CN109182354A - A kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof - Google Patents

A kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof Download PDF

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CN109182354A
CN109182354A CN201811128112.XA CN201811128112A CN109182354A CN 109182354 A CN109182354 A CN 109182354A CN 201811128112 A CN201811128112 A CN 201811128112A CN 109182354 A CN109182354 A CN 109182354A
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shelled turtle
adenovirus
trionyx sinensis
turtle trionyx
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钱国英
徐程
徐洁皓
刘佳铤
陈宇
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Zhejiang Wanli University
Zhejiang Wanli College
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Abstract

The present invention detects and adenovirus homologous sequence from illness Shelled Turtle Trionyx Sinensis vitro samples for the first time, it is compared by high-flux sequence identification method with sequence similarity, it is finally recovered out a kind of nucleic acid molecules of Shelled Turtle Trionyx Sinensis adenovirus penton protein (penton), its nucleotide sequence encodes the amino acid sequence of penton protein as shown in SEQ ID NO:2 as shown in SEQ ID NO:1.Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules of the present invention can be used for preparing the specific molecular probes or kit of detection Shelled Turtle Trionyx Sinensis adenovirus, or be used to prepare the antibody or vaccine of Shelled Turtle Trionyx Sinensis adenovirus.

Description

A kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of nucleic acid molecules of Shelled Turtle Trionyx Sinensis adenovirus penton protein and Its purposes.
Background technique
Shelled Turtle Trionyx Sinensis (Pelodiscus sinensis) is due to its unique nutritive and medicinal value, its aquaculture in recent years It rapidly develops, national cultured output reaches 330,000 tons of peaks within 2013, wherein 140,000 tons of Zhejiang, accounts for national nearly 50% yield. With the development of aquaculture, the disease of Shelled Turtle Trionyx Sinensis is on the rise, it has also become restricts the key factor for supporting the development of soft-shelled turtle industry.
Adenovirus is a kind of diameter in 90-100nm, by the icosahedron nucleocapsid and double-stranded DNA gene group group of no cyst membrane At virus.Complete virion is icosahedral symmetry structure, and capsid contains 240 hexons (hexon), 12 five neighbours Body (penton), 12 ciliums (fiber) and some little albumens such as VI, VIII, Ⅸ, III a etc..Adenovirus passes through its dynein C The globular domain and cell surface specific receptor (CAR or CD46) at end combine, while the penton protein and cell of virus The integrin on surface interacts, and the internalization of virus is completed by cell endocytic.Adenovirus into cell is micro- in the cell It is transported to nucleus under the auxiliary of pipe, and finally completes shell process of undressing before nuclear Pore Complex, then viral genome quilt It is discharged into nucleus.Adenoviral gene group end binding protein TP has an effect with nuclear matrix, and promotor gene group turns Record.The great expression of virus genomic duplication and structural proteins can induce the viral assembly rank for initially entering progeny virion Section.The structural proteins ingredient such as hexon, penton for translating in endochylema is assembled into complete virus coat after entering nucleus, Under the packaging signal of genome left end and the assistance of multiple protein, viral DNA is packed since left end into virus coat, formed at Ripe virion.In virus infection advanced stage, by the intermediate filament structure lytic cell destroyed in cytoskeleton, to discharge son Generation virus.The host range of adenovirus is very wide, including the vertebrates such as people, ox, horse, pig, sheep, dog, mouse, poultry.Adenovirus can Cause different degrees of disease, the multiple organ lesion from slight infection in respiratory system to lethal, especially in immune deficiency Individual is easy to cause a disease.Whether adenovirus, which infects reptile at present, there is no research to report.
The early diagnosis of virus and the research and development of antiviral vaccine play a key effect to control viral disease, viral gene Sequence can be used as specific molecular marker viral inside and outside detection animal body, and the albumen of viral gene coding is also that development is antiviral The important molecule of vaccine.For example, sign hydropericardium syndrome (HPS) is a kind of poultry disease as caused by family's aviadenovirus -4, MS Shah etc. has studied the penton protein of HPS virus in expression in escherichia coli and can be used as the subunit vaccine (MS of broiler chicken Shah,A Ashraf,M Rahan,et al.A subunit vaccine against hydropericardium syndrome using adenovirus penton capsid protein.Vaccine,2012,30(50):7153- 7156)。
Summary of the invention
The present invention detected from illness Shelled Turtle Trionyx Sinensis in vitro tissue for the first time with adenovirus homologous sequence, isolate a kind of China The nucleic acid molecules of soft-shelled turtle adenovirus penton protein (penton).
The present invention provides a kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules, nucleotide sequence such as SEQ ID Shown in NO:1 or its antisense sequences.
The present invention also provides a kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein by above-mentioned nucleic acid molecule encoding, amino acid Sequence is as shown in SEQ ID NO:2.
The present invention also provides a kind of Shelled Turtle Trionyx Sinensis adenovirus, the penton with the amino acid comprising SEQ ID NO:2 The capsid of albumen.
The present invention also provides the Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules to be used to prepare detection Shelled Turtle Trionyx Sinensis gland The specific molecular probes of virus or the purposes of kit, or it is used to prepare the antibody of Shelled Turtle Trionyx Sinensis adenovirus or the purposes of vaccine.
The present invention also provides the high-flux sequence identification method of the Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules, It comprises the following steps that (1) completes the LncRNA sequencing of 1 sample, guarantees that each sample sequencing generates the clean for being not less than 10G Data, each sample Q30 are not less than 85%；(2) sequencing quality Distribution value counts；(3) it is compared to related RNA biometric database, point Class annotates RNA；(4) qualified RNA reads is spliced, and obtains virus contigs sequence；(5) virus contigs sequence Column are compared with virus database to identify viral species.
