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標題: Expression of hepatitis B virus surface antigen protein in a cell line and silkworm larvae with recombinant bombyx mori nuclear polyhedrosis virus
利用重組家蠶核多角體病毒在細胞株及蠶體表現B型肝炎表面抗原蛋白之 研究
作者: 彭淑貞
Peng, Shu-Jen
關鍵字: 家蠶
Bombyx mori
Hepatitis B surface antigen protein
Nuclear polyhedrosis virus
出版社: 昆蟲學系
摘要: Expression of Hepatitis B Virus Surface Antigen Protein in a Cell Line and Silkworm Larvae with Recombinant Bombyx mori Nuclear polyhedrosis virusThe silkworm (Bombyx mori) larvae and BmN cell line were infected with the recombinant virus BmYFHBs containing hepatitis B surface antigen (HBsAg) gene which was constructed from B. mori nuclear polyhedrosis virus (BmNPV). the cellular aberration was variable in infected cells. Histological sections of larval tissues stained with immunoenzymic method showed HBsAg protein at 48 hr post-infection and polyhedral inclusion bodies at 72 hr post-infection. The epidermal cells became swollen and lysed, and inclus-ion bodies liberated into body cavity at 84 hr post-infection. At frist, the recombinant virus formed in fat body, epidermis, trachea membrane and hemocytes. Later midgut, Malpighian tubules and silk glands were infected. The virus was not detected in muscles. Various silkworm strains were infected with BmYFHBs virus by feeding to the 5th instar. The more susceptible the more HBsAg protein were produced in hemolymph among these strains. The same was found among strains in the same group. Determination of susceptibility to the recombinant virus and production of HBsAg showed that the susceptibility of artificial diet-fed larvae and extracts from whole larva were higher than those fed on mulberry leaves but were not significantly different in hemolymph. Injection of different recombinant viruses into larvae showed increasing HBsAg production with incubation time until the peak on the 4th day post-infection. Microbial contamination in silkworms was measured under aseptically and non-aseptically rearingconditions.More samples were found contaminated under non-aseptically rearing condition. Decrease in feeding number of larvae and diet changing time under aseptical condition may lower microbial contamination. Injection of 5th instars with BmYFHBs resulted in similar HBsAg production in hemolymph and larval body extracts under aseptically and non-aseptically rearing conditions.
由家蠶核多角體病毒(BmNPV) 所構築含B型肝炎表面抗原基因之重組病毒 BmYFHBs,感染家蠶細胞株(BmN) 或幼蟲,其細胞病變呈相異性。幼蟲組 織切片以酵素免疫間接法染色,於48小時始測得B型肝炎表面抗原(HBsAg) 蛋白,72小時出現重組病毒,84小時真皮細胞腫大並開始破裂,多角體釋 出於體腔。重組病毒最初感染部位為脂肪體、真皮層、氣管被膜細胞、血 球細胞等組織,其後中腸細胞、馬氏管、絲腺也會受感染,但肌肉均無發 現含有病毒。不同家蠶品系經口接種BmYFHBs,感受性越高者,其血液中 HBsAg產量亦越多,反之亦然;同一系統之各品系間亦有此種趨勢。不同 飼料育蠶後,測定對重組病毒之感受性及HBsAg產量,結果發現人工飼料 育蠶之感受性及蠶體萃取液之產量較桑葉育為高,但血液中之產量則未有 顯著差異。家蠶幼蟲經注射接種不同表現載體之重組病毒,結果顯示隨感 染天數的增加,HBsAg產量亦逐漸提高,且於第四天達最高。測定無菌飼 育和非無菌飼育家蠶受微生物污染之結果,發現在非無菌飼育環境下,各 檢體受微生物污染較高,在無菌飼育環境下減少飼育隻數及置換飼料次數 ,可降低或抑制微生物污染,另取其五齡家蠶幼蟲,經注射接種BmYFHBs ,其血液及蠶體萃取液中HBsAg之產量均無差異。
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