CN108220249A - Siphovirus and its preparation method and application - Google Patents
Siphovirus and its preparation method and application Download PDFInfo
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- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 claims description 2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2795/00—Bacteriophages
- C12N2795/00011—Details
- C12N2795/10011—Details dsDNA Bacteriophages
- C12N2795/10311—Siphoviridae
- C12N2795/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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Abstract
The invention discloses a kind of siphovirus, which includes：Encode the nucleotide sequence of amino acid sequence shown in SEQ ID NO.1.Preparation method and application the invention also discloses above-mentioned siphovirus.The siphovirus of the present invention is that the one plant of new virus obtained is detached from patients with periodontitis oral cavity using viral metagenomics method, new foundation is provided for the cause of disease of mouth disease, treatment and preventative strategies.
Description
Technical field
The present invention relates to dentistry and virology technical fields more particularly to one kind to detach from patients with periodontitis oral cavity
The siphovirus and its preparation method of acquisition and application.
Background technology
Periodontosis is a complex set of infectious diseases, it is generally recognized that it is caused by bacterium stimulation, is more by human immunity etc.
Factor participates in.Periodontosis is with absorption of alveolar bone, tooth mobility and comes off for main clinic symptoms, has become adult and loses tooth
Main cause.Severe periodontitis can not only influence oral health, also with diabetes, angiocardiopathy, under-weight newborn early
The systemic diseases such as production are related, and serious negative effect is generated to whole body health.Traditional periodontosis Study of Etiology is usually recognized
Morbidity for periodontosis is related to particular kind of bacterium infection, however bacterium is caused a disease, theory but can not completely explain periodontal
The various Clinical symptoms of disease.For example, Umeda etc. the study found that Periodontal Pathogens are widely present among saliva, contacts all teeth
Tooth, but periodontosis is but often not full mouth morbidity, and a limited number of tooth is only influenced, there is locus specificity；Although tooth
Always there are the purpose Periodontal Pathogens persistent infection of variety classes sum number and field planting in all environment, but periodontosis is but typically short-term hair
Disease has self limiting；Virus gradually attracts attention as another potential periodontosis pathogenic factor, in periodontosis pathogenic process,
The immunological function repression of the anti-Periodontal Pathogens of host caused by Colonization, virus with the relevant Periodontal Pathogens of virus, with
The mechanism such as the relevant activated viral of periodontosis can explain bacterial plaque amount and periodontitis severity, the pass of tempo relatively reasonablely
System.Viral metagenomics can fully excavate viral group and unknown virus in specific environment as an emerging technology.
Styloviridae is a kind of distrand DNA virus, and only meeting bacterial infection, appearance have the tail of long shrinkage-void
Sheath.Bacteriophage in Human Oral Cavity is that belong to tailed phages purpose Stylovinidae (be usually all lysogeny phagocytosis mostly
Body and the host range for having medium-width), Myoviridae (mainly lytic phage and host range it is slightly wide by one
A bit) and Podoviridae (mainly lytic phage and host range slightly narrower).About bacteriophage in periodontal
Status and effect in the occurrence and development of disease, current correlative study is not very much, and still has arguement.There is researcher comparing
It is found after bacteriophage quantity in the saliva and bacterial plaque of patients with periodontitis and periodontal health crowd, in the bacterial plaque of patients with periodontitis
Bacteriophage quantity and type be very different compared with the bacterial plaque of periodontal health person, and related to Periodontal Status, and periodontal is good for
Bacteriophage structure inside Kang Renqun then stablizes (Ly, M., et al., Altered oral viral ecology in relatively
association with periodontal disease.MBio,2014.5(3):P.e01133-14), it is thus regarded that phagocytosis
The morbidity of body and periodontitis may be closely related.
Invention content
The technical problem to be solved in the present invention is to provide a kind of siphovirus, which is from patients with periodontitis oral pocket
The new virus of acquisition is detached in inner wall gingival epithelium, treatment and the prevention for mouth disease provide new solution.
In addition, it is also desirable to provide preparation method and the application of a kind of above-mentioned siphovirus.
In order to solve the above-mentioned technical problem, the present invention is achieved through the following technical solutions:
In one aspect of the invention, a kind of siphovirus, the amino acid sequence such as SEQ ID of TMP albumen are provided
Shown in NO.3.
Preferably, the amino acid sequence of the siphovirus archaeal dna polymerase B area albumen is as shown in SEQ ID NO.4.
