以GP21及GP46單株抭體分析昆蟲細胞生?人類第一型T細胞白血病/淋巴瘤病毒外套膜蛋白特性

Abstract

以桿狀病毒表現載體系統（baculovirus expression vector system）的兩個不同時期之?動子-鹼性蛋白?動子（basic protein promoter）及多角體蛋白?動子（polyhedrin protein promoter)，皆能夠成功地表現出完整的HTLV-I外套膜蛋白序列。但由酵素連結免疫分析、放射標定或西方點墨法得知，HTLV-I外套膜蛋白中之gp21蛋白僅能累積於秋行軍蟲細胞內而無法分泌至培養液中。由鹼性蛋白?動子所生產之外套膜蛋白有較完全的醣化作用，使未經醣化及部份醣化的蛋白幹擾觀察之現象得以減少。此外，以免疫螢光法觀觀測的結果可知，較完全醣化之外套膜蛋白能大量地聚集於秋行軍蟲的細胞表面，因此醣化應與外套膜蛋白的運送有關。 醣化作用抑制劑加tunicamycin明顯地抑制了HTLV-I穿透膜蛋白gp21在昆蟲細胞之生成，glucosidase的抑制劑deoxynojirimycin亦可阻止小分子量膜蛋白產物生成，由此可推測醣化作用與膜蛋白的切割有關。此外，以Endoglycosidase H及N-Glycosidase F處理蛋白產物後得知，核多角體蛋白?動子生產之46?62kDa附近之醣蛋白屬於high-mannose醣化作用之產物，而鹼性蛋白?動子生產之27、30及46?62kDa醣蛋白亦屬於high-mannose的醣蛋白。 利用酵素連結免疫分析偵測核多角體蛋白及鹼性蛋白?動子生產之HTLV-I外套膜蛋白之相對產量，可知後者產量較低。綜合各項優缺點，可作為使用此兩種?動子時取拾的參考。

The entire envelope gene of human T cell lymphotropic virus type I (HTLV-I) has been successfully expressed in insect cell by using two baculovirus vectors, under the control of the polyhedrin and basic protein promoters respectively. Enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis revealed that the recombinant envelope proteins were accumulated within the cells. Even with radioimmunoprecipitation analysis, the recombinant envelope proteins were still not detectable in the culture media of recombinant baculovirus infected Sf 21-AE cells. The glycosylated precursor was detected, which was then cleaved into two mature products of respective molecular weights 46kDa and 21kDa. In comparison to polyhedrin promoter-driven HTLV-I envelope gene expression, the glycosylation of expressed HTLV-I envelope proteins driven by basic protein promoter were more completely glycosylated. The more completely glycosylated HTLV-I envelope proteins tended to accumulated abundantly on the insect cell surface. Thus it is most likely that the glycosylation is important for the HTLV-I envelope proteins being transported to the cell surface. Treatment of a N-linked glycosylation inhibitor (tunicamycin) and a glycosid?se inhibitor (deoxynojirimycin) has been shown to result in the absence of 21kDa HTLV-I envelope protein in recombinant baculovirus infected Sf 21-AE cells. The evidences suggest that the HTLV-I envelope protein precursor can be cleaved only after glycosylation. Endoglycosidase H and N-glycosidase F treatment revealed a high-mannose type glycosylation having occurred in 46?62kDa proteins produced by polyhedrin promoter-driven expression and the 27,30 and 46?62kDa proteins by basic protein promoter-driven expression.