CN103882097A - 高产蛋白酶枯草芽孢杆菌的鉴定方法 - Google Patents

高产蛋白酶枯草芽孢杆菌的鉴定方法 Download PDF

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CN103882097A
CN103882097A CN201210561060.1A CN201210561060A CN103882097A CN 103882097 A CN103882097 A CN 103882097A CN 201210561060 A CN201210561060 A CN 201210561060A CN 103882097 A CN103882097 A CN 103882097A
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bacterial strain
pcr amplification
identification method
primer
bacillus subtilis
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张述智
夏伟
朱绍辉
张�浩
王晓丽
徐权汗
李之详
许团辉
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Qingdao Zhongren Pharmaceutical Coltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

本发明公开了一种高产蛋白酶枯草芽孢杆菌的鉴定方法，取待测菌株接种于脱脂奶粉平板培养基上35℃培养24h后，革兰氏染色，镜检；再分别取待测菌株1.0ml用基因组DNA提取试剂盒提取DNA模板，设计合成PCR扩增引物，上游引物P1：5＇-AGAGTTTGATCCTGGCTCAG-3＇；下游引物P2：5＇-AGTAAGGAGGTGATCCAACCGCA-3＇，进行16SrDNA的PCR扩增。对菌株选择性强，鉴定方法科学，时间周期短，效率高。

Description

高产蛋白酶枯草芽孢杆菌的鉴定方法
  
技术领域
本发明属于生物技术领域，具体涉及菌株的鉴定方法。 
  
背景技术
催化蛋白质水解的酶类有很多种类，重要的有胃蛋白酶、胰蛋白酶、组织蛋白酶、木瓜蛋白酶、枯草杆菌蛋白酶和地衣芽孢杆菌蛋白酶等。蛋白酶对所作用的反应底物有严格的选择性，一种蛋白酶仅能作用于蛋白质分子中一定的肽键，如胰蛋白酶催化水解碱性氨基酸所形成的肽键。蛋白酶广泛存在于动物内脏、植物茎叶、果实和微生物中。微生物蛋白酶主要由霉菌、细菌，其次由酵母、放线菌生产。 
现有技术鉴定枯草芽孢杆菌的方法普遍存在对菌株选择性低，鉴定方法不科学，时间周期长，效率低的问题。 
  
发明内容
为了克服现有技术领域存在的上述缺陷，本发明的目的在于，提供一种高产蛋白酶枯草芽孢杆菌的鉴定方法，解决现有技术对菌株选择性低，鉴定方法不科学，时间周期长，效率低的问题。 
本发明提供的高产蛋白酶枯草芽孢杆菌的鉴定方法，包括以下步骤： 
一、形态学鉴定，取待测菌株接种于脱脂奶粉平板培养基上35℃培养24h后，革兰氏染色，镜检；
二、PCR鉴定，分别取待测菌株1.0ml用基因组DNA提取试剂盒提取DNA模板，设计合成PCR扩增引物，上游引物P1：5＇- AGAGTTTGATCCTGGCTCAG -3＇；下游引物P2：5＇- AGTAAGGAGGTGATCCAACCGCA -3＇，进行16S rDNA的PCR扩增，PCR反应条件为：94℃预变性10min，然后 94℃变性1min，54℃退火1min，72℃延伸2min，共25个循环，最后72℃延伸10min。 
本发明提供的高产蛋白酶枯草芽孢杆菌的鉴定方法，其有益效果在于，对菌株选择性强，鉴定方法科学、时间周期短、效率高。 
  
具体实施方式
下面结合一个实施例，对本发明提供的高产蛋白酶枯草芽孢杆菌的鉴定方法进行详细的说明。 
  
实施例
本实施例的高产蛋白酶枯草芽孢杆菌的鉴定方法，包括以下步骤： 
一、形态学鉴定，取待测菌株接种于脱脂奶粉平板培养基上35℃培养24h后，革兰氏染色，镜检；
二、PCR鉴定，分别取待测菌株1.0ml用基因组DNA提取试剂盒提取DNA模板，设计合成PCR扩增引物，上游引物P1：5＇- AGAGTTTGATCCTGGCTCAG -3＇；下游引物P2：5＇- AGTAAGGAGGTGATCCAACCGCA -3＇，进行16S rDNA的PCR扩增，PCR反应条件为：94℃预变性10min，然后 94℃变性1min，54℃退火1min，72℃延伸2min，共25个循环，最后72℃延伸10min。 
PCR产物纯化后连接克隆载体并转化，酶切鉴定后送至上海Invitrogen公司测序，所测序列提交GenBank进行同源性比较。所得序列为： 
agagtttgat cctggctcag gacgaacgct ggcggcgtgc ctaatacatg caagtcgagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat gcttgtttga accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag tgttaggggg tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat gggcagaaca aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct tttaggagcc agccgccgaa ggtgggacag atgattgggg tgaagtcgta acaaggtagc cgtatcggaa ggtgcggttg gatcacctcc ttact。

Claims (1)

1.一种高产蛋白酶枯草芽孢杆菌的鉴定方法，其特征在于：包括以下步骤：
一、形态学鉴定，取待测菌株接种于脱脂奶粉平板培养基上35℃培养24h后，革兰氏染色，镜检；
二、PCR鉴定，分别取待测菌株1.0ml用基因组DNA提取试剂盒提取DNA模板，设计合成PCR扩增引物，上游引物P1：5＇- AGAGTTTGATCCTGGCTCAG -3＇；下游引物P2：5＇- AGTAAGGAGGTGATCCAACCGCA -3＇，进行16S rDNA的PCR扩增，PCR反应条件为：94℃预变性10min，然后 94℃变性1min，54℃退火1min，72℃延伸2min，共25个循环，最后72℃延伸10min。
CN201210561060.1A 2012-12-21 2012-12-21 高产蛋白酶枯草芽孢杆菌的鉴定方法 Pending CN103882097A (zh)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016118864A1 (en) * 2015-01-23 2016-07-28 Novozymes A/S Bacillus strains improving health and performance of production animals
US11331351B2 (en) 2015-01-23 2022-05-17 Novozymes A/S Bacillus strains improving health and performance of production animals
US11473052B2 (en) 2015-01-23 2022-10-18 Novozymes A/S Bacillus subtilis subspecies

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016118864A1 (en) * 2015-01-23 2016-07-28 Novozymes A/S Bacillus strains improving health and performance of production animals
CN107567493A (zh) * 2015-01-23 2018-01-09 诺维信公司 改进生产动物的健康和性能的芽孢杆菌属菌株
AU2016209132B2 (en) * 2015-01-23 2021-07-01 Novozymes A/S Bacillus strains improving health and performance of production animals
US11166989B2 (en) 2015-01-23 2021-11-09 Novozymes A/S Bacillus strains improving health and performance of production animals
CN107567493B (zh) * 2015-01-23 2021-11-16 诺维信公司 改进生产动物的健康和性能的芽孢杆菌属菌株
US11331351B2 (en) 2015-01-23 2022-05-17 Novozymes A/S Bacillus strains improving health and performance of production animals
US11473052B2 (en) 2015-01-23 2022-10-18 Novozymes A/S Bacillus subtilis subspecies
US11801272B2 (en) 2015-01-23 2023-10-31 Novozymes A/S Bacillus strains improving health and performance of production animals

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Application publication date: 20140625