牛泡沫病毒跨膜蛋白的穿膜区具有细胞毒性作用

Membrane Association of Bovine Foamy Virus Transmembrane (TM) Determines its Cytotoxicity

泡沫病毒(FV)具有广泛的宿主范围,可以感染从人到鱼几乎所有脊椎动物细胞,可见其包膜蛋白具有极强的膜融合能力.分析牛泡沫病毒(BFV)的跨膜蛋白(TM),在其C端存在一个穿膜螺旋区,其中含有连续的疏水氨基酸.本文以BFV3026原病毒DNA为模板,利用PCR分段扩增TM编码区,克隆原核表达载体pET32a(+),序列分析证实后,转化E.coliBL21(DE3),经IPTG诱导,仅有穿膜区完全缺失的TM3获得表达,其余含有完整或部分穿膜区的TM1及TM2均未表达.表达菌生长状态测定显示:TM穿膜区C端疏水氨基酸串联区对细胞具有强烈致死效应.同时利用金属螯合层析对所表达TM3蛋白进行初步纯化,Westernblot证实其有良好的抗原性.

Foamy viruses have large cellular tropism, which can infect all vertebrates from fish to human being, indicating that Env protein has the high capability of membrane fusion. Conserved features in all FVs TM protein include a region of hydrophobic domain called membrane spanning domain (MSD) and stretches of hydrophobic amino acids in MSD. To investigate whether theses feature are associated with the cytotoxicity effect of TM to the E. coli, a series of mutants were constructed and expressed in the E. coli BL21 (DE3) using pET-32a( + ) as expressing vector. Our results showed that only TM3 that had not MSD was expressed in E. coli, whereas other two, which contain full or part of the MSD (TM1 and TM2) did not get expression. Furthermore, the bacterial amount and living bacterial analysis revealed that the cytotoxicity of TM was dependent on its MSD, especially on the stretches of hydrophobic amino acids in MSD. Western-blotting analysis showed that TM3 protein was purified with affinity purification.