It is furthermore preferred that the amino acid sequence of its bacteriophage structural proteins is as shown in SEQ ID NO.5.It is further preferred that the bacteriophage
Comprising：Encode the nucleotide sequence of amino acid sequence shown in SEQ ID NO.1.Most preferably, which has SEQ ID
Full length nucleotide sequence shown in NO.2.
In another aspect of this invention, a kind of siphovirus, the amino of archaeal dna polymerase B area albumen are additionally provided
Acid sequence is as shown in SEQ ID NO.4.
Preferably, the amino acid sequence of bacteriophage structural proteins is as shown in SEQ ID NO.5.
In another aspect of this invention, a kind of siphovirus, the amino acid sequence of bacteriophage structural proteins are additionally provided
Row are as shown in SEQ ID NO.5.
Preferably, the amino acid sequence of TMP albumen is as shown in SEQ ID NO.3.
In another aspect of this invention, a kind of pharmaceutical composition for including above-mentioned siphovirus is additionally provided.
In another aspect of this invention, a kind of thimerosal for including above-mentioned siphovirus is additionally provided.
In another aspect of this invention, a kind of kit for detecting siphovirus is additionally provided, is directed to comprising specificity
The primer pair of all or part of nucleotide sequence design shown in SEQ ID NO.2.It is anti-by simple PCR using the kit
It should can detect the siphovirus of the present invention.
In another aspect of this invention, a kind of preparation method of siphovirus is additionally provided, is included the following steps：
Patients with periodontitis gingiva tissue sample is taken, extracts the viral genome in the sample；
The viral genome reverse transcription is subjected to PCR amplification into viral cDNA, then with random primer, the product after amplification
By high-flux sequence, sequencing library is built；
It is contig short sequence assembly of the length more than 100bp after high-flux sequence result is carried out data prediction；
The sequence of splicing with the known array data in database is compared, finds out the similitude between sequence；
It analyses and compares as a result, according to known array design amplimer, by nested PCR amplification, obtains unknown virus
Full-length gene order, the amino acid sequence shown in gene order coding SEQ ID NO.1；
Through homology analysis, species taxonomy is carried out to unknown virus, which is classified as siphovirus.
In another aspect of this invention, a kind of application of above-mentioned siphovirus is additionally provided, is used to prepare or screens and is pre-
Anti- or treatment mouth disease drug.
The siphovirus of the present invention is to use viral metagenomics method from patients with periodontitis oral pocket inner wall gum
One plant of new virus of acquisition is detached in epithelium, new foundation is provided for the cause of disease of mouth disease, treatment and preventative strategies.
Description of the drawings
The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
Fig. 1 is the gene structure figure of 1 siphovirus Siphoviridae 29632 of the embodiment of the present invention.
Specific embodiment
In the following example, test method without specific conditions, usually routinely condition, such as《Fine works molecular biosciences
Learn experiment guide》(chief editors such as F.M. Ao Sibai, R.E. James Kingstons, J.G. Sai Deman, Ma Xuejun, Su Yuelong's translates Beijing：Section
Learn publishing house, 2004) described in method carry out.
Traditional periodontosis Study of Etiology focus is mostly periodontal pathogenic bacteria, and bacterium cause a disease it is theoretical be not enough to it is complete
Explain the relevant clinical symptoms of periodontosis, the present invention will pay close attention to the viral factor being transferred in periodontosis generation, evolution, power
Figure discloses the viral group in periodontal environment, filters out the suspicious Causative virus of periodontosis, is successive treatment and prevention periodontosis
Basis is provided.Due to the special biological characteristics of virus, traditional viruses indentification method can not be met the requirements, and the present invention is first
The secondary Study of Etiology that the technologies such as viral metagenomics, high-flux sequence and bioinformatics are applied to periodontosis, is established
The viral metagenomics skill of unknown bacteriophage in viral group and diagnosis periodontal environment in one analysis periodontal environment
Art platform.
The present invention is fished from patients with periodontitis oral pocket inner wall gingival epithelium tissue using viral metagenomics method and taken
The sequence of doubtful new virus through sequence assembly, gene walking, PCR amplification, clones the whole genome sequence of new virus, overall length
29632bp, through homology analysis, multiple segments and Siphovirus contig89 segment homologys are higher, which belongs to
The new virus is named as 29632 (GenBank of siphovirus Siphoviridae by Styloviridae：KY053532).