Detailed description of the invention
Fig. 1: viruses indentification sequencing analysis experiment flow figure.
Fig. 2: there is host with reference to genomic viral sequencing information analysis flow chart diagram.
Fig. 3: FASTQ formatted file schematic diagram.
Fig. 4: the probability distribution graph of initial data base identification mistake.
Fig. 5: the contigs staple diagram of splicing.
Specific embodiment
In order to better explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but The contents of the present invention are not limited solely to following embodiment.It in following technical steps if not otherwise specified, is this field Conventional scheme, following reagents or raw material derive from commercial channel if not otherwise specified.
The present invention obtains the in vitro liver organization of illness Shelled Turtle Trionyx Sinensis first, is extracted with RNeasy mini (Qiagen) kit After cell total rna, high-flux sequence analysis is carried out by biotechnology service company.
The RNA sample carries out sequence similarity by high-flux sequence identification method, with adenovirus and compares, and is finally recovered out It is determined as a kind of nucleic acid molecules of Shelled Turtle Trionyx Sinensis adenovirus penton protein (penton), nucleotide sequence such as SEQ ID NO:1 Shown, the amino acid sequence of coding is as shown in SEQ ID NO:2.
Trionyx Sinensis Virus identifies sequencing analysis experiment flow:
Sample detection, Jian Ku, each link is sequenced quality of data sum number amount can be had an impact, the quality of data is direct Influence that follow-up is analyzed as a result, obtaining quality data is to guarantee correct, comprehensive, the believable premise of analysis of biological information. In order to guarantee accuracy, the reliability of sequencing data from source, the laboratory Biomarker is every to sample detection, Jian Ku, sequencing The all stringent control of one production stage, fundamentally ensures the output of quality data.Experiment flow figure is as shown in Fig. 1.
Sample detection:
Detection to RNA sample mainly includes 4 kinds of methods:
(1) agarose gel electrophoresis analyzes RNA palliating degradation degree and whether has pollution；
(2) Nanodrop is detected: detecting purity (OD260/280 >=1.8 of RNA sample；OD260/230≥1.0)
(3) Qubit 2.0 is detected: the concentration (total rna concentration >=250ng/ul) of accurate quantification RNA sample
(4) Agilent 2100bioanalyzer is detected: the integrality of accurate detection RNA sample, guarantee uses qualified (value >=8.0,28S/18S >=1.5 RIN of total serum IgE are sequenced in sample；The supreme lift of map baseline；The peak 5S is normal).
Library construction:
(1) sample rRNA is removed using epicentre Ribo-ZeroTM kit；
(2) Fragmentation Buffer is added to interrupt rRNA-depleted RNA at random；
(3) using rRNA-depleted RNA as template, with hexabasic base random primer (random hexamers) synthesis the Then buffer, dATP, dUTP, dCTP, dGTP, RNase H and DNA polymerase I synthesis the is added in one cDNA chain Two cDNA chains purify cDNA using AMPure XP beads；
(4) the double-strand cDNA purified carries out end reparation plus A again and connects sequence measuring joints, then uses AMPure XP Beads carries out clip size selection；
(5) finally degrade chain containing U, be enriched with to obtain cDNA library finally by PCR.
Library Quality Control:
After the completion of library construction, Library Quality is detected, testing result can carry out machine sequencing after reaching requirement, Detection method is as follows:
(1) tentatively quantitative using Qubit2.0 progress, it is examined using insert size of the Agilent 2100 to library It surveys, insert size can just carry out next step experiment after meeting expection.
(2) Q-PCR method carries out accurate quantitative analysis (library effective concentration > 2nM) to the effective concentration in library, completes library inspection.
Upper machine sequencing:
After library inspection is qualified, different libraries carry out pooling according to machine data volume under target, with Illumina HiSeq Xten platform is sequenced.
Analysis of biological information:
Host species have with reference to genome, using having host with reference to genomic viral analysis process, schematic diagram such as Fig. 2.
Sequencing data and its quality control:
The original image data file that Illumina HiSeq platform is sequenced identifies (Base Calling) through base It is converted into primitive sequencer sequence (Raw Data or Raw Reads), as a result with FASTQ stored in file format, wherein including sequencing The sequence information of sequence and its corresponding sequencing quality information.FASTQ formatted file schematic diagram such as Fig. 3 (note: FASTQ file In usual corresponding sequence units of every 4 row: the first row is started with@, followed by sequence identifier (ID) and sequencing coordinate Description information；Second behavior base sequence, i.e. Reads；The third line is started with "+", is followed by optional description information；Fourth line The sequencing quality of corresponding sequence, the corresponding ASCII value of each character subtracts 33 in fourth line, the survey of as corresponding second row base Sequence mass value).
It is as follows that identifier details are sequenced in Illumina:
Identifier is sequenced in table 1:Illumina
Base mass value is sequenced:
Base mass value (Quality Score or Q-score) is the probability of base identification (Base Calling) error Integer mapping.Usually used Phred base mass value formula [1] are as follows: Q-score=-10*log10 (P), wherein P is alkali The probability of base identification error.Following table gives the corresponding relationship of the probability of base mass value and base identification error:
Table 2: the mapping table of base mass value and the probability of base identification error
Base mass value is higher to show that base identification is more accurate, and base identifies that error rate is lower.As shown in Table 1, for alkali The base that matrix magnitude is Q20 identifies there is 1 wrong identification base in 100 bases；The alkali for being Q30 for base mass value Base identifies there is 1 wrong identification base in 1,000 base；Q40 indicates there is 1 wrong identification base in 10,000 bases.
