猪圆环病毒2型湖南分离株全基因扩增及感染性克隆构建

发布时间：2018-03-15 18:47

  本文选题：猪圆环病毒　切入点：MPRCA　出处：《湖南农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：猪圆环病毒(Porcine circo virus,PCV)是圆环病毒科(Circoviridae)圆环病毒属(Circo virus)的成员,是目前发现的最小的哺乳动物病毒。PCV2主要有两种基因型(亚型)：PCV2a和PCV2b。当前研究表明PCV2的变异速度加快,其中PCV2b逐渐成为优势毒株。临床上常见多种PCV2亚型共感染和PCV2与多病原混合感染现象。PCV2呈现全球性分布,能损伤猪免疫系统,引起猪严重的免疫抑制,给养猪业造成重大经济损失。本研究首先采用常规PCR法对31份湖南省某地区猪病料进行PCV2检测和序列分析,选取PCV2 capsid蛋白氨基酸序列突变株HNLYYA1用于多引物滚环复制扩增(Multiply-primed rolling-circle amplification, MPRCA)技术体系的建立。利用特异性引物和随机引物的组合对PCV2突变株HNLYYA1的总DNA进行MPRCA反应,最后利用限制性内切酶(EcoR Ⅰ和Nco Ⅰ)酶切、PCR和测序反应来鉴定MPRCA产物。结果显示,酶切后的MPRCA产物条带与PCV2基因组或片段的大小一致。将回收的目的条带与pSP72载体连接,转化DH5a菌,最后对提取的重组质粒进行测序和序列比对分析,并将PCV2突变株HNLYYA1提交于GenBank(登录号KJ867555),其全基因组大小为1767 bp,基因型为PCV2b-1C。该株PCV2 Cap蛋白与其他毒株进行比对,发现其具有独特氨基酸突变。其中NLS区34位组氨酸(H)突变为酪氨酸(Y)、LOOP GH区169位精氨酸(R)突变为甘氨酸(G)、LOOP HI区206位赖氨酸(K)突变为异亮氨酸(I)。后续将PCV2突变株HNLYYA1的MPRCA产物EcoR Ⅰ单酶切和重新连接环化,获得该株PCV2的全长环化基因组,并将其体外转染PK-15细胞,经过细胞传代培养完成PCV2突变株HNLYYA1感染性克隆的病毒拯救。本研究成功建立了PCV2 MPRCA的技术体系,并揭示了湖南PCV2分离株HNLYYA1的全基因组特征,利用细胞转染法构建了PCV2感染性克隆。所形成的研究结果为PCV2毒株突变进化提供序列信息基础,也为PCV2感染性克隆构建、PCV2毒株的复制和致病机理的研究奠定实验基础。PCV2 MPRCA技术体系的建立为其他环状病毒的全基因组分离和感染性克隆的构建研究提供新的技术途径。
[Abstract]:Porcine circo virus.PCV2 is a member of the genus Circoviridae.PCV2, the smallest mammalian virus found at present, has two main genotypes (subtypes: PCV2a and PCV2b.current studies have shown that the mutation rate of PCV2 is increasing. Among them, PCV2b gradually became the dominant virus strain. In clinic, many kinds of PCV2 subtype co-infection and mixed infection phenomenon of PCV2 and multi-pathogen. PCV2 showed a global distribution, which can damage the immune system of pigs and cause serious immunosuppression in pigs. In this study, 31 samples of pig disease in Hunan Province were detected by PCV2 and sequenced by routine PCR method. PCV2 capsid amino acid sequence mutant (HNLYYA1) was selected for the establishment of multiply-primed rolling-circle amplification (MPRCA) technique system. The total DNA of PCV2 mutant HNLYYA1 was reacted with specific primers and random primers. Finally, the restriction endonuclease Ecor 鈪，

本文编号：1616504