Embodiment 1 is detached from patients with periodontitis oral cavity using viral metagenomics method and obtains the complete of siphovirus
Long gene order
Gingiva tissue about 2g is taken, is put into 2ml centrifuge tubes, sealed membrane seals after adding in 1mlPBS and 3 diameter 0.3cm steel ball
Mouthful, after be homogenized (multigelation three times and be homogenized 5min).Under the conditions of 4 DEG C, 13000g is centrifuged 10 minutes, final proof in extraction
Product, again under the conditions of 4 DEG C, 12000g is centrifuged 5 minutes, extracts Supernatant samples, twice centrifugation removal cell fragment and other micro- lifes
Object；Supernatant samples are handled by 0.45m filters, 7500rpm centrifugation 1min filterings remove the non-viral capsomere in suspension again；
Water-bath 90min at 37 DEG C removes virus using Turbo DNase, RNase, Baseline Zero Dnase and Benzonase
Inhereditary material outside particle package.Current processed 66 parts of samples of case group, 32 parts of samples of control group.And extract its hereditary object
Matter (including DNA, RNA, dsDNA, ssDNA, dsRNA and ssRNA etc.).With QIAamp Viral RNA extraction Kit
The viral genome in sample is extracted, passes through the inhereditary material of DNA enzymatic removal DNA virus and reverse transcription and Klenow
Fragment enzymes use III Reverse Transcriptase kits of SuperScript at once after having extracted viral genome
Reverse transcription operation is carried out, the primer of reverse transcription is Fixed-Primer-8N, and the design of the primer is in website (http://
Www.changbioscience.com/primo/primor.html it is completed on), random primer is generated in website, it is then logical
It crosses and is compared with the database of NCBI progress BLAST, the primer of people or bacterium can not wherein be matched as Fixed- by selecting
Primer；The Fixed-Primer-8N of 1 μ l 100pmol is added in into the 12 μ l of viral genome of extraction, is turned upside down 5-6 times
It is uniformly mixed；70 DEG C of water-bath 5min；2min is stood on ice；After carry out reverse transcription；Reverse transcription product is placed in 5min at 94 DEG C and is denaturalized,
Cooled on ice 2min；The Klenow enzymes that 1 μ l are added in backward above-mentioned product carry out the synthetic reaction of the second chain, synthesize viral double-strand
CDNA, while primer label is added in respectively on the viral double-strand cDNA of different samples；Pass through single-primed PCR viral genetic
Substance, the primer of PCR amplification is the corresponding Fixed-Primer primers of Fixed-Primer-8N, and template is to be obtained in previous step
The double-strand cDNA templates arrived；PCR product passes through 1% agarose gel electrophoresis, cuts the DNA for meeting high-flux sequence；Setting
48 multiple holes, and voltage is set as 100 volts.Smear bands were obtained after electrophoresis through 90 minutes, 48 multiple holes is cut respectively and corresponds to
Length range is placed in 2ml centrifuge tubes, weighs in the gel of 500-1000bp.Product is purified through PCR Purification Kit,
Gel extraction after electrophoresis；The operating instruction of kit is built according to Miseq high-throughput sequencing libraries, builds Miseq sequencing libraries,
It send to sequencing company and is sequenced.Sequencing result carries out data processing and sequence analysis by Stanford University's genome project center, according to
Each sequence information is classified to each sample according to primer label, then removes primer and label.Length is short more than 100bp
Sequence is spliced into contig (contigs) through NEWBLER2.5 softwares.The biological heredity information in ncbi database is downloaded, is established
Local BLAST library, and the nucleotide and amino acid sequence that will splice and translate carry out in local library and ncbi database
Multiple BLAST is compared.Setting E-value≤0.00001 is critical parameters, by size point of the comparison result according to E-Value values
For eucaryote, bacterium, virus and unknown nucleotide sequence.Comparison result is uploaded to visualization website, analyzes virus sequence information, finds
Doubtful completely new bacteriophage sequences.
It analyses and compares as a result, splice with 8.1 softwares of geneious (U.S.) to the bacteriophage segment in library,
After obtaining bacteriophage sequences, 34 pairs of verification primer amplifications (see the table below 1) are designed.After PCR amplification, product is through gel electrophoresis, use
It is sequenced after AxyPrep DNAGel Extraction Kit (Axygene, Silicon Valley, USA) gel extraction, clone.
