家蚕微孢子虫感染BmN细胞的差异表达基因筛选及Akirin的原核表达

为探究家蚕微孢子虫(Nosema bombycis,Nb)感染宿主细胞的分子机制,以感染Nb的家蚕卵巢上皮细胞(BmN)为材料,通过GeneFishing技术进行基因差异表达分析,发现感染Nb的BmN细胞有10个基因被诱导或抑制表达,这些差异表达基因功能涉及蛋白质合成、细胞信号转导、线粒体呼吸链电子传递、细胞能量代谢、细胞免疫及一些未知功能。运用RT-PCR方法克隆在感染Nb的BmN细胞中差异表达的免疫相关基因Akirin,获得一个长588 bp的完整ORF,编码195个氨基酸,预测蛋白质分子质量21.98 kD,等电点为8.97,属于碱性蛋白,该蛋白质经SignalP在线预测未见信号肽。通过原核表达获得家蚕Akirin的外源融合表达蛋白,为进一步研究家蚕Akirin在家蚕先天免疫系统中的功能奠定了基础。

Identification of Differentially Expressed Genes in BmN Cells Infected by Nosema bombycis and Prokaryotic Expression of Akirin Gene

In order to clarify the molecular mechanism about infection of Nosema bombycis to host cells,we made use of GeneFishing technology to analyze the differentially expressed genes in Bombyx mori ovarian epithelial cells(BmN) infected by N.bombycis.The results revealed that expressions of 10 genes were induced or inhibited in BmN cells infected by N.bombycis.These differentially expressed genes were involved in protein synthesis,cellular signal transduction,electron transport in mitochondrial respiratory chain,cellular energy metabolism,cellular immunity and unknown functions.We cloned one of the differentially expressed genes named Akirin,a cellular immunity related gene,by using RT-PCR.Sequence analysis showed that it contains an ORF of 588 bp encoding 195 amino acids with deduced protein molecular mass of 21.98 kD and isoelectric point of 8.97,belonging to alkaline protein.The deduced protein was predicted to have no signal peptide by online SignalP program.Exogenous fusion protein was obtained from prokaryotic expression of Bombyx mori Akirin gene,establishing a good basis for further studies on functions of Akirin gene in Bombyx mori innate immune system.