利用核醣體去氧核糖核酸之內轉錄區間鑑定昆蟲細胞株和細胞株的交互污染

Abstract

昆蟲細胞株 (insect cell lines) 為研究工業化之體外 (in vitro) 增殖病毒或生產重組蛋白的有效工具，特別適用於昆蟲桿狀病毒表現載體 (baculovirus expression vector system, BEVS)。就多數的細胞培養研究室而言，昆蟲細胞被經常性的使用、操作和維持時，需要定期的偵測和鑑定，以避免發生細胞株交互污染 (cell line cross-contamination, CLCC) 的事件。因此我們利用分子標示法，針對核醣體去氧核糖核酸 (ribosomal DNA, rDNA; or nuclear rDNA) 之內轉錄區間 (internal transcribed spacer, ITS)，進行昆蟲細胞株 (NTU-LY, NTU-PN, IPLB-LD-652Y, and Sf9 cell lines) 之種源鑑定和細胞株交互污染 (Cell line cross-contamination, CLCC) 的檢測。係以增幅的 ITS 加上 5.8S 區間，若蟲體與其同源細胞株的序列相似度 (identity) 高達 95% 至 99% 者，如此這些被增幅的複製子序列 (amplicon’s sequences)，可用於證實該細胞株的物種來源。根據這些增幅的複製子序列，配合兩種限制酶，PstI 和 HindII 的篩選，利用聚合酶連鎖反應—限制酶片段長度多態型 (PCR-restriction fragment length polymorphism, PCR-RFLP) 的技術，建立一套快速鑑定昆蟲細胞株的方法。同時，也透過DNA 逢機增幅多態型聚合酶連鎖反應 (random amplified polymorphic DNA-PCR, RAPD-PCR) 的分析，利用隨機的核苷酸引子—OPU-10，進行本研究之細胞株的篩選和分析。另外，設計了各別的專一性引子對：Ly-ITS1/Ly-ITS2、ITS1-1/Ld-ITS1 及 Sf9-F2/ITS4，分別針對 LY、LD 及 Sf9 等三細胞株，進行種特異性聚合酶連鎖反應 (species-specific polymerase chain reaction, SS-PCR) 的鑑定。上述的三個分子標示法：PCR-RFLP、RAPD-PCR 和 SS-PCR的技術輔助確認，可鑑別細胞種源和避免發生細胞株交互污染。除此之外，在確認 NTU-LY 細胞株的種源無誤，並無發生細胞株交互污染後，則利用細胞株進行大量生產的工作。之後透過昆蟲細胞的懸浮培養，進行昆蟲病毒的產量工作，最適條件分別為：細胞密度為 1 x 106 cell/ml，培養總體積為 200 ml，轉速為 65 rpm，培養於 28℃。透過懸浮培養量病毒的平均每顆細胞的黑角舞蛾核多角體包體產量 (LyxyNPV OBs)，高達 70.5 ~ 97 OBs/cell，遠高於單層培養的 27.7 OBs/cell。

Insect cell lines are useful tools in the study of virus basic biology and production of recombinant proteins, especially by baculovirus expression vector system (BEVS). In most cell culture laboratories, several different cell lines are routinely used, maintained, or handled, these cell lines should be regularly monitored to avoid cell line cross-contamination (CLCC). We developed a technique for identification of species origin and CLCC detection for insect cell lines (NTU-LY, NTU-PN, IPLB-LD652Y, and Sf9 cell lines) based on the sequence of internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The identity of the amplicon’s sequences between insect and its homologous cell line was up to 95 %~99 %, thus these amplicon’s sequences could be used to confirm the species origin of a cell line. Two endonucleases, PstI and HindII based on these sequences of amplicons, were selected for rapid identification of insect cell lines by PCR-RFLP (PCR-restriction fragment length polymorphism). The homologous cell lines could be examined by RAPD-PCR (random amplified polymorphic DNA – PCR) method with a selected oligonucleotide primer, OPU-10. Three species-specific primer sets, Ly-ITS1/Ly-ITS2, ITS1-1/Ld-ITS1, and Sf9-F2/ITS4, were then designated for identification of LY, LD, and Sf9 cells, respectively, by SS-PCR (species-specific PCR). Therefore three methods, RFLP-PCR, RAPD and SS-PCR, can be used to define cell origin and avoid CLCC. Otherwise, the mass production of NTU-LY cells was also undertaken by suspension culture after confirmations of cell origin and free from CLCC. The optimal condition of LY cell suspension culture was 1 x 106 cells/ml, in 200 ml culture volume and 65 RPM at 28℃. The LyxyNPV OBs (occlusion bodies) production reached to 70.5 ~ 97 OBs/cell that was much higher than monolayer infection (27.7 OBs/cell).