中文摘要：

家蚕（Bombyx mori）体内存在一种半胱氨酸蛋白酶抑制剂（Bombyx cysteine proteinase inhibitor,BCPI）,其可抑制半胱氨酸蛋白酶的活性,并且在Ras1CA家蚕的丝腺组织中高量表达。为了进一步阐明BCPI在家蚕体内的生物学功能,本研究利用分子生物学的手段对bcpi基因进行了研究,对其在不同组织和时期的表达特征和响应蜕皮激素的情况进行了分析。结果表明,bcpi基因CDS全长315个碱基,编码105个氨基酸,N端具有19个氨基酸的信号肽结构域。反转录PCR（reverse transcription PCR,RT-PCR）和实时荧光定量PCR（quantitative Real-time PCR,q RT-PCR）的结果显示,bcpi基因主要在血细胞中表达,在头部、体壁等组织中也有微量表达。从发育时期上来看,该基因在上蔟期和羽化变态期存在上调表达。Western blot检测结果表明,BCPI蛋白主要存在于血淋巴中,并且在上蔟期和蛹后期血淋巴中的含量明显升高。当用蜕皮激素处理家蚕时,bcpi基因发生明显上调表达,但是双荧光素酶报告系统的结果显示,在细胞水平bcpi上游1 500 bp启动子的活性受到蜕皮激素的抑制,推测其在体内有更复杂的调控机制。以上结果为深入研究BCPI的功能提供了更多的参考信息。

英文摘要：

A cysteine proteinase inhibitor from silkworm（Bombyx mori）, named Bombyx cysteine proteinase inhibitor（BCPI）, demonstrated the inhibitor activity for Bombyx cysteine proteinase（BCP） and cathespin L in vitro. By proteomics tools, BCPI was also identified from silk gland of silkworm, which showed the abundant expressional level in the silk gland of Ras1 CAsystem of transgenic silkworm. In order to explore the expression pattern and its regulation in silkworm in vivo, in this study, we cloned the gene of bcpi and investigated the spatial and temporal expression at transcriptional level and translational level. The result showed that bcpi had a length of 315 bp, encoding a protein of 105 amino acid with a signal peptide of 19 amino acid at the Nterminal and a domain of inhibitor I29 at the C-terminal. Though BCPI was a protein from silkworm with low molecular weight, it had the high similarity with N- terminal domain of the cysteine proteinase from the baculovirus of lepidopteran insect. By reverse transcription PCR（RT- PCR） and quantitative Real- time PCR（qR T- PCR）, the gene demonstrated a strong expression in the haemocyte of silkworm, and had a weak expression in head and epidermis in the 3rdday of 5thinstar, respectively. In addition, differentially expressional level of bcpi were analyzed during the developmental stages from 1st day of 4th instar to 10 th day of pupa stage by qR T-PCR. An up-regulated expression of bcpi was found in the pre-pupal stage and late pupa stage before eclosion. The expression pattern of bcpi suggested it perhaps was involved in development and metamorphosis of silkworm. By the Western blot, the high level of BCPI was accumulated in the hemolymph of silkworm. The strongest expressional level of BCPI in hemolymph appeared in the wander stage and the stage before eclosion.The result showed the BCPI functions in the hemolymph of silkworm and regulated the activity of Bombyx cysteine proteinase in different developmental stage. In order to investigate whether expressio