WO2020113843A1 - 一种海洋噬菌体的逆转录酶序列及其应用 - Google Patents

一种海洋噬菌体的逆转录酶序列及其应用 Download PDF

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WO2020113843A1
WO2020113843A1 PCT/CN2019/078408 CN2019078408W WO2020113843A1 WO 2020113843 A1 WO2020113843 A1 WO 2020113843A1 CN 2019078408 W CN2019078408 W CN 2019078408W WO 2020113843 A1 WO2020113843 A1 WO 2020113843A1
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reverse transcriptase
bacteriophage
phage
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汪岷
李慧芳
刘云丹
梁彦韬
刘昭阳
邵红兵
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中国海洋大学
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  • the invention belongs to the technical field of microbial engineering, and particularly relates to a reverse transcriptase sequence derived from marine bacteriophage and its application.
  • Reverse transcription also known as reverse transcription, uses RNA as a template to synthesize cDNA through reverse transcriptase. It is a special way of DNA biosynthesis and is catalyzed by reverse transcriptase. American scientists H.M.Temin and D.Baltimore discovered the reverse transcriptase in 1970, and thus won the 1975 Nobel Prize in Physiology and Medicine. The discovery of reverse transcription has greatly promoted the development of molecular biology and genetic engineering technology. It is indispensable in molecular cell biology experiments such as constructing and expressing eukaryotic or prokaryotic target genes, constructing cDNA libraries, and detecting gene sequences. The tools, together with Taq enzymes, etc., constitute the basic tools of modern biotechnology.
  • Phages are a large number of viruses that are ubiquitous in the ocean.
  • Basic research on the isolation, identification, biological characteristics, and genome of marine bacteriophages plays a vital role in understanding marine bacterial mortality, biological carbon cycle, microbial population structure, and horizontal transfer of marine microbial genes.
  • the research started later than other organisms.
  • humans began to have a preliminary understanding of bacteriophages, and its ecological significance was gradually recognized.
  • our understanding of marine phages is only the tip of the iceberg. Especially in the isolation of marine phages, only nearly 2,000 strains of tailed phages have been isolated.
  • the currently known reverse transcriptases are mainly derived from animal RNA viruses, and no reverse transcriptases derived from marine phages have been found.
  • the purpose of the present invention is to provide a marine bacteriophage reverse transcriptase sequence to make up for the shortcomings of the existing reverse transcriptase technology, to provide a new analytical biology research tool, and to promote its research in molecular biology, biochemistry and virology And application.
  • bacteriophages can only lyse one or similar bacteria, so it can be used for the identification and typing of bacteria.
  • bacteriophages have been used to divide Staphylococcus aureus into four groups. This method of phage typing is very helpful in tracking and analyzing the source of infection of these bacterial infections in epidemiological investigations.
  • the bacteriophage titer increase test should be used to check the corresponding bacteria in the specimen. If a certain bacteriophage is detected in the test material, it is often suggested that the corresponding bacteria are present.
  • a reverse transcriptase of marine bacteriophage its gene sequence:
  • the seawater filtered with a 0.22 ⁇ m filter membrane was used for phage separation.
  • the filtered water sample was mixed with the fresh bacterial solution obtained above at 1:1 (v/v), and after infection, the phage separation was performed by the double-layer plate method , Constant temperature cultivation, observe the presence or absence of transparent plaque;
  • Genomic DNA was extracted from the host bacteria in the above-mentioned host bacterial solution, and 16SrDNA identification was performed to obtain that the host bacteria was Marinomonas dokdonensis;
  • the marine bacteriophage was named Marinomonas phage LX, and the whole genome was sequenced; reverse transcriptase was found at the ORF 4 position of its genome, which is an RNA-dependent DNA polymerase.
  • bacteriophage genes An important tool for molecular biology research is the small number of bacteriophages genes. Some bacteriophages are genetically defective strains. Some bacteriophages are easy to control and identify through artificially induced mutations and inheritance, and can be used for gene transduction and transformation studies. In recent years, bacteriophage has become the main gene carrier tool in genetic research.
  • Marine bacteriophage Marinomonas phage LX and its reverse transcriptase can be used in molecular biology, especially in the preparation of medical clinical drugs.
  • Figure 1 is the electron micrograph of marine bacteriophage Marinomonas phage LX.
  • the surface water of the coastal seawater in Qingdao Luxun Park was used as the isolation source of bacteriophage host bacteria.
  • the surface seawater stored at 4°C was diluted with physiological saline according to the actual bacterial concentration, and the plate was cultured on the LB solid medium. on. After sealing, it was placed in a 28°C environment and incubated overnight. Afterwards, it was separated and purified several times by the zoning method, and the separated bacteria were stored in a refrigerator at 4°C until use.
  • the surface seawater collected in part is filtered with a 0.22 ⁇ m filter membrane to remove impurities such as large particles, micro-plankton and bacteria in the water, and a relatively pure virus water sample is obtained.
  • Genomic DNA was extracted from the resulting host bacteria, and PCR amplification was performed using primer 16S primer sequences 27F (5'-AGAXXXXX-3') and 1492R (5'-AGAXXXXX-3'). After PCR amplification, electrophoresis was performed on PCR. The products are identified and sequenced. The 16S rDNA results obtained by sequencing were compared in NCBI, and the host strain was determined to be Marinomonas dokdonensis.
  • the marine bacteriophage Marinomonas phage LX was subjected to whole-genome sequencing, and reverse transcriptase was found at the ORF 4 position of its genome, which is an RNA-dependent DNA polymerase.
  • the reverse transcriptase sequence is as follows:

