CDNA Cloning, Expression And Location Of Pagumogonimus Skrjabini Cysteine Protease

Background: P.skrjabini is epidemic in 15 provinces of China including Sichuan,Chongqing,etc. It does great harm to people's health. Cysteine protease exists in many animals including human and parasite. As a main kind of digestive enzyme of parasite, cysteine protease plays an important role in the relationship between parasite and host. More and more studys were focused on cysteine protease because the protease has extensive prospect of use, such as research of immunodiagnosis, vaccine and chemotherapy. However there wasn't any study on cysteine protease of P.skrjabini, which is only prevalent in China.Object: 1. To clone P.skrjabini'1?, cysteine protease cDNA fragments. 2. To express the cDNA in E.coli and observe the immunoreaction with immune serum of fusion protein. 3. To locate the tissue of the adult worm where cysteine protease is expressed in P.skrjabini.Methods: 1 .Metacercarias of P.skrjabini were collected from Xingwen County of Sichuan and the adult worms were isolated from dog infected by metacercarias of the parasite. After the total RNA of the parasite had been isolated with Tripure? RT-PCR was carried out with the degenerated primers designed according to the conserved amino acid sequences of the related parasite's cysteine proteases. Then the amplified fragments were cloned into pUCm-T vector and sequenced after being purified. Subsequently the sequences were analysed with the program of DNASIS. 2. After the ligation of PinPoint?Xa-1 T-vector and PCR product, the clones were screened to determine the fragment orientation prior to protein expression. The expression was carried out in E.coli induced by adding IPTG and analyzed on SDS-PAGEfollowed by Coomassie blue staining. The expressed fusion protein was identified and its immunoreactive ability was determined by Western blotting. 3. The digoxin labeled cRNA probe was synthesised by in vitro transcription with the cloned cDNA as template. And the frozen sections of the adult worms were analysed by hybridization in situ to locate the gene expression.Results:1. A 500bp band appeared in the gel electrophoresis after RT-PCR. Three cDNA fragments of metacercaria and one cDNA fragment of adult were obtained by cloning. And after the cDNA fragments being sequenced and submitted to GenBank, four accession numbers were obtained. Alignment of the deduced amino acid sequences with the sequences of the other known eukaryotic cysteine protease revealed significantly high levels of homology. The alignment is particularly strong around the putative active site Cys,His and Asn residues of the enzyme. 2. In the identification of the expressed fusion protein, Western blot illustrated two biotinylated bands of 33kDa and 22.5kDa in cells containing the PinPoint?Xa-1 T-Vector plus the insert of interest. JM109 cells containing the vector without insert showed two visible bands of 13kDa and 22.5kDa. Cells with no vector showed a single band of 22.5kDa. In immunoreaction observation, Western blot analysis showed a single band of 33kDa. 3. In the hybridization in situ analysis, intestinal epithelium was stained positively on transverse section of adult worms.Conclusion: 1 .Four cDNA fragments of P.skrjabini cysteine protease are cloned and sequenced. And the sequences containe the important sites which are correlated to the activity of cysteine protease. 2. A fusion protein with 33kDa molecular weight is expressed. The expressed protein has immunoreactive ability as it reacted with immune serum. 3. Cysteine protease is mainly expressed in intestinal epithelium of P.skrjabini.