- 學位論文
木毒蛾核多角體病毒（LyxyMNPV）量產用之舞毒蛾細胞（IPLB-LD652Y）次選殖株系篩選
Screening of sub-clonal cell lines from IPLB-LD652Y for the mass production of Lymantria xylina multiple nucleopolyhedrovirus（LyxyMNPV）
摘要
利用次選殖（sub-clone）技術從舞毒蛾 IPLB-LD652Y 母細胞株選殖出 IPLB-LD652Y-5、IPLB-LD652Y-7、IPLB-LD652Y-b、IPLB-LD652Y-d、IPLB-LD652Y-f、IPLB-LD652Y-g、及IPLB-LD652Y-h 共 7 株次選殖株。此 7 株次選殖株與母株在形態上皆可以區分為四型：圓形、鱗狀、梭狀及多形態細胞，其中以圓形細胞所佔之比例為最高。黑角舞蛾核多角體病毒（LyxyNPV）感受性試驗結果，顯示對照組 SL-7B 細胞株對各株供試病毒（Ly5、LyxyExp-DsRed、LyxyExp-EGFP） 均無感受性；而陽性對照組 NTU-LY 系列細胞株則對各株供試病毒均呈現高感受性。至於 IPLB-LD652Y 系列細胞株對 Ly5 皆具高感受性，但對 LyxyExp-DsRed 則均呈現低感受性；對 LyxyExp-EGFP 之感受性除 IPLB-LD652Y-h 較低外，其餘各株均呈現高感受性。依產 Ly5 胞外病毒能力，選出產量高的 3 株次選殖株（IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f）與其母株作進一步細胞特性之探討。選出之 3 株次選殖細胞株之生長速率均顯著高於母株，而 IPLB-LD652Y-f 又顯著高於 IPLB-LD652Y-5。染色體數分析結果顯示這 4 株細胞株染色體數分布皆很相近，其平均值介於 78.02 至 87.28。同功異構酶分析顯示 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f 皆有相同的 EST Esterase （EST） 、 Malate dehydrogenase ( MDH ) 及 Lactate dehydrogenase ( LDH ) 電泳圖譜，且明顯不同於 NTU-LY1 的電泳圖譜。ITS （Internal transcribed spacer） 序列分析顯示 IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f 細胞株之 ITS 片段序列並無任何差異。感染 LyxyExp-EGFP 後之 EGFP 相對螢光分析顯示出4 株細胞株 （IPLB-LD652Y、IPLB-LD652Y-5、IPLB-LD652Y-7 及 IPLB-LD652Y-f） 之 EGFP 相對螢光值，隨著感染時間的增加會有明顯的改變。綜合以上結果可篩選出對 LyxyMNPV 感受性、產量最高及生長速率快之IPLB-LD652Y-7細胞株。
並列摘要
Seven new cell lines, IPLB-LD652Y-5, IPLB-LD652Y-7, IPLB-LD652Y-b, IPLB-LD652Y-d, IPLB-LD652Y-f, and IPLB-LD652Y-h, have been sub-cloned from the parental cell line, IPLB-LD652Y. There were four kinds of cell shape within these cell lines including round, squamous, spindle, and polymorphous shape. Among them, the round shape cells were the most preponderant. According to the virus infection results, SL-7B cannot been infected with Ly5, LyxyExp-DsRed, and LyxyExp-EGFP while the NTU-LY cell lines had high susceptibility with the same viruses infection. Moreover, the IPLB-LD652Y and subcloned cell lines showed similar results as the NTU-LY cell lines, but them showed lower susceptibility when infected with LyxyExp-DsRed, and only IPLB-LD652Y-h had lower susceptibility when infected with LyxyExp-EGFP. According to the results, we chose three sub-clonal cell lines with high production of Ly5 virus, IPLB-LD652Y-5, IPLB-LD652Y-7, and IPLB-LD652Y-f for the further comparison with the parental cell line. We can observe that the chosen cell lines have higher growth rate than the parental cell line, and the growth rate of IPLB-LD652Y-f were higher than that of IPLB-LD652Y-5.The distribution of chromosome numbers of these four cell lines were very close, between 78.02 to 87.28. In the result of isozyme, whether on EST Esterase （EST）、 Malate dehydrogenase ( MDH ) or Lactate dehydrogenase ( LDH ), the electrophoresis patterns of the chose cell lines were all the same and significant different from that of the NTU-LY1. The ITS （Internal transcribed spacer） sequences of the four cell lines, and they showed no significant difference. In the relative fluorescent unit (RFU) comparison, significant curve growth and decline with the increasing with the time post LyxyExp-EGFP inoculation were observed. Integrating the results, the IPLB-LD652Y-7 could be sifted as the highest virus susceptibility, production, and speedy growth cell line when infected with LyxyMNPV.