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Abstract

提供了一种海洋噬菌体的逆转录酶序列及其在分子生物学中的应用。

Description

一种海洋噬菌体的逆转录酶序列及其应用 技术领域
本发明属于微生物工程技术领域，具体涉及一种海洋噬菌体来源的逆转录酶序列及其应用。
背景技术
逆转录又称为反转录，是以RNA为模板，通过逆转录酶，合成cDNA的过程，是DNA生物合成的一种特殊方式，由逆转录酶进行催化。美国科学家H.M.Temin和D.Baltimore在1970年发现了逆转录酶，并因此获得了1975年的诺贝尔生理学医学奖。逆转录的发现对分子生物学、基因工程技术的发展起到了巨大的推动作用，是构建、表达真核或者原核生物目的基因，构建cDNA文库，检测基因序列等分子细胞生物学实验中不可或缺的工具，与Taq酶等共同构成了现代生物技术的的基础工具。
噬菌体是在海洋中普遍存在并且数量巨大的一类病毒。对海洋噬菌体的分离、鉴定、生物学特性和基因组等方面的基础研究对于理解海洋细菌死亡率、生物碳循环、微生物种群结构、海洋微生物基因水平转移有着至关重要的作用。但是由于噬菌体本身结构的特殊性和技术的限制，较于其他生物，研究起步较晚。20世纪90年代初，人类对噬菌体开始有了初步的认识，其生态学意义也逐渐被认可。但目前我们对海洋噬菌体的认识还只是冰山一角，特别是海洋噬菌体的分离方面，仅有近2000株有尾噬菌体得到分离，在得到的全基因组信息中，大部分为未知序列，这极大的限制了未来对海洋噬菌体的深入研究及利用，因此对海洋噬菌体进一步的多角度深入认识已经成为亟待解决的海洋生态学热点问题。
并且，目前已知的逆转录酶主要来自动物RNA病毒，尚未发现任何的海洋噬菌体来源的逆转录酶。
发明内容
本发明的目的是提供一种海洋噬菌体的逆转录酶序列，以弥补现有逆转录酶技术的不足，以提供新型分析生物学研究工具，促进其在分子生物学、生物化学和病毒学的研究与应用。
细菌的鉴定与分型噬菌体的作用具有高度特异性。一种噬菌体只能裂解一种或与该种相近的细菌，故可用于细菌的鉴定和分型。目前已利用噬菌体将金黄色葡萄球菌分为四个群数，这种用噬菌体分型的方法，在流行病学调查上，对追查和分析这些细菌性感染的传染源很有帮助。
检测标本中的细菌应用噬菌体效价增长试验检查标本中的相应细菌，若在检材中检出某种噬菌体时，常提示有相应细菌存在。
为达到上述目的，本发明采取的具体技术方案为：
一种海洋噬菌体的逆转录酶，其基因序列：
Figure PCTCN2019078408-appb-000001
Figure PCTCN2019078408-appb-000002
上述逆转录酶的具体筛选和提取过程如下：
(1)多种菌液制备
将表层海水进行梯度稀释，涂布于LB固体培养基平板上，培养；随后采用分区划线法经多次纯化分离，将分离出的多种细菌保存于4℃中备用，得到了多种新鲜菌液；
(2)海洋噬菌体的分离纯化
用0.22μm滤膜过滤后的海水进行噬菌体分离，取过滤后的水样与上述得到的新鲜菌液分别进行1：1(v/v)混合，侵染后，用双层平板法进行噬菌体分离，恒温培养，观察有无透明噬菌斑的出现；
如果有透明噬菌斑，提取该噬菌斑，置于SM buffer中，震荡、过滤，用得到的噬菌体样品继续进行双层板实验，观察结果，重复3至5次，直至得到的噬菌斑形态大小均相似，此为分离出来的海洋噬菌体，与之对应的新鲜菌液为宿主菌液；
