Donpudsa Suchao, Visetnan Suwattana, Supungul Premruethai, Tang Sureerat, Tassanakajon Anchalee, Rimphanitchayakit Vichien
Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand.
Center of Excellence for Molecular Biology and Genomics of Shrimp, Department of Biochemistry, Faculty of Science, Chulalongkorn University, Phyathai Road, Bangkok 10330, Thailand.
Dev Comp Immunol. 2014 Nov;47(1):95-103. doi: 10.1016/j.dci.2014.06.015. Epub 2014 Jul 9.
An antimicrobial protein, crustin, is involved in the innate immunity of crustacean by defending the host directly against the microbial pathogens. By data mining the Penaeus monodon EST database, two type I crustins, carcininPm1 and 2, and ten type II crustins, crustinPm1-10, were identified. The abundant crustins were crustinPm1, 4 and 7, each with variation in the length of Gly-rich repeat among its members. A few crustinPm1, 4 and 7 with deletion in the Cys-rich region were also observed. Furthermore, the crustinPm4 with the longest N-terminal Gly-rich region was characterized. The crustinPm4 allelic genes were expressed mainly from the hemocytes. Its expression was up-regulated readily by WSSV infection and gradually decreased to normal level afterwards. The recombinant crustinPm4-1 (rcrustinPm4-1) isoform was produced using the Escherichia coli expression system and tested for its antimicrobial activity. The rcrustinPm4-1 was able to inhibit the growth of a Gram-positive bacterium, Bacillus megaterium but not Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus. It also inhibited the growth of two Gram-negative bacteria, E. coli 363 and Vibrio harveyi 639 at lower potency. The rcrustinPm4-1 affected the WSSV infection because the expression of an intermediate early gene ie1 in WSSV-infected hemocyte cell culture was reduced. It was shown further that the rcrustinPm4-1 could delay by about one and a half days the manifestation of disease by WSSV.
一种抗菌蛋白——甲壳素，通过直接抵御宿主免受微生物病原体侵害，参与甲壳类动物的先天免疫。通过对斑节对虾EST数据库进行数据挖掘，鉴定出两种I型甲壳素（carcininPm1和2）以及十种II型甲壳素（crustinPm1 - 10）。含量丰富的甲壳素是crustinPm1、4和7，其每个成员富含甘氨酸重复序列的长度存在差异。还观察到一些crustinPm1、4和7在富含半胱氨酸区域存在缺失。此外，对具有最长N端富含甘氨酸区域的crustinPm4进行了表征。crustinPm4等位基因主要在血细胞中表达。其表达在感染白斑综合征病毒（WSSV）后迅速上调，随后逐渐降至正常水平。使用大肠杆菌表达系统制备了重组crustinPm4 - 1（rcrustinPm4 - 1）异构体，并对其抗菌活性进行了测试。rcrustinPm4 - 1能够抑制革兰氏阳性菌巨大芽孢杆菌的生长，但对枯草芽孢杆菌、藤黄微球菌和金黄色葡萄球菌无抑制作用。它对两种革兰氏阴性菌大肠杆菌363和哈维氏弧菌639的生长也有较低程度的抑制作用。rcrustinPm4 - 1影响WSSV感染，因为在WSSV感染的血细胞培养物中，中间早期基因ie1的表达降低。进一步表明，rcrustinPm4 - 1可使WSSV疾病的表现延迟约一天半。
