CN114717212B - Glutathione sulfhydryl transferase as AFB control agent 1 Application of detoxication enzyme for duck liver injury - Google Patents

Glutathione sulfhydryl transferase as AFB control agent 1 Application of detoxication enzyme for duck liver injury Download PDF

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CN114717212B
CN114717212B CN202210349010.0A CN202210349010A CN114717212B CN 114717212 B CN114717212 B CN 114717212B CN 202210349010 A CN202210349010 A CN 202210349010A CN 114717212 B CN114717212 B CN 114717212B
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孙铝辉
张宇
赵玲
邓江
曹可欣
晏依琴
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Huazhong Agricultural University
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Abstract

The present invention discloses a glutathione sulfydryl transferase as a medicine for preventing and curing AFB 1 The invention tests 10 GST in duck GSTs enzyme family, screens GST and GST3 with detoxification effect, provides GST and GST3 as AFB prevention and treatment 1 Use of an actuator liver injury target enzyme. The glutathione thiol transferase (GST or GST 3) of the invention can effectively inhibit or relieve AFB 1 The induced liver toxicity of duckling is mainly related to the metabolism of AFBO-GSH which can catalyze AFBO to generate non-toxic AFBO. GST and GST3 of the invention can be used for treating AFB 1 The development of medicines and novel feed additives for causing duck liver injury provides a novel target gene.

