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Portunus trituberculatus anti-lipopolysaccharide factor PtALF-3 gene and encoding proteins and application thereof

A technology for resisting lipopolysaccharide factor and swimming crab, which is applied in the field of molecular biology and can solve the problem of less anti-lipopolysaccharide factor in swimming crab

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-3 gene and encoding proteins and application thereof
  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-3 gene and encoding proteins and application thereof
  • Portunus trituberculatus anti-lipopolysaccharide factor PtALF-3 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-3 gene of Portunus trituberculatus is shown in SEQ ID No.1.

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

[0027] ggggagtggg tgaggagcta ccagaaataa taacaacagc atcaaaagca

[0028] acgcccacca gttttcatca agtgttgccc ttgcttccct ccacgagctt

[0029] ccctcaag atg cgg aaa gga gtg gtg gcc ggc ctg tgc ctg

[0030] gca ctg gtg gtg atg tgc ctg tac ctg ccc cag cct tgc gag

[0031] gct cag tat gag gct ctg gta act tcc att ctt gga aaa ctc

[0032] act gga ctg tgg cac aac gac tcg gtg gac ttc atg ggc cac

[0033] att tgc tac ttc cgc cgc cgc cct aag atc aga aga ttt aag

[0034] ctg tac cac gag ggc aag ttt tgg tgt cct ggt tgg gcg cct

[0035] ttc gag ggc agg tcg agg aca aag ag agg agg tcg ggg tca tcc

[0036] agg gag gcc acc aag gac ttc gtg cgc aaa gct tta cag aac

[0037] gga ctc gtc aca cag cag gat gct tct ctg tgg ctg aat aac

[0038] taa agcagaggaa gtgagtgctg tgtacgacga ggaggagaaa

[0039] gaggataaca tgaaagtaac tgtct...

Embodiment 2

[0082] The base sequence of Portunus trituberculatus anti-lipopolysaccharide factor PtALF-3 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.

[0083] The sequence listing SEQ ID No.2 is:

[0084] Met Arg Lys Gly Val Val Ala Gly Leu Cys Leu Ala Leu Val Val Met

[0085] Cys Leu Tyr Leu Pro Gln Pro Cys Glu Ala Gln Tyr Glu Ala Leu Val

[0086] Thr Ser Ile Leu Gly Lys Leu Thr Gly Leu Trp His Asn Asp Ser Val

[0087] Asp Phe Met Gly His Ile Cys Tyr Phe Arg Arg Arg Pro Lys Ile Arg

[0088] Arg Phe Lys Leu Tyr His Glu Gly Lys Phe Trp Cys Pro Gly Trp Ala

[0089] Pro Phe Glu Gly Arg Ser Arg Thr Lys Ser Arg Ser Gly Ser Ser Arg

[0090] Glu Ala Thr Lys Asp Phe Val Arg Lys Ala Leu Gln Asn Gly Leu Val

[0091] Thr Gln Gln Asp Ala Ser Leu Trp Leu Asn Asn

[0092]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

[0096] In vitro antibacterial test of the anti-lipopolysaccharide factor PtALF-3 recombinant protein of Portunus trituberculatus:

[0097] 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 .

[0098] 2. Determination of the antibacterial activity of recombinant protein PtALF-3: Utilize the recombinant protein PtALF-3 obtained in the above examples to use Tris-HCl (50mM, pH=8.0) gradient dilution (1, 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, ...

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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-3 gene and encoding proteins and application thereof. In the invention, a PtALF-3 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-3 protein has obvious antibacterial activity to Gram-negative Vibrio alginolyticus, P. Aeruginosa, Edwardsiella tarda, Gram-positive Staphylococcus aureus, Micrococcus luteus and fungus Pichia pastoris, and the minimal inhibitory concentrations are respectively 0.58 mu M, 0.58 mu M, 9.26 mu M, 74.08 mu M, 18.52 mu M and 9.26 mu M. 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-3 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, traditional antib...

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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