Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 gene and encoding proteins and application thereof

A technology of anti-lipopolysaccharide factor and portunus trituberculatus, which is applied in the field of molecular biology, can solve the problem of low anti-lipopolysaccharide factor of portunus trituberculatus

Inactive Publication Date: 2012-02-01
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 reports on the anti-lipopolysaccharide factors of Portunus trituberculatus

Method used

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  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 gene and encoding proteins and application thereof
  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 gene and encoding proteins and application thereof
  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 gene and encoding proteins and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The base sequence of the anti-lipopolysaccharide factor PtALF-5 gene of Portunus trituberculatus is shown in SEQ ID No.1.

[0026] The sequence listing SEQ ID No.1 is:

[0027] ggggggagtta ccctcggaca ctgcaagggg aaggttgttc gctcctacta

[0028] acttaatcca tcttgtgagc ttgttccctt caac atg gtc cgc gga

[0029] ttt ctg gcg gtg gtg gtg gtc ggc gtg tgt cta cat ccc act

[0030] cca gca gct tcc ttt gac atc tcc agc ttc atc tcc act acg

[0031] gct gac acg gtg atc aag cgt ctc tac gtg gac cgg gaa atc

[0032] aat ctt ttc gat cac tac tgt atc ata agt cgc agt cct cac

[0033] atc agc agg tgg gaa ctc aag tgg caa gcg act gtt acc tgc

[0034] ccc gga tgg acg cct gtg aaa gga aaa gtg cgg ggc tac tcc

[0035] aac cct ttg tca gct gag cga gag gcc acc aga gac ttt gtg

[0036] cag aga atc ​​gta cag cga gga ctg gtc aca agg gac gag gcg

[0037] agt gag tgg ctg tga gctgcacgcc accacaccac cacaccgcta

[0038] cactacagca tcaccacacc acaccaccac tacactaagg ctcgtttttt

[0039] tttaagcact cctgcacttc...

Embodiment 2

[0081] The base sequence of Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 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.

[0082] The sequence listing SEQ ID No.2 is:

[0083] Met Val Arg Gly Phe Leu Ala Val Val Val Val Gly Val Cys Leu His

[0084] Pro Thr Pro Ala Ala Ser Phe Asp Ile Ser Ser Phe Ile Ser Thr Thr Thr

[0085] Ala Asp Thr Val Ile Lys Arg Leu Tyr Val Asp Arg Glu Ile Asn Leu

[0086] Phe Asp His Tyr Cys Ile Ile Ser Arg Ser Pro His Ile Ser Arg Trp

[0087] Glu Leu Lys Trp Gln Ala Thr Val Thr Cys Pro Gly Trp Thr Pro Val

[0088] Lys Gly Lys Val Arg Gly Tyr Ser Asn Pro Leu Ser Ala Glu Arg Glu

[0089] Ala Thr Arg Asp Phe Val Gln Arg Ile Val Gln Arg Gly Leu Val Thr

[0090] Arg Asp Glu Ala Ser Glu Trp Leu

[0091] It has a complete coded protein containing 125 amino acids, of which 1-20 amino acids of the coded sequence are signal peptides, and the mature pe...

Embodiment 3

[0095] In vitro antibacterial test of the anti-lipopolysaccharide factor PtALF-5 recombinant protein of Portunus trituberculatus:

[0096] 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 .

[0097] 2. Determination of antibacterial activity of recombinant protein PtALF-5: Utilize the recombinant protein PtALF-5 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), 50 μl was added to a sterile flat-bottomed 96-w...

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Abstract

The invention belongs to the technical field of molecular biology, in particular relates to a Portunus trituberculatus anti-lipopolysaccharide factor PtALF-5 gene and encoding proteins and application thereof. In the invention, a PtALF-5 gene cDNA is amplified from a Portunus trituberculatus by utilizing a cDNA (Complementary Deoxyribonucleic Acid) library and an RACE (Rapid-amplification of cDNA Ends) technology and plays an important role in immune defense of the Portunus trituberculatus. A recombinant PtALF-5 protein has stronger antibacterial activity to Gram-negative Vibrio alginolyticus and P. Aeruginosa, and the minimal inhibitory concentrations are respectively 7.77 mu M and 31.08 mu M; but the recombinant PtALF-2 protein has no obvious inhibitory action to Gram-positive Staphylococcus aureus, Micrococcus luteus and fungus Pichia pastoris. The invention lays a foundation for disease prevention and control, gene-assisted selection and development of feed additives of the Portunus trituberculatus.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a PtALF-5 gene of Portunus trituberculatus anti-lipopolysaccharide factor and its encoded protein and application. Background technique [0002] Portunus trituberculatus (Portunus trituberculatus) is an important marine economic crab. Due to its rich nutritional value and rapid growth, it has become an important marine aquaculture species in my country. However, with the continuous expansion of the breeding scale and the continuous improvement of the degree of intensification, various diseases caused by bacteria, fungi, etc. frequently broke out in the Portunus trituberculatus breeding population, causing huge economic losses and seriously restricting the development of Portunus trituberculatus aquaculture industry. Healthy and sustainable development. [0003] Due to the lack of in-depth understanding of the immune system of Portunus trituberculatus, tradi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C07K14/435A61K38/17A61P31/04A61P37/04A23K1/17A23L3/3544A23K20/195
Inventor 崔朝霞刘媛
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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