Screening Interactional Factor Of SjGCP Protein And Study Of SjActin And SjTWF

Schistosomiasis is a serious parasitic disease infected by schistosome, which infects people and livestock and widely distributed in the world. Schistosome is a unique dioecious fluke, SGCP is the only protein which was found expressed especially at the gynaecophoral canal of male worms and transfer from male onto female, and SGCP was thought have some function on the development and mature of female worms. So the studies on SGCP and proteins interacted with SGCP would show some light on the mechanism of growth and development of schistosome.一.Screening interactional factor of SjGCP proteinRecombinant plasmid pGBKT7-BD-SjGCP and 18d of Schistosoma japonicum cDNA library have been constructed ready for yeast Two-hybrid system. To screen of protein interact with SjGCP. , 131 inserted fragments was acquired from 383 positive recombinant plasmid, sequencing analysis of these fragments show that 45 different genes sequence we have got. Then these genes have been cloned into the plasmid of pGBKT7-BD-SjGCP to verify interaction of SjGCP protein by repeating Two-Hybrid, also Negative Control is done in order to remove false positive. Finally 16 genes sequence of the protein which interact with SjGCP were finally got. Because of using repeat subcultured yeast cell and repeat Two- Hybrid to remove false positive ,the results of 16 protein have be screened is reliable in this study .二.SjACT and SjTWF protein preliminary researchIn order to study of SjACT and SjTWF protein, screened by yeast Two-Hybrid. First, Recombinant plasmids of pET28a-SjACT and pET28a-SjTWF were constructed and SjACT and SjTWF proteins were expressed in E. coli. Good immunogencity was be verified using antiserum by western-bolt method, this results also confirmed that SjACT is a Smooth Muscle Actin (SMA,Actin-α2) of actin protein family. Then, sjACT and sjGCP protein was observed expression upper Schistosoma japonicum epidermis by using Laser Scanning Confocal Microscope, so this study shows reliability of interaction of SjGCP and SjACT protein. Additionally, Transcript level of SjACT and sjTWF is highest on 23d, 21d was detected out individually by using real-time PCR method. Finally we detected IL-10,TNF-α,IFN-γand IL-4 production following splenocyte activation with SjTWF protein,TNF-αand IFN-γproduction shows Significant differences from stimulated in relation to non-stimulated cells( P<0.001), but IL-10 production shows no significant differences (P>0.5), and IL-4 shows differences(P<0.5).This finding suggests a tendency of Th1 and Th2 type of immune response.三. RNA interference SjACT and SjTWF proteinTo determine the role of SjACT and SjTWF in Schistosoma japonicum growth and development, three siRNA was design to interference of SjACT and SjTWF protein expression on japonicum cultivated in vitro. analysising from transcriptional level and production of protein express, this experiment was succeed, and founding mortality of japonicum have positive correlation to effect of interference by siRNA. Observation form of Morphology, movement affected when interference of SjACT and SjTWF protein expression of japonicum. Conclusion that SjACT and SjTWF protein have some relationship to Schistosoma japonicum growth and development.