细胞融合病毒全长cDNA克隆的构建与鉴定

Construction of full-length genomic cDNA clone of cell fusing agent virus

目的分析细胞融合病毒基因组同源性,构建细胞融合病毒全长cDNA克隆,为进一步构建该病毒株的感染性全长cDNA克隆奠定基础。方法通过DNAstar和Mega7.0构建了细胞融合病毒的系统发生树,对其基因和氨基酸序列同源性进行分析比对。选取细胞融合病毒Galveston株的全基因序列,通过体外基因合成的手段获得覆盖病毒全基因的3个DNA片段,并分别克隆至p BR002载体。利用限制性内切酶和T4连接酶将分片段基因组连入pACNR载体,最终获得含有pACNR的细胞融合病毒Galveston全长cDNA克隆。根据Galveston病毒株基因组序列和pBR002载体的序列,利用DNAstar及Oligo6软件设计5对引物,用来对其连接处进行测序及鉴定。结果与结论构建了细胞融合病毒系统发生树,明确了其进化地位和其他黄病毒的同源性关系。构建出细胞融合病毒Galveston株全长cDNA克隆,经过酶切鉴定和序列测定表明所获得cDNA克隆为该病毒的全长序列。

Objective To analyze cell fusing agent virus genome homology,obtain the cDNA clone spanning the entire genome of cell fusing agent virus,and lay the foundation for construction of full-length infectious cDNA clone. Methods The phylogenetic tree of the cell fusing virus was constructed using DNAstar and Mega7. 0,and the homology of cell fusing agent virus gene and amino acid sequence was analyzed and compared. According to the whole genomic sequence of Galveston cell fusing agent virus,three 3 DNA fragments were obtained,which covered the whole gene of virus by gene synthesis in vitro. Each fragment was cloned in pBR002 vector,using restriction enzymes and T4 ligase before BC＇ was ligated into fragments containing pACNR-Avector. Five primer pairs were designed by DNA star software and Oligo6 software to identify the connection sequence. Results and Conclusion The phylogenetic tree of the cell fusing virus was constructed,and its evolutionary status and homology with other flaviviruses were clarified. After being identified by enzyme digestion and sequencing analysis,the full-length cDNA clone of cell fusing agent virus was obtained.