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Portunus trituberculatus antilipopolysaccharide factor PtALF-4 gene and coding protein and applications 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: 2012-01-25
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 antilipopolysaccharide factor PtALF-4 gene and coding protein and applications thereof
  • Portunus trituberculatus antilipopolysaccharide factor PtALF-4 gene and coding protein and applications thereof
  • Portunus trituberculatus antilipopolysaccharide factor PtALF-4 gene and coding protein and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The anti-lipopolysaccharide factor PtALF-4 gene of Portunus trituberculatus is the base sequence shown in SEQ ID No.1.

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

[0027] ggggactccc tcaccggcta caacacaggc acgtgaaaca cacacacagg

[0028] tgactccacg gacaccttat cttaaacagg atg cat gtg tcc cgg

[0029] cca tgg gca gtg gtg gtg aca gtg gtg gtg gtg atg gcc cta

[0030] ccc aca ccc acg caa ggg ggg tgg cta gac att gta aaa gcc

[0031] atc gta gta gta cct gca gcg aga gaa acc atc aag aca caa gaa

[0032] atc acg ctg ctg gat cac tac tgc acg ttg tcg agg agt ccc

[0033] tac att aag agc ctt gag ctg cac tac aga gcg gaa gtg acc

[0034] tgc cct ggc tgg acc atc att agg gga aga ggc tct aat cac

[0035] aga aac cca acc aac tcc ggg aaa gac gct ctg aag gac ttt

[0036] atg acg cag gcg gtg gcg gca gga cta gtc act aag gaa gaa

[0037] gcg gca ccc tgg ctc aac cac cgc taa gccacaccca

[0038] gcacaccctt cactccccctt ctacacttct tccttggact gtatttctaa

[0039] gcgtttcggc gactaatctc gactgca...

Embodiment 2

[0088] The base sequence of Portunus trituberculatus anti-lipopolysaccharide factor PtALF-4 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.

[0089] The sequence listing SEQ ID No.2 is:

[0090] Met His Val Ser Arg Pro Trp Ala Val Val Val Thr Val Val Val Val

[0091] Met Ala Leu Pro Thr Pro Thr Gln Gly Gly Trp Leu Asp Ile Val Lys

[0092] Ala Ile Val Val Pro Ala Ala Arg Glu Thr Ile Lys Thr Gln Glu Ile

[0093] Thr Leu Leu Asp His Tyr Cys Thr Leu Ser Arg Ser Pro Tyr Ile Lys

[0094] Ser Leu Glu Leu His Tyr Arg Ala Glu Val Thr Cys Pro Gly Trp Thr

[0095] Ile Ile Arg Gly Arg Gly Ser Asn His Arg Asn Pro Thr Asn Ser Gly

[0096] Lys Asp Ala Leu Lys Asp Phe Met Thr Gln Ala Val Ala Ala Gly Leu

[0097] Val Thr Lys Glu Glu Ala Ala Pro Trp Leu Asn His Arg

[0098] It has a complete coded protein containing 125 amino acids, of which 1-25 amino acids of the coding sequence are signal peptides, an...

Embodiment 3

[0102] In vitro antibacterial test of the anti-lipopolysaccharide factor PtALF-4 recombinant protein of Portunus trituberculatus:

[0103] 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 .

[0104] 2. Determination of the antibacterial activity of recombinant protein PtALF-4: Utilize the recombinant protein PtALF-4 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, and particularly relates to a portunus trituberculatus antilipopolysaccharide factor PtALF-4 gene and coding protein and applications thereof. The gene is amplified to the PtALF-4 gene cDNA from the portunus trituberculatus by utilizing a cDNA library and an RACE (rapid-amplification of cDNA ends) technology and plays an important role in the aspect of the immunologic defense of the portunus trituberculatus. The recombined PtALF-4 protein has stronger bacteriostatic activity for Gram-negative bacterium vibrio alginolyticus, pseudomonas aeruginosa, wdwardsiella tarda and fungal pichia pastoris, and the minimum inhibitory concentrations are 3.32muM, 53.14muM, 13.28muM and 106.28muM, however, the recombined PtALF-4 protein has no obvious inhibitory action on the Gram-positive bacterium staphylococcus aureus and micrococcus luteus. The invention lays the foundation for the disease control of the portunus trituberculatus, the auxiliary breeding of the gene and the development of the feed additive.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a PtALF-4 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, trad...

Claims

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Application Information

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IPC IPC(8): A23K1/16A23L3/3544C12N15/12A61P31/04A61P31/10A61P37/04A61K38/17C07K14/435A23K20/147
Inventor 崔朝霞刘媛
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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