猪带绦虫六钩蚴45W基因家族分子克隆

Molecular Cloning of 45W Gene Family of Taenia solium Oncosphere

利用反转录聚合酶链式反应(RT-PCR)从激活的猪带绦虫六钩蚴克隆到45W基因家族。通过测序及DNAStar软件分析证实,共得到8个A型转录本、3个与A型转录本相应的B型转录本和4个其它B型转录本,以及1个C型转录本,提示45W相关基因可能更多。在本研究中,发现45W-4B非常保守,有望以此为基础研制出有效的抗猪囊尾蚴病的疫苗。在大肠杆菌中以融合蛋白(GST)的形式对经改造的A型转录本(A3)和B型转录本(B2)进行表达,SDS-PAGE证明,A3呈可溶性表达;B2的表达产物主要形成了包涵体,用强变性剂(6M尿素)处理后再经SDS-PAGE,证明B2也得到了高效表达。Western分析结果表明,二者均与囊虫患者血清不反应或反应很弱;B2能与猪抗六钩蚴血清反应,说明B2可能具有免疫反应性,且其抗原表位可能是线性的。从预测的结果看,猪带绦虫45W蛋白可能是跨膜糖蛋白,跨膜区位于C末端,在它的结构中可能还存在FnⅢ组件。同时,A3和B2蛋白结构中还存在Cn-Em重复单元,以及由16～17个氨基酸构成的α-螺旋的结构单元。在45W蛋白的加工过程中,磷酸化修饰可能是一项重要内容。由此推测,45W蛋白的作用很复杂,可能在调节基因的表达和维持正常的细胞形态等过程中发挥重要的作用。

45W related genes were amplified by RT-PCR from the hatched and activated oncospheres of Taenia solium, cloned and sequenced. A total of 16 transcripts were obtained, suggesting that 45W related genes had far more than five members as previously reported. The 45W-4B clone in our study was found to be highly conversed. The protein encoded by 4B transcript would be a good candidate for the development of recombinant vaccine against cysticercosis in pigs. The modified transcripts A3E/X and B2E/X were expressed in the form of GST fusion protein in E.coli. The fusion proteins of GST-A3E/X and GST-B2E/X could not be detected using sera from humans infected with cysticercosis. Also, GST-A3E/X fusion protein could not be detected by sera raised from pigs infected with oncospheres. Transcripts of this family encode glycoproteins with a transmembrane region located at the C terminal. The secondary structures of 45W proteins were characterized with Cn-Em repeat units and a 16-17 amino acids comprised a-helix structural unit. In the processing of 45W proteins, the phosphorylated modification on which some functions are depended may be important. These results suggest that 45W proteins＇functions are complicated, related with the regulation of gene expression and maintance of the cellular cytoskeleton and so on.