小麦Bowman-Birk型蛋白酶抑制因子的克隆表达及其抑制鱼糜凝胶软化的效果研究

Cloning,Expression and Purification of Wheat Bowman-Birk Inhibitor and the Inhibitory Effect on Surimi Gel Softening

为探究重组小麦(Triticum turgidum)Bowman-Birk蛋白酶抑制剂(BBI)抑制活性特征及其对鲢鱼肌原纤维蛋白自溶反应的抑制效果,采用TA克隆技术克隆小麦BBI成熟肽基因片段,并对原核表达的小麦BBI蛋白进行镍亲和层析纯化,利用SDS-PAGE、TSK-GEL G2000SWxl高效液相色谱检测诱导及纯化效果。在测定重组小麦BBI对胰蛋白酶的抑制活性的基础上,进一步通过SDS-PAGE分析了重组BBI对鲢鱼肌原纤维蛋白自溶反应的抑制作用。结果表明,经双酶切鉴定证实成功获得了BBI成熟肽基因片段,该基因与野生二粒小麦BBI相关基因(GenBank登录号EU346892.1)序列相似性为99.63%。目的蛋白在SDSPAGE及TSK-GEL G2000SWxl高效液相色谱分析中,分别表现为单一带和单一峰,产物得到有效纯化,分子量约10.5kD。该重组小麦BBI对胰蛋白酶有明显的抑制活性。添加量5μg·mg-1重组BBI可显著抑制鲢鱼肌原纤维蛋白(尤其是肌球蛋白重链)在pH 7.5,50℃下的自溶。

In order to explore the inhibitory activity and inhibitory effect on autolysis reaction of silver carp myofibrillar protein of recombinant wheat BBI, TA cloning was used to clone BBI gene fragment which encoded mature peptide of wheat. The target protein was purified with Ni^2＋-NTA agarose af- finity chromatography after prokaryotic expression system. SDS-PAGE and HPLC of TSK-GEL G2000SWxl were conducted to examine the purified product. On the basis of inhibitory activity of recombinant BBI to trypsin, SDS-PAGE was conducted to analyze the inhibitory effects of BBI on au- tolysis reaction of silver carp myofibrillar protein. The results of double enzyme cutting indicated that BBI gene fragment was successfully cloned, which shared the sequence identity of 99.63% with the gene related to BBI of wild emmer （GenBank accession No. EU346892.1）. The target protein of wheat BBI was purified highly and performed a single band about 10.5 kD and a sharp peak respectively on SDS-PAGE and HPLC of TSK-GEL G2000SWxl. Activity assay revealed that wheat BBI can inhibit trypsin. The analysis about autolysis reaction of silver carp myofibrillar protein at pH 7.5 and 50℃ on SDS-PAGE indicated that the degradation of the major textural protein in surimi, myosin heavy chain,was suppressed significantly by the recombinant inhibitor with the concentration of 5 μg·mg^-1.