关键词: RT-PCR, 日本血吸虫, 组织蛋白酶L, 基因克隆

Abstract: 　Objective To analyze the full coding sequence of proteinase cathepsin L1 (SjCL1) of Schistosoma japoni-cum, and clone it into the eukaryotic expression vector pcDNA3. Methods Total RNA was isolated from adult worms of S. japanicum, the sequence of SjCL1 gene 5'-end was attained by performing averse nested PCR, and the sequence of SjCL1 gene 5'-end was determined by sequencing after being cloned into T vector. The coding region gene of SjCL1 was amplified by PCR, and the fragment from PCR was cloned into eukaryotic expression vector pcDNA3 via Bam HI and Xho I sites. The resulting construct was determined by PCR, restriction analysis and sequencing. Results A 320 bp sequence of SjCL1 gene 5'-end of Schistosoma japonicum was obtained by using averse nested PCR. After combined with the reported segment of SjCL1 gene, an integral coding sequence was obtained. The coding region of SjCL1 gene was specifically amplified by PCR, with a size of about 1 kb. The expression plasmid pcDNA-SjCL1 contained the amplified fragment, which is validated by PCR, restriction analysis and sequencing. Conclusion The eukaryotic expression plasmid containing the coding sequence of SjCL1 gene was constructed.

Key words: RT-PCR, Schistoscma japonicum, cathepsin L, gene cloning