Identification and analysis of co-receptor for hepatitis C virus

我们首次通过聚合酶链式反应克隆了与丙型肝炎病毒包膜蛋白结合的人CD81基因。然后将该基因亚克隆到稳定表达载体中，用G418筛选出CD81高表达的COS7细胞克隆，制成36个大的大肠杆菌琼脂平板，每个平板代表1×10~(-4)个不同的克隆，过夜培养后制备DNA。以文库DNA为载体，用脂质体转染法将CD81基因导入表达CD81的COS7细胞。24小时后，我们将含有丙型肝炎病毒的患者血清加入到培养液中。4d后收获细胞，提取总RNA。我们通过链特异性多步RT-PCR检测了丙型肝炎病毒的负链RNA，即病毒复制。在第一轮筛查中，36人中有6人呈阳性。我们将600个基因库划分为子库，并以同样的方式继续筛选。最终，我们在最后一轮获得了8个阳性克隆。然而，据我们所知，所有克隆似乎都是假阳性。我们现在使用基于逆转录病毒的表达文库和使用TTH聚合酶的更特异的RT-PCR来重新进行筛选。

We first cloned human CD81 gene that had been reported to bind to the envelope protein of hepatitis C virus (HCV) by PCR. Then, we subcloned the gene into a stable expression vector and selected COS7 cell clones highly expressing CD81 with G418.We made 36 large E.coli agar plates, each representing 1x10^4 different clones, and made DNA preparation after overnight culture. After that, we transfected CD81-expressing COS7 cells with library DNA preparations by lipofection. Twenty-four hours later, we added HCV containing patient serum to the culture media. After 4 days, cells were harvested and total RNA was extracted. We examined negative strand RNA of HCV, meaning viral replication, by strand-specific multi-step RT-PCR. There were six positive pools among 36 at the first round screening. We divided the pool by 600 genes into subpools, and continued the screening in the same way. Finally, we obtained 8 positive clones in the final round. However, as far as we checked, all clones seemed false-positive. We now re-try the screening by using retrovirus-based expression library and by more specific RT-PCR with Tth polymerase.