CN114717212B - 谷胱甘肽巯基转移酶作为防治afb1致鸭肝脏损伤解毒酶的应用 - Google Patents
谷胱甘肽巯基转移酶作为防治afb1致鸭肝脏损伤解毒酶的应用 Download PDFInfo
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Abstract
本发明公开了一种谷胱甘肽巯基转移酶作为防治AFB1致鸭肝脏损伤解毒酶的应用,本发明对鸭GSTs酶家族中10种GST进行了试验,筛选出了有解毒效果的GST和GST3,提供了GST和GST3作为防治AFB1致动物肝脏损伤靶标酶的应用。本发明的谷胱甘肽巯基转移酶(GST或GST3)能有效抑制或缓解AFB1诱导的雏鸭肝脏毒性作用,主要与其能够催化AFBO生成无毒的AFBO‑GSH的代谢有关。本发明的GST和GST3可以作为治疗AFB1致鸭肝脏损伤药物及新型饲料添加剂开发提供新的的靶标基因。
Description
技术领域
本发明涉及AFB1解毒领域,具体涉及一种谷胱甘肽巯基转移酶作为防治AFB1致鸭肝脏损伤解毒酶的应用。
背景技术
据***粮食和农业组织的统计数据显示,全球大约1/4的农作物及农产品会受到霉菌毒素的污染(Mohajeri M et al.,2018;孙飞和方热军,2014)。其中黄曲霉毒素B1(aflatoxin B1,AFB1)污染分布范围极广,毒性极强,禽类对其尤为敏感,雏鸭是常见畜禽中对AFB1最敏感的物种之一(Rushing B R and Selim M I,2019)。畜禽采食黄曲霉毒素污染的饲料后,肝脏作为其的主要靶器官和重要的解毒器官,会出现严重的病理变化,并伴随着生产性能下降、生殖***损伤、免疫机能抑制等情况,甚至导致毒素残留于肉类产品中,长期食用被黄曲霉毒素污染的食物会导致癌症等疾病的发生(Williams et al,2004;黄志伟等,2015)。
当AFB1进入动物肝脏中时,在CYP450s(细胞色素酶P450家族)的作用下,AFB1被转化为高毒性的AFB1-外8,9-环氧化物(AFBO)和其余低毒性的代谢产物。之后AFBO在GSTs(谷胱甘肽巯基转移酶家族)、mEH(微粒体环氧化物水解酶)和AFAR(黄曲霉毒素B1醛还原酶)的作用下代谢为AFBO-GSH和AFB1-dialcohol达到解毒效果(Deng et al.,2018)。畜禽中,AFBO的代谢主要是依靠II相药物代谢酶系GSTs,它是具有多种生理功能的一组同工酶,广泛存在于生物体内,位于胞浆、线粒体及微粒体。值得注意的是,不同物种体内催化AFBO结合GSH生成AFBO-GSH而解毒的关键GSTs酶系存在差异性(Deng et al.,2018)。
鸭AFB1中毒造成的肝脏损伤尚无有效的治疗或缓解方法,GSTs作为其关键的解毒酶,目前在NCBI数据库中记录的十余种GSTs酶中哪些亚型能缓解AFB1诱导的鸭肝脏毒性尚不清楚。
发明内容
本发明针对现有技术对防治AFB1致家禽肝脏毒性的不足,提供了一种谷胱甘肽巯基转移酶作为防治AFB1致鸭肝脏损伤靶标酶的应用,本发明对鸭GSTs酶家族中10种GST进行了试验,筛选出了有解毒效果的GST和GST3,提供了GST和GST3作为防治AFB1致鸭肝脏损伤靶标酶的应用。
为实现上述目的,本发明所涉及一种谷胱甘肽巯基转移酶作为防治AFB1致鸭肝脏损伤靶标酶的应用;所述谷胱甘肽巯基转移酶为GST和GST3。
本发明还提供了一种谷胱甘肽巯基转移酶的基因在防治AFB1致鸭肝脏损伤的药物或饲料添加剂中的应用,所述谷胱甘肽巯基转移酶的基因为GST和GST3;其中,
所述GST的核苷酸序列为:
ATGTCAGGGAAGCCCAGGCTCACCTACATTAATGGAAGGGGACGAATGGAGCCGATCCGATGGCTGCTGGCAGCAGCCGGCGTGGAGTTTGAAGAAAACTTTGTGGAAACAAAAGAACAGTTAGAAAAGTTAATCAAAGGTGGAGACCTGCTGTTCCAGCAAGTGCCCATGGTGGAGATTGATGGGATGAAGATGGTGCAGACCAGAGCCATCCTCCGCTACATAGCGGGGAAATACAACCTCTATGGGAAGGACCTGAAGGAGAGAGCCCTGATCGACATGTATGTGGAAGGAATAACAGATCTGATGCAAATGATTATGATGTTTCCTTTCGCTCCAGCTGAGGCAAAGCCAAAAAATCTTGCCTCAATTGAAGAGAAGGCAACAAAGAGATACTTCCCAGTCTTTGAAAAGATTTTGAAACAACACGGCCAAGACTTTCTTGTGGGGAACCGACTCAGCTGGGCAGATGTTCAGCTATTGGAAGCCATTTTAGCAGTGGAGGAGAAAGTACCTGCTGTGCTTTCTGGGTTTCCTCAGCTGCAGGCCTTTAAAACAAAAATGAGCAACGTGCCTACAATTAAGAAGTTCCTGCAGCCTGGCAGCGCAAGGAAGCCCCCACCAGATGAGAATTATGTGGCACTTGTGATGTCGATTTTTAATCTAAGCTGA,其如SEQ ID No.1所示;
所述GST3的核苷酸序列为:
ATGGCTGGAAAACCCAAACTTTACTACTTTGATGGAAGAGGCAAAATGGAATCCATTCGCTGGTTGTTAGCTGCAGCCGGGGTTGAGTTTGAAGAGGAGTTTTTGGAAACACGAGAACAGTATGAGAAGCTCCTGCAAAGTGGATCCCTGCTGTTCCAGCAAGTTCCCCTGGTGGAGATCGATGGGATGAAGATGGTGCAGACCAGAGCCATCCTCAACTACATTGCAGCAAAGTACAACCTCTACGGAAAGGACCTGAAGGAGAGAGCCCTGATTGATATGTATGTTGGAGGAACTGAGGACCTTATGGGCTTTATTTTGATGTTCCCATTCTTATCGGCTGAGGATAAAGAGAAGCAACGTGCCTTTATAGTTCAGAAGGCCACCAGCAGGTACTTCCCAGTATATGAAAAGGTTCTGAAAGACCATGGGCAGGACTTCCTTGTTGGCAACAGTTTTAGCTGGGCAGACATTCATCTTCTTGAAGCCATTTTAATGGTAGAAGAGAAGAAGTCGGACGTTCTCTCGAGCTTTCCTCAGTTGCAGGCATTTAAAAGAAGGATAAGCAGCATCCCCACAATCAAGAAGTTTCTAGAGCCTGGAAGCCAGAGAAAACCTGTTCCTGATGATAAATATGTGGAGACTGTGAGGAAAGTTCTCCGCATGTATTATGATATAAAAGCGAATTAG,如SEQ ID No.2所示。
本发明GST和GST3是基于下表1中基因筛选得到。
表1 GSTs Accession Number
本发明还提供了一种重组表达载体,它含上述GST或GST3的表达载体,其中,所述表达载体为pcDNA3.1(+)。
本发明还提供了一种上述重组表达载体在防治AFB1致鸭肝脏损伤的药物或饲料添加剂中的应用。
本发明还提供了一种谷胱甘肽巯基转移酶防治AFB1致鸭肝脏损伤的药物或饲料添加剂中的应用;所述谷胱甘肽巯基转移酶为GST和GST3,所述GST的氨基酸序列为:
MSGKPRLTYINGRGRMEPIRWLLAAAGVEFEENFVETKEQLEKLIKGGDLLFQQVPMVEIDGMKMVQTRAILRYIAGKYNLYGKDLKERALIDMYVEGITDLMQMIMMFPFAPAEAKPKNLASIEEKATKRYFPVFEKILKQHGQDFLVGNRLSWADVQLLEAILAVEEKVPAVLSGFPQLQAFKTKMSNVPTIKKFLQPGSARKPPPDENYVALVMSIFNLS,其如SEQ ID No.3所示;
所述GST3的氨基酸序列为:
MAGKPKLYYFDGRGKMESIRWLLAAAGVEFEEEFLETREQYEKLLQSGSLLFQQVPLVEIDGMKMVQTRAILNYIAAKYNLYGKDLKERALIDMYVGGTEDLMGFILMFPFLSAEDKEKQRAFIVQKATSRYFPVYEKVLKDHGQDFLVGNSFSWADIHLLEAILMVEEKKSDVLSSFPQLQAFKRRISSIPTIKKFLEPGSQRKPVPDDKYVETVRKVLRMYYDIKAN,其如SEQ ID No.4所示。
本发明原理:
