水产学报2011，Vol.35Issue(9)：1310-1319,10.DOI:10.3724/SP.J.1231.2011.17473
中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal)的重组表达及活性分析
Recombinant expression and bioactivity assay of Kazal-type serine proteinase inhibitor(Fc-Kazal) from Fenneropenaeus chinensis
摘要
Abstract
Chinese shrimp (Fenneropenaeus chinensis) is one of the most important aquaculture animals in China. The studies on the innate immune responses of shrimp, especially on immune defense against the main crustacean pathogens, will provide more knowledge of shrimp immunity to prevent infectious diseases. Arthropod innate defence responses[e. G. Prophenoloxidase(proPO) activation and Toll pathway initiation] and many other biological processes are mediated by serine proteinase( SP) cascades. If the activity of SPs is out of control, it will be fatal to organisms. Serine protease inhibitors ( SPI) play a critical role in precise regulation of SP activity, and also directly participate in the selection and digestion of pathogen. One of the well known SPI is the Kazal-type SPI which are usually multi-domain proteins containing more than one Kazal domain. Each domain contains 50-60 amino acids with six cysteine residues forming a 1 -5,2 -4, 3-6 disulphide bridges resulting in a characteristic three-dimensional structure. The inhibitory specificity of a Kazal domain varies with a different reactive PI amino acid, which is the second amino acid after the second Cys. However,the knowledge about the Kazal-type SPI in aquatic animals' innate immunity is limited. In this paper, the fragment encoding the Kazal-type SPI (Fc-Kazal) mature peptide was amplified. It was predicted that the mature peptide was composed of 114 amino acids with a calculated molecular mass of 16.25 ku and a theoretical isoelectric point of 8. 9. Fc-Kazal contains two tandem Kazal domains. Each of the domain contains six conserved cysteine residues forming three disulfide bridges(Cl -C5,C2 -C4,C3 -C6), which are identical to those of the classical Kazal-type proteinase inhibitor family members. Semi-quantitative RT-PCR revealed that transcripts of Fc-Kazal were highly expressed in the gills, haemocytes and lymphoid organ. Low expressions could be found in the heart,intestine and hepatopancreas. No expressions were observed in eyestalks, ventral nerve cord and muscle. The recombinant protein was expressed by Pcr(R) T7/NT TOPO(R) TA system. The result showed that the fusion protein (rFc-Kazal) was expressed in the form of inclusion bodies. The LC-ESI-MS analysis showed that three peptide fragments of rFc-Kazal were identical to the corresponding sequence of Fenneropenaeus chinensis Kazal-type proteinase inhibitor (GI83638451). Fusion protein was purified by immobilized-metal affinity chromatography and Ni-NTA technology. The concentration of purified rFc-Kazal was 0. 4g/L. The effect of refolded rFc-Kazal on bacteriostatic activity was assayed in this research. The results indicated that the rFc-Kazal has antimicrobial activity against Vibrio anguillarum, Staphylococcus aureus, Aeromonas salmonicida and Bacillus thurigiensis. The results reveal that Fc-Kazal may play an important role in the innate immunity of the shrimp.关键词
中国明对虾/Kazal型丝氨酸蛋白酶抑制剂/先天免疫/重组表达/抑菌活性Key words
Fenneropenaeus chinensis/Kazal serine proteinase inhibitor/innate immunity/recombinant expression/bacteriostatic activity分类
农业科技引用本文复制引用
黄明,刘逸尘,张亦陈,孙妍,孙金生..中国明对虾Kazal型丝氨酸蛋白酶抑制剂基因(Fc-Kazal)的重组表达及活性分析[J].水产学报,2011,35(9):1310-1319,10.基金项目
国家自然科学基金项目(30871907,30901119,30901094) （30871907,30901119,30901094）
天津市科委(10JCZDJC18200,10JCYBJC09200,09JCYBJC15000) （10JCZDJC18200,10JCYBJC09200,09JCYBJC15000）
