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肌原纤维结合型丝氨酸蛋白酶(myofibril-bound serine proteinase,MBSP)是最近发现的一种蛋白酶。该酶参与肌原纤维蛋白的降解及鱼糜制品弹性的下降。但是,对该酶一级结构的研究,迄今为止,未有报道。本文根据已测定的鲤MBSP N-末端氨基酸序列以及丝氨酸蛋白酶活性中心保守序列设计兼并引物,结合RT-PCR技术实现了MBSP基因片段的扩增。再根据克隆到的MBSP片段序列设计基因特异引物,用于MBSP基因的5′和3′末端快速扩增。综合以上结果,鲤MBSP的全长被确定。序列分析表明,MBSP cDNA含有一732 bp的开放阅读框,编码243个氨基酸残基,其中信号肽长度为21个氨基酸残基。组成丝氨酸蛋白酶活性中心的氨基酸残基(His61,Asp107和Ser197)在MBSP中保守存在。成熟MBSP含有222个氨基酸残基,分子量为24.5 ku,比其天然蛋白的分子量30 ku略小。成熟MBSP的等电点为10.43。鲤MBSP与鲫MBSP,猪胰蛋白酶,牛胰蛋白酶,美洲鲽胰蛋白酶的同源性分别为80.6%,55.8%,55.3%和53.9%。而与仓鼠肌肉中具有胰凝乳蛋白酶性质的蛋白酶的同源性为39.2%。MBSP有高含量的赖氨酸残基(11.93%),此特性可能与该酶的肌原纤维结合特性有关。
Myofibril-bound serine proteinase (MBSP) is a recently discovered protease. This enzyme is involved in the degradation of myofibrillar proteins and the reduction of the elasticity of surimi products. However, the research on the primary structure of this enzyme has not been reported so far. In this paper, according to the determined MBSP N-terminal amino acid sequence and serine protease activity center conservative sequence design annex primers, combined with RT-PCR technology to achieve MBSP gene fragment amplification. Gene-specific primers were designed according to the cloned MBSP fragment sequences for rapid amplification of the 5 ’and 3’ ends of the MBSP gene. Based on the above results, the total length of the carp MBSP was determined. Sequence analysis showed that the MBSP cDNA contained a 732 bp open reading frame encoding 243 amino acid residues, of which the signal peptide was 21 amino acids in length. The amino acid residues that make up the serine protease active center (His61, Asp107 and Ser197) are conserved in MBSP. The mature MBSP contains 222 amino acid residues with a molecular weight of 24.5 ku, which is slightly smaller than the native protein of 30 ku. The isoelectric point of mature MBSP is 10.43. The homology between MBSP of carp and MBSP of pig carp, porcine trypsin, bovine trypsin and tryptase of American trypsin were 80.6%, 55.8%, 55.3% and 53.9%, respectively. While the homology with the chymotrypsin protease in hamster muscle was 39.2%. MBSP has a high content of lysine residues (11.93%), which may be related to the myofibrillar binding properties of the enzyme.