Chitin binding protein CBP58, and encoding gene and application thereof

A technology of CBP58-R and CBP58-F, which is applied to chitin-binding protein CBP58 and its coding gene and application fields, can solve the problem of unsatisfactory biological activity and expression of chitin protein, harsh conditions for chitin protein activity, Chitin protein application limitations and other issues, to achieve huge industrial benefits and economic value, good inhibition effect, low production cost effect

Inactive Publication Date: 2015-09-02
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, people's understanding of Pseudoalteromonas and its chitinase protein is still limited at present. The biological activity and expression of some existing chitin proteins are still not ideal, and some chitin proteins have conditions for the existence of activity. Harsh, high cost of preparation and storage, these factors limit the application of chitin protein

Method used

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  • Chitin binding protein CBP58, and encoding gene and application thereof
  • Chitin binding protein CBP58, and encoding gene and application thereof
  • Chitin binding protein CBP58, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The preparation of embodiment 1CBP58 protein solution

[0033] 1. Amplification of CBP58 gene

[0034] Using PCR technology, the genomic DNA of Pseudoalteromonas sp. DL-6 strain was used as a template, and PCR amplification was performed with upstream primers and downstream primers to obtain PCR amplification products. Using Primer5.0 software to design primers, respectively: upstream primer CBP58-F (SEQ ID No.2): 5-GGAATTC CATATG CATGGTTATATGGATAGCCCTAAAG-3, the underline marks the NdeI restriction site, the downstream primer CBP58-R (SEQ ID No.3): 5-CCGCTCGAG CAGTGT GGTCCATACATCGGCTTG-3, the XhoI restriction site is underlined.

[0035] PCR reaction system:

[0036]

[0037]

[0038] PCR reaction conditions: use 94°C for 2min, 1 cycle, then 30 cycles of 94°C for 30s, 55°C for 30s and 68°C for 7min; finally use 68°C for 15min, 1 cycle for amplification. The end of the PCR reaction was detected by 1% agarose gel electrophoresis, such as figure 1 As shown, t...

Embodiment 2

[0096] Chitinase activity of embodiment 2CBP58 protein

[0097] 1. Preparation of colloidal chitin: Weigh 1g of powdered α-type chitin from shrimp shells, add 4ml of acetone in a mortar, and grind until it becomes a paste. If the powder is not very fine and relatively rough, you can add acetone during the process. Grind it thoroughly until it becomes a paste; add 40ml of concentrated hydrochloric acid while grinding at 4°C and filter it with glass fiber. Then the filtrate was slowly added to 50% ethanol placed on ice which was vigorously stirred in a beaker, and the beaker was placed on a magnetic stirrer in a chromatographic cabinet at 4°C and stirred overnight. Use a small molecular weight dialysis bag for overnight dialysis and wash to neutrality, and finally store it in PBS buffer at 4°C for future use.

[0098] 2. Establishment of standard curve for enzyme activity assay

[0099] Take 1mL sample solution, add 1mL potassium ferricyanide reagent (to prepare 1L 0.5M sodium...

Embodiment 3

[0107] Example 3 Scanning Electron Microscopy (SEM) Observation of CBP58 Protein Binding to Powdered Chitin

[0108] 1mL reaction system includes 2mg / mL α-chitin or β-chitin, 0.5mg / mL CBP58, incubate at room temperature for 24h, centrifuge at 12,000×g for 10min, pour off the supernatant, precipitate and dry naturally at 37°C to fix the sample , without adding CBP58 was set as a control, and the substrate binding was observed under a scanning electron microscope.

[0109] The combination of CBP58 and powdered chitin observed by scanning electron microscope is as follows: Figure 5 shown. from Figure 5 A-B and Figure 5 From E-F, it can be seen that the edges and surfaces of α-chitin and β-chitin treated with CBP58 are rough, loose and porous, which is more conducive to the contact degradation of chitinase and substrate; from Figure 5 C-D and Figure 5 In G-H, it can be seen that the control buffer is smooth and dense, and it is not easy to combine the enzyme with the sub...

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Abstract

The invention relates to a chitin binding protein CBP58, and an encoding gene and an application thereof, and belongs to the technical field of gene engineering. The encoding gene of the chitin binding protein CBP58 is cloned from a strain pseudoalteromonas sp.DL-6 for the first time; and a high-activity expression product is obtained by construction of a prokaryotic expression vector and imidazole elution. The preparation process is simple; a simple and convenient method is provided for subsequent research on the chitin protein CBP58; the chitin protein CBP58 is low in production cost and easy to store, has a chitin binding function, is applied to purification and auxiliary identification of chitin, has a good inhibiting effect on aspergillus niger (Aspergillus niger) and cytospora mandshurica miura (Cytospora mandshurica miura), has a potential biological control function and has wide application value; a novel research direction is provided for research and development of anti-fungal medicines in the field of agriculture; and great industrial benefits and economic value are generated.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a chitin-binding protein CBP58 and its coding gene and application. Background technique [0002] Chitin, commonly known as chitin and chitin, is a linear polymer connected by N-acetylglucosamine monomers with β-1,4-glycosidic bonds. It is the most abundant in nature. It is found in the marine environment. The second largest renewable resource after cellulose. Chitin widely exists in the crustaceans of shrimps and crabs and insects, as well as in the cell walls of fungi (yeast, mold), algae and plants. It is estimated that more than 10 11 Tons of chitin is formed, and its degradation is mainly accomplished by chitinase-producing microorganisms. [0003] The microbial chitin degradation system mainly consists of endochitinase (chitinase, EC 3.2.1.14), exochitinase (chitinase, EC 3.2.1.14), β-N-acetylhexosaminidase (β- N-acetylhexosaminidase, EC 3.2.1.52...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31C12N15/70A01P3/00C12R1/01
CPCC07K14/195
Inventor 王晓辉迟乃玉张庆芳
Owner DALIAN UNIV
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