Obtain Of The Recombinant Adenovirus Serotype5Earring CARex And PTDtat Gene And Study Of The Infection Efficiency In Schistosomula Schistosoma Japonicum

Objective RNAi has become an invaluable tool for the functional analysis of genes in awide variety of organisms. Gene knockdown has been reported for Schistosomamansoni by soaking or electroporating different life-stages in dsRNA. However, thecurrent utility of this technology in parasite research is questionable, including howdelivery dsRNA, siRNA or siRNA expression vector to Schistosome efficiently, reducemortality caused by delivery method and get interference effect for a long time. Thefuture application of RNAi in Schistosoma functional genomics will greatly depend onhow we can overcome these difficulties. The main purpose of this study was thatwhether Ad5infect the schistosomulum of S.j and the infection efficiency, we alsoreconstruct the recombinant Ad5by inserting genes which could improve infectionefficiency of Ad5, as a preliminary study of application of adenovirus in RNAi ofschistosomula S.j.Method recombinant adenovirus plasmids carring CARex, PTDtat and CARex-PTDtat fused gene were constructed via vitro ligation and linearized by Pac I,transfected to293A cells mediated by liposome and packaged successfully, PCRidentification was performed after three generations of amplification and viral titer wasdetected by TCID50method, titer were108-109, in order to improve the virus titer thatrecombinant adenovirus were purified by CsCl density gradient centrifugation, the titerwere1011. Three recombinant adenovirus and wild adenovirus infected tumor cellsexpressing different CAR on the surface, high-CAR expressing cells(A549), medium-CAR expressing cells (HepG2) and low-CAR expressing cells(T24),expression quantity of GFP in a variety of cells was detected by inverted fluorescencemicroscope after48h, percentage of GFP positive cells and mean fluorescence intensitywere detected by flow cytometry. Finally cercaria of50positive snails S.j werecollected and converted into schistosomula using mechanical method in vitro, infectedwith recombinant adenoviruses and investigated by inverted fluorescence microscope in24h,48h and72h.Results Construction and package of recombinant adenovirus carring pAd-CARex、pAd-PTDtat and pAd-CARex-PTDtat, cells experiment showed the expression quantityof GFP in recombinant adenovirus groups would be higher than wild type, but effect ofthe pAd-CARex-PTDtat group was a litter worse than the pAd-CARex and pAd-PTDtatgroup, results of percentage of GFP positive cells in A549, HepG2, T24cells wereshowed as below: pAd group was54.8%,38.2%and8.52%; pAd-CARex group was90.4%,79.2%and32.1%; pAd-PTDtat group was95.8%,98.8%and93.9%;pAd-CARex-PTDtat group was70.5%,53.4%and13.3%; GFP positive cells ofpAd-CARex group showed a33%increase compared with pAd group, also withoutobvious distinction in three group, pAd-PTDtat group was61.6%, GFP positive cellswas increased alone with the decline of CAR, there was near85%in T24cell,pAd-CARex-PTDtat group was only10%. Mean fluorescence intensity of A549cellswas strongest, T24cell was weakest in each group, pAd-PTDtat group was strongest,pAd-CARex-PTDtat group was weakest, but all of them were higher than pAd.Experiment of schistosomula showed that wild pAd had no infection in schistosomulaS.japonicum, but pAd-PTDtat had a weakly infection. These indicated that PTDtat genecould improve the infection efficiency of adenovirus in tumor cells and schistosomula.Conclusion Cells experiment results showed that CARex and PTDtat gene couldimprove infection efficiency of adenovirus on tumors cells, especially low-CAR cells,PTDtat gene effect was better than CARex, but CARex-PTDtat fused gene effect was bad; Schistosomula experiment results showed wild pAd, pAd-CARex and pAd-CARex-PTDtat could not infect Schistosomula s.j, pAd-PTDtat had a faint infection. Theseindicate that PTDtat gene not only improved the infection of adenovirus on tumors butalso Schistosomula.