBL gene cDNA [complementary DNA (deoxyribonucleic acid)] can be obtained from portunus trituberculatus by means of amplification by the aid of unigene and RACE technologies obtained by means of transcriptome sequencing, and obvious bacterial inhibition, bacterial binding and bacterial agglutination activity of recombinant PtMBL proteins are discovered; obvious inhibition effects can be realized by the recombinant PtMBL proteins for gram-negative bacteria (vibrioalginolyticus and pseudomonas aeruginosa) and gram-positive bacteria (staphylococcus aureus and micrococcus luteus), and the minimum inhibitory concentration is 0.81-1.61 micro-M, 0.81 micro-M, 0.81-1.61 micro-M and 3.22-6.44 micro-M; the recombinant protein PtMBL is obvious in binding activity for the vibrio alginolyticus, the pseudomonas aeruginosa, the staphylococcus aureus, the micrococcus luteus and pichia pastoris; in addition, obvious agglutination effects can be realized by the recombinant protein PtMBL for the vibrio alginolyticus, the pseudomonas aeruginosa, the staphylococcus aureus, the micrococcus luteus and the pichia pastoris in the presence of Ca2+; a foundation can be laid for controlling diseases for the portunus trituberculatus, and the portunus trituberculatus mannose-binding lectin PtMBL gene and the encoding protein have potential application values in the aspects of antibacterial medicine development, bacterial agglutination preparations, novel immunological 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 Applications(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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