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目的通过天然分子疫苗免疫猪血清作为探针筛选日本血吸虫尾蚴cDNA文库,应用表达序列标签获得日本血吸虫新基因。方法制备天然分子免疫猪血清,将筛选的尾蚴cDNA文库中大肠杆菌XL1-Blue侵入大肠杆菌BM25.8后环化成质粒,筛选出已转入质粒的菌株。提取质粒DNA,进行测序,得到一个表达序列标签(EST)。通过NCBI网站的BLASTx及BLASTn公共数据库,编辑分析EST序列并进行同源性比较。结果从26个克隆样品中筛选出8个阳性克隆,其中有一个新基因(GenBank登录号DQ097517),该基因全长861bp,有完整的阅读框架,与蜜蜂(Apismellifera)SMC3蛋白编码的mRNA基因序列同源性最高。结论疫苗免疫猪血清可作为筛选日本血吸虫新基因的有效探针,新发现1个日本血吸虫相关基因。
Objective To screen the cDNA library of Schistosoma japonicum cercaria by immunizing porcine serum with natural molecular vaccine as a probe and obtain the new gene of Schistosoma japonicum by expression sequence tagging. Methods The natural molecular immunized porcine serum was prepared. Escherichia coli XL1-Blue in the selected cDNA library of cercariae was invaded into E. coli BM25.8 and then circularized into plasmids to screen out the plasmids which had been transformed into plasmids. Plasmid DNA was extracted and sequenced to obtain an expressed sequence tag (EST). The BLASTx and BLASTn public databases of the NCBI website were used to edit and analyze EST sequences and compare homology. Results Eight positive clones were screened from 26 clones. Among them, a new gene (GenBank accession number DQ097517) was obtained. The full length cDNA was 861bp in length and had a complete reading frame. Compared with the mRNA sequence of Apis mellifera SMC3 protein Highest homology. Conclusions Vaccination of pig serum can be used as a valid probe to screen new genes of Schistosoma japonicum, and a new gene of Schistosoma japonicum has been found.