Identification Of The Proteins Interacting With SjGCP And Studies On Two Of Them

Schistosomiasis is an important global public health problem; millions of people are still at risk of acquiring this infection. An ideal method for sustainable control of schistosomiasis is using vaccine alone or in combination with drugs. Gynecophoral canal protein (GCP) is especially expressed in the adult male worm and its protein shows wide distribution in the adult female worm after paring. It is proved that the protein could impact on the process of paring and play important roles in the development of Schistosoma. So the study about the GCP protein and the proteins interacting with it is significant.In this study, we analyzed the function of the gene SjGCP and confirmed a series of proteins interacting with SjGCP used yeast two-hybrid system, and two of them were selected for further study. The following major progresses were achieved in this work.(1) Microarray was used to analyze the function of SjGCP and the result showed that SjGCP might be involved in binding and play important roles in the process of growth, development, reproduction and metabolism for S. japonicum. In preliminary work of our group, yeast two-hybrid cDNA library from 18d worms of S. japonicum was constructed, and the sequences of 45 different genes interacting with SjGCP was obtained. In order to validate the interaction, we performed a serious of experiments to exclude false positive clones using BD-lamin as a negative control. As a result, we got 16 positive clones, 12 of which were uploaded in GenBank and the other four were unknown genes.(2) We cloned the gene SjGALE and generated the expression product in E. coli. The expression level of SjGALE during different developmental stages of S. japonicum was evaluated by real-time RT-PCR and Western blot. The transcription profile demonstrated that SjGALE mRNA was expressed at all developmental stages anyalyzed in my study; we observed that the SjGALE transcription level increased gradually in the 7 and 18-day-old schistosomula, peaking at the 23-day-old parasites, and then, decreased in the 31 and 42-day-old adult worms. The Western blot had a similar profile. Immunolocalization indicated that the protein is mainly located on the tegument of the parasite. Infection of mice immunized with rSjGALE demonstrated a 34% and 49% reduction of the mean worm burden and liver egg burden, respectively, in two independent experiments, indicating immune protection. The liver egg count from each female adult worm was significantly reduced by 63% in two trials. The cytokine profile and IgG isotype analysis demonstrated the induction of a Th1 immune profile in response to immunization with this protein, further suggesting protection against infection. In conclusion, these findings indicate that SjGALE is a potential vaccine against S. japonicum.(3) It was validated the interaction between the Sj34.9 and the SjGCP, in order to study the biological function of the molecule, the complete sequence of Sj34.9 was amplified by RACE technique. The sequence is 1514 bp, displays an ORF of 966 bp and encodes a protein of 321 amino acids, with a predicted molecular mass of approximately 34.9 kDa and an isoelectric point of 9.38. The sequece was uploaded to GenBank and the accession number is JF907494. The expression level of Sj34.9 during different developmental stages of S. japonicum was evaluated by real-time RT-PCR. The transcription profile demonstrated that Sj34.9 mRNA was expressed stably at all developmental stages anyalyzed in my study. Mouse immunization with rSj34.9 induced no protection against worm burden infection. However, vaccination with rSj34.9 reduced by 28.9% of liver eggs number. Microarray was used to explore the genes differentially expressed between the RNAi group and the control. The result showed 378 genes expressed differently which included 202 up-regulated genes and 176 down-regulated genes; Pathway analysis indicated that genes expressed differently were mainly related to metabolism and signal transduction; The gene ontology category analysis showed that most of these genes might be involved in binding, membrane and cellular process. So we predicted that the gene Sj34.9 might play important roles in the process of growth, development, reproduction and metabolism for Schistosoma japonicum.