Transfer of the E6 and E7 Genes of Human Papillomavirus Type 18 into Human Epithelial Cells via Recombinant Retrovirus Infection

　　　　　攜帶乳突狀腫瘤病毒第十八型之E6及E7基因的反轉錄病毒質體，以轉染DNA方式，送入包裝細胞Amphoψ2。利用質體上所含的neo□基因，經過G418篩選，共得到13個重組反轉錄病毒，並由感染BALB/c3T3來測定效價。此十三株重組病毒的效價約為0.2-1.2*10□CFU/ml。利用E6/E7基因之特定引子及PCR方法，確定這些基因存在於受感染之細胞，並用RT-PCR及E7基因之特定引子，進一步証明E7之表現。且這些細胞株可以在soft agar中生，表示E6及E7基因使細胞轉形之活性存在。再以重組病毒去感染人類鼻咽癌細胞株，NPC-TW039及NPC-TW076。所得的G418□細胞利用西方點墨法及乳突狀腫瘤病毒第十八型之E6專一性抗體HPVC1P5偵測。結果感染細胞可測到約18kDa E6蛋白存在，表示此帶有E6/E7基因之重組病毒可感染人類細胞株。至於是否可以此類重組病毒感染人類原生細胞，使其成為細胞株，則尚需更進一步地探討。
　　　　　A retroviral vector carrying the E6 and E7 genes of HPV type 18 was transfected into a packaging cell line, Ampho ψ 2. Thirteen recombinant viruses carrying thc E6 and E7 genes were obtained. The titers of these recombinant viruses were estimated by infecting BALB/c3T3 cells and then counting thc number or G418□ colonies. Presence of HPV E6/E7 gcnes was confirmed by the PCR method and sequence-specific primers. The expression of E7 gene was examined by RT-PCR method. Results showed that the titers were ranged between 0.2 and 1.2*10□ CFU/ml and the E7 transcripts were detected in all 13 cell clones. These E6 and E7-containing cell clones were able to grow in soft agar, indicating the E6/E7 delivered by the recombinant retroviruses retained their transformation function. These recombinant viruses were then used to infect human NPC cell lines, NPC-TW076 and -TW039 and cell clones resistant to G418 were obtained. Using Western blot analysis and HPV type 18 E6-specific monoclonal antibody. HPV-ClP5, these cells were shown to contain a protein with a molecular mass of 18 kDa. Our data indicated that the HPV E6/E7-containing recombinant retroviruses were capable of infecting human cell lines. The potential of using these recombinant retroviruses to immortalize human primary epithelial cells was discussed.