CONSTRUCTION OF FULL LENGTH HEPATITIS A VIRUS CDNA FOR TRANSFECTION

甲型肝炎病毒(HAV)基因已克隆到pBR322中。六个cDNA 共同跨越整个基因组的克隆被分离和连接 共同形成一个克隆，被认为代表了全长的甲型肝炎病毒cDNA. 组织培养细胞(体外)和绒猴(在体内)的转基因 Vivo)，但未能产生甲型肝炎病毒。精细构造作图 对甲型肝炎病毒基因的分析表明，大约40个碱基对在 结扎过程。缺失已被修复，但转染 绒猴和组织培养细胞仍未能产生甲型肝炎病毒。一个 额外的修饰结构，不同的是两个被认为是 对传染性很重要，也不能在绒猴中产生甲型肝炎病毒。这个 对整个全长构建体进行测序；与 用于创建该构造的父克隆没有显示任何 不同之处。 将该cDNA插入到RNA转录载体中，并加入链RNA 是由该基因在体外合成的。用该RNA对恒河猴的转基因研究 无法生成甲型肝炎病毒。甲型肝炎病毒正链RNA的制备 来自负链甲型肝炎病毒RNA和脊髓灰质炎病毒RNA聚合酶的体外研究，已在 进步。

cDNA from hepatitis A virus (HAV) has been cloned into pBR322. Six cDNA clones which together span the entire genome were isolated and ligated together to form a single clone thought to represent full length HAV cDNA. Transfection of both tissue culture cells (in vitro) and marmosets (in vivo) with these plasmids failed to generate HAV. Fine structure mapping of the HAV cDNA indicated that about 40 base pairs had been deleted during the ligation process. The deletion was repaired, but transfection of marmosets and tissue culture cells still failed to generate HAV. An additional modified construct, differing by two nucleotides thought to be important for infectivity, also failed to generate HAV in marmosets. The entire full length construct was sequenced; comparison with the sequence of the parent clones used to create the construct did not reveal any differences. The cDNA was placed into an RNA transcription vector and plus strand RNA was made in vitro from the cDNA. Transfection of marmosets with the RNA failed to generate HAV. Preparation of plus strand HAV RNA, synthesized in vitro from minus strand HAV RNA and poliovirus RNA polymerase, is in progress.