1.谷胱甘肽巯基转移酶(GST)被认为是参与亲电化合物代谢的最重要的酶(R.N.Armstrong,1991)。这些酶催化药物代谢途径中最重要的II期反应之一,该途径包括GSH与有害细胞亲电物质的结合,例如一些强致癌物(L.G.Higgins and J.D.Hayes,2011)以及内源性代谢物、激素和酚类微量营养素(F.Galli,2007)。作为药物代谢基因功能的一部分,GST介导的与GSH的结合使亲脂性亲电子试剂增加在水中的溶解度,从而促进其细胞转运和***。胞质中的GST的其他功能包括亲脂性小分子的结合、硫醇异构酶活性以及蛋白质硫醇的GSH依赖性保护和氧化还原调节参与信号转导(Z.Y.V.Adler et al.,1999;D.M.Townsend et al.,2009)。
2.本发明运用鸭原代肝细胞为模型,通过鸭原代肝细胞过表达GSTs,检测GSTs在缓解AFB1致鸭肝脏毒性作用中的功能。通过CCK-8试验、LDH试验以及AFBO代谢物AFBO-GSH液相色谱检测,结果表明在十种GSTs中,仅有GST和GST3均能够显著提高攻毒条件下鸭原代肝细胞活率,显著降低细胞培养液中的LDH含量,显著提高AFBO-GSH的产量,说明了GST和GST3在抑制AFB1毒性上的作用。
通过上述研究发现,鸭原代肝细胞中超表达GST和GST3可以显著缓解AFB1对其造成的细胞活率下降、LDH释放和提高AFBO-GSH的产量,而其他GSTs超表达无明显效果。因此确定GST和GST3为鸭肝中参与AFB1解毒的关键酶。
本发明的有益效果:
本发明的谷胱甘肽巯基转移酶(GST或GST3)能有效抑制或缓解AFB1诱导的雏鸭肝脏毒性作用,主要与其能够提高剧毒的AFBO的代谢效率有关。本发明的GST和GST3可以作为治疗AFB1致鸭肝脏损伤药物及新型饲料添加剂开发的靶标酶。
附图说明
图1为75ppbAFB1攻毒24h后,超表达GSTs后的鸭原代肝细胞活率图;
图2为150ppbAFB1攻毒24h后,超表达GSTs后的鸭原代肝的细胞活率图;
图3为75ppbAFB1攻毒24h后,细胞培养液中的LDH活性的变化图;
图4为150ppbAFB1攻毒24h后,细胞培养液中的LDH活性的变化图;
图5为75ppbAFB1攻毒24h后细胞中的AFBO-GSH含量的变化图。
具体实施方式
下面结合具体实施例对本发明作进一步的详细描述,以便本领域技术人员理解。
实施例1 AFB1处理空载质粒转染后的鸭原代肝细胞和GSTs过表达鸭原代肝细胞后的细胞活率变化
从鸭肝cDNA中克隆得到的下表1中GSTs的基因任意一种的CDs片段,构建pcDNA3.1(+)-GSTs重组表达载体,转染;并通过qPCR验证GST转染效率。对照组为鸭原代肝细胞pcDNA3.1(+)转染组,实验组则为不同pcDNA3.1(+)-GSTs转染组;以GST为例:
从鸭肝cDNA中克隆得到的GST CDs片段,胶回收后被同源重组到pcDNA3.1(+)上,测序比对成功后得到pcDNA3.1(+)-GST重组表达载体。通过原位灌注从鸭肝中分离得到鸭原代肝细胞,细胞贴壁培养24h后别转染分pcDNA3.1(+)和pcDNA3.1(+)-GST重组表达载体,并通过qPCR验证GST转染效率。对照组为鸭原代肝细胞pcDNA3.1(+)转染组,实验组则为pcDNA3.1(+)-GST转染组;
表1 GSTs Accession Number
在细胞转染表达24h后,根据之前的实验结果,选取75ppb和150ppb两个不同浓度的含AFB1的培养液进行攻毒试验。在攻毒24h后,按照同仁CCK-8试剂盒说明书对其进行细胞活率检测,在检测吸光值时,采取双波长检测(450nm和630nm)。
结果如图1和图2所示:在75ppb和150ppb的AFB1的作用下,鸭原代肝细胞的细胞活率均出现了显著下降,分别下降40%和50%。在所转染的十种GSTs中,过表达GST和GST3后的鸭原代肝细胞对AFB1的表现出了更强的耐受能力,其细胞活率显著高于对照攻毒组5%-10%,说明GST和GST3能够有效缓解AFB1的毒性。
实施例2 AFB1处理空载质粒转染后的鸭原代肝细胞和GSTs过表达鸭原代肝细胞后的细胞培养液中LDH活性的变化
对照组为鸭原代肝细胞空载质粒转染组(pcDNA3.1(+)),实验组为不同GSTs转染组。转染24h后,选取75ppb和150ppb两个不同浓度的AFB1的培养液进行攻毒试验。在攻毒24h后,取细胞培养皿中培养液上清进行LDH(乳酸脱氢酶)检测,检测方法严格按照南京检测LDH检测试剂盒说明书进行。
结果如图3和图4所示:在75ppb和150ppb的AFB1的作用下,细胞培养液中的LDH的活性显著上升,说明细胞受到显著损伤,在所转染的十种GSTs中,过表达GST和GST3后的鸭原代肝细胞对AFB1的表现出了更强的耐受能力,其LDH活性显著低于对照攻毒组12%-17%,说明GST和GST3能够有效缓解AFB1的毒性。
