Molecular cloning and partial sequencing of hepatitis A viral cDNA
Molecular cloning and partial sequencing of hepatitis A viral cDNA
复制标题
甲型肝炎病毒 cDNA 的分子克隆和部分测序
作者：
D. Linemeyer;J. Menke;A. Martín;J. Hughes;A. Young;S. Mitra
从感染的猴肾细胞培养物中纯化甲型肝炎病毒，并将病毒RNA用于合成双链cDNA。将该cDNA插入质粒引发的合成系统后或插入pBR 322的Pst I位点后克隆。通过限制性内切酶分析和病毒插入片段的交叉杂交对所得克隆进行作图，以产生代表至少97%的病毒基因组的复合图谱，缺少ca。从基因组的5'端开始220个碱基。基于其与病毒RNA和来自异源绒猴宿主系统的总甲型肝炎病毒感染的细胞RNA的阳性杂交，证实克隆是甲型肝炎病毒特异性的。确定了与病毒基因组5'端同源的3，054个碱基对的cDNA的核苷酸序列，并鉴定了854个连续编码三联体的开放阅读框。此外，编码VP-1和VP-3病毒结构蛋白的序列位于核苷酸序列中。
Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.