CN103074337A - Herpes simplex virus DNA sequence and its application - Google Patents
Herpes simplex virus DNA sequence and its application Download PDFInfo
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- CN103074337A CN103074337A CN2012105136225A CN201210513622A CN103074337A CN 103074337 A CN103074337 A CN 103074337A CN 2012105136225 A CN2012105136225 A CN 2012105136225A CN 201210513622 A CN201210513622 A CN 201210513622A CN 103074337 A CN103074337 A CN 103074337A
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Abstract
The invention relates to a herpes simplex virus DNA sequence and its application, and belongs to the technical field of the biomedicine. The specificity and the PCR amplimer of herpes simplex virus DNA are determined through searching and analyzing gene database data, and the length of the sequence is 217bp. The sequence is obtained through the PCR amplification and the cloning of a human herpes simplex virus infection sample, and sequencing tests prove that the sequence is consistent with a predicted sequence. The specificity of the sequence to the herpes simplex virus DNA is verified by the results of bioinformatics method analysis and gene chip hybridization analysis. The herpes simplex virus DNA sequence can be used for herpes simplex virus diagnosis as a gene chip diagnosis probe and a PCR amplification detection sequence. A method used by the clinical detection technology established in the invention has the advantages of rapidness, accuracy, simple operation, low cost and the like.
Description
Technical field:
Invention relates to dna sequence dna and the application of one section hsv HSV, belongs to field of biomedicine technology.
Background technology:
(Herpes simplexvirus, HSv) is spherical in shape for hsv, and intact virus forms core by core, capsid, tunicle (Tegument) and cyst membrane and contains distrand DNA, is wound in the fibril spool.Capsid is the icosahedron symmetry, by 162 shell ultrafine particles compositions, diameter 100nm.The outer one deck tunicle of capsid covers, and gage distortion, outermost layer are typical double-layer of lipoid cyst membrane, on projection is arranged.The viral diameter that cyst membrane is arranged is 150～200nm.The cyst membrane surface contains gb, gC, gD, gE, gG, gH glycoprotein, and that cell is adsorbed/penetrate (gB gC gD ge), control virus is relevant from sprout releases (gH) and inducing cell fusion (gbgC gD gH) of nuclear membrane with virus.And have and induce neutralizing antibody (gd is the strongest) and cytotoxicity (known HSV glycoprotein all can).
The HSV genome is a linear DNA molecule, is comprised of covalently bound long segment (L) and short-movie section (S).Every fragment all contains unique sequence and Inverted repeat.72 genes are arranged in the genome, and the 70 different protein of encoding altogether wherein except the characteristic of 24 kinds of albumen is not clear, form viral DNA in conjunction with albumen and various enzyme by 18 kinds of proteins encoded, and it is synthetic to participate in viral DNA, the metabolism of packing and Nucleotide etc.The different albumen of kind more than 30 form virus structural protein (such as capsid protein, envelope protein), and at the DNA of protection HSV, and the pathogenic effects of HSV plays an important role with inducing in the immune response.
HSV can grow in various kinds of cell, and clone commonly used has bhk cell, Vero cell, Hep-2 cell etc.Virus is first when separating, and former generation, breast rabbit kidney cell, human embryonic lung cell were responsive.The HSV infection animal is in extensive range, the many animals intracranial inoculation can cause herpes simplex encephalitis, small white mouse is that footpad inoculation can cause the central nervous system lethal infection, and the rabbit cornea inoculation causes herpetic keratitis, and the inoculation of cavy intravaginal can cause cervicitis and cervical cancer.On the inoculated into chick embryo chorioallantoic membrane, form proliferative white patch.
Virus is main by direct close contact and transmission through sex.The number of ways such as HSV direct oral cavity, respiratory tract, reproductive tract mucous membrane and damaged skin are invaded body.The people infects very general, and infection rate reaches 80～90%, and common clinical manifestation is the bleb that mucous membrane or local skin gather, and also serious systemic disease can occur occasionally, involves internal organ.
Obtained antibody from parent by placenta so that interior baby is many in 6 months, primary infection about 90% mostly is inapparent infection without clinical symptom.Primary infection often betides 1～15 years old, common are gingivostomatitis, ties up to buccal mucosa and in groups bleb occurs at the gums place, after breaking, and many lid layers necrotic tissue.Can cause in addition herpes labialis, eczema sample bleb, herpetic keratitis, herpes simplex encephalitis etc.After genital herpes was more common in 14 years old, more serious, local severe pain was with heating general malaise and poradenolymphitis.
