关键词: 斯氏狸殖吸虫, 半胱氨酸蛋白酶, 克隆, 原位杂交

Abstract: 　Objective To clone the cysteine protease cDNA fragment from Pagumogonimus skrjabini adults and lo- cate the tissue of the adult worm where cysteine protease is expressed. Methods The cysteine protease cDNA fragment was amplified by reverse transcription-polymerase chain reaction
RT-PCR with degenerated primers. The production was TA-cloned into the pUCm-T vector and sequenced. DNASIS program was used to analyse the nucleotide sequence and de- duce the amino acid sequence, which was aligned with the correlated parasite cysteine protease afterwards. The digoxin la- beled cRNA probe was synthesised by in vitro transcription with the cloned cDNA as template. The frozen sections of the adult worms were analysed by hybridization in situ to locate the gene expression. Results A 495 bp cDNA fragment was amplified by RT-PCR and sequenced. An amino acid sequence was deduced by DNASIS. Sequence analysis and align- ment showed significant homologies with the correlated parasite cysteine proteinases and conservation of Cys, His and Asn residues that from a catalytic triad. In the hybridization in situ analysis, intestinal epithelium was stained positively on trans- verse section of adult worms. Conclusion The cysteine proteinase cDNA fragment from Pagumogonimus skrjabini adults was cloned. There are some key sites which are correlated to the function of cysteine protease in the cDNA fragment. Cysteine protease is mainly expressed in intestinal epithelium of P. skrjabini.

Key words: Pagumogonimus skrjabini, cysteine protease, clone, hybridization in situ