Description

Glutathione sulfhydryl transferase as AFB control agent 1 Application of detoxication enzyme for duck liver injury
Technical Field
The invention relates to AFB 1 The detoxification field, in particular to a glutathione sulfydryl transferase used for preventing and treating AFB 1 Application of detoxication enzyme for duck liver injury is provided.
Background
Statistics from the national food and agricultural organizations have shown that about 1/4 of the world's crops and agricultural products are contaminated with mycotoxins (Mohajeri M et al, 2018; sun Fei and Fang Rejun, 2014). Wherein aflatoxin B 1 (aflatoxin B 1 ,AFB 1 ) Has wide pollution distribution range and strong toxicity, is particularly sensitive to birds, and duckling is common to AFB in livestock and poultry 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 is taken as a main target organ and an important detoxification organ of the livestock and poultry, serious pathological changes can occur, and the conditions of production performance reduction, reproductive system damage, immune function inhibition and the like are accompanied, even toxins remain in meat products, and diseases such as cancers can occur after long-term eating of aflatoxin-contaminated food (Williams et al 2004; huang Zhiwei and the like, 2015).
When AFB 1 When entering animal liver, AFB is prepared by CYP450s (cytochrome P450 family) 1 Converted to highly toxic AFB 1 -exo 8, 9-epoxide (AFBO) and the remaining low toxic metabolites. AFBO was then found in GSTs (glutathione thiol transferase family), mEH (microsomal epoxide hydrolase) and AFAR (aflatoxin B) 1 Aldehyde reductase) into AFBO-GSH and AFB 1 Dialcohol achieves detoxification (Deng et al, 2018). In livestock and poultry, AFBO metabolism mainly depends on phase II drug metabolizing enzyme GSTs, which is a group of isozymes with multiple physiological functions, widely exists in organisms and is located in cytoplasm, mitochondria and microsomes. Notably, there is a variability in the key GSTs enzyme lines that catalyze the formation of AFBO-GSH from AFBO-bound GSH in vivo in different species to detoxify (Deng et al, 2018).
Duck AFB 1 There is no effective treatment or alleviation of liver injury caused by poisoning, GSTs are used as key detoxification enzymes thereof, and currently, more than ten GSTs are recorded in NCBI database, which subtypes can alleviate AFB 1 The induced duck liver toxicity is not yet clear.
Disclosure of Invention
The invention aims at the prior art for preventing and treating AFB 1 The deficiency of fowl liver toxicity is caused by providing a glutathione sulfhydryl transferase as a treatment for AFB 1 The invention tests 10 GSTs in duck GSTs enzyme family, screens GST and GST with detoxification effectGST3 provides the application of GST and GST3 as target enzyme for preventing and treating duck liver injury caused by AFB 1.
To achieve the above object, the present invention relates to a glutathione thiol transferase as a prophylactic and therapeutic agent for AFB 1 Application of duck liver injury target enzyme; the glutathione sulfhydryl transferase is GST and GST3.
The present invention also provides the gene of glutathione sulfydryl transferase for preventing and treating AFB 1 The application of the glutathione-transferase gene is GST and GST3 in medicines or feed additives for causing duck liver injury; wherein, the liquid crystal display device comprises a liquid crystal display device,
the nucleotide sequence of GST is:
ATGTCAGGGAAGCCCAGGCTCACCTACATTAATGGAAGGGGACGAATGGAGCCGATCCGATGGCTGCTGGCAGCAGCCGGCGTGGAGTTTGAAGAAAACTTTGTGGAAACAAAAGAACAGTTAGAAAAGTTAATCAAAGGTGGAGACCTGCTGTTCCAGCAAGTGCCCATGGTGGAGATTGATGGGATGAAGATGGTGCAGACCAGAGCCATCCTCCGCTACATAGCGGGGAAATACAACCTCTATGGGAAGGACCTGAAGGAGAGAGCCCTGATCGACATGTATGTGGAAGGAATAACAGATCTGATGCAAATGATTATGATGTTTCCTTTCGCTCCAGCTGAGGCAAAGCCAAAAAATCTTGCCTCAATTGAAGAGAAGGCAACAAAGAGATACTTCCCAGTCTTTGAAAAGATTTTGAAACAACACGGCCAAGACTTTCTTGTGGGGAACCGACTCAGCTGGGCAGATGTTCAGCTATTGGAAGCCATTTTAGCAGTGGAGGAGAAAGTACCTGCTGTGCTTTCTGGGTTTCCTCAGCTGCAGGCCTTTAAAACAAAAATGAGCAACGTGCCTACAATTAAGAAGTTCCTGCAGCCTGGCAGCGCAAGGAAGCCCCCACCAGATGAGAATTATGTGGCACTTGTGATGTCGATTTTTAATCTAAGCTGA, which is shown in SEQ ID No. 1;
the nucleotide sequence of GST3 is:
ATGGCTGGAAAACCCAAACTTTACTACTTTGATGGAAGAGGCAAAATGGAATCCATTCGCTGGTTGTTAGCTGCAGCCGGGGTTGAGTTTGAAGAGGAGTTTTTGGAAACACGAGAACAGTATGAGAAGCTCCTGCAAAGTGGATCCCTGCTGTTCCAGCAAGTTCCCCTGGTGGAGATCGATGGGATGAAGATGGTGCAGACCAGAGCCATCCTCAACTACATTGCAGCAAAGTACAACCTCTACGGAAAGGACCTGAAGGAGAGAGCCCTGATTGATATGTATGTTGGAGGAACTGAGGACCTTATGGGCTTTATTTTGATGTTCCCATTCTTATCGGCTGAGGATAAAGAGAAGCAACGTGCCTTTATAGTTCAGAAGGCCACCAGCAGGTACTTCCCAGTATATGAAAAGGTTCTGAAAGACCATGGGCAGGACTTCCTTGTTGGCAACAGTTTTAGCTGGGCAGACATTCATCTTCTTGAAGCCATTTTAATGGTAGAAGAGAAGAAGTCGGACGTTCTCTCGAGCTTTCCTCAGTTGCAGGCATTTAAAAGAAGGATAAGCAGCATCCCCACAATCAAGAAGTTTCTAGAGCCTGGAAGCCAGAGAAAACCTGTTCCTGATGATAAATATGTGGAGACTGTGAGGAAAGTTCTCCGCATGTATTATGATATAAAAGCGAATTAG as shown in SEQ ID No. 2.
GST and GST3 of the present invention were obtained based on the gene screening in Table 1 below.
Table 1 GSTs Accession Number
The invention also provides a recombinant expression vector which contains the GST or GST3 expression vector, wherein the expression vector is pcDNA3.1 (+).
The invention also provides a recombinant expression vector for preventing and treating AFB 1 Application of the medicine or feed additive for causing duck liver injury.
The invention also provides a glutathione sulfydryl transferase for preventing and treating AFB 1 Application of medicines or feed additives for causing duck liver injury; the glutathione sulfhydryl transferase is GST and GST3, and the amino acid sequence of the GST is as follows:
MSGKPRLTYINGRGRMEPIRWLLAAAGVEFEENFVETKEQLEKLIKGGDLLFQQVPMVEIDGMKMVQTRAILRYIAGKYNLYGKDLKERALIDMYVEGITDLMQMIMMFPFAPAEAKPKNLASIEEKATKRYFPVFEKILKQHGQDFLVGNRLSWADVQLLEAILAVEEKVPAVLSGFPQLQAFKTKMSNVPTIKKFLQPGSARKPPPDENYVALVMSIFNLS, which is shown in SEQ ID No. 3;
the amino acid sequence of GST3 is:
MAGKPKLYYFDGRGKMESIRWLLAAAGVEFEEEFLETREQYEKLLQSGSLLFQQVPLVEIDGMKMVQTRAILNYIAAKYNLYGKDLKERALIDMYVGGTEDLMGFILMFPFLSAEDKEKQRAFIVQKATSRYFPVYEKVLKDHGQDFLVGNSFSWADIHLLEAILMVEEKKSDVLSSFPQLQAFKRRISSIPTIKKFLEPGSQRKPVPDDKYVETVRKVLRMYYDIKAN, which is shown in SEQ ID No. 4.
The principle of the invention is as follows:
1. glutathione thiol transferase (GST) is considered to be the most important enzyme involved in the metabolism of electrophiles (R.N.Armstrong, 1991). These enzymes catalyze one of the most important phase II reactions in the metabolic pathways of drugs, including the binding of GSH to harmful cell electrophiles, such as some strong carcinogens (l.g. higgins and j.d. hayes, 2011) as well as endogenous metabolites, hormones, and phenolic micronutrients (f.galli, 2007). As part of the drug metabolizing gene function, GST-mediated binding to GSH increases the solubility of lipophilic electrophiles in water, thereby facilitating their cellular transport and excretion. Other functions of GST in the cytoplasm include binding of lipophilic small molecules, thiol isomerase activity, GSH-dependent protection of protein thiols and redox regulation involved in signal transduction (z.y.v.adler et al, 1999;D.M.Townsend et al, 2009).
