[cDNA cloning and location of Pagumogonimus skrj abini cysteine protease].
OBJECTIVE
To clone the cysteine protease cDNA fragment from Pagumogonimus skrjabini adults and locate 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 deduce the amino acid sequence, which was aligned with the correlated parasite cysteine protease afterwards. The digoxin labeled 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 alignment 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 transverse section of adult worms.


CONCLUSIONS
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.
Wang
Ying
Y
Department of Pathogenic Biology, Third Military Medical University, Chongqing 400038.
Zhang
Xi-lin
XL
Zhang
Yan-ling
YL
Duan
Jian-hua
JH
Zhang
Jing-ru
JR
Huang
Fu-sheng
FS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010238:Paragonimus / Q000201:enzymology* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D003546:Cysteine Endopeptidases