After the HSV primary infection produced immunizing power, part virus can arrive in gasserian ganglion and the spinal nerve section cell or on every side in the star-shaped glial cell, with the latent state sustainable existence, is in relative equilibrium with body along neural myelin, does not cause clinical symptom.When organism fever, catch cold, Exposure to Sunlight, menstruation, nervous, use hypophysis or adrenocortical hormone, suffer some bacteriovirus infection etc., the activated viral propagation of hiding, come downwards to sensory nerve ending along the nerve fiber rope, to near epidermic cell, continue propagation, cause the local bleb of recurrent.Be characterized in recurring pathology at every turn and often betide same position.The most common between the lip nose skin and mucous membrane intersection in groups exanthema vesiculosum appears.Herpetic keratitis, herpetic cervicitis etc. also can be shown effect repeatedly.
HSV affects embryonic cell mitotic division by placental infection, easily miscarries, causes the congenital disorders such as fetal anomaly, mental retardation.About 40～60% newborn infant can be infected when the birth canal by infecting, and high heat, expiratory dyspnea and central nervous system pathological change occur, wherein 60～70% be contaminted the newborn infant can be therefore and dead, sequela can reach 95% among the survivor.
Summary of the invention:
The invention reside in by information biology and Protocols in Molecular Biology, determined dna sequence dna and the PCR primer thereof of one section hsv HSV, this sequence can be used for setting up gene chip with corresponding PCR primer and PCR method is carried out the diagnosis of one section hsv.
Technical scheme of the present invention is:
The present invention has determined that length is one section hsv (HSV) dna sequence dna and the primer thereof of 217bp, carries out pcr amplification with described primer, and its sequence is as follows:
GTCTGTTTGG GCTTGTCGTT ATGGGAGCCT GGGGGGCGTG GGGTGGGTCA CAGGCAACCG 60
AATATGTTCT TCGTAGTGTT ATTGCCAAAG AGGTGGGGGA CATACTAAGA GTGCCTTGCA 120
TGCGGACCCC CGCGGACGAT GTTTCTTGGC GCTACGAGGC CCCGTCCGTT ATTGACTATG 180
CCCGCGTAGA CGGAATATTT CTTCGCTATC ACTGCCC 217
Amplification PCR primer is:
A．5’-GTCTGTTTGG GCTTGTCGTT-3’
B．5’-GGGCAGTGAT AGCGAAGAAA-3’。
Above-mentioned one section hsv (HSV) dna sequence dna is used for the probe sequence of the gene chip of diagnosis HSV, and carries out pcr amplification by above-mentioned primer sequence, carries out hybridization check behind the mark or is directly used in the PCR diagnosis.
The employed method of clinical detection technique that the present invention sets up has the advantages such as quick, accurate, easy and simple to handle, with low cost.
Description of drawings:
Fig. 1 is the pcr amplification collection of illustrative plates of clinical samples.
Fig. 2 is the order-checking collection of illustrative plates of this sequence.
Fig. 3 is with the sequencing result and the comparative analysis of estimating sequence of Blast software to pcr amplification product.
Fig. 4 is Blast Search Results in the gene database.
Fig. 5 and Fig. 5 are continuous to be the gene chip hybridization result.
Embodiment:
At first take hsv as keyword, in gene database, search for the nucleotide sequence of simplexvirus.Obtain altogether several nucleotide sequences in close relations, these nucleotide sequences are used nucleotide sequence (nr) in the software comparison gene database online with the online Blast of NCBI, get rid of nonspecific nucleotide sequence, obtained the specific nucleotide sequence of 217bp, with this section sequences Design 1 pair of PCR primer, extract DNA with clinical samples, carry out pcr amplification, screen a pair of sequence and the primer that is fit to carry out pcr amplification, the product that pcr amplification obtains is seen Fig. 1 in estimating in the same size, and sequence number is among the figure: 1. standard molecular weight DNA, molecular size range is respectively under upper: 600,500,400,300,200,100; 2. clinical positive sample, amplified production is very pure, and size about 200 and is estimated in the same size; 3. clinical ' negative ' specimens does not have amplified production.The collection of illustrative plates result shows with the special amplification clinical sample of the energy of the primer among the present invention.Then clone with T-vector, and order-checking, sequencer address is seen Fig. 2,46 beginnings from the way, to 262,217bp is cloned sequence altogether, all the other sequences are the cloning vector sequence, sequence is 217bp, estimates sequence by the comparison of Blast software, shows consistent with the sequence of estimating, see Fig. 3, all sequences is all consistent with the expectation sequence, shows that this sequence can be by specific amplification, and the PCR that can be used for HSV detects.Sequence comprises cDNA with the DNA(of the Blast muca gene database of NCBI website) sequence, this sequence is only consistent with the sequence of HSV, and do not have homology with other species sequence, see Fig. 4, search result shows to only have the plasmid dna sequence of HSV in this sequence homology, and be to match fully, and other sequence only has short sequence to match with it, and because these sequences do not have primer to match therewith, so in detection, can not be detected.With behind this sequence mark as the gene chip hybridization in 80 sites of probe with the other 18 kinds of Viral infection pathogens that contain urogenital infections disease and 15 hypotype HPV, only have sequence of the present invention that hybridization signal is arranged therewith, and other all sequences amixia signal therewith, see that Fig. 5, Fig. 5 are continuous, four repetitions among the figure, results of hybridization shows to only have this sequence that special signal is arranged, and other equal amixia signal shows that this sequence is the distinguished sequence of HSV virus.
