Pagumogonmus Skrjabini Adult Worm Of Clinical Diagnosis Related Antigen Of Identification, Location And PsMt01 Protein Expression In Different Stage

Objective:The recognition between patient sera and soluble antigens of Pagumogonimus skrjabini helps us to know bio-characters of specific antigens. Also, the study on the expression of PsMt01 in different stage and antigens of tegument and intestine of Pagumogonimus skrjabini adult worm provides useful information to identify effective antigens for clinical and screening for paragonimiasis. Methods:(1) The soluble antigens of adult were analyzed by SDS-PAGE, two-dimensional gel electrophoresis (2-DE), western blotting and-immunogold-labeling technique.(2) The total RNA of metacercaria of P.skrjabini was reverse transcripted into cDNA, The cysteine protease cDNA was amplified, cloned into T vector and sequenced (PsMt 01). And then,the expression of PsMt 01 in the egg, larva and audlt stage were analyzed by RQT-PCR. (3) The adlut worm of P.skrjabini were made into frozen section 15μm in thickness. The antigen on the sections were localized with specific antibody in sera from patient infected by Pagumogonimus skrjabini. The immunodiagnosis-associated antigens were identified by using immonogold techniques, immune electron microscopy, enzyme-labeled immunoassay and laser scanning confocal microscopy techniques The dult worm of intestine of Pagumogonimus skrjabini was reconstructed by three-dimensional reconstruction.Results:(1) Three protein spots were identified by De Novo sequencing. A protein spot matches six kinds of Paragonimus related-protein. The coefficient of protein matching is the highest with a new type of protein westerpain-1/10 of Paragonimus westermani, B protein spot matches five kinds of Paragonimus related-protein. The coefficient of protein matching is the highest with cysteine proteinase of Paragonimus westermani, C protein spot matches no Paragonimus related-protein. Moreover antigen of adult worm was located in the adult worm membrane and intestinal tract by immunogold labeling technique. (2) Both immonogold techniques and enzyme-labeled immunoassay showed that specific antgen was concentrated in the tegumental and intestine. Confocal laser scanning microscope observations that semi-quantitative analysis of tegument antigen of average gray value is 80.65 and semi-quantitative analysis of intestine antigen of average gray value is 71.25. At the ultrastructural level, the immunogold labeling showed deposit of gold particles on the tegumental and on the surface membrane and intestine. (3) PsMt 01 protein was not detected in eggs,while it was detected from the metacercarial to adult stages. The maximum transcription levels was in the 0-to 7-week-old juveniles. Conclusion:(1) The main components of identified protein which analyzed by tandem mass spectrometry and immunogold labeling technique is pellicle of polypide and excretory-secretory, these protein play important role in invasion and immune escape. (2) PsMt 01 was detected the early development of larvae of Pagumogonimus skrjabini, a period during which the parasite migrates actively through the host tissues, which means it is important for invasion into host tissues. (3) Adult worm specific antigens of Pagumogonimus skrjabini identificated by using sera from the Patients with Paragonimiasis are tegument and intestine-associated antigens. Therefore, immunodiagnostic antigen of paragonimiasis are the adlut components of the surface membrane and intestine.