Application of glutathione mercaptotransferase as detoxifying enzyme for preventing and treating duck liver injury caused by AFB1

A thiol transferase, glutathione technology, applied in the direction of transferase, application, gene therapy, etc., can solve the problem of unclear liver toxicity

Active Publication Date: 2022-07-08
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Duck AFB 1 There is no effective treatment or mitigation method for liver damage caused by poisoning. GSTs are the key detoxification enzymes. Among the more than ten GSTs enzymes recorded in the NCBI database, which subtypes can alleviate AFB 1 Induced duck liver toxicity is unknown

Method used

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  • Application of glutathione mercaptotransferase as detoxifying enzyme for preventing and treating duck liver injury caused by AFB1
  • Application of glutathione mercaptotransferase as detoxifying enzyme for preventing and treating duck liver injury caused by AFB1
  • Application of glutathione mercaptotransferase as detoxifying enzyme for preventing and treating duck liver injury caused by AFB1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 AFB 1 Changes of cell viability in duck primary hepatocytes transfected with empty plasmid and after GSTs overexpressed duck primary hepatocytes

[0034] The CDs fragments of any of the GSTs genes in Table 1 below were cloned from duck liver cDNA to construct pcDNA3.1(+)-GSTs recombinant expression vector and transfected; and the GST transfection efficiency was verified by qPCR. The control group was the pcDNA3.1(+) transfection group of duck primary hepatocytes, and the experimental group was the transfection group with different pcDNA3.1(+)-GSTs; taking GST as an example:

[0035] The GST CDs fragments cloned from duck liver cDNA were recovered by gel and were homologously recombined into pcDNA3.1(+), and the pcDNA3.1(+)-GST recombinant expression vector was obtained after successful sequencing and alignment. Duck primary hepatocytes were isolated from duck liver by in situ perfusion. After 24 hours of adherent culture, the cells were respectively transfect...

Embodiment 2

[0040] Example 2 AFB 1 Changes of LDH activity in cell culture medium of duck primary hepatocytes transfected with empty plasmid and GSTs overexpressed duck primary hepatocytes

[0041] The control group was the duck primary hepatocyte empty plasmid transfection group (pcDNA3.1(+)), and the experimental group was the transfection group with different GSTs. 24h after transfection, two different concentrations of AFB, 75ppb and 150ppb, were selected 1 The culture medium was used for the challenge test. Twenty-four hours after the challenge, the culture medium supernatant in the cell culture dish was taken for LDH (lactate dehydrogenase) detection, and the detection method was carried out in strict accordance with the instructions of Nanjing detection LDH detection kit.

[0042] The result is as image 3 and Figure 4 Shown: AFB at 75ppb and 150ppb 1 The activity of LDH in the cell culture medium was significantly increased under the action of GST3, indicating that the cells...

Embodiment 3

[0043] Example 3: AFB 1 Changes of AFBO-GSH content in duck primary hepatocytes transfected with empty plasmid and in duck primary hepatocytes overexpressing GSTs

[0044] The control group was the duck primary hepatocyte empty plasmid transfection group (pcDNA3.1(+)), and the experimental group was the transfection group with different GSTs. 24h after transfection, with 75ppb of AFB 1 The culture medium was used for the challenge test. After 24 hours of challenge, the cells and culture medium were collected into 2ml centrifuge tubes and frozen at -80°C. The collected samples were freeze-dried, extracted and derivatized, and then the content of AFBO-GSH was detected by liquid chromatography.

[0045] The results of liquid chromatography showed that among the 10 transfected GSTs, only GST and GST3 could significantly increase the content of AFBO detoxification metabolite AFBO-GSH (46% and 13%), indicating that they can significantly alleviate AFB. 1 Toxicity to duck primary...

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Abstract

The invention discloses an application of glutathione mercaptotransferase as a detoxifying enzyme for preventing and treating duck liver injury caused by AFB1, and provides an application of GST and GST3 as target enzymes for preventing and treating animal liver injury caused by AFB1 by testing 10 GST in a duck GSTs enzyme family and screening out GST and GST3 with detoxifying effects. The glutathione mercaptotransferase (GST or GST3) provided by the invention can effectively inhibit or relieve AFB1-induced liver toxicity of ducklings, and is mainly related to the metabolism of catalyzing AFBO to generate nontoxic AFBO-GSH. The GST and the GST3 can be used as drugs for treating duck liver injury caused by AFB1 and provide new target genes for development of novel feed additives.

Description

technical field [0001] The present invention relates to AFB 1 The field of detoxification, in particular to a kind of glutathione thiol transferase as the prevention and treatment of AFB 1 Application of detoxification enzymes in duck liver injury. Background technique [0002] According to the statistics of the Food and Agriculture Organization of the United Nations, about a quarter of the world's crops and agricultural products are contaminated with mycotoxins (Mohajeri M et al., 2018; Fei Sun and Fang Rejun, 2014). of which aflatoxin B 1 (aflatoxin B 1 ,AFB 1 ) The pollution has a wide distribution range and is extremely toxic, and poultry is particularly sensitive to it. 1 One of the most sensitive species (Rushing B R and Selim M I, 2019). After livestock and poultry eat aflatoxin-contaminated feed, the liver, as its main target organ and an important detoxification organ, will experience severe pathological changes, accompanied by decreased production performance...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/85A61K38/45A61K48/00A61P39/02A23K20/189A23K50/75
CPCC12N9/1088C12N15/85C12Y205/01018A61K38/45A61K48/005A61P39/02A23K20/189A23K50/75
Inventor 孙铝辉张宇赵玲邓江曹可欣晏依琴
Owner HUAZHONG AGRI UNIV
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