兰州大学机构库 >生命科学学院
O型FMDV开放阅读框编码基因重组腺病毒的构建
Alternative TitleConstruction of Recombinant Adenovirus Containing the ORF Coding Genes of O-type FMDV
张兴旺
Thesis Advisor王勤 ; 柳纪省
2004-06-01
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name硕士
Keyword口蹄疫病毒 开放阅读框 同源重组 复制缺陷型腺病毒
Abstract口蹄疫是由口蹄疫病毒引起偶蹄动物的一种急性 高度接触性 发热性传染病 以传播迅速 感染率高而著称 目前该病的最主要防治手段是采用疫苗接种 为了研制出高效 安全 经济的FMD基因工程疫苗 本论文进行了FMDV的开放阅读框 ORF编码基因活载体疫苗的前期工作基础研究 FMDV ORF编码基因的克隆 选用牛源O型FMDV 在PK细胞中传代 提取总RNA 反转录成cDNA后 用高保真PCR系统扩增病毒ORF编码基因 并将其克隆测序及分析对比 结果表明 成功地一次扩增 克隆了牛源 O 型 FMDV 的 ORF 编码基因 该基因是由L基因 P1结构蛋白基因 P2和P3非结构蛋白基因以及起始密码子和终止密码子组成 长6954 nt 共同编码一个多聚蛋白 L基因 P1 P2和P3区分别涵盖603 2208 1464和2679个nt 与原始强毒株Akesu/58的ORF编码基因的核酸序列进行比较 它们的同源性分别为98.8% 97.9% 99.0%和97.6% ORF编码基因重组腺病毒的构建 首先 将O型FMDV的ORF编码基因定向克隆入腺病毒穿梭载体 pAdTrack-CMV 通过双酶切 PCR 扩增鉴定 确认其插入有目的基因 获得了重组穿梭质粒rpAdTrack-ORF 其次 将重组穿梭质粒电击转化含有腺病毒骨架载体质粒的RecA+ 大肠杆菌BJ5183 以分子量测定和酶切图谱鉴定证实实现了同源重组 克隆获得了 FMDV 的 ORF 编码基因复制缺陷性重组腺病毒质粒rpAd-ORF最后 经 PacI 酶切释放病毒反转末端重复序列 ITR 为自由末端以此重组感染性腺病毒基因组DNA转染细胞后得到了FMDV的ORF编码基因重组腺病毒 rAd-ORF rAd-ORF 的特征是 E1 E3 区缺失的复制缺陷型重组人腺病毒 外源基因均插入至E1区中 外源基因以CMV为启动子 以SV40polyA为加尾信号 还带有增强型绿色荧光蛋白示踪基因 rAd-ORF是以O型FMDV的ORF编码基因作为外源基因的复制缺陷型重组腺病毒 迄今国内外尚未见报道 这为将其开发成为新一代的基因工程口蹄疫病毒疫苗奠定了坚实的基础
Other AbstractFoot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. Presently, inoculating vaccine is the main means to prevent and cure FMD. To develop effective safe and economical gene-engineering vaccines of FMD , we construct recombinant adenovirus which containing the ORF coding genes of O-type FMDV. Cloning of ORF coding genes: The total RNA was extracted from PK15 cell which infected by O-type FMDV. The cDNA of ORF coding genes obtained by RT-PCR, followed by amplification of cDNA fragment with high fidelity PCR system. Then the cDNA fragment was inserted into pMD18-T vector and sequenced. The sequenced results indicated that the cDNA coding genes is 6954 bp. Gene L, P1, P2 and P3 were 603,2208, 1464 and 2679 bp, respectively. Compared with the stain Akesu/58, the homologies of nucleotide sequence of gene L, P1, P2 and P3 were 98.8%, 97.9%,99%and 97.6% ,respectively. This genes was used to construct recombinant adenovirus. Construction of recombinant adenovrus: First, the interesting gene was cloned into shuttle vector pAdTrack-CMV , Recombinants was selected with kanamycin and screened by restriction endonuclease digestion and PCR .Then a recombinant shuttle plasmid was obtained; Second, the resultant construct was linearized and transformed into E. coli strain BJ5183 carried with a supercoiled adenoviral vector pAdEasy-1. Recombinants were selected with kanamycin and screened by restriction endonuclease digestion. Third, the recombinant adenoviral constructs were cleaved with Pac I to expose their inverted terminal repeats and transfected into 293 packaging cell lines. The strain of recombinant adenovirus containing ORF of O-type FMDV was successfully obtained. The characteristics of the recombinant adenovirus: it is E1 and E3 deleted replication defective adenovirus with exogenous gene intergrateded into E1 region; it has CMV IE promoter and SV40 polyA signal. It has GFP as report gene in its genome. Recombinant adenovirus ORF holds great potential to be developed as recombinant FMDV vaccines.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221903
Collection生命科学学院
Recommended Citation
GB/T 7714
张兴旺. O型FMDV开放阅读框编码基因重组腺病毒的构建[D]. 兰州. 兰州大学,2004.
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