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Portunus trituberculatus anti-lipopolysaccharide factor PtALF-2 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-2 gene and encoding proteins and application thereof
  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-2 gene and encoding proteins and application thereof
  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-2 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-2 gene of Portunus trituberculatus is shown in SEQ ID No.1.

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

[0027] ggggagtcac tatccgaggt accccaaaga gggacatcaa aagcattgaa

[0028] gactacgcaa ctaaacttcg ctcaag atg cgg aaa ggg gtg gtg

[0029] acc ggc ctg ttc gtg gcg ctg gtg gtg atg tgt ctg tac ttg

[0030] ccc cag ccc tgc gag gct cag tat gaa gct ctg acg gct gct

[0031] att ttg aca aag ctg tca aaa atg tgg cac agt gac act ctg

[0032] aat ttc ttg ggc cac acc tgc cac gtc agc cgt act ccg acg

[0033] gtc aag cgc ttt aag ctg tac tgg aag ggc aag ttt tgg tgt

[0034] cct ggt tgg gcg cct ttc agt ggc act tcg agg acc aag agc

[0035] agg tca gga tca gcc aga gag gcc acc aag agt ttt gtg gac

[0036] caa gct tta cag cgc aga ctt atc acg cag caa gaa gcg gac

[0037] ctc tgg ctg aag ggg taa tgggtgctca cgccgatgca tggggaggag

[0038] gccgagtggg aggagagtga agataaagaa gaaatgactt gtgtctgatc

[0039] taaaacgagt ttttaaaatt...

Embodiment 2

[0081] The base sequence of Portunus trituberculatus anti-lipopolysaccharide factor PtALF-2 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 Arg Lys Gly Val Val Thr Gly Leu Phe Val Ala Leu Val Val Met

[0084] Cys Leu Tyr Leu Pro Gln Pro Cys Glu Ala Gln Tyr Glu Ala Leu Thr

[0085] Ala Ala Ile Leu Thr Lys Leu Ser Lys Met Trp His Ser Asp Thr Leu

[0086] Asn Phe Leu Gly His Thr Cys His Val Ser Arg Thr Pro Thr Val Lys

[0087] Arg Phe Lys Leu Tyr Trp Lys Gly Lys Phe Trp Cys Pro Gly Trp Ala

[0088] Pro Phe Ser Gly Thr Ser Arg Thr Lys Ser Arg Ser Gly Ser Ala Arg

[0089] Glu Ala Thr Lys Ser Phe Val Asp Gln Ala Leu Gln Arg Arg Leu Ile

[0090] Thr Gln Gln Glu Ala Asp Leu Trp Leu Lys Gly

[0091]It has a complete coded protein containing 123 amino acids, of which 1-26 amino acids of the coding sequence are signal peptides, and the m...

Embodiment 3

[0095] In vitro antibacterial test of the anti-lipopolysaccharide factor PtALF-2 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 Edwardsiella tarda at 28°C, and LB medium for Staphylococcus aureus 37°C, LB medium for Micrococcus luteus at 37°C, YPD medium for Pichia pastoris at 28°C, culture each of the above strains on a shaker at 220rpm / min to make the bacterial concentration reach the logarithmic growth phase, respectively use 50mM Tris-HCl (pH=8.0) the buffer solution dilutes the thalline so that the number of colonies per milliliter of the bacterium solution is about 1×10 3 .

[0097] 2. Determination of antibacterial activity of recombinant protein PtALF-2: Utilize the recombinant protein PtALF-2 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, ...

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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-2 gene and encoding proteins and application thereof. In the invention, a PtALF-2 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-2 protein has obvious antibacterial activity to Gram-negative Vibrio alginolyticus, P. Aeruginosa, Edwardsiella tarda, Gram-positive Staphylococcus aureus and Micrococcus luteus, and the minimal inhibitory concentrations are respectively 1.02 mu M, 2.04 mu M, 4.07 mu M, 16.30 mu M and 65.19 mu M; but the recombinant PtALF-2 protein has no obvious inhibitory action to 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, in particular to a PtALF-2 gene of Portunus trituberculatus anti-lipopolysaccharide factor, its encoded protein and its 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, traditional anti...

Claims

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Application Information

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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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