实施例3:AFB1处理空载质粒转染后的鸭原代肝细胞和GSTs过表达鸭原代肝细胞后的细胞中AFBO-GSH含量的变化
对照组为鸭原代肝细胞空载质粒转染组(pcDNA3.1(+)),实验组为不同GSTs转染组。转染24h后,用75ppb的AFB1的培养液进行攻毒试验。攻毒24h后,将细胞及培养液收集至2ml离心管中,放入-80℃中冷冻。将收集得到的样品进行冷冻干燥,萃取衍生,然后用液相色谱检测AFBO-GSH的含量。
液相色谱结果显示:在所转染的10种GSTs中,只有GST和GST3能够显著提高AFBO解毒代谢产物AFBO-GSH的含量(46%和13%),说明其能显著缓解AFB1对鸭原代肝细胞的毒性(图5)。
其它未详细说明的部分均为现有技术。尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例,而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
序列表
<110> 华中农业大学
<120> 谷胱甘肽巯基转移酶作为防治AFB1致鸭肝脏损伤解毒酶的应用
<160> 4
<170> SIPOSequenceListing 1.0
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atgtcaggga agcccaggct cacctacatt aatggaaggg gacgaatgga gccgatccga 60
tggctgctgg cagcagccgg cgtggagttt gaagaaaact ttgtggaaac aaaagaacag 120
ttagaaaagt taatcaaagg tggagacctg ctgttccagc aagtgcccat ggtggagatt 180
gatgggatga agatggtgca gaccagagcc atcctccgct acatagcggg gaaatacaac 240
ctctatggga aggacctgaa ggagagagcc ctgatcgaca tgtatgtgga aggaataaca 300
gatctgatgc aaatgattat gatgtttcct ttcgctccag ctgaggcaaa gccaaaaaat 360
cttgcctcaa ttgaagagaa ggcaacaaag agatacttcc cagtctttga aaagattttg 420
aaacaacacg gccaagactt tcttgtgggg aaccgactca gctgggcaga tgttcagcta 480
ttggaagcca ttttagcagt ggaggagaaa gtacctgctg tgctttctgg gtttcctcag 540
ctgcaggcct ttaaaacaaa aatgagcaac gtgcctacaa ttaagaagtt cctgcagcct 600
ggcagcgcaa ggaagccccc accagatgag aattatgtgg cacttgtgat gtcgattttt 660
aatctaagct ga 672
<210> 2
<211> 690
<212> DNA
<213> 绿头鸭(Anas platyrhynchos)
<400> 2
atggctggaa aacccaaact ttactacttt gatggaagag gcaaaatgga atccattcgc 60
tggttgttag ctgcagccgg ggttgagttt gaagaggagt ttttggaaac acgagaacag 120
tatgagaagc tcctgcaaag tggatccctg ctgttccagc aagttcccct ggtggagatc 180
gatgggatga agatggtgca gaccagagcc atcctcaact acattgcagc aaagtacaac 240
ctctacggaa aggacctgaa ggagagagcc ctgattgata tgtatgttgg aggaactgag 300
gaccttatgg gctttatttt gatgttccca ttcttatcgg ctgaggataa agagaagcaa 360
cgtgccttta tagttcagaa ggccaccagc aggtacttcc cagtatatga aaaggttctg 420
aaagaccatg ggcaggactt ccttgttggc aacagtttta gctgggcaga cattcatctt 480
cttgaagcca ttttaatggt agaagagaag aagtcggacg ttctctcgag ctttcctcag 540
ttgcaggcat ttaaaagaag gataagcagc atccccacaa tcaagaagtt tctagagcct 600
ggaagccaga gaaaacctgt tcctgatgat aaatatgtgg agactgtgag gaaagttctc 660
cgcatgtatt atgatataaa agcgaattag 690
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Met Ser Gly Lys Pro Arg Leu Thr Tyr Ile Asn Gly Arg Gly Arg Met
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Glu Pro Ile Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Phe Glu Glu
20 25 30
Asn Phe Val Glu Thr Lys Glu Gln Leu Glu Lys Leu Ile Lys Gly Gly
35 40 45
Asp Leu Leu Phe Gln Gln Val Pro Met Val Glu Ile Asp Gly Met Lys
50 55 60
Met Val Gln Thr Arg Ala Ile Leu Arg Tyr Ile Ala Gly Lys Tyr Asn
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Leu Tyr Gly Lys Asp Leu Lys Glu Arg Ala Leu Ile Asp Met Tyr Val
85 90 95
Glu Gly Ile Thr Asp Leu Met Gln Met Ile Met Met Phe Pro Phe Ala
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Pro Ala Glu Ala Lys Pro Lys Asn Leu Ala Ser Ile Glu Glu Lys Ala
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Thr Lys Arg Tyr Phe Pro Val Phe Glu Lys Ile Leu Lys Gln His Gly
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Gln Asp Phe Leu Val Gly Asn Arg Leu Ser Trp Ala Asp Val Gln Leu
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Leu Glu Ala Ile Leu Ala Val Glu Glu Lys Val Pro Ala Val Leu Ser
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Gly Phe Pro Gln Leu Gln Ala Phe Lys Thr Lys Met Ser Asn Val Pro
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Thr Ile Lys Lys Phe Leu Gln Pro Gly Ser Ala Arg Lys Pro Pro Pro
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Asp Glu Asn Tyr Val Ala Leu Val Met Ser Ile Phe Asn Leu Ser
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<211> 229
<212> PRT
<213> 绿头鸭(Anas platyrhynchos)
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Met Ala Gly Lys Pro Lys Leu Tyr Tyr Phe Asp Gly Arg Gly Lys Met
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Glu Ser Ile Arg Trp Leu Leu Ala Ala Ala Gly Val Glu Phe Glu Glu
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Glu Phe Leu Glu Thr Arg Glu Gln Tyr Glu Lys Leu Leu Gln Ser Gly
35 40 45
Ser Leu Leu Phe Gln Gln Val Pro Leu Val Glu Ile Asp Gly Met Lys
50 55 60
Met Val Gln Thr Arg Ala Ile Leu Asn Tyr Ile Ala Ala Lys Tyr Asn
65 70 75 80
Leu Tyr Gly Lys Asp Leu Lys Glu Arg Ala Leu Ile Asp Met Tyr Val
85 90 95
Gly Gly Thr Glu Asp Leu Met Gly Phe Ile Leu Met Phe Pro Phe Leu
100 105 110
Ser Ala Glu Asp Lys Glu Lys Gln Arg Ala Phe Ile Val Gln Lys Ala
115 120 125
Thr Ser Arg Tyr Phe Pro Val Tyr Glu Lys Val Leu Lys Asp His Gly
130 135 140
Gln Asp Phe Leu Val Gly Asn Ser Phe Ser Trp Ala Asp Ile His Leu
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.谷胱甘肽巯基转移酶在制备防治AFB1致鸭肝脏损伤的药物或饲料添加剂中的应用;其特征在于:所述谷胱甘肽巯基转移酶为GST和GST3,其中,所述GST的氨基酸序列如SEQ IDNo.3所示,所述GST3的氨基酸序列如SEQ ID No.4所示。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US6136605A (en) * | 1994-08-26 | 2000-10-24 | Wisconsin Alumni Research Foundation | Glutathione S-transferase isoforms |
CN104099251A (zh) * | 2014-04-11 | 2014-10-15 | 江南大学 | 一种新型黑曲霉菌株及其在多种真菌毒素降解中的应用 |
CN106860878A (zh) * | 2017-01-13 | 2017-06-20 | 华中农业大学 | Mt基因作为防治afb1致鸭肝脏损伤靶标基因的应用 |
CN113755440A (zh) * | 2021-09-10 | 2021-12-07 | 广东海洋大学 | 一种afb1诱导鸡肝脏损伤的细胞模型建立方法 |
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Patent Citations (4)
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 (zh) * | 2014-04-11 | 2014-10-15 | 江南大学 | 一种新型黑曲霉菌株及其在多种真菌毒素降解中的应用 |
CN106860878A (zh) * | 2017-01-13 | 2017-06-20 | 华中农业大学 | Mt基因作为防治afb1致鸭肝脏损伤靶标基因的应用 |
CN113755440A (zh) * | 2021-09-10 | 2021-12-07 | 广东海洋大学 | 一种afb1诱导鸡肝脏损伤的细胞模型建立方法 |
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