CONSTRUCTION OF FULL LENGTH HEPATITIS A VIRUS CDNA FOR TRANSFECTION

用于转染的全长甲型肝炎病毒 CDNA 的构建

• 批准号: 3960614
• 负责人: J I COHEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3960614
• 关键词: Hepatovirus; complementary DNA; genetic manipulation; genetic mapping; genetic transcription; genome; molecular cloning; nucleic acid sequence; plasmids; tissue /cell culture; virus RNA; virus genetics

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.

将甲型肝炎病毒（HAV）的cDNA克隆到pBR 322中。 六个cDNA 分离并连接一起跨越整个基因组的克隆 一起形成被认为代表全长HAV cDNA的单个克隆。 组织培养细胞（体外）和绒猴（体内）的转染 体内）不能产生HAV。 精细结构制图 结果表明，在HAV感染过程中，约有40个碱基被缺失， 连接过程。 缺失得到修复，但转染 绒猴和组织培养细胞仍然不能产生HAV。 一个 另外的修饰的构建体，其被认为是 重要的传染性，也未能产生HAV的绒猴。 的 对整个全长构建体进行测序;与 用于产生构建体的亲本克隆没有显示任何 差异 将cDNA置于RNA转录载体中， 是在体外从cDNA中制备的。 用RNA转染绒猴 无法生成HAV。 制备正链HAV RNA，在 从负链HAV RNA和脊髓灰质炎病毒RNA聚合酶体外， 中求进工作总