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Preparation of recombinant protein of long oyster cell wall hydrolase and application of the obtained recombinant protein

A technology of recombinant protein and oyster, which is applied in the fields of molecular biology and biochemistry, and achieves the effects of potential application value, simple genetic manipulation, and favorable expression.

Active Publication Date: 2020-10-09
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the preparation, function and application of invertebrate cell wall hydrolase

Method used

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  • Preparation of recombinant protein of long oyster cell wall hydrolase and application of the obtained recombinant protein
  • Preparation of recombinant protein of long oyster cell wall hydrolase and application of the obtained recombinant protein
  • Preparation of recombinant protein of long oyster cell wall hydrolase and application of the obtained recombinant protein

Examples

Experimental program
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experiment example 1

[0028] Experimental example 1: Prokaryotic recombinant expression and purification of long oyster CgCLE-1 in vitro

[0029] 1. Construction of recombinant vector

[0030] The recombinant vector used in the present invention is the pPICZαA prokaryotic expression vector of Invitrogen Company. Total RNA was extracted from fresh long oyster hemolymphocytes, and cDNA was obtained by reverse transcription PCR. By PCR technology, using primers P1 (5'-GAATTCAGAGGAGAACCGATGGAAGCTA-3') and P2 (5'-GCGGCCGCCACAAAGTACAGACCCCGAGTT-3') with EcoRI and Not I restriction enzyme sites added at the 5' end to amplify the long oyster cell wall Coding region of the hydrolase Hydolase_2 domain. The reaction conditions were as follows: 94°C pre-denaturation for 5 minutes, followed by the following cycles: 94°C denaturation for 30 seconds, 65°C annealing for 30 seconds, 72°C extension for 2 minutes, a total of 35 cycles, and finally 72°C extension for 10 minutes. The amplified fragment was purified ...

experiment example 2

[0034] Experimental Example 2: Antibacterial Activity Detection of CgCLE-1 Recombinant Protein from Long Oyster

[0035]Escherichia coli (Escherichia coli), Aeromonas hydrophila (Aeromonas hydrophila) and Staphyloccocus aureus (Staphyloccocus aureus) were cultured in LB medium at 37°C and 220rpm until logarithmic growth phase; Vibrio splendidus (Vibrio splendidus), Vibrio vulnificus, Vibrio alginolyticus, Pseudomonas putida and Pseudomonas aeruginosa were respectively cultured in 2216E medium at 28°C and 220rpm until logarithmic growth phase The obtained cells were collected by centrifugation and washed with TBS (50mM Tris-HCl, 100mM NaCl, pH=7.4) and resuspended to a concentration of 10 4 CFU. 50 μ L above-mentioned embodiment obtains recombinant protein CgCLE-1 (160 μ g mL -1 ) and an equal volume of each bacterial suspension obtained above were incubated at room temperature for 2 h. In blank group (Blank group), 50 μL TBS was used instead of recombinant protein. Take 20...

experiment example 3

[0036] Experimental results: CgCLE-1 can significantly inhibit Vibrio splendidus ( figure 2 ), Escherichia coli ( image 3 ), Vibrio vulnificus ( Figure 4 ), Pseudomonas putida ( Figure 5 ), Vibrio alginolyticus ( Figure 6 ), Pseudomonas aeruginosa ( Figure 7 ), Aeromonas hydrophila ( Figure 8 ) and Staphylococcus aureus ( Figure 9 ) growth (P<0.01). Experimental Example 3: Determination of the minimum inhibitory concentration (MIC) of CgCLE-1 recombinant protein of long oyster

[0037] The CgCLE-1 recombinant protein was diluted in multiples, and the antibacterial activity was tested according to the method of Experimental Example 2. The lowest drug concentration that can inhibit the obvious growth of a certain microorganism is the MIC of CgCLE-1.

[0038] Experimental results: See Table 1 for the minimum inhibitory concentrations of CgCLE-1 on the tested bacteria.

[0039] Table 1. The minimum inhibitory concentration of CgCLE-1 against different bacteria

[...

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Abstract

The invention belongs to the technical field of molecular biology and biological chemistry and in particular relates to preparation of recombinant protein of crassostrea gigas cell wall hydrolase and application of the prepared recombinant protein. The recombinant protein of the crassostrea gigas cell wall hydrolase is prepared by utilizing a yeast expression system. The recombinant protein of the crassostrea gigas cell wall hydrolase, which is prepared by utilizing a yeast expression system, has a remarkable inhibition effect on the growth of escherichia coli, aeromonas hydrophila, staphylococcus aureus, vibro splendidus, vibrio vulnificus, vibrio alginolyticus, pseudomonas putida and pseudomonas aeruginosa. The recombinant protein provided by the invention can be used for developing bacteriostatic agents.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and biochemistry, and specifically relates to the preparation of a recombinant protein derived from oyster oyster cell wall hydrolase and the application of the prepared recombinant protein. Background technique [0002] Long oyster is one of the most important mariculture shellfish in the world, and has long occupied a pivotal position in my country's aquaculture economy. In recent years, diseases caused by various pathogenic microorganisms have continuously broken out in oyster culture groups, causing huge losses to the culture industry. Therefore, research on the immune defense mechanism of long oyster and the discovery of effective disease-resistance-related functional proteins will help humans effectively prevent and control its diseases. [0003] Long oyster belongs to mollusk, lacks adaptive immune system, and only relies on innate immune system to resist pathogen invasion. Ant...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/36C12N15/56C12N15/81C12N1/19A61K38/47A61P31/04C12R1/84
CPCA61K38/00C12N9/2462C12N15/815C12Y302/01017Y02A50/30
Inventor 张峘王玲玲王昊刘瑞贾志浩邱丽梅宋林生
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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