(3)宿主菌鉴定
将上述宿主菌液中的宿主菌进行基因组DNA提取，进行16SrDNA鉴定，得到宿主菌为海单孢菌Marinomonas dokdonensis；
(4)海洋噬菌体测序
将海洋噬菌体命名为Marinomonas phage LX，进行全基因组测序；在其基因组ORF 4位点处发现逆转录酶，是一种RNA依赖性的DNA聚合酶。
逆转录酶序列的发现说明了噬菌体需要通过逆转录把自身基因整合进入宿主菌的DNA 序列当中，在这个过程中很有可能出现基因的缺失，替换，甚至是大量序列的重排，导致宿主菌的某些功能发生改变，例如使一些毒性细菌丧失感染能力、减缓或加速某些细菌代谢的速率等。
分子生物学研究的重要工具噬菌体基因数量少，有些噬菌体为某种遗传基因缺陷株，有些噬菌体经人工诱导的变异和遗传容易控制和辩认，并且可用于基因的转导和变换等研究。近年来，噬菌体已成为遗传研究中的主要的基因载体工具。
海洋噬菌体Marinomonas phage LX及其逆转录酶能够应用于分子生物学，尤其是在制备医学临床药品上的应用。
本发明的优点和技术效果：
逆转录酶序列的发现填补了在海洋噬菌体研究领域的空白。在分子生物学领域具有巨大的应用前景；能够为一些临床疾病的治疗开展一些新思路。同时，也可以利用噬菌体来改变海洋中物质、能量的循环过程。
附图说明
图1为海洋噬菌体Marinomonas phage LX的电镜图。
具体实施方式
以下通过具体实施例并结合附图对本发明进一步解释和说明。
实施例1：
本实施例以青岛鲁迅公园的近岸表层海水水体为噬菌体宿主菌分离源，用生理盐水将保存于4℃的表层海水根据实际细菌浓度情况进行梯度稀释，涂布培养在LB固体培养基的平板上。密封后放在28℃环境中，过夜培养。随后采用分区划线法经多次纯化分离，将分离出的多种细菌保存于4℃冰箱中备用。用0.22μm滤膜过滤部分采集到的表层海水，去除掉水中较大的颗粒物、微微型浮游生物以及细菌等杂质，得到较为纯净的病毒水样。取200μL过滤后的水样与200μL的新鲜菌液分别混合，侵染20min后，用双层平板法进行噬菌体分离实验。28℃恒温培养24h后，观察有无透明噬菌斑的出现。用移液枪将观察到的噬菌斑戳出，置于SM buffer中，震荡，过滤，用得到的噬菌体样品继续进行双层板实验，观察结果，重复3至5次，直至得到的噬菌斑形态大小均相似，此为分离出来的海洋噬菌体，命名为Marinomonas phage LX，与之对应的菌株为宿主菌。将得到的宿主菌进行基因组DNA提取，使用引物16S引物序列27F(5’-AGAXXXXX-3’)和1492R(5’-AGAXXXXX-3’)进行PCR扩增，PCR扩增后，电泳，对PCR产物进行鉴定、测序。将测序得到的16S rDNA结果在NCBI里比对，测得宿主菌为海单孢菌Marinomonas dokdonensis。
取20uL纯化后效价达到10 10-11pfu/mL的噬菌体原液滴于铜网上，待其自然沉淀15min，加1滴2％磷钨酸(PTA)于铜网上，染色10min，干燥后用透射电子显微镜观察。经电镜观察，此噬菌体为无尾球状噬菌体，大小为23nm(图1)。
将海洋噬菌体Marinomonas phage LX进行全基因组测序，在其基因组ORF 4位点处发现逆转录酶，是一种RNA依赖性的DNA聚合酶。
逆转录酶序列如下：
Figure PCTCN2019078408-appb-000003

