CN103074337B

CN103074337B - Herpes simplex virus DNA sequence and its application

Info

Publication number: CN103074337B
Application number: CN201210513622.5A
Authority: CN
Inventors: 马明星
Original assignee: KUNMING HUANJI BIOLOGICAL CHIP INDUSTRY Co Ltd
Current assignee: Jilin Huanji Biological Technology Co ltd
Priority date: 2012-12-04
Filing date: 2012-12-04
Publication date: 2014-07-09
Anticipated expiration: 2032-12-04
Also published as: CN103074337A

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

One section of herpes simplex virus dna sequence and application

Technical field:

Invention relates to DNA sequence dna and the application of one section of hsv HSV, belongs to field of biomedicine technology.

Background technology:

(Herpes simplexvirus, HSv) is spherical in shape for hsv, and intact virus forms core containing distrand DNA by core, capsid, tunicle (Tegument) and cyst membrane, is wound in fibril spool.Capsid is icosahedron symmetry, by 162 shell ultrafine particles compositions, diameter 100nm.The outer one deck tunicle of capsid covers, gage distortion, and outermost layer is typical double-layer of lipoid cyst membrane, above has projection.The viral diameter that has cyst membrane is 150～200nm.Cyst membrane surface is containing gb, gC, gD, gE, gG, gH glycoprotein, with virus to cell adsorb/penetrate (gB gC gD ge), control virus from nuclear membrane sprout releases (gH) and inducing cell fusion (gbgC gD gH) relevant.And have and induce neutralizing antibody (gd is the strongest) and cytotoxicity (known HSV glycoprotein all can).

HSV genome is a linear DNA molecule, is made up of covalently bound long segment (L) and short-movie section (S).Every fragment all contains unique sequence and Inverted repeat.In genome, have 72 genes, 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, participate in viral DNA synthetic, the metabolism of packaging and Nucleotide etc.The different albumen composition virus structural proteins of kind more than 30 (as capsid protein, envelope protein), at the DNA of protection HSV, and play an important role in the pathogenic effects of HSV and induction immune response.

HSV can grow in various kinds of cell, and conventional clone has bhk cell, Vero cell, Hep-2 cell etc.Virus is first while separating, and primary newborn rabbit kidney cell, human embryonic lung cell are more responsive.HSV infection animal is in extensive range, many animals intracranial inoculation can cause herpes simplex encephalitis, small white mouse is that footpad inoculation can cause central nervous system lethal infection, and rabbit cornea inoculation causes herpetic keratitis, and the inoculation of cavy intravaginal can cause cervicitis and cervical cancer.On 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.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 serious systemic disease also can occur occasionally, involves internal organ.

Within 6 months, obtain antibody so that interior baby is many from parent by placenta, primary infection approximately 90%, without clinical symptom, mostly is inapparent infection.Primary infection often betides 1～15 years old, common are gingivostomatitis, ties up to buccal mucosa and bleb in groups occurs at gums place, after breaking, and many lid layers necrotic tissue.In addition can cause herpes labialis, eczema sample bleb, herpetic keratitis, herpes simplex encephalitis etc.After genital herpes is more common in 14 years old, more serious, local severe pain, with heating general malaise and poradenolymphitis.

HSV primary infection produces after immunizing power, and part virus can arrive in gasserian ganglion and spinal nerve section cell or around in star-shaped glial cell, with latent state sustainable existence, in relative equilibrium, not cause clinical symptom with body along neural myelin.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 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 lip nose skin and mucous membrane intersection there is exanthema vesiculosum in groups.Herpetic keratitis, herpetic cervicitis etc. also can be shown effect repeatedly.

HSV, by placental infection, affects embryonic cell mitotic division, easily miscarries, causes the congenital disorders such as fetal anomaly, mental retardation.Approximately 40～60% newborn infant can be infected in the time of the birth canal by infecting, and occurs high heat, expiratory dyspnea and central nervous system pathological change, wherein 60～70% be contaminted newborn infant can be therefore and dead, in survivor, sequela can reach 95%.

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 of hsv HSV, this sequence and accordingly PCR primer can be used for setting up gene chip and PCR method is carried out the diagnosis of one section of hsv.

Technical scheme of the present invention is:

One section of hsv (HSV) DNA sequence dna that the present invention has determined that length is 217bp and primer thereof, carry 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 of hsv (HSV) DNA sequence dna is used for the probe sequence of the gene chip of diagnosing HSV, and carries out pcr amplification by above-mentioned primer sequence, carries out hybridization check or be directly used in PCR diagnosis after mark.

The method that the clinical detection technique that the present invention sets up uses has the advantages such as quick, accurate, easy and simple to handle, with low cost.

Brief description of the 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 to the sequencing result of pcr amplification product and the comparative analysis of estimating sequence with Blast software.

Fig. 4 is Blast Search Results in gene database.

Fig. 5 and Fig. 5 are continuous is gene chip hybridization result.

Embodiment:

First taking 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 to the nucleotide sequence (nr) in software comparison gene database online with the online Blast of NCBI, get rid of nonspecific nucleotide sequence, obtain the specific nucleotide sequence of 217bp, with this section of 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 applicable to carrying out pcr amplification, the product that pcr amplification obtains is in the same size in estimating, see Fig. 1, in figure, sequence number is: 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 big or small 200 left and right are in the same size with expectation, 3. clinical ' negative ' specimens, does not have amplified production.Collection of illustrative plates result shows amplification clinical sample that can be special with the primer in the present invention.Then clone with T-vector, and order-checking, sequencer address is shown in Fig. 2, from way, 46 start, and to 262,217bp is cloned sequence altogether, all the other sequences are 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 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 there is no 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 completely, and other sequence only has short sequence to match with it, and because not having primer, these sequences match therewith, therefore can not be detected in detection.With after this sequence mark as probe the gene chip hybridization with 80 sites of the other 18 kinds of viral pathogenic infection things that contain urogenital infections disease and 15 hypotype HPV, only have sequence of the present invention to have therewith hybridization signal, and other all sequences amixia signal therewith, see that Fig. 5, Fig. 5 are continuous, in figure, be four repetitions, results of hybridization shows to only have this sequence to have special signal, and other equal amixia signal shows that this sequence is the distinguished sequence of HSV virus.

The present invention can be used for PCR method or method for gene chip and carries out the clinical detection of urogenital infections disease and 15 hypotype HPV.Carry out pcr amplification by sequence provided by the invention, obtain according to PCR method whether product occurs and the big or small infection that determines whether HSV virus.Also can be used for this sequence is probe, as a detection site in gene chip, then with the PCR primer sequence amplification clinical samples in the present invention, and after mark, carries out hybridization check.

HSV sequence table

Specification sheets sequence table

Organization Applicant

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Street: 6th floors, 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:kmydsh1999@163.com

<110>OrganizationName: Kunming Huanji Biochip Development Co., Ltd

Application Project

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<120>Title:

<130>AppFileReference:

<140>CurrentAppNumber:

<141>CurrentFilingDate:____-__-__

Sequence

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<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 of hsv (HSV) DNA detection sequence.

Claims

1. one section of 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 of herpes simplex virus dna sequence as claimed in claim 1 in the reagent for the preparation of detection hsv, it is characterized in that the probe sequence of this sequence as the gene chip for detection of hsv, and carry out pcr amplification by primer A and B, after mark, carry out hybridization check, described pcr amplification primer is:

A.5’-GTCTGTTTGG GCTTGTCGTT-3’

B.5’-GGGCAGTGAT AGCGAAGAAA-3’。