日本血吸虫特异性膜蛋白的筛选及其中sj15蛋白编码序列的克隆

Screening for specific membrane protein of Schistosoma japonicum and cloning of sequence encoding protein sj15

目的:筛选日本血吸虫可能的特异性膜蛋白,并根据筛选结果在日本血吸虫成虫中尝试克隆某一可能具有免疫保护功能的预测蛋白的编码序列,为血吸虫病防治研究寻找新的疫苗候选分子或药物靶标。方法:以"Schistosoma japonicum"和"membrane or tegument"为关键词,在公共数据库NCBI进行查找并下载数据,建立本地化的日本血吸虫膜蛋白序列数据库,与本实验室建立的曼氏血吸虫、人类、大鼠及小鼠蛋白质数据库等进行多序列比对,筛选日本血吸虫特异性膜蛋白,同时通过CBS网站相应分析工具软件预测目的蛋白的特征和功能。针对其中可能有免疫保护功能的预测蛋白的预测编码序列设计引物,采用RT-PCR技术从日本血吸虫成虫中扩增出该编码序列,并克隆入pGEX-4T-1构建原核表达重组质粒。结果:共检索到153个理论推测的日本血吸虫膜蛋白序列,筛选出28个可能的日本血吸虫特异性膜蛋白;成功从日本血吸虫成虫中扩增出相对分子质量为15 000的预测蛋白的预测编码序列,暂命名为sj15基因,同时成功构建pGEX-4T-1-sj15重组质粒。结论:成功筛选出多个日本血吸虫特异性膜蛋白;从日本血吸虫成虫中成功克隆出GenBank公布的预测的sj15基因,证实了成虫中该预测基因的存在,并成功构建原核表达载体,为进一步的功能研究奠定了基础。

Objective：To screen for the potential specific membrane protein from the protein database built by our Lab and to clone the encoding sequence of a potential immunoprotective protein according to the screening result, so as to search for new vaccine candidate or drug targets for Schistosomiasis control. Methods： The related data were retrieved using the key words ＂Schistosorna japonicurn＂ and ＂membrane or tegument＂ from the NCBI database to construct local database of membrane protein of S. japonicurn. The specific membrane proteins were screened by multi-alignment with the protein sequences from our local database of Schistosorna rnansoni, Homo sapiens, Rattus norvegicus and Mus rnusculus. Meanwhile, the character and function of the predicted membrane protein were analyzed with public software in the website CBS. The specific primer for the sequence of the predicted protein with potential immuno-protective function were designed and synthetized; the coding sequence was then amplified from the adult worm of S. japonicurn by PCR and was cloned into prokaryotic expression vector pGEX-4T-1. Results： A total of 153 predicted membrane protein of S. japonicurn were retrieved and 28 potentially specific for S. japonicurn were screened out. The coding sequence of the predicted protein, with a relative molecular weight of about 15 000, was successfully amplified from the adult worm of S. japonicurn and was temporarily named gene sj15; the recombinant plasmid pGEX-4T-1- sj15 was also successfully constructed. Conclusion： Many specific membrane proteins of S. japonicurn have been successfully screened out. The predicted sj15 gene （announced as AY223457 in GenBank） has been successfully cloned, proving the existence of sj15 gene in adult worm of S. japonicurn. The prokaryotic expression vector pGEX-4T-1 for sj15 has also been successfully constructed, providing a foundation for further study of sj15 function.