As unit of each sequencing reaction, distribution map is done to the base error rate of each sample sequencing reads, can reflect The sequencing quality of each sequencing reaction.Sequencing error rate is related with base quality, by sequenator itself, sequencing reagent, sample etc. Multiple factors joint effect.For RNA-seq technology, there are two features for sequencing error rate distribution tool: (1) sequencing error rate can be with The increase of sequencing sequence (Sequenced Reads) length and increase, this is because in sequencing procedure chemical reagent consumption Caused by, and the feature all having for Illumina high-flux sequence platform.(2) position of several bases can also occur before Higher sequencing error rate, be will because of during RNA-seq builds library reverse transcription need random primer, thus it is speculated that preceding several bases Sequencing error rate is higher the reason is that random primer base and RNA template are not fully integrated.Sample base error rate distribution map 4 (note: abscissa is the base positions of reads, and ordinate is the average value of the base error rate of each sequencing reaction).
Raw sequencing data quality condition summarizes:
Table 3: data output quality situation list
Note: #SampleID: sample ID；Raw_reads: statistics original sequence data, with four behaviors, one unit, statistics The number of the sequencing sequence of each primitive sequencer file；Raw_Base: the number of primitive sequencer sequence multiplied by sequencing sequence length Degree；GC (%): the quantity summation for calculating bases G and C accounts for the percentage of overall base quantity；N (%): the number of the base containing N is calculated Amount summation accounts for the percentage of overall base quantity；Q20 (%): base of the mass value greater than 20 accounts for the percentage of overall base；Q30 (%): base of the mass value greater than 30 accounts for the percentage of overall base.
RNA classification annotation:
NcRNA and repetitive sequence annotation:
Bowtie software is a kind of short sequence alignment program, and the reads for being particularly suitable for high-flux sequence acquisition is compared, hundred Step visitor utilize Bowtie software, by Clean Reads respectively with Silva database, GtRNAdb database, Rfam database and Repbase database carry out sequence alignment, filtering rRNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), NcRNA and the repetitive sequences such as small nucleolar RNA (snoRNA), after the sRNA that residue does not compare these non-coding databases is carried out Continuous analysis.SRNA annotation category statistics see the table below:
Table 4:sRNA classification annotation statistics
Note: Types: measurement type；Total: filtered Reads；RRNA: the reads for rRNA is annotated； ScRNA: the reads for small cytoplasmic RNA is annotated；SnRNA: the reads for small nuclear RNA is annotated；SnoRNA: annotate for kernel it is small The reads of RNA；TRNA: the reads for transfer RNA is annotated；Repbase: the reads for repetitive sequence is annotated； Unannotated: it is remaining that Silva database, GtRNAdb database, Rfam database and Repbase database are not compared Reads will be used for subsequent analysis；Number: number；Percentage: the reads percentage of annotation.
RNA splicing:
The reads segment being sequenced by viral RNA can be assembled into the contigs of virus, we will filter core Sugared body RNA, tRNR, the sequence after repetitive sequence etc. obtain longer contigs, pass through the virus of assembling by assembling Contigs obtains more accurate sample virus infection information.By velvet software, acquired RNA is spliced, below It is the displaying for splicing statistical result:
Table 5: assembling contigs sequence N50 statistical form
Note: #Samples: sample names；The N50 length of Contigs_N50:contigs；Contigs_Num: assembling Contigs number.
The contigs distribution of lengths of splicing is as shown in Figure 5.Abscissa indicates the length of interval distribution of assembling contigs, indulges Coordinate representation corresponds to the number of contigs in section.
Splice contigs classification annotation:
Splicing gained contigs is subjected to classification annotation, detects its species distribution situation, the database used: GenBank Virus RefSeq nucleic acid database (is represented simply as " Virus RefSeq Nucletide "), GenBank Virus RefSeq albumen database (is represented simply as " Virus RefSeq Protein "), NCBI NR (NCBI non-redundant Protein sequences), NCBI NT (NCBI non-redundant nucleotide sequences), host gene Group sequence (Host Genome) (does not annotate host genome) if referring to genome without host.Alignment algorithm Using Blast alignment algorithm, evalue (1e-5) is limited using parameter.
Identify that virus, screening criteria is as follows according to the priority orders that virus database annotates:
If 1, annotation arrives GenBank Virus RefSeq viral nucleic acid database, the virus annotated retains；
2, it does not annotate to 1, but annotates and arrive GenBank Virus RefSeq virus protein database, then the annotation Virus retains；
3,1 and 2 Virus Info all is arrived without annotation, but annotation arrives Virus Info in NCBI NT and NR virus base, and For there is the host that also do not annotate for referring to host genome to refer on genome, then the virus of the annotation retains.
Go out a kind of nucleotide sequence Shelled Turtle Trionyx Sinensis gland as shown in SEQ ID NO:1 by BLAST sequence alignment screening and identification The nucleic acid molecules of pentomer albumen (penton), the amino acid sequence of coding is as shown in SEQ ID NO:2.
The Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules can be used for preparing the specificity of detection Shelled Turtle Trionyx Sinensis adenovirus The purposes of molecular probe or kit, or it is used to prepare the antibody of Shelled Turtle Trionyx Sinensis adenovirus or the purposes of vaccine.
Sequence table
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<120>a kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof
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gacttcaccc acagccgcct gagcaacttg ttgggcatcc gcaagcggca acccttccag 120
gagggcttta ggatcaccta cgatgacctg gagggtggta acattcccgc actgttggat 180
gtggacgcct accaggcaag c 201
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35 40 45
Asp Leu Glu Gly Gly Asn Ile Pro Ala Leu Leu Asp Val Asp Ala Tyr
50 55 60
Gln Ala Ser
65

