CN103074337A - 一段单纯疱疹病毒dna序列及应用 - Google Patents
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Abstract
本发明涉及一段单纯疱疹病毒DNA序列及应用，属于生物医学技术领域。本发明通过基因数据库资料的搜索和分析，确定了一段单纯疱疹病毒DNA的特异性及PCR扩增引物，该序列长度为217bp。通过对人类单纯疱疹病毒感染标本的PCR扩增，克隆，获得该序列，测序证明与预计序列一致。再经生物信息学方法分析，基因芯片杂交分析，结果证实了该序列对于单纯疱疹病毒DNA的特异性。本发明作为基因芯片诊断的探针及PCR扩增检测序列，用于单纯疱疹病毒的诊断。用本发明建立的临床检测技术所使用的方法具有快速、准确、操作简便、成本低等优点。
Description
技术领域：
发明涉及一段单纯疱疹病毒HSV的DNA序列及应用，属于生物医学技术领域。
背景技术：
单纯疱疹病毒（Herpes simplexvirus,HSv）呈球形，完整病毒由核心、衣壳、被膜(Tegument)及囊膜组成核心含双股DNA，缠绕成纤丝卷轴。衣壳呈二十面体对称，由162壳微粒组成，直径100nm。衣壳外一层被膜覆盖，厚薄不匀，最外层为典型的脂质双层囊膜，上有突起。有囊膜的病毒直径为150～200nm。囊膜表面含gb、gC、gD、gE、gG、gH糖蛋白，与病毒对细胞吸附/穿入（gB gC gD ge）、控制病毒从细胞核膜出芽释放（gH）及诱导细胞融合（gbgC gD gH）有关。并有诱生中和抗体(gd最强)和细胞毒作用（已知的HSV糖蛋白均可）。
HSV基因组为一线性DNA分子，由共价连接的长片段（L）和短片段（S）组成。每片段均含有单一序列和反转重复序列。基因组中有72个基因，共编码70各异的蛋白质，其中除24种蛋白的特性还不清楚外，有18种编码蛋白组成病毒DNA结合蛋白及各种酶类，参予病毒DNA合成，包装及核苷酸的代谢等。30多种不同蛋白组成病毒结构蛋白（如衣壳蛋白、囊膜蛋白），在保护HSV的DNA，以及HSV的致病作用和诱导机体免疫应答中起重要作用。
HSV可在多种细胞中生长，常用的细胞系有BHK细胞，Vero细胞、Hep-2细胞等。病毒初次分离时，原代乳兔肾细胞、人胚肺细胞较敏感。HSV感染动物范围广泛，多种动物脑内接种可引起疱疹性脑炎，小白鼠是足垫接种可引起中枢神经系统致死性感染，家兔角膜接种引起疱疹性角膜炎，豚鼠阴道内接种可引起宫颈炎和宫颈癌。接种鸡胚绒毛尿囊膜上，形成增殖性白色斑块。
病毒主要通过直接密切接触和性接触传播。HSV经口腔、呼吸道、生殖道粘膜和破损皮肤等多种途径侵入机体。人感染非常普遍，感染率达80～90%，常见的临床表现是粘膜或皮肤局部集聚的疱疹，偶而也可发生严重的全身性疾病，累及内脏。
6个月以内婴儿多从母体通过胎盘获得抗体，初次感染约90%无临床症状，多为隐性感染。原发感染常发生于1～15岁，常见的有龈口炎，系在口颊粘膜和齿龈处发生成群疱疹，破裂后，多盖一层坏死组织。此外可引起唇疱疹、湿疹样疱疹、疱疹性角膜炎、疱疹性脑炎等。生殖器疱疹多见于14岁以后，比较严重，局部剧痛，伴有发热全身不适及淋巴结炎。
HSV原发感染产生免疫力后，部分病毒可沿神经髓鞘到达三叉神经节和脊神经节细胞中或周围星形神经胶质细胞内，以潜伏状态持续存在，与机体处于相对平衡，不引起临床症状。当机体发热、受寒、日晒、月经、情绪紧张，使用垂体或肾上腺皮质激素，遭受某些细菌病毒感染等，潜伏的病毒激活增殖，沿神经纤维索下行至感觉神经末梢，至附近表皮细胞内继续增殖，引起复发性局部疱疹。其特点是每次复发病变往往发生于同一部位。最常见在唇鼻间皮肤与粘膜交界处出现成群的小疱疹。疱疹性角膜炎、疱疹性宫颈炎等亦可反复发作。
HSV通过胎盘感染，影响胚胎细胞有丝分裂，易发生流产、造成胎儿畸形、智力低下等先天性疾病。约40～60%的新生儿在通过感染的产道时可被感染，出现高热、呼吸困难和中枢神经系统病变，其中60～70%受染新生儿可因此而死亡，幸存者中后遗症可达95%。
发明内容：
本发明在于通过生物信息学和分子生物学技术，确定了一段单纯疱疹病毒HSV的DNA序列及其PCR引物，该序列和相应的PCR引物可用于建立基因芯片和PCR方法进行一段单纯疱疹病毒的诊断。
本发明的技术方案为：
本发明确定了长度为217bp的一段单纯疱疹病毒（HSV）DNA序列及其引物，用所述的 引物进行PCR扩增，其序列如下：