2. According to the invention, the duck primary liver cells are used as a model, and the functions of the GSTs in relieving the toxic effect of the duck liver caused by AFB1 are detected through the overexpression of the GSTs by the duck primary liver cells. The CCK-8 test, the LDH test and AFBO metabolite AFBO-GSH liquid chromatography detection show that in ten GSTs, only GST and GST3 can obviously improve the primary hepatocyte activity of ducks under the condition of toxicity attack, obviously reduce the LDH content in cell culture solution, obviously improve the yield of AFBO-GSH, and prove that GST and GST3 inhibit AFB 1 Toxicity.
Through the research, the over-expression of GST and GST3 in the primary liver cells of the ducks can obviously relieve the AFB 1 The cell activity rate is reduced, LDH is released, the yield of AFBO-GSH is improved, and other GSTs are over-expressed without obvious effect. Thus, GST and GST3 were determined to be involved in AFB in duck liver 1 Key enzymes for detoxification.
The invention has the beneficial effects that:
the glutathione thiol transferase (GST or GST 3) of the invention can effectively inhibit or relieve AFB 1 The induced liver toxicity of duckling is mainly related to the fact that the duckling can improve the metabolism efficiency of highly toxic AFBO. GST and GST3 of the invention can be used for treating AFB 1 Drug for causing duck liver injury and target enzyme developed by novel feed additive.
Drawings
FIG. 1 is 75ppbAFB 1 After 24h of toxin attack, a primary hepatocyte activity rate map of ducks after GSTs are overexpressed;
FIG. 2 is 150ppbAFB 1 After 24h of detoxification, GSTs are overexpressedCell viability map of duck primary liver;
FIG. 3 is 75ppbAFB 1 Graph of change in LDH activity in cell culture fluid 24h after challenge;
FIG. 4 is 150ppbAFB 1 Graph of change in LDH activity in cell culture fluid 24h after challenge;
FIG. 5 is 75ppbAFB 1 Change in AFBO-GSH content in cells 24h after challenge.
Detailed Description
The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
EXAMPLE 1 AFB 1 Treatment of Duck Primary liver cells transfected with empty plasmid and Change in cell Activity after GSTs overexpressing Duck Primary liver cells
Constructing pcDNA3.1 (+) -GSTs recombinant expression vector from CDs fragments of any one of the GSTs genes in the following table 1 cloned from duck liver cDNA, and transfecting; and GST transfection efficiency was verified by qPCR. The control group is a duck primary liver cell pcDNA3.1 (+) transfection group, and the experimental group is a different pcDNA3.1 (+) -GSTs transfection group; taking GST as an example:
the GST CDs fragment cloned from duck liver cDNA is recovered by glue and is homologous recombined onto pcDNA3.1 (+), and sequencing and comparison are successful to obtain pcDNA3.1 (+) -GST recombinant expression vector. Duck primary liver cells are separated from duck liver through in-situ perfusion, and are subjected to cell wall-attached culture for 24 hours, and then the pcDNA3.1 (+) and pcDNA3.1 (+) -GST recombinant expression vectors are transfected respectively, and the GST transfection efficiency is verified through qPCR. The control group is a duck primary liver cell pcDNA3.1 (+) transfection group, and the experimental group is a pcDNA3.1 (+) -GST transfection group;
table 1 GSTs Accession Number
After 24h of cell transfection expression, two different concentrations of 75ppb and 150ppb of AFB-containing cells were selected based on the results of the previous experiments 1 And (3) carrying out a toxicity attack test on the culture solution of the strain. After 24h of detoxification, the kit pair is according to the specification of the syn CCK-8 kitIt was subjected to cell viability assay, and at the time of absorbance detection, dual wavelength assays (450 nm and 630 nm) were used.
The results are shown in fig. 1 and 2: AFB at 75ppb and 150ppb 1 Under the action of the (2), the cell viability of the primary hepatocytes of the ducks is obviously reduced by 40% and 50% respectively. Of the ten GSTs transfected, duck primary hepatocytes overexpressing GST and GST3 were used for AFB 1 Shows stronger tolerance, the cell activity rate is significantly higher than that of the control virus attack group by 5 to 10 percent, which proves that GST and GST3 can effectively relieve AFB 1 Is a toxic property.
EXAMPLE 2 AFB 1 Treatment of changes in LDH Activity in Duck Primary hepatocytes transfected with empty plasmid and cell culture Medium after overexpression of Duck Primary hepatocytes by GSTs
The control group is a duck primary hepatocyte empty plasmid transfection group (pcDNA3.1 (+)), and the experimental group is a different GSTs transfection group. After 24h of transfection, two different concentrations of AFB of 75ppb and 150ppb were selected 1 And (3) carrying out a toxicity attack test on the culture solution of the strain. After 24h of detoxification, the supernatant of the culture solution in the cell culture dish is taken for LDH (lactate dehydrogenase) detection, and the detection method is strictly carried out according to the instruction of the Nanjing detection LDH detection kit.
The results are shown in fig. 3 and 4: AFB at 75ppb and 150ppb 1 Under the action of the above, the activity of LDH in the cell culture solution is obviously increased, which indicates that cells are obviously damaged, and in ten transfected GSTs, duck primary liver cells which overexpress GST and GST3 have the effect on AFB 1 Shows stronger tolerance, and the LDH activity of the compound is significantly lower than that of a control virus attack group by 12% -17%, which proves that GST and GST3 can effectively relieve AFB 1 Is a toxic property.
Example 3: AFB (alpha-fetoprotein) 1 Treatment of changes in AFBO-GSH content in Duck primary hepatocytes transfected with empty plasmid and cells after GSTs overexpressing Duck primary hepatocytes
The control group is a duck primary hepatocyte empty plasmid transfection group (pcDNA3.1 (+)), and the experimental group is a different GSTs transfection group. 24h after transfection, 75ppb of AFB was used 1 And (3) carrying out a toxicity attack test on the culture solution of the strain. After 24h of detoxification, the cells and the culture solution are collected into a 2ml centrifuge tube and are put into a-80 ℃ for coolingFreezing. And freeze-drying the collected sample, extracting and deriving, and detecting the content of AFBO-GSH by liquid chromatography.
The liquid chromatography results showed that: of the 10 GSTs transfected, only GST and GST3 were able to significantly increase the content of AFBO detoxification metabolite AFBO-GSH (46% and 13%), indicating that it was able to significantly alleviate AFB 1 Toxicity to duck primary hepatocytes (fig. 5).
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Sequence listing
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145 150 155 160
Leu Glu Ala Ile Leu Met Val Glu Glu Lys Lys Ser Asp Val Leu Ser
165 170 175
Ser Phe Pro Gln Leu Gln Ala Phe Lys Arg Arg Ile Ser Ser Ile Pro
180 185 190
Thr Ile Lys Lys Phe Leu Glu Pro Gly Ser Gln Arg Lys Pro Val Pro
195 200 205
Asp Asp Lys Tyr Val Glu Thr Val Arg Lys Val Leu Arg Met Tyr Tyr
210 215 220
Asp Ile Lys Ala Asn
225

