吉普賽舞蛾之次選殖細胞株IPLB-LD-652Y-a ~ -f的特性及對黑角舞蛾核多角體病毒之感受性分析

Abstract

昆蟲體外細胞培養系統的發展，已將近一世紀，至今此技術廣泛的應用於桿狀病毒的研究以及重組蛋白的生產。吉普賽舞蛾 (Lymantria dispar) 細胞株IPLB-LD-652Y經本實驗選殖後，一共得到六個子細胞株IPLB-LD-652Y-a~f (簡稱LD-a ~ -f)。這些子細胞具有不同比例的細胞形態，包括圓形 (round-shaped)、紡錘形 (spindle-shaped)、鱗形 (squamous-shaped) 與多態形 (polymorphous-shaped)。 LD-a細胞以紡錘形細胞最多；其中 LD-d細胞具有雙臂很長的紡錘形細胞 (類感覺神經細胞)；LD-b、-c和-f則有較多圓形細胞。這些子細胞株與其母株具有相同的酯酶圖譜 (esterase pattern)。 LD-b與 -f的細胞質含有較多的脂肪顆粒，但兩者的分布狀況不同，LD-b脂肪顆粒均勻散布在細胞質中，而LD-f則呈聚集狀。所有的細胞子株皆對野生型黑角舞蛾核多角體病毒株 (Lymantria xylina multiple nucleopolyhedrovirus)，LyxyMNPV 和LyxyNPV-2具有高感受性，感染後14天之平均感染率可達98%。其中LD-a 感染黑角舞蛾核多角體病毒產生較高多角體產量，較低胞外病毒效價；反之，LD-d感染黑角舞蛾核多角體病毒為較少多角體產量與較低胞外病毒效價。另外，在螢光重組病毒LyxyExp-EGFP和LyxyExp-DsRed感染實驗中，LD-a產生最高紅、綠螢光蛋白產量；LD-d則最少。本實驗結果顯示子細胞株系之形態、特性以及病毒感受性上不同於母株，尤其是子細胞LD-a可產生較母株高之重組蛋白產量，具有生產重組蛋白的潛力。

Insect in vitro culture system has been developed for a century and wild applied in study of baculovirus and the recombinant protein production.The gypsy moth (Lymantria dispar) cell line, IPLB-LD-652Y, was subcloned six cell strains, IPLB-LD-652Y-a~f (LD-a ~ -f). These cell strains contain different proportions of four morphological different cells: round, spindle, squamous, and polymorphous cells. LD-a cells contain more spindle-shaped cells. The predominant cells in LD-d cells are elongate spindle cells (sensory neurve-like cells), and in LD-b, -c and -f cells are round cells. The growth rates of cell strains were faster than parental cells. These cell strains showed the same esterase isozyme pattern with that of parental cell line. LD-b and -f cells contained more lipid droplets in cytoplasm than other cell strains, while the lipid droplets were dispersed evenly in LD-b cells and aggregated in LD-f cells. All the cell strains and the parental cells were highly susceptible to LyxyMNPV-5 and LyxyNPV-2, which were isolated from the moribund larvae of L. xylina with nucleopolyhedrosis, the average infection rate was almost 98% susceptible to the viruses at 14 dpi. LD-a had high yields of polyhedra, but lower virus titer of ECV among cell strains. And the cell strains also showed a high susceptibility to fluorescent recombinant viruses, LyxyExp-EGFP and LyxyExp-DsRed. LD-a cells produced the highest amount of DsRed and EGFP, respectively, but LD-d produced the lowest recombinant proteins. The results of this study showed that sub-cloned cell strains had different morphology, characteristics and virus susceptibility compared with parental cells. We conclude that LD-a cells will be potentially useful for producing recombinant proteins.