Preliminary Analysis Of Biological Functions Of Schistosoma Japonicum Female Reproductive Development Related Proteins

Schistosomes have evolved separate sexes,the mating of male and female worms can stimulate the female sexual mature,eggs produced by the mature female worms are important in transmission of the parasite and responsible for pathogenesis.Females from single-sex infections are underdeveloped in that they are stunted and exhibit an immature reproductive system,can't lay normal eggs.In our previous study,the i TRAQ proteomics technology was used to analyze the differentially expressed proteins of unisexual and bisexual females at 18,21,23,and 25 days after infection.The seven high expressed proteins of bisexual females were analyzed to study their biological functions.The seven proteins are Sj TCTP,Sj Annexin,Sj UL30,CAX72463,Sj GST,CAX70849,Sj UDP-4.Real-time PCR was used to analyze the corresponding genes transcript profiles of these seven proteins at different times(post infection of 7,14,18,21,25,28,35,42d),females and males of 18,21,25,28,35,42 d post infection,and 18,21,23,25 d female worms come from unisexual and bisexual infections.The results showed that:Sj GST(glutathione-S-transferase isoenzyme)and Sj UDP-4(glucose isomerase)were highly expressed during the period of strong metabolism of Schistosoma(7 d,21 d);the expression of Annexin increased gradually with the growth and development of schistosoma;the expression of other genes was highest at 25 d.Their transcriptal expression levels in male worms were stable,their expression in female worms changed with the growth and development of females.Their transcription levels were higher in bisexually infected females than those in unisexually infected females.To study the biological function of these proteins in schistosoma,short-term interference was used to select the most effective si RNA molecules.the long-term interference of the target genes were performed by inject periodically the si RNA molecules to infected mice through tail vein.interference.Collect the disturbedworms and count them,and observe the morp Hological changes of some worms using scanning electron microscope and transmission electron microscope.Meanwhile,the number of charge eggs in the liver of mice and the hatching status of eggs and hatching were counted.The results showed that the highest worm reduction and egg hatching reduction of the Sj TCTP gene interference were 55.9% and 69.5%,respectively;Sj Annexin had highest egg burden reduction reach up to 82.8%;electron microscopy analysis found that the gene expression was interfered with the morp Hology of the worm.The changes,especially the gene expression of CAX70849 is interfered to cause the worm body,forming gaps and large voids or even ulceration between the body surface.The immunoprotection of r Sj ELAV-like 1 and r Sj ELAV-like 2 were evaluated,these two genes are highly expressed at 25 day in unisexual infected females than bisexual infected females.The genes were cloned and expressed,the purified proteins r Sj ELAV-like 1 or r Sj ELAV-like 2 were mixed with 206 adjuvants and injected subcutaneously into mice for immunoprotection tests.Both had certain effects in reducing worms and eggs borden.The r Sj ELAV-like 1 immunization has 17.81%worm reduction,44.52% egg reduction and-20.38% egg hatching reduction,the r Sj ELAV-like 2 reduced 36.03% worm reduction,51.34% egg reduction and 46.83%egg hatching reduction.The ELISA test showed that the specific Ig G antibody level of r Sj ELAV-like 2 changed significantly after the first immunization.In this study,the transcriptional expression profiles of schistosome female reproductive development-related proteins at different times,different genders,and different infection statest were tested,These proteins effection on schistosome survive,female worms fecundity and egg hatching capacity were analysised by long term RNA interference.The results laid a theoretical foundation for further research and use of these genes to prevent and control schistosomiasis.Result shows r Sj ELAV-like2 is more suitable as a vaccine candidate molecule for schistosomiasis japonicum through evaluation of immune protection effect,which provides a certain data basis for schistosomiasis prevention research.