柞蚕感染微孢子虫后血淋巴免疫应答蛋白质的分离与鉴定

了解柞蚕Antheraea pernyi感染微孢子虫初期血淋巴内免疫系统及刺激应答相关蛋白质种类, 本研究以柞蚕5龄雌幼虫的起蚕（结束4眠, 刚完成蜕皮的幼虫）添食柞蚕微孢子虫Nosema pernyi为材料, 对感染后血淋巴利用SDS-PAGE进行分离后, 利用LC-MS/MS质谱技术和蛋白质组学分析对差异蛋白质条带进行鉴定。结果显示： 感染微孢子虫144 h后, 血淋巴中分子量约为44 kD （AP44）和28 kD （AP28）的蛋白质条带表达量增高。质谱分析AP28和AP44蛋白质条带样品, 共鉴定117个不重复蛋白质, 其中2个样品共有蛋白质12个, AP28独有蛋白质52个, AP44独有蛋白质53个。对质谱数据利用COG数据库进行搜寻鉴定, 显示AP28和AP44的鉴定蛋白质中涉及柞蚕免疫系统及刺激应答生物过程的蛋白质共有29个, 其中AP28中包括热激蛋白、 泛素样蛋白、 泛素结合酶E2、 保幼激素环氧水解酶、 微管结合蛋白、 溶菌酶、 ADP-核糖基化因子、 防御蛋白、 肽聚糖识别蛋白等15个, AP44中包括DRK、 酚氧化酶原、 类免疫球蛋白等10个; 二者共有热激蛋白hsp21.4、 酚氧化酶原、 抗菌肽等4个。本研究结果可以为今后研究柞蚕对微孢子虫的免疫应答及防御机制提供参考。

Separation and identification of haemolymph proteins involved in immune response to Nosema pernyi infection in Antheraea pernyi (Lepidoptera: Saturniidae) larvae

This study was aimed to preliminarily identify the proteins involved in the early immune response in the haemolymph from Antheraea pernyi larvae infected by Nosema pernyi. The 5th instar female larvae of A. pernyi were fed with N. pernyi spores and the proteins in the haemolymph were separated by SDS-PAGE, and the proteome profiles of the differential protein bands were further analyzed and identified by LC-MS/MS and proteomics analysis. One dimensional SDS-PAGE analysis revealed that protein bands at 44 kD (AP44) and 28 kD (AP28) were enriched in the haemolymph of the female larvae at 144 h after feeding with the spores of N. pernyi. A total of 117 non-redundant proteins were identified by LC-MS/MS and searching the COG database, including 52 unique proteins from AP28, 53 unique proteins from AP44, and 12 proteins from both of them. Among them, 29 proteins are immune-related, including 15 proteins from AP28, such as heat shock proteins, ubiquitin-like proteins, ubiquitin-conjugating enzyme E2, juvenile hormone epoxide hydrolase, microtubule-associated protein RP/EB family member 3, lysozyme-like protein, ADP-ribosylation factor, putative defense protein and peptidoglycan recognition protein-like protein, and 10 proteins from AP44, such as DRK, prophenoloxidase and hemolin. Four proteins were identified from both AP28 and AP44, including heat shock protein hsp21.4, prophenoloxidase and basic attacin. The results provide the basis for further study on the mechanism of immune response and defense against N. pernyi infection in A. pernyi.