Expression, Identifacation And Histological Location Of Pagumogonimus Skrjabini Adult Cysteine Protease Gene And Its Fusion Protein's Application In Clinic Diagnosis

Background: P.skrjabini is epidemic in 15 provinces of China including Sichuan,Chongqing,etc. As an unsuitable host, people infected with P.skrjabini often exhibit extrapulmonary type of paragonimiasis with multiple organs and tissue damage that do great harm to the people's health and economy. 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. Recently, more and more studies were focused on cysteine protease because the protease has extensive prospect of use, such as research of immunodiagnosis, vaccine and chemotherapy. However there was little study on cysteine protease of P.skrjabini, which is only prevalent in China. Object: 1. To construct PinPointTM Xa-1 T- adult cysteine proteinase gene segment recombinant molecule. 2. To transform the recombinant molecule into JM109 strain. 3. To express the recombinant molecule in E.coli JM109 and primarily investigate the immunoreactivity of the fusion protein for further usage on serodiagnosis of the fluke disease. 4. Using the crude extraction of the fusion protein, we have detected 15 serums from patients (5 infected with C.sinensis ,5 infected with P.skrjabini,5 infected with S.japonicum) by cystatin capture ELISA to evaluate the effect on immunodiagnosis. 5. Also we have located the tissue of the worm where cysteine protease is expressed in P.skrjabini.Methods: The E coli DH5α containing target gene was inoculated in 5 ml LB medium that contained 100μg/ml ampicillin, incubated overnight at 37°C with shaking. The plasmid is extracted by Alkaline Lysis and the gene fragment is amplified with PCR. After purification with Gel-Purification Recovery, the target gene segment ligate with PinPointTM Xa-1 T vector, and is transformed into JM109 strain. All the clones, which have ampicilline resistance, are screened by amplifying specified segment with PCR. The positive clones, which can produce specified 496bp gene fragment in PCR, are sequenced to confirm having the proper orientation . Then the clone, in which target gene fragment is in the proper orientation, is inoculated with the overnight cultures in 1:100 with LB containing 2μMbiotin and 100μg/ml ampicillin. Incubate for 1 hour at 37°C with shaking and then induced by adding IPTG in final concentration of 100μM in LB medium which contains 100μg/ml ampicillin.After incubated for 4~5 hours at 37°C with shaking, the expressed fusion protein sample is prepared with Alkaline Lysis Solution ,electrophoresised by 12% SDS-PAGE , stained with Coomb's blue stain .The same sample is also transfer the proteins to nitrocellulose membrane following SDS-PAGE electrophoresis, to identify the biotinylated fusion protein by the Streptavidin Alkaline Phosphatase stain, and the fusion protein's immunoreactive property was examined with Western blotting. Using the crude extraction of the fusion protein, we have detected 15 serum from patients (5 infected with C．sinensis ;5 infected with P.skrjabini ;5 infected with S. japonicum) by cystatin capture ELISA. Also, we have located the distributing of cysteine protease expression by immunohistochemistry.Results: Twenty one ampicillin resistance strain were harvested in the transformation test . PinPointTM Xa-1 T- cysteine protease gene fragment recombinant molecule were successfully constructed and also verified by sequencing results .A positive band was clearly fond in 32 kDa by Streptavidin-Alkaline Phosphatase stain .In the same position, the fusion protein was also detected in western blotting. Using the crude extraction of the fusion protein,we have detected 15 serum from patients (5 infected with C.sinensis ;5 infected with P.skrjabini ;5 infected with S.japonicum) by cystatin capture ELISA the results show clearly that only the samples from the patients infected with P.skrjabini have the strong positive reaction(P﹤0.01),and the others show negative reaction. Immunohistochemical study show that the po...