家蚕基质金属蛋白酶基因Bm-MMP的克隆、序列分析及表达研究

Cloning,sequence analysis and expression of a matrix metalloproteinase gene(Bm-MMP)in the silkworm,Bombyx mori

基质金属蛋白酶(matrix metalloproteinases,MMPs)家族是一类蛋白水解酶,能够降解基底膜和细胞外基质中大部分蛋白质。为了研究MMPs对家蚕Bombyxmori基本生理功能的影响,本文利用RACE和RT-PCR方法,首次从家蚕蛹中克隆了一个MMP基因的全长cDNA,命名为Bm-MMP。序列分析表明,Bm-MMP的mRNA存在两个选择性剪切变体,分别命名为Bm-MMP-V1和Bm-MMP-V2。其中Bm-MMP-V1cDNA全长为2257bp,包含一个1686bp的开放阅读框,编码561个氨基酸,预测蛋白质分子量约为62.3kD;Bm-MMP-V2cDNA全长为2188bp。同源性分析表明,Bm-MMP-V1和Bm-MMP-V2的氨基酸序列与蜡螟Galleria mellonella的Gm1-MMP的氨基酸序列同源性最高,均为88.8%;与黑腹果蝇Drosophila melanogaster的Dm1-MMP的氨基酸序列同源性,分别为61.2%和64.3%。将Bm-MMP-V1的编码区连接到表达载体pET28a(+)上,并在大肠杆菌BL21中进行原核表达,SDS-PAGE和Western blot分析结果表明,带有6×His标签的融合蛋白被成功表达。半定量RT-PCR分析表明,Bm-MMP-V1和Bm-MMP-V2在4龄眠蚕、熟蚕、吐丝后36及48h、预蛹中的表达量比5龄中食期与化蛹后的表达量高,推测该基因与家蚕幼虫蜕皮变态有关;LPS诱导5龄3d的幼虫,其Bm-MMP-V1和Bm-MMP-V2在血液中的表达量升高,推测Bm-MMP可能与免疫相关。本研究为进一步研究Bm-MMP在家蚕体内的作用机制奠定了基础。

Matrix metalloproteinases （ MMPs）, a degradation of extracellular matrix and/or basement family of proteolytic enzymes, are involved in the 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-VI 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 resuhs 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.