Overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA, coded amino acid and application

A microcystin and enzyme-degrading technology, which is applied in the fields of biotechnology applications and environmental protection, can solve the problems of high cost, inability to completely remove MC, and heavy workload, and achieve the goal of maintaining vitality, maintaining detoxification ability, and reducing damage Effect

Inactive Publication Date: 2010-07-14
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These degradation technologies have many problems such as high cost, heavy workload, and inability to completely remove MC.

Method used

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  • Overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA, coded amino acid and application
  • Overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA, coded amino acid and application
  • Overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA, coded amino acid and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Obtaining of cDNA sequence of MlrA gene and its encoded amino acid sequence

[0059] 1. Sample Collection and Processing

[0060] The water samples used in the experiment were collected from Xiangang Reservoir, Huizhou City, Guangdong Province, Minghu Lake, Jinan University, Guangzhou City, and Guangdong Tilapia Breeding Farm, and fish gut samples were collected from Xiangang Reservoir, Huizhou City, Guangdong Province.

[0061] Water samples were collected from surface water bodies at various locations with sampling bottles.

[0062] The fish intestines were killed on the spot, and the hindguts (about 10 cm) were taken, and the excreta in the intestines were picked out and mixed with 15 ml of distilled water to obtain fish intestine samples.

[0063] After mixing the above water sample and fish intestinal sample, filter through a medium-speed qualitative filter paper to remove impurities, centrifuge at 12000rpm for 1min, collect the microbial mixture, and st...

Embodiment 2

[0081] Example 2 MlrA detection results in water bodies and fish bodies in different seasons

[0082] 1. Sample Collection and Processing

[0083] The water samples used in the experiment were collected from Xiangang Reservoir, Huizhou City, Guangdong Province, Minghu Lake, Jinan University, Guangzhou City, and Guangdong Tilapia Breeding Farm, and fish gut samples were collected from Xiangang Reservoir, Huizhou City, Guangdong Province.

[0084] In March, April, May, July, August, October, November and December, water samples and fish intestinal samples were collected respectively.

[0085]Water samples were collected from surface water bodies at various locations with sampling bottles.

[0086] The fish gut samples were killed on the spot, and the hindguts (about 10 cm) were taken, and the excreta in the guts were picked out and mixed with 15 ml of distilled water to obtain the fish gut samples.

[0087] After the water samples and fish intestinal samples in the water envir...

Embodiment 3

[0105] Example 3 Preparation of MlrA crude enzyme preparation expressing MlrA protein

[0106] 1. Construction of recombinant expression vector pET3c-MlrA

[0107] The full-length cDNA sequence of the Sphingomonas MlrA gene obtained in Example 1 was cloned, and the PCR product was purified using H.Q.&.Q.Gel Extraction Kit II.

[0108] Take 50 μL of the E.coli DM5α preservation solution containing the pET3c plasmid, inoculate it in 4 mL of LB liquid medium (containing Amp), and cultivate overnight at 37°C with shaking at 280 rpm. The pET3c plasmid was extracted using HP Plasmid Mini Kit.

[0109] 2. Expression of Sphingomonas MlrA and preparation of MlrA crude enzyme preparation

[0110] PCR purified product and pET3c plasmid were used to construct recombinant expression vector pET3c-MlrA. The recombinant expression vector pET3c-MlrA was identified and transformed into the expression strain Escherichia coli BL21(DE3)pLysS. The resulting recombinant bacteria were expanded an...

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Abstract

The invention provides an overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA, a coded protein and application. The nucleotide sequence of the overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA is shown as SEQ IDNO:1, and the coded amino acid sequence s shown as SEQ ID NO:3. The overall length cDNA sequence of micro-capsule algae toxins degrading enzyme MlrA in the invention has uniqueness and specifity for degradation of algae toxins, and can be used for detecting the situation of micro-capsule algae toxins in water body or fish products. The micro-capsule algae toxins degrading enzyme MlrA can be used as crude enzyme preparation in optimizing aquaculture water to effectively prevent and control damage of algae toxins on eating safety of bred aquatic products, and also can be used as fish bait additive to mitigate damage of micro-capsule algae toxins on fish hepatic cell DNA, maintain detoxication capacity of hepatic cells on algae toxins during blue-green algae bloom outbreak, and greatly improve eating safety of bred fish.

Description

technical field [0001] The invention relates to the field of biotechnology application and environmental protection, in particular to the full-length cDNA sequence, coded amino acid and application of microcystin degrading enzyme MlrA. Background technique [0002] Microcystin (MC) is a type of algal toxin that occurs most frequently in cyanobacterial blooms and causes the most serious harm. It can cause poisoning and death of wild animals, livestock, poultry, etc., and cause human liver damage and high incidence of liver cancer. . More than 60 isomers of this toxin have been identified, the most common being MC-LR, MC-RR and MC-YR. [0003] Regarding the biodegradation of MC, Bourne et al. believed that the degradation of MC-LR was at least caused by three hydrolytic enzymes in the cell. Bourne et al. (Bourne D G, Riddles P, Jones G J, et al. 2001. Characterization of a gene cluster involved in bacterial degradation of the cyanobacterial toxin microcystin LR [J]. Environ ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N15/63C12N15/70C12N1/00C12N1/21C02F3/34A23K1/18C02F103/00
Inventor 梁旭方胡永乐陈小佳谢秋玲姚煜程炜轩曹亮何珊
Owner JINAN UNIVERSITY
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