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Development of paramyxovirus vector

副粘病毒载体的研制

基本信息

批准号：
07670357
负责人：
TAKEUCHI Kaoru
金额：
$ 1.41万
依托单位：
National Institute for Infectious Diseases
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670357/
关键词：
mumps virus vector infectious cDNA paramyxovirus

项目摘要

Paramyxoviruses are negative-stranded RNA virus and have interesting characteristic of cell tropisms and replication modes and may be usuful for vector for introducing exogeneous genes into cells. For this porpose, we intended to make mumps virus rescue system from cDNA.As a initial step, we constructed a minigenome containing reporter cat (chloramphenicol acetyl transferase) gene flanked by the mumps virus 5' and 3' end Sequences. When a minigenome was transfected into the cells expressing the L,NP and P protein from cDNAs, unfortunately, no CAT activity was detected in the cell lysates indicating no replication of the minigenome. We determined the nucleotide sequences of the NP,P,L plasmids and also the nuclotide sequences of the RT-PCR fragments synthesized from the mumps virus genomic RNA and any problem could not be detected. As it was reported that the cat minigenome system of some paramixoviruses did not work for unknown reason, this may be also the case for mumps virus. During the course of experiments, we found tha mumps virus SH gene is not essential for virus growth and SH gene unit may be deleted and replaced with other genes of interst, and we established cell lines stably expressing the mumps virus F or HN proteins and these cell may be useful to recover the mumps virus without the F or HN genes. To identify the reasons for inability of the replication of the mumps minigenome, we tried the measles virus rescue system developed by Dr.Billeter. As we were able to recover infectious measles virus from the measles full-length cDNA and detect CAT activity from the cell lysate transfected with measles minigenome, we do not believe that basic techniques we used are problem. We are now constructing complete cDNA of the mumps virus genome.

副粘病毒是一种负链RNA病毒，具有有趣的细胞趋向性和复制模式特征，可作为外源基因导入细胞的载体。为此，我们拟从cDNA中制备腮腺炎病毒拯救系统。作为第一步，我们构建了一个包含报告猫（氯霉素乙酰转移酶）基因的小基因组，该基因位于腮腺炎病毒5‘和3’端序列的两侧。不幸的是，当将一个小基因组从cdna中转染到表达L、NP和P蛋白的细胞中时，在细胞裂解物中没有检测到CAT活性，这表明小基因组没有复制。我们测定了流行性腮腺炎病毒基因组RNA合成的NP、P、L质粒的核苷酸序列和RT-PCR片段的核苷酸序列，均未发现任何问题。据报道，一些副粘病毒的猫小基因组系统由于未知的原因不起作用，这可能也是腮腺炎病毒的情况。在实验过程中，我们发现腮腺炎病毒SH基因不是病毒生长所必需的，SH基因单元可能被删除并被其他感兴趣的基因所取代，我们建立了稳定表达腮腺炎病毒F或HN蛋白的细胞系，这些细胞可能有助于恢复没有F或HN基因的腮腺炎病毒。为了找出腮腺炎小基因组无法复制的原因，我们尝试了比勒特博士开发的麻疹病毒抢救系统。由于我们能够从麻疹全长cDNA中恢复传染性麻疹病毒，并从转染麻疹小基因组的细胞裂解液中检测CAT活性，我们不认为我们使用的基本技术是有问题的。我们现在正在构建腮腺炎病毒基因组的完整cDNA。