Cell Cuiture System Supporting Hepatitis C Virus Replication by Using Infections RNA

在我们以前的报告中使用的接种血清含有丙型肝炎病毒的异质克隆，所谓的准种。然而，我们发现，有限的物种在培养的Mr-2C细胞系统中成为优势。为了获得这种优势的HCV种类，我们通过长RT-PCR扩增2个HCV基因组区域，即结构和非结构区域，用于来自HCV感染的MT-2C细胞接种后8天的RNA样品。然后将这些PCR产物插入质粒载体pBR 322中，得到质粒文库。全长HCV基因组大小的克隆为196个，其中60个可用于合成完整大小和足够量的HCV RNA。将HCV RNA转染MT-2C细胞，分别于接种后7天和14天检测到HCV RNA。然后从三个阳性克隆中筛选出一个可能具有感染性克隆可行性的克隆。最后，我们通过无细胞传播确认了三个感染性克隆。然而，这些克隆的感染性都很弱，因此，为了获得更强的感染性，我们重新构建了一个新的克隆，该克隆具有最后三个克隆中的一致氨基酸序列。将该克隆插入表达载体中，置于CMV启动子下。因此，我们显示了HCV蛋白的表达，即，核心、包膜2、NS 3、NS 4A和NS 5A。这是第一次使用来自HCV感染的培养细胞的克隆证明HCV蛋白的表达。通过使用该克隆，我们正在对MT-2C细胞和其他细胞系进行RNA转染的过程中，并确认了明确的感染性。

The inoculated serum which was used in our previous report contained heterogeneous clones of hepatitis C virus, so called quasi-species. However, we found that Limited species became predominant during the cultured Mr-2C cell system. In order to obtain such a predominant HCV species, we amplified 2 HCV genomic regions, the structure and the non-structure regions, by long RT-PCR for the RNA sample from HCV-infected MT-2C cells 8 days after inoculation. Then these PCR products were inserted into plasmid vector pBR322 leading to a plasmid library. Clones of full-length HCV genomic size were 196, of which 60 were available in the synthesis of HCV RNA of complete size and sufficient quantity. RNA in a set (3 to 5 cloned a set) was transfected into MT-2C cells .In three sets, HCV RNA was detected from media of transfected cells at both 7 and 14 days post-inoculation. Then we identified an individual clone which might have feasibility of infectious clone from three positive sets . Finally we confirmed three infectious clones by cell free transmission. However, the infectivities of these clones were faint.Thus, in order to obtain a stronger infectivity, we reconstructed a new clone which has a consensus amino acid sequence among the last three clones. This clone was inserted into an expression vector under the CMV promoter. Consequently, we showed the expression of HCV protein, i.e., core, envelope 2, NS3, NS4A and NS5A. It is the first time to demonstrate the expression of HCV proteins using the clone originated from HCV-infected culture cells. By using this clone, we are on the process of performing RNA transfection to MT-2C cells and other cell lines and confirm a definite infectivity.