蟹类原肌球蛋白基因的克隆与表达

原肌球蛋白是甲壳类动物的主要过敏原之一。本文通过分子生物学技术方法，分别克隆得到锯缘青蟹(Scylla serrata)、中华绒鳌蟹(Eriocheir sinensis)和三疣梭子蟹(Portunus trituberculatus)的原肌球蛋白基因序列。测序结果表明，三个基因的序列长度均为855 bp，编码284个氨基酸残基，三者氨基酸序列同源性为99.3%。三种蟹的原肌球蛋白基因序列与GenBank中其他甲壳类动物的原肌球蛋白具有很高的同源性。将锯缘青蟹的原肌球蛋白基因与pGEX-4T-3载体连接后，经IPTG诱导得到分子量约为61 kDa的融合表达蛋白。通过与甲壳类过敏患者血清的免疫印迹反应，证实融合表达的原肌球蛋白具有过敏原性，表明原肌球蛋白是蟹类的主要过敏原之一。该融合蛋白有望用于甲壳类食物过敏诊断试剂的开发与应用。

Cloning and expression of tropomyosins from crabs

Although tropomyosin (TM) is known as a major allergen of crustaceans, few study was carried out on it in China. In this study, three tropomyosin genes were amplified by polymerase chain reaction (PCR) from three species of crab (Mud crab, Scylla serrata; Chinese mitten crab, Eriocheir sinensis; Japanese blue crab, Portunus trituberculatus), respectively. Sequence analysis showed that all the three cloned DNA fragments had the open reading frame (ORF) of 855 bp, encoding proteins with 284 amino acid residues and molecular weight of approximately 34 kDa. The three TMs revealed extremely high identity to TMs from other crustaceans. The tropomyosin of Mud crab was further studied by recombining with the vector pGEX-4T-3, and over expressed in E.coli JM109. A major protein band with size of about 61 kDa was observed on SDS-PAGE, which is close to the predicted molecular weight of the target fusion protein GST-TM. The expressed protein was water soluble and was further purified by GST affinity column. Western-blot analysis both using anti-mud crab TM polyclonal antibody and sera from subjects with crustacean allergy revealed positive reaction to the GST-TM fusion protein, strongly suggesting that the expressed protein is allergenic and can be used as a potential antigen for allergy diagnosis.