RESUMO
Objective: To perform a proteomics analysis for metacercariae, juvenile and adult worms of Paragonimus skrjabini. Methods: Crabs were collected in P. skrjabini endemic areas of Kaiyang and Bijie in Guizhou Province. Metacercariae were isolated, placed in PBS, and were used to infect three SD rats(10 metacercariae/rat) by intragastric administration and infect three male dogs(20 metacercariae/dog) through feeding. The rats were sacrificed at 1 month after infection to obtain juvenile worms. The dogs were sacrificed at 3 months after infection to obtain adult worms. The metacercariae, juvenile and adult worms were lysed, and total protein was extracted by ultrasonication. The total protein content was determined by Bradford method and separated by SDS-PAGE and two dimensional gel electrophoresis. Images of two dimensional gel electrophoresis were analyzed using the PDquest 8.0 software. The dots with difference were digested and analyzed with mass spectrometry. Finally, online searches in NCBI and local databases were performed. Results: Results of SDS-PAGE showed that the total protein of metacercariae, juvenile and adult worms was concentrated within Mr of 25 000-116 000. Fifty-one protein dots with difference were found by two dimensional gel electrophoresis, comprising of 20 dots for metacercariae, 25 for juvenile worms and 6 for adult worms. Thirty-six peptide sequences of metacercariae and juvenile worms were analyzed. They were basically determined to be Achrornobacter lyticus protease â (a lysine-specific serine protease), ascorbate reductase protein, glutathione s-transferase DHAR2 analogue, heat shock proteins Hsp82 and Hsp96-ß, actin, cystatin, etc., by online searches, and cysteine, actin and heat shock protein by local searches in the diginea database(downloaded to a local computer from NCBI). Mass spectrometry was not performed for adult worms, as the variation of grayscale value between their spots was far less than those for metacercariae and juvenile worms. Conclusion: One difference is that the metacercariae of P. skrjabini have actin, while the juvenile worms have detoxification proteins and stress proteins. But they both have hydrolases and cysteine enzymes.