The present invention can be used for the clinical detection that PCR method or method for gene chip carry out urogenital infections disease and 15 hypotype HPV.Carry out pcr amplification with sequence provided by the invention, obtain product according to PCR method and whether occur determining whether the HSV Viral infection with size.Also can be used for this sequence is probe, as a detection site in the gene chip, then with the amplification of the PCR primer sequence among the present invention clinical samples, and carries out hybridization check behind the mark.
The HSV sequence table
The specification sheets sequence table
Organization Applicant
----------------------
Street: Building 6, Economic Development Zone, Kunming, Yunnan Province Information Technology Industry Base returned student undertaking area Building 3
City: Kunming
State: Yunnan Province
Country: China
PostalCode:650000
PhoneNumber:0871-8314895
FaxNumber:0871-8318952
EmailAddress:kmydsh1999163.com
＜110〉OrganizationName: Kunming Huanji Biochip Development Co., Ltd
Application Project
-------------------
<120>Title:
<130>AppFileReference:
<140>CurrentAppNumber:
<141>CurrentFilingDate:____-__-__
Sequence
--------
<213>OrganismName:Herpes simplex virus
<400>PreSequenceString:
GTCTGTTTGG GCTTGTCGTT ATGGGAGCCT GGGGGGCGTG GGGTGGGTCA CAGGCAACCG 60
AATATGTTCT TCGTAGTGTT ATTGCCAAAG AGGTGGGGGA CATACTAAGA GTGCCTTGCA 120
TGCGGACCCC CGCGGACGAT GTTTCTTGGC GCTACGAGGC CCCGTCCGTT ATTGACTATG 180
CCCGCGTAGA CGGAATATTT CTTCGCTATC ACTGCCC 217
<212>Type:DNA
<211>Length:217
SequenceName: one section hsv (HSV) DNA detection sequence.
Claims (2)
1. one section herpes simplex virus dna sequence is characterized in that, the length of this sequence is 217bp, and sequence is as follows:
GTCTGTTTGG GCTTGTCGTT ATGGGAGCCT GGGGGGCGTG GGGTGGGTCA CAGGCAACCG 60
AATATGTTCT TCGTAGTGTT ATTGCCAAAG AGGTGGGGGA CATACTAAGA GTGCCTTGCA 120
TGCGGACCCC CGCGGACGAT GTTTCTTGGC GCTACGAGGC CCCGTCCGTT ATTGACTATG 180
CCCGCGTAGA CGGAATATTT CTTCGCTATC ACTGCCC 217
Amplification PCR primer is:
A．5’-GTCTGTTTGG GCTTGTCGTT-3’
B．5’-GGGCAGTGAT AGCGAAGAAA-3’。
2. the application of one section herpes simplex virus dna sequence as claimed in claim 1, it is characterized in that this sequence conduct is for detection of the probe sequence of the gene chip of hsv, and carry out pcr amplification by above-mentioned primer sequence, carry out hybridization check behind the mark or be directly used in PCR detecting.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011132078A2 (en) * | 2010-04-23 | 2011-10-27 | Genomic Vision | Diagnosis of viral infections by detection of genomic and infectious viral dna by molecular combing |
| CN102458463A (en) * | 2009-05-22 | 2012-05-16 | 健诺西生物科学公司 | Vaccines against herpes simplex virus type 2: compositions and methods for eliciting an immune response |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102458463A (en) * | 2009-05-22 | 2012-05-16 | 健诺西生物科学公司 | Vaccines against herpes simplex virus type 2: compositions and methods for eliciting an immune response |
| WO2011132078A2 (en) * | 2010-04-23 | 2011-10-27 | Genomic Vision | Diagnosis of viral infections by detection of genomic and infectious viral dna by molecular combing |
Non-Patent Citations (1)
| Title |
|---|
| CONEJERO GODBERG ET AL: "Infectious pathogen detection arrays: viral detection in cell lines and postmortem brain tissue", 《BIO TECHNIQUES》, vol. 39, no. 5, 30 November 2005 (2005-11-30), pages 741 - 751 * |
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Application publication date: 20130501 Assignee: Fujian atlas biological science and Technology Co.,Ltd. Assignor: KUNMING HUANJI BIOLOGICAL CHIP INDUSTRY Co.,Ltd. Contract record no.: 2018530000011 Denomination of invention: Herpes simplex virus DNA sequence and its application Granted publication date: 20140709 License type: Exclusive License Record date: 20180620 |
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