Spandau D F, Wang H G, Fraser M J, Lee C H
Department of Pathology, Indiana University School of Medicine, Indianapolis 46202.
Virology. 1991 Dec;185(2):938-41. doi: 10.1016/0042-6822(91)90577-x.
We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV-1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.
我们从苜蓿银纹夜蛾核型多角体病毒构建了一种重组杆状病毒，称为AcX，它在感染的草地贪夜蛾（Sf21AE）昆虫细胞中表达编码乙型肝炎病毒X蛋白的基因。使用多克隆抗X抗体在免疫印迹上检测到来自AcX感染细胞提取物中的X蛋白的16.5 kDa单体和33 kDa二聚体。通过将AcX感染的Sf21AE细胞与非洲绿猴肾（CV-1）细胞融合，然后用报告质粒pSV2cat转染融合细胞，来测定昆虫细胞产生的X蛋白的生物活性。在CV-1:Sf21AE(AcX)融合细胞中cat基因的表达比来自CV-1:Sf21AE(AcMNPV)融合细胞的表达高7倍，表明昆虫细胞产生的X蛋白具有功能。昆虫细胞产生的X蛋白的反式激活功能也通过将AcX感染的Sf21AE细胞的核提取物刮入pSV2cat转染的CV-1细胞中得到验证。在刮入之前用抗X抗血清处理AcX感染的细胞核提取物消除了提取物的反式激活活性。我们得出结论，昆虫细胞产生的X蛋白在功能上与在哺乳动物细胞中产生的X蛋白相同。
