关键词: 家蚕, 基质金属蛋白酶, RACE, 序列分析, 表达谱, 半定量RT-PCR

Abstract: Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, are involved in the degradation of extracellular matrix and/or basement membrane protein components. To study the essential function of MMPs in Bombyx mori, the full-length cDNA of Bm-MMP gene（Bm-MMP） was obtained with RACE and RT-PCR methods. Two alternative splice variants of Bm-MMP (Bm-MMP-V1 and Bm-MMP-V2) were obtained. The full-length cDNA of Bm-MMP-V1 is 2 257 bp, containing a 1 686 bp open reading frame, which encodes 561 amino acid residues, with calculated molecular mass of 62.3 kD. The full-length cDNA of Bm-MMP-V2 is 2 188 bp. The deduced amino acid sequences of Bm-MMP-V1 and Bm-MMP-V2 share the same high similarity (88.8%) with sequences of Gm1-MMP in Galleria mellonella, and their identities with Dm1-MMP in Drosophila melanogaster are 61.2% and 64.3%, respectively. The Bm-MMP-V1 was then constructed into vector pET28a(+) for prokaryotic expression. The results of SDS-PAGE and Western blot analysis indicated that a 62 kD protein with 6×His·tag was expressed in Escherichia coli BL21. The RT-PCR assay showed that the Bm-MMP was highly expressed in 4th-instar moulting larvae, mature larvae, 36 and 48 h of mounted silkworms, and prepupae. It is supposed that Bm-MMP is relative to the molting and metamorphosis. In addition, we found that LPS challenge induces a higher expression of Bm-MMP in hemocytes. The results suggest that Bm-MMP may be involved in the process of immunity. The successful cloning and expression of Bm-MMP provide a basis for the further study on its function.

Key words: Bombyx mori, matrix metalloproteinases (MMPs), RACE, sequence analysis, expression profile, semi-quantitative RT-PCR