Claims (5)

1. a kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules, nucleotide sequence is as shown in SEQ ID NO:1 or it is anti- Adopted sequence.
2. a kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein, amino acid sequence is as shown in SEQ ID NO:2.
3. a kind of Shelled Turtle Trionyx Sinensis adenovirus, the capsid of the penton protein with the amino acid comprising SEQ ID NO:2.
4. the spy that Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules described in claim 1 are used to prepare detection Shelled Turtle Trionyx Sinensis adenovirus The purposes of opposite molecule probe or kit, or it is used to prepare the antibody of Shelled Turtle Trionyx Sinensis adenovirus or the purposes of vaccine.
5. the high-flux sequence identification method of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules described in claim 1, including step It is rapid as follows: (1) the LncRNA sequencing of 1 sample is completed, guarantees that each sample sequencing generates the clean data for being not less than 10G, Each sample Q30 is not less than 85%；(2) sequencing quality Distribution value counts；(3) it is compared to related RNA biometric database, classification annotation RNA；(4) qualified RNA reads is spliced, and obtains virus contigs sequence；(5) virus contigs sequence and disease Malicious database is compared to identify viral species.
CN201811128112.XA 2018-09-27 2018-09-27 A kind of Shelled Turtle Trionyx Sinensis adenovirus penton protein nucleic acid molecules and application thereof Pending CN109182354A (en)

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CN110551721A (en) * 2019-08-29 2019-12-10 山东省农业科学院家禽研究所 Long-chain non-coding RNA for inhibiting serum IV (avian adenovirus) replication and application thereof
CN110551721B (en) * 2019-08-29 2022-03-08 山东省农业科学院家禽研究所 Long-chain non-coding RNA for inhibiting serum IV (avian adenovirus) replication and application thereof

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