Forstová J, Krauzewicz N, Griffin B E
Department of Virology, Royal Postgraduate Medical School, London, UK.
Nucleic Acids Res. 1989 Feb 25;17(4):1427-43. doi: 10.1093/nar/17.4.1427.
Two different recombinant baculoviruses have been generated for expressing the middle T antigen (MT) of polyoma virus in insect (Sf9) cells. One (pAcI-PyMT) produces moderate levels of MT and the other (pVL-PyMT) high levels. Indirect immunofluorescence and cellular fractionation studies with pAcI-PyMT infected Sf9 cells give results similar to those observed with wild type polyoma virus infected mouse cells, and show MT to be mainly associated with cytoplasmic membranes in the insect cell. In the latter, a sub-population of MT is phosphorylated in in vitro protein kinase assays. The yields of MT from pVL-PyMT infected cells are high enough to suggest that this protein can now be produced by this method in sufficient amounts for definitive biochemical and crystallographic analyses.
已构建了两种不同的重组杆状病毒，用于在昆虫（Sf9）细胞中表达多瘤病毒的中T抗原（MT）。一种（pAcI-PyMT）产生中等水平的MT，另一种（pVL-PyMT）产生高水平的MT。用pAcI-PyMT感染的Sf9细胞进行间接免疫荧光和细胞分级分离研究，得到的结果与野生型多瘤病毒感染的小鼠细胞所观察到的结果相似，表明MT主要与昆虫细胞的细胞质膜相关。在后者中，MT的一个亚群在体外蛋白激酶测定中被磷酸化。来自pVL-PyMT感染细胞的MT产量足够高，表明现在可以通过这种方法生产足够量的这种蛋白质，用于确定性的生化和晶体学分析。