Claims (1)

1. Preparation of glutathione sulfhydryl transferase for preventing and treating AFB 1 Application of medicines or feed additives for causing duck liver injury; the method is characterized in that: the glutathione sulfhydryl transferase is GST and GST3, wherein the amino acid sequence of the GST is shown as SEQ ID No.3, and the amino acid sequence of the GST3 is shown as SEQ ID No. 4.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136605A (en) * 1994-08-26 2000-10-24 Wisconsin Alumni Research Foundation Glutathione S-transferase isoforms
CN104099251A (en) * 2014-04-11 2014-10-15 江南大学 New aspergillus niger strain and application thereof in degradation of a plurality of kinds of fungaltoxin
CN106860878A (en) * 2017-01-13 2017-06-20 华中农业大学 MT genes are used as preventing and treating AFB1Cause the application of duck hepar damnification target gene
CN113755440A (en) * 2021-09-10 2021-12-07 广东海洋大学 AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6136605A (en) * 1994-08-26 2000-10-24 Wisconsin Alumni Research Foundation Glutathione S-transferase isoforms
CN104099251A (en) * 2014-04-11 2014-10-15 江南大学 New aspergillus niger strain and application thereof in degradation of a plurality of kinds of fungaltoxin
CN106860878A (en) * 2017-01-13 2017-06-20 华中农业大学 MT genes are used as preventing and treating AFB1Cause the application of duck hepar damnification target gene
CN113755440A (en) * 2021-09-10 2021-12-07 广东海洋大学 AFB (active carbon boron)1Cell model establishing method for inducing chicken liver injury

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