Claims (2)

  1. 一种海洋噬菌体的逆转录酶，其特征在于，该逆转录酶的基因序列：
    UUAAAAAUGUAAGUUUGCUUCAUUUUGACCCCUCUUGAGUUUUAUGCUCAGUAGUUUUUGCGUUAAUUGCUUGGUGCUAGCGUGGCGCGCAUGGCCCAGCCAGCUUUGAAUAUUUUGCUGUAUGUCGAUUAGAUCGACCUCGCCAGCGGCGUAUUUCUUUUGAAAUUUACGCAGCUUGGUUUUAAUUCUUUUCACGCUGCAUUUGCGUAACAAACGAUGCGUUGAGUAAAUCCGAUAGCCGAGAAAAUCCAACGCCCUCCCGUUGUGUUUUGCAAUCGGGAAUACUUGCGUUUUGCUGUUUGUUUGCAGUCUCAAUUGUGACCACAGAAACCGCUCGAUCUGUUCGCGCCACUCAUGCAACUGCGCUUUGUCGUGAUGAAUGACUGUGAAAUCAUCCAUAUAACGCAUGUAAUUUGGCGCUUUUAAUGUGUGUUUUACGAACCAAUCCAACUCAUGCAAAUACACAUUGGCAAAAACUUGGCUAGUCAAAUUGCCCAGCGGUAUGCCGACGCCUGCCGCAUCGCUUGGGCUGUUGUCGAUAAUGUAAAACAACAGUGCUUUCGUGCGUUGGCACCUUAAUUUUUUAUCUAAUAUCGAUUUCAAUAUACUGUGAUCGAUGCUCGAAAAAUACUUGCUAAUGUCCGCUUUUAGCGCGAACGCUUUGCCGUGUUUACGCUCCACUUGCUGUAUAAACGAUUGCGCCCGAUCCGCGCCUUUAUGCGUACCUUUGUUUUUGCGGCACGCAUAGGAAUCAUAGAUAUAUUGGCGAUCAAACAGCGGUUCGAUGAUGUUAUAAAUUGCCCUAUGCACAACUCUAUCCCGAAAAUGCGGUGCGCUAAUUAGCCGACGUUUUGGCUCAAACACAUAAAAAUGAUGAUAGAGCGACAUUUGAUACAUCUGCCAAACUAACUCGUUUUGUAUGCUGAUCACGUUUUCUUCGAGAUUGUUAAAAAACGACAAUACCGCUGUGUUUGUUGUUUUACCGAGCCGACACUGAUAUGCCGCAUUCAGUAUGUUUUCAAACUGAUAAAUCUGUUCGUAUAAACACUCGCUCGACGCAUCGAGCGAGGUAAUUCGUGUUUCGGCAAUAGCCGAGGAUGCUGCAUCCUUUUCAUGACUGCACUGACAGCACGCCUUGGCGUUGCUGUUUCUGGCAUUAUCAAGAGCGGGGCGGAACCCGAUGUUCGUGUUCGAAUUCGAACGAGCAUUGUUCAGAUUGAGCGCGCCCAACCCGGCAUUCGAGCCGUUGUUCCAAUUGCCACCGCGUAGCGGGAAUCGGUUCCGCUUAUUCAUCAU。
  2. 权利要求1所述的海洋噬菌体及其逆转录酶能够应用于分子生物学，能够用于制备医学临床药品。
PCT/CN2019/078408 2018-12-04 2019-03-16 一种海洋噬菌体的逆转录酶序列及其应用 WO2020113843A1 (zh)

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