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PtPLC (protein tyrosine phosphatase-like (proline instead of catalytic arginine), member c) gene of Pacifastin type serine protease inhibitor of portunus trituberculatus and a coding protein and application thereof

A technology of serine protease and Portunus trituberculatus, which is applied in the field of molecular biology, can solve the problems of no reports of Portunus trituberculatus Pacifastin-type serine protease inhibitors and the loss of Portunus trituberculatus aquaculture

Inactive Publication Date: 2013-04-03
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Portunus trituberculatus (Portunus trituberculatus) is an important mariculture species in my country. However, with the continuous expansion of aquaculture scale and the continuous improvement of the degree of intensification of aquaculture, various diseases caused by bacteria, fungi and viruses are becoming more and more serious. Swimming crab farming has caused huge losses
[0005] So far, there is no report about Pacifastin-type serine protease inhibitors in Portunus trituberculatus

Method used

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  • PtPLC (protein tyrosine phosphatase-like (proline instead of catalytic arginine), member c) gene of Pacifastin type serine protease inhibitor of portunus trituberculatus and a coding protein and application thereof
  • PtPLC (protein tyrosine phosphatase-like (proline instead of catalytic arginine), member c) gene of Pacifastin type serine protease inhibitor of portunus trituberculatus and a coding protein and application thereof
  • PtPLC (protein tyrosine phosphatase-like (proline instead of catalytic arginine), member c) gene of Pacifastin type serine protease inhibitor of portunus trituberculatus and a coding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The PtPLC gene of Portunus trituberculatus Pacifastin-type serine protease inhibitor is the base sequence shown in SEQ ID No.1.

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

[0027] ggacttgcgg gcctgattct ccagagggtc gac atg aag gct caa gtg

[0028] gtg ctg ctc ctg ctg ggg gct gct gtg acg gcg gca acc agt

[0029] ccg cct ttt gtg gaa ctc cct agt gat cct gat gct cct gag

[0030] tgt gag gga cgc cca ctg gtt gac cgc tgg agg aag gac tgc

[0031] aac tgg tgc tcc tgc aat gag ggg aga gtg agg tgc tcc cga

[0032] caa ctg tgt cct gaa ggt cag cag gat cca gaa cca cag tgt

[0033] gag ggc agc ccc act tgg aag gat gac tgc aac acg tgc cgc

[0034] tgt gct ggt ggc cga gct gtg tgc acc gcc aag cac tgt gat

[0035] caa ctt ggt cca gag cag cag att gtg gaa gtg cag gtg gag

[0036] agt gca gag tgt aag gaa ggc tcc cgg tgg cgt gtg gag tgc

[0037] aat tgg tgc acc tgt cgg gga ggg aag ggc gcc tgc aca gag

[0038] atg gcc tgt tta aac tgg gat gag gac cag gca cgg gag gat

[0039] gga atc ​​...

Embodiment 2

[0096] The base sequence of Portunus trituberculatus Pacifastin-type serine protease inhibitor PtPLC 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.

[0097] The sequence listing SEQ ID No.2 is:

[0098] MKAQVVLLLLGAAVTAATSPPFVELPSDPDAPECEGRPVDRWRKDCNWCSCNEGRVRCSRQLCPEGQQDP

[0099] EPQCEGSPTWKDDCNTCRCAGGRAVCTAKHCDQLGPEQQIVEVQVESAECKEGSRWRVECNWCTCRGGKGA

[0100] CTEMACLNWDEDQAREDGILECHGSSRWKKDCNWCRCAEGRGFCTKKACPQTGPFDNLPEDMCVPGSRWL

[0101] VDCNWCGCSDDGRSSFCTLMACIPGYVHEGPTCEDGSVWKTDDCNICRCIDGMSACTKRLCATPNQEPRTP

[0102] QVPNEPECQGELGIDRWRQDCNWCNCRNGAGACTKRQCLEGVKDDQPECEGNPSWMKDCNRCHCVDGRVC

[0103] TTKFCGEFRK

[0104] It has a complete coded protein containing 365 amino acids, of which 1-17 amino acids of the coding sequence are signal peptides, and the mature peptide contains 348 amino acids, with a predicted molecular weight of 38.86 kDa and an isoelectric point of 4.99. The mature pep...

Embodiment 3

[0108] Inhibitory activity test of PtPLC recombinant protein of Portunus trituberculatus Pacifastin type serine protease inhibitor:

[0109] 1. Preparation of protease solution and reagents: Bovine trypsin and bovine α-chymotrypsin were respectively dissolved in 1M Tris-HCl solution (pH=7.0) to make the protease concentration reach 0.1mg / ml, and set aside at 4°C. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) was dissolved in 1M Tris-HCl solution (pH=7.0) to make the concentration of AEBSF reach 100mM, set aside at 4°C. A 1% Azocasein substrate solution with a mass fraction of 1% was prepared with a phosphate buffer solution with a pH value of 7.2. The operation was carried out under sterile conditions, and it was stable in a refrigerator at 4° C. for 2-3 days before use. Take 10g of trichloroacetic acid (TCA) and dissolve it in 50ml of double-distilled water, and finally dilute to 100ml to prepare 10% TCA, and store it at 4°C for later use.

[0110] 2. Determ...

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Abstract

The invention belongs to the technical field of molecular biology, and specifically relates to a PtPLC (protein tyrosine phosphatase-like (proline instead of catalytic arginine), member c) gene of a Pacifastin type serine protease inhibitor of portunus trituberculatus and a coding protein and application thereof. The PtPLC gene of the portunus trituberculatus is shown in a base sequence in a sequence table SEQ ID No.1. The recombined PtPLC (protein tyrosine phosphatase-like (proline instead of catalytic arginine), member c) protein has prominent inhibitory ability on bovine trypsin and bovine alpha-chymotrypsin. When the concentration of the recombined protein PtPLC is respectively 1mum, 2mum and 3mum, the inhibitory ability on the bovine trypsin is respectively 88.73%, 90.73% and 97.18%; and when the concentration of the recombined protein PtPLC is respectively 2mum and 3mum, the inhibitory ability on the bovine alpha-chymotrypsin respectively reaches 31.1% and 99.69%. The PtPLC gene has potential application value in terms of medicine development, feed additives and the like and lays the foundation of disease control of the portunus trituberculatus and gene-assisted selection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a PtPLC gene of Portunus trituberculatus Pacifastin type serine protease inhibitor, its encoded protein and its application. Background technique [0002] Serine protease inhibitors are a class of proteins that have inhibitory activity on serine proteases. They combine with target enzymes to form stable complexes to prevent harmful hydrolytic activation of serine proteases in the body. They are important components of the immune system in organisms. Serine protease inhibitors can be divided into 74 families according to the similarity of sequence features, topological structures and functions, but there are few reports on their structures and functions in crustaceans such as shrimps and crabs. [0003] Pacifastin is a new family of serine protease inhibitors, which were first discovered in the hemolymph of the freshwater crayfish Pacifastus leniusculus in 198...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/15C07K14/81A61K38/57A23K1/16A23L3/3526A23B7/154
Inventor 崔朝霞刘媛王双艳
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI