CN113462698A - 林可霉素的新型抗性基因及其应用 - Google Patents
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- 229960005287 lincomycin Drugs 0.000 title claims abstract description 42
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- 238000007476 Maximum Likelihood Methods 0.000 description 1
- 238000012952 Resampling Methods 0.000 description 1
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Abstract
本发明涉及林可霉素的抗性基因及其应用,具体公开了林可霉素的新型抗性基因erm41_like基因及其应用,其中基因序列如SEQ ID No.1所示。经序列相似度比对,没有在基因组上发现目前已知的抗性基因。本发明还公开了该基因的检测方法。本发明对了解林可霉素的耐药机制和补充耐药数据库提供了帮助,也为之后对抗林可霉素耐药的研究提供了理论支持,为解决林可霉素耐药提供靶点,为对抗细菌耐药研究奠定基础。
Description
技术领域
本发明属于生物技术领域,尤其涉及林可霉素的抗性基因及其应用。
背景技术
林可霉素是由林可霉素链霉菌变异菌株发酵产生的一种天然广谱抗生素,可作用于细菌核糖体50S亚基,抑制肽链延长,进而阻挠蛋白质合成。因其对革兰氏阳性菌和阴性菌具有良好的抑菌作用,在动物及人类疾病的预防和控制方面被广泛应用。其耐药机理及代表性抗性基因主要有:外排泵(如lmrA、lmrB、lsaA)、去甲基化(如ermA、ermB、ermF)、核苷酸转移(如lnuA、lnuB、lnuC)和磷酸化(mphA、mphB)。
隐马尔可夫模型(Hidden Markov Model,HMM)是一种基于概率模型,从可观察的参数中确定该过程中隐含的参数的方法。在抗生素抗性基因(Antibiotic resistancegenes,ARGs)的研究领域,HMM用于构建抗性基因的数据库模型,实现抗性基因的注释,对传统的基于基因序列相似度的注释方法(如BLAST)上进行了补充,而且具有发现新型抗性基因的潜力。目前,应用广泛的数据库,如Pfam、TIGERFAM、Resfam等都是基于HMM模型构建的数据库。
ARGs-OAP是一个在线ARGs分析平台,可实现宏基因组数据的ARGs快速注释和定量,其内含的数据库(Structured Antibiotic Resistance genes,SARG)整合了两大ARGs数据库,ARDB和CARD,并通过对ARGs序列的逐级分类注释,为全面分析ARGs奠定了坚实的基础。在SARG数据库基础上,该平台应用HMM模型构建的方法,建立了基于模型比对的数据库SARGfam,可用于开发潜在的新型抗性基因。
发明内容
针对上述背景技术,本发明目的是提供林可霉素的抗性基因及其应用,本发明是从一株Conexibacter(无对应中文名称)中发现了林可霉素的新型抗性基因erm41_like基因,补充了林可霉素耐药的数据库,为深入研究细菌耐药性打下了坚实基础。
本发明第一方面是提供林可霉素的抗性基因,其基因序列如SEQ ID No.1所示,命名为erm41_like基因。
本发明第二方面是提供erm41_like基因表达抑制剂在制备降低受试者对林可霉素类抗生素耐药性的产品中的应用。
本发明第三方面是提供检测SEQ ID No.1序列的试剂在制备检测林可霉素类抗生素耐药性的试剂盒中的应用,进一步地,所述检测SEQ ID No.1序列的试剂为以PCR方法对SEQ ID No.1序列进行定性或定量检测构建的试剂。
本发明第四方面是提供erm41_like基因作为靶基因在提高受试者对林可霉素类抗生素敏感度方面的应用。
本发明第五方面是提供一种检测林可霉素类抗生素耐药性的方法,进一步地,基于SEQ ID No.1序列信息检测林可霉素类抗生素耐药性,所述方法不以诊断为目的。
在本发明的技术方案中,所述林可霉素类抗生素耐药性是指革兰氏阳性菌和革兰氏阴性菌对林可霉素类抗生素的耐药性。
上述技术方案具有如下优点或者有益效果:
本发明提供林可霉素的抗药基因erm41_like基因及其应用,其中erm41_like基因序列如SEQ ID No.1所示。经序列相似度比对,没有在基因组上发现目前已知的抗性基因。本发明对了解林可霉素的耐药机制和补充耐药数据库提供了帮助,也为之后对抗林可霉素耐药的研究提供了理论支持,为解决林可霉素耐药提供靶点,为对抗细菌耐药研究奠定基础。
附图说明
图1是本发明实施例1中erm41_like基因与已知参考基因序列之间系统发育树图,用于描述erm41_like基因序列与已知参考序列之间的差异度。
具体实施方式
下述实施例仅仅是本发明的一部分实施例,而不是全部的实施例。因此,以下提供的本发明实施例中的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。基于本发明的实施例,本领域技术人员在没有作出创造性劳动的前提下所获得的所有其他实施例,都属于本发明的保护范围。
实施例1
1.基因组开放阅读框(open reading frame,ORF)预测
应用prodigal软件对分装得到的纯菌基因组进行ORF预测,得到该基因组的ORF文件。
2.基于HMM模型比对
应用HMMER软件中的hmmscan功能,将步骤1得到的ORF文件与SARGfam数据库进行比对(参数设置:--cut_ga-tblout),得到基于模型比对的ORF注释结果。其中比对结果如表1所示。
表1
Target name | Query id | E-value | sore | Bias |
macrolide_lincosamide_streptogramin__erm_41__train_msa | 11-205_41 | 1.60E-24 | 80.2 | 11.2 |
3.基于序列相似度比对
应用blastp,将步骤1得到的ORF文件与SARG数据库进行比对(参数设置:-evalue1e-10-max_target_seqs 1),得到基于序列相似度的ORF注释结果。其中比对结果如表2所示。
表2
4.潜在新型抗性基因的确定
将步骤3的结果中序列相似度高于70%,覆盖度高于65%的ORF注释结果定义为真,即为已知的ARGs,而不满足该阈值的ORF若在步骤2中存在注释结果,则将该ORF定义为潜在的新型基因;若某一ORF基于序列相似度比对后,不被注释为ARG,但在HMM模型的比对结果中,被注释为ARG,该ORF同样认为是潜在的新型基因。经过筛选,发现该纯菌基因组中存在潜在的新型林可霉素抗性基因erm41_like。将SARG数据库中erm41基因对应的参考序列与erm41_like的基因序列利用ClustalW进行多重序列比对,并用MEGA构建最大似然系统发育树,通过自举法重抽样1000次评估树的拓扑结构,进一步确定潜在新型基因erm41_like与已知参考基因序列之间的差异度(如图1),由图可见,erm41_like基因序列为单独的分支,与数据库中erm41的参考序列之间存在明显差异。
实施例2
将含有erm41_like基因的菌株进行纯化分离,检测其对林可霉素的最小抑菌浓度MIC值之后发现,该菌株对林可霉素的MIC值高达200mg/L,且该菌株的基因组序列经BLAST比对发现,无其他林可霉素的抗性基因,说明erm41_like基因使得菌株对林可霉素具有抗性,erm41_like是林可霉素的耐药基因。
以上所述仅为本发明的优选实施例,并非因此限制本发明的专利范围,凡是利用本发明说明书及附图内容所作的等效变换,或直接或间接运用在其他相关的技术领域,均同理包括在本发明的专利保护范围内。
SEQUENCE LISTING
<110> 清华大学深圳国际研究生院
<120> 林可霉素的新型抗性基因及其应用
<130> 2021
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 780
<212> DNA
<213> Conexibacter
<400> 1
atggcccgag ccgcccagcg gcgccggacg ccgcgccgca cgaaccagtc aggcgtccac 60
atccttcatt cacatgccgt cgtccgtgct ctcgtgcgct cggcggagct cggcgaggac 120
gaccacgtcg ttgacctcgg cgccgggccc ggcgcgctga ccgcggcgct cgccgcgatc 180
ggcgcgcgcg tgacggcggt cgagcgcgac gaggcgttcg tgcgcacgct gcgccggcgc 240
ttcgccgacc ggccgcgcgt gcgcgtggtg cacggcgacc tgctgcaggt gccgatcccg 300
cgcgccgcga aggtcgtcgc gaacatcccc ttcgggacct ccagcgcgct gctggcgcgg 360
ctgttgaacc cggcgggccg gctgcgcccc ggggccgatc tcgtcgtcga gcgcggcttc 420