Sequence after sequencing passes through the shearing and splicing of carrier, finds out and is inserted into base and mutational site, and carries out error correction.The base cloned
Its code area (Coding Sequence, CDS) is analyzed because sequence is compared by blast, the results are shown in Figure 1, new phage
Full-length gene order is 29632bp (SEQ ID NO.2), and the amino acid sequence of coding is as shown in SEQ ID NO.1
(2499AA), wherein, the amino acid sequence (SEQ ID NO.3) of 6085-7005 nucleotide sequence coded 306AA is length
Cauda-bactivirus TMP albumen；The amino acid sequence (SEQ ID NO.4) of 20599-20970 nucleotide sequence coded 123AA
For archaeal dna polymerase B area；Amino acid sequence (the SEQ ID of 21842-22369 nucleotide sequence coded 175AA
NO.5 it is) bacteriophage structural proteins, these genes and albumen play an important role in bacteriophage.
After the gene order cloned is by comparing analysis of encoding area, then through homology analysis, multiple segments with
Siphovirus contig89 segment homologys are higher, which belongs to Styloviridae.The new phage is named as
Siphoviridae 29632(GenBank：KY053532), overall length 29632bp belongs to siphovirus.
1 34 pairs of primer sequences of table
Embodiment described above only expresses embodiments of the present invention, and description is more specific and detailed, but can not
Therefore it is interpreted as the limitation to the scope of the claims of the present invention.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection model of the present invention
It encloses.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (13)
1. a kind of siphovirus, which is characterized in that the amino acid sequence of its TMP albumen is as shown in SEQ ID NO.3.
2. siphovirus according to claim 1, which is characterized in that the amino acid sequence of its archaeal dna polymerase B area albumen
Row are as shown in SEQ ID NO.4.
3. siphovirus according to claim 2, which is characterized in that the amino acid sequence of its bacteriophage structural proteins is such as
Shown in SEQ ID NO.5.
4. siphovirus according to claim 3, which is characterized in that the bacteriophage includes：Encode SEQ ID NO.1 institutes
Show the nucleotide sequence of amino acid sequence.
5. siphovirus according to claim 4, which is characterized in that the bacteriophage has shown in SEQ ID NO.2
Full length nucleotide sequence.
A kind of 6. siphovirus, which is characterized in that the amino acid sequence of its archaeal dna polymerase B area albumen such as SEQ ID NO.4
It is shown.
7. siphovirus according to claim 6, which is characterized in that the amino acid sequence of its bacteriophage structural proteins is such as
Shown in SEQ ID NO.5.
A kind of 8. siphovirus, which is characterized in that the amino acid sequence of its bacteriophage structural proteins such as SEQ ID NO.5 institutes
Show.
9. siphovirus according to claim 8, which is characterized in that the amino acid sequence of its TMP albumen such as SEQ ID
Shown in NO.3.
10. a kind of pharmaceutical composition or thimerosal for including any one of the claim 1-9 siphovirus.
11. a kind of kit for detecting siphovirus, which is characterized in that comprising specificity for complete shown in SEQ ID NO.2
Portion or the primer pair of partial nucleotide sequence design.
12. a kind of preparation method of siphovirus, which is characterized in that include the following steps：
Patients with periodontitis gingiva tissue sample is taken, extracts the viral genome in the sample；
The viral genome reverse transcription is subjected to PCR amplification into viral cDNA, then with random primer, the product after amplification passes through
High-flux sequence builds sequencing library；
It is contig short sequence assembly of the length more than 100bp after high-flux sequence result is carried out data prediction；
The sequence of splicing with the known array data in database is compared, finds out the similitude between sequence；
It analyses and compares as a result, according to known array design amplimer, by nested PCR amplification, obtains the overall length of unknown virus
Gene order, the amino acid sequence shown in gene order coding SEQ ID NO.1；
Through homology analysis, species taxonomy is carried out to unknown virus, which is classified as siphovirus.
13. the application of any one of the claim 1-9 siphovirus, which is characterized in that be used to prepare or screen prevention or
Treat the drug of mouth disease.
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| CN201611139393 | 2016-12-12 | ||
| CN201611139393X | 2016-12-12 |
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Cited By (3)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020077559A1 (en) * | 2018-10-17 | 2020-04-23 | 深圳华大生命科学研究院 | Method and device for finding temperate bacteriophages from whole bacterial genome sequence and storage medium |
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| CN110211628A (en) * | 2019-06-12 | 2019-09-06 | 湖南大学 | A kind of lysogenic phage prediction technique based on high-flux sequence data |
| CN110211628B (en) * | 2019-06-12 | 2022-06-07 | 湖南大学 | Lysogenic phage prediction method based on high-throughput sequencing data |
| CN114300046A (en) * | 2021-12-28 | 2022-04-08 | 武汉百奥维凡生物科技有限公司 | Identification method of new macrovirome viruses |
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