家蚕亲环蛋白A基因(BmCyPA)的克隆与表达分析

Cloning and Expression Analysis of Cyclophilin A Gene(BmCyPA) from the Silkworm,Bombyx mori

亲环蛋白(CyP)能与免疫抑制剂环孢霉素A(CsA)结合而作为CsA的胞内受体。在已构建的家蚕蛹cDNA文库中获得了家蚕亲环蛋白A(BmCyPA)基因的cDNA序列。利用生物信息学方法对此序列的开放阅读框(ORF)和序列同源性等进行分析,并以家蚕蛹总RNA反转录的cDNA为材料克隆了BmCyPA,通过原核表达目的蛋白后,将纯化的BmCyPA融合蛋白免疫新西兰兔得到抗血清,Western blotting检测目的蛋白在蚕体中得到表达。利用荧光定量PCR方法检测家蚕5龄幼虫各组织中BmCyPA mRNA的转录水平,由高到低依次为脂肪体、丝腺、中肠、马氏管、表皮、头部、气门、卵巢;Western blotting检测BmCyPA在5龄幼虫各组织中的表达水平,由高到低依次为脂肪体、气门、表皮、马氏管、中肠、丝腺、头部和卵巢。亚细胞定位显示Bm-CyPA在细胞质与细胞核中均有分布。推测BmCyPA与家蚕的生长发育以及促进蛋白质折叠和介导免疫应答等有密切联系。

Cyclophilin（CyP）,as an intracellular receptor of cyclosporine A（CsA）,is the binding protein to immune inhibitor CsA.The sequence of Bombyx mori cyclophilin A（BmCyPA） cDNA was identified from the constructed cDNA library from silkworm pupae.Bioinformatic methods were applied to analyze its open reading frame（ORF） and sequence homology etc.BmCyPA was cloned from the cDNAs which were reverse transcribed from total RNA of silkworm pupae.After prokaryotic expression of the target protein,the purified BmCyPA fusion protein was used to immunize a New Zealand rabbit for antiserum preparation.Western blotting analysis showed that the target protein was expressed in silkworm body.Fluorescence quantitative PCR analysis on mRNA transcriptional levels of BmCyPA in different tissues of the 5th instar silkworm larvae indicated that the mRNA level was in the order of fat bodysilk glandmidgutMalpighian tubuleepidermisheadspiracleovary.Western blotting analysis on translational levels of BmCyPA in different tissues of the 5th instar silkworm larvae displayed that the translational level was in the order of fat bodyspiracleepidermisMalpighian tubulemidgutsilk glandheadovary.Subcellular localization result showed that BmCyPA protein existed in both cytoplasm and nucleus.It is suggested that BmCPA is closely related to the regulation of growth and development,the facilitation of protein folding,and mediation of immune response in silkworm.