中华绒螯蟹螺原体类螺旋蛋白SLP25对中华绒螯蟹和RAW264.7细胞免疫反应的研究

Immune Responses of Eriocheir Sinensis and RAW264.7 Cells Induced by Spiralin-like Protein SLP25 from Spiroplasma Eriocheiris

中华绒螯蟹螺原体是一种引起中华绒螯蟹颤抖病的新型病原微生物,对中华绒螯蟹养殖业造成了很大的损失.本文研究了螺原体的一个特有蛋白——螺旋蛋白所引起的中华绒螯蟹和小鼠RAW264.7细胞的免疫反应.克隆和原核表达中华绒螯蟹螺原体类螺旋蛋白SLP25后,用以感染中华绒螯蟹和小鼠巨噬细胞RAW264.7,用实时定量PCR和Western Blot的方法检测宿主免疫相关基因或蛋白表达量的变化.类螺旋蛋白SLP25刺激后,中华绒螯蟹的抗脂多糖因子、酚氧化酶原、消极素轻链和Peroxiredoxin 6等基因的mRNA表达量变化显著;RAW264.7细胞内的TNF-α、IL-1β和IL-10等基因的mRNA表达量变化显著,而TGF-β1和CD40的mRNA表达量变化不显著;同时RAW264.7细胞内P38和ERK蛋白的磷酸化水平变化显著,而JNK蛋白的磷酸化水平变化不显著.通过本文的研究表明类螺旋蛋白SLP25可以引起宿主广泛的免疫变化,进而证明其是重要的毒力相关因子.

Spiroplasma eriocheiris caused mass mortality of Chinese mitten crab Eriocheir sinensis. This paper studied the immune responses of E. sinensis and RAW264.7 cells induced by a unique gene, spiralin-like protein of S. eriocheiris. After cloning and expression the spiralin-like protein in E. coli, the role of this protein in promoting immune responses was characterized through analyzing the interactions between S. eriocheiris and E. sinensis as well as RAW264.7 cells. Real-time PCR and western blot were used for detection of expression levels of immune genes in both hosts. The mRNA expressions of anti-lipopolysaccharide factor, prophenoloxidase, pacifastin light chain and peroxiredoxin 6 in E. sinensis and TNF-α,IL-1β, IL-10 in RAW264.7 cells were induced by spiralin-like protein, whereas TGF-α and CD40 in RAW264.7 cells were not. In RAW264.7 cells, the levels of phosphorylated P38 and phosphorylated ERK increased after spiralin-like protein induced, whereas the level of phosphorylated JNK did not change. This study reveals that spiralinlike protein of S. eriocheiris can induce the immune responses of E. sinensis and RAW264.7 cells and the protein may be an important vimlence factor of S. eriocheiris. This study gives light to the pathogenesis of the spiroplasmas and provides knowledge of immune reactions of hosts from both invertebrate and vertebrate challenged with spiroplasmas unique protein.