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
扩增PCR引物为：
A．5’-GTCTGTTTGG GCTTGTCGTT-3’
B．5’-GGGCAGTGAT AGCGAAGAAA-3’。
上述一段单纯疱疹病毒（HSV）DNA序列用于诊断HSV的基因芯片的探针序列，并通过上述的引物序列进行PCR扩增，标记后进行杂交检测或直接用于PCR诊断。
本发明建立的临床检测技术所使用的方法具有快速、准确、操作简便、成本低廉等优点。
附图说明：
图1为临床标本的PCR扩增图谱。
图2为该序列的测序图谱。
图3是用Blast软件对PCR扩增产物的测序结果与预计序列的比较分析。
图4是基因数据库中Blast搜索结果。
图5及图5续是基因芯片杂交结果。
具体实施方式：
首先以单纯疱疹病毒为关键词，在基因数据库中搜索疱疹病毒的核苷酸序列。共获得数条关系密切的核苷酸序列，将这些核苷酸序列用NCBI网上的Blast在线使用软件比对基因数据库中的核苷酸序列（nr），排除非特异性的核苷酸序列，获得了217bp特异性的核苷酸序列，以这段序列设计了1对PCR引物，以临床标本提取DNA，进行PCR扩增，筛选到一对适合进行PCR扩增的序列及引物，PCR扩增获得的产物于预计大小一致，见图1，图中序号为：1.标准分子量DNA，分子量大小从上之下分别为：600,500,400,300,200,100；2.临床阳性标本，扩增产物很纯，大小200左右，与预计大小一致；3.临床阴性标本，没有扩增产物。图谱结果显示用本发明中的引物能特异的扩增临床样品。然后用T-vector进行克隆，并测序，测序报告见图2，从途中46开始，到262位，共217bp为克隆序列，其余序列为克隆载体序列，序列为217bp，通过Blast软件比对预计序列，显示与预计的序列一致，见图3，所有序列均与预计序列一致，表明该序列可以被特异性的扩增，可用于HSV的PCR检测。序列用NCBI网站的Blast搜索基因数据库的DNA（包含cDNA）序列，该序列只与HSV的序列一致，而与其它物种序列没有同源性，见图4，搜寻结果表明，只有HSV的质粒DNA序列于本序列同源，并且是完全配对，而其它序列只有短序列与之配对，而且由于这些序列没有引物与此配对，故在检测中不会被检测到。用这一序列标记后作为探针与含有泌尿生殖道感染性疾病及15亚型HPV的另外18种病毒的感染病原物的80个位点的基因芯片杂交，只有本发明序列与此有杂交信号，而其它所有序列与此无杂交信号，见图5、图5续，图中是四个重复，杂交结果表明只有本序列有特异信号，而其它均无杂交信号，表明该序列为HSV病毒的特异序列。
本发明可用于PCR方法或基因芯片方法进行泌尿生殖道感染性疾病及15亚型HPV的临床检测。用本发明提供的序列进行PCR扩增，根据PCR方法获得产物是否出现和大小判断是否为HSV病毒的感染。也可用于本序列为探针，作为基因芯片中的一个检测位点，然后用本发明中的PCR引物序列扩增临床标本，并标记后进行杂交检测。
HSV序列表
说明书序列表
Organization Applicant
----------------------
Street:云南省昆明市经开区信息产业基地海归创业园3幢6楼
City:昆明市
State:云南省
Country:中国
PostalCode:650000
PhoneNumber:0871-8314895
FaxNumber:0871-8318952
EmailAddress:kmydsh1999163.com
<110>OrganizationName:昆明寰基生物芯片开发有限公司
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:一段单纯疱疹病毒（HSV）DNA检测序列。
Claims (2)
1.一段单纯疱疹病毒DNA序列，其特征在于，该序列的长度为217bp，序列如下：
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
扩增PCR引物为：
A．5’-GTCTGTTTGG GCTTGTCGTT-3’
B．5’-GGGCAGTGAT AGCGAAGAAA-3’。
2.如权利要求1所述的一段单纯疱疹病毒DNA序列的应用，其特征在于该序列作为用于检测单纯疱疹病毒的基因芯片的探针序列，并通过上述的引物序列进行PCR扩增，标记后进行杂交检测或直接用于PCR检测。
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