gcgctgcgcg caagcgcgcc ggtgcagcgc tcggcggagg ccgcgtggcg cagcgcccgc 480
tacgagatcc gcctggccgg tccgatcccc cgcgcgagct tcgggccgcc accggccgtc 540
gacgctgcgc acctgcggat ccgcccgcgt cggatgctcg actccgaggc ggaggcgcgc 600
ctgcgggcgc tgctcgccgc cgcctacgac gggcgcgtcc gcagcgccgg ttcggtcgcc 660
cgccgcgtcg tgggtccggc gcgcgcgagg cgcggcctgc ttgcggccgg cgtcgacgcg 720
ggcacgccgg cgacgctggt gcccgcgtgg gcgtggggcg cggtcgcaga gggcgggtaa 780
Claims (9)
1.林可霉素的抗性基因,其特征在于,其基因序列如SEQ ID No.1所示,命名为erm41_like基因。
2.erm41_like基因表达抑制剂在制备降低受试者对林可霉素类抗生素耐药性的产品中的应用。
3.检测SEQ ID No.1序列的试剂在制备检测林可霉素类抗生素耐药性的试剂盒中的应用。
4.根据权利要求3所述的应用,其特征在于,所述检测SEQ ID No.1序列的试剂为以PCR方法或QPCR方法对SEQ ID No.1序列进行定性或定量检测构建的试剂。
5.erm41_like基因作为靶基因在提高受试者对林可霉素类抗生素敏感度方面的应用。
6.根据权利要求2-5任一所述的应用,其特征在于,所述林可霉素类抗生素耐药性是指革兰氏阳性菌和革兰氏阴性菌对林可霉素类抗生素的耐药性。
7.一种检测林可霉素类抗生素耐药性的方法,其特征在于,基于SEQ ID No.1序列信息检测林可霉素类抗生素耐药性。
8.根据权利要求7所述的方法,其特征在于,所述方法不以诊断为目的。
9.根据权利要求7所述的方法,其特征在于,所述林可霉素类抗生素耐药性是指革兰氏阳性菌和革兰氏阴性菌对林可霉素类抗生素的耐药性。
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US20100317677A1 (en) * | 2007-09-11 | 2010-12-16 | Hassel Bret A | Methods of Treating a Microbial Infection by Modulating RNase-L Expression and/or Activity |
US20120190708A1 (en) * | 2009-07-10 | 2012-07-26 | University Of Maryland, Baltimore | Targeting nad biosynthesis in bacterial pathogens |
CN109852714A (zh) * | 2019-03-07 | 2019-06-07 | 南京世和基因生物技术有限公司 | 一种肠癌早期诊断和腺瘤诊断标志物及用途 |
CN112111502A (zh) * | 2020-09-25 | 2020-12-22 | 清华大学深圳国际研究生院 | 氯霉素的新型抗性基因及其应用 |
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US20100317677A1 (en) * | 2007-09-11 | 2010-12-16 | Hassel Bret A | Methods of Treating a Microbial Infection by Modulating RNase-L Expression and/or Activity |
US20120190708A1 (en) * | 2009-07-10 | 2012-07-26 | University Of Maryland, Baltimore | Targeting nad biosynthesis in bacterial pathogens |
CN109852714A (zh) * | 2019-03-07 | 2019-06-07 | 南京世和基因生物技术有限公司 | 一种肠癌早期诊断和腺瘤诊断标志物及用途 |
CN112111502A (zh) * | 2020-09-25 | 2020-12-22 | 清华大学深圳国际研究生院 | 氯霉素的新型抗性基因及其应用 |
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