人呼吸道合胞病毒兰州株基因组全长cDNA克隆的构建及鉴定  

Construction and identification of full-length genomic cDNA clone from RSV LZ01/09 strain

作　　者：朱传凤 瓦晓霞 包红 寇桂英 傅生芳 马超 ZHU Chuan-feng;WA Xiao-xia;BAO Hong;KOU Gui-ying;FU Sheng-fang;MA Chao(The Second Department of Research, Lanzhou Institute of Biological Products Co., Ltd., Center for Gansu Provincial Vaccine Engineering Research, Lanzhou 730046, Gansu Province, China)

机构地区：[1]兰州生物制品研究所有限责任公司第二研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2019年第1期1-7,共7页Progress In Microbiology and Immunology

基　　金：中国生物技术集团科研基金(NBG-LZ-014)

摘　　要：目的构建用于呼吸道合胞病毒(respiratory syncytial virus,RSV)体外拯救的RSV基因组全长cDNA克隆,并进行鉴定。方法根据RSV Long株基因组序列设计并合成引物,利用RT-PCR技术分6段扩增RSV LZ01/09基因组序列并构建克隆载体;测序后,利用重叠PCR与酶切连接技术,根据基因组序列选择特异性酶切位点,引入Kpn I、Xma I和Sal I酶切位点,构建成4个亚克隆载体;将亚克隆载体的插入片段连接至经过改造且包含T7启动子、锤头状核酶、多克隆位点、丁肝核酶、T7终止子的p RSV1载体中,构建RSV基因组全长cDNA克隆;对克隆全长cDNA序列进行测定,与亲本RSV LZ01/09基因组进行同源性比对分析,并与RSV实验参比株进行系统进化树分析。结果测序结果显示,RSV LZ 01/09的基因组全长为15 204 bp,与GenBank公布的RSV基因组序列长度相当,将完整的序列提交GenBank,登录号为KY782635;酶切及测序结果显示,用于RSV全长cDNA克隆构建的基本载体p BSKS-MCS(简称p RSV1)与预期相符,RSV全长基因组cDNA克隆质粒(简称转录载体p RSV1-4F)酶切片段大小与预期一致;同源性比对结果显示,全长cDNA序列与亲本RSV LZ01/09基因组序列同源性高达99.83%;系统进化树分析结果显示,其与RSV-A亚型序列同属于一个分支。结论测序及酶切分析结果表明已成功构建RSVLZ01/09基因组全长cDNA克隆,为建立拯救RSV重组病毒的反向遗传学系统平台奠定了基础。Objective The study aims to construct a full-length genomic cDNA clone to rescue recombinant RSV (respiratory syncytial virus) in vivo. Methods RT-PCR was performed to amplify the fragments of the reverse-transcribed cDNA from RSV LZ01/09 strain, the fragments were sequenced and assembled to the complete genome. The overlap PCR and restriction enzyme digestion-ligation were employed to construct a full-length genomic cDNA clone. After sequencing, the full-length genomic cDNA was analyzed in homogenity with genomic sequence from parent strain and compared phylogenetically with the complete genomic sequences from RSV reference strains. Results The sequence analysis showed a full length of genome for RSV LZ01/09 strain with 15 204 bp in accordance with the published date by GenBank, which was presented to GenBank with a registration NO. KY782635. The primer pairs were designed and synthesized based on the genome sequence from RSV Long strain. Six fragments were amplified using cDNA from RSV LZ01/09 strain as a template, cloned and sequenced and assembled to generate the complete genomic sequence. The single and specific restriction enzyme sites were selected and new sites such as Kpn I, Xma I and Sal I were introduced in designed prime pairs, and 4 fragments were amplified for construction of sub-clone vectors. The inserts were recovered and ligated with the modified vector (pRSV1) bearing T7 promoter, hammer-head ribozyme, multicloning sites, hepatitis delta ribozyme, and T7 terminator. The full-length genomic cDNA clone (pRSV1-4F) was generated stepwise through digestion-ligation. It was shown by sequence analysis that the full-length genomic cDNA and whole-genome for the parent strain shared 99.83% identity for nucleotide sequence. The phylogenetic analysis showed that the clone was clustered with the strains of RSV-A subgroup. Conclusion The full-length genomic cDNA clone (pRSV1-4F) has been successfully constructed, on which a solid foundation could be provided for recovery of recombinant RSV through reverse ge

关 键 词：呼吸道合胞病毒 拯救 全长CDNA克隆 序列分析 

分 类 号：R373.14[医药卫生—病原生物学]