Identification Of Phage Display Peptides With Affinity For The Tegument Of Schistosoma Japonicum Schistosomula

Section One:Differential Panning and Identify of peptides specifically binding to the tegument of live S.j schistosomulaObjective:The specific peptide with targeting and/or antagonism to the Schistosoma japonicum was gained by the techniqe of differential panning with liver polypides in vitro from Ph.D.-12TM Phage Displayed Peptide Library.Method:The specific peptides binding to schistosomula but cercarie were isolated by the techniqe of differential panning in vitro:The12peptide library without bingding to the cercarie was incubated and amplified with the schistosomula after force-absorbed with the cercarie, and the gained peptide library was eluted and amplified. After3times reverse absorption-amplification-panning-amplification, the DNA of15random phage clones was extract for sequencing. The sequences were analyzed through amino acids-translation and bioinformatics-analysis. The gained phage clones binding to schistosomulum and adult were detected and analyzed by the recovery rate of phage, Western-blot and immunohistochemistry.Result:The four M13phage peptide clones with different sequences MppZL6, MppZL4, MppZL1and MppZLO, without the insertion element in MppZLO, were gained by3time’s differential panning in vitro. Through bioinformatics-analysis, the peptide inserts from MppZL6, MppZL4and MppZL1, all containing arginine residue, were overlaped of8or9hydrophil polar amino acid residues and hydrophobe nonpolar amino acid residues, whose isoelectric point was6.07,8.75,8.50, respectively. BLAST analysis indicated that MppZL1showed53%(7/13) identities to apolipoprotein B of Mus musculus, MppZL6showed69%(9/13) identities to S.j CHGC07116protein and MppZL4showed80%(8/10) identities to surface antigen BspA-like influencing the functions of the hydrogenosome of T. v G3. The recovery rate of phage showed that the level of the phage clone MppZL4binding to schistosomulum was significantly higher than MppZL6and MppZL1. By Western blot, only the membrane protein of S.j adult binding to MppZL4clone displayed the specific band,45.0kDa. The results of immunohistochemistry suggested that ZL4could binding to Sj schistosomulum, lung-schistosomulum, hepatic-schistosomulum and adult worm.Conclusion:The specific M13phage peptide MppZL4, using the techniqe of differential panning with liver polypides in vitro from Ph.D.-12TM Phage Displayed Peptide Library, could specifically binded to the tegument of live Sj schistosomulum and adult worms in vitro of mouse. Section Two:Functional testing of the MppZL4and RhB-ZL4Objective:The biological functions of MppZL4and RhB-ZL4were detected by the killing-S.j-experiments in vivo and vitro, and the mechanism was preliminary study from the cellular level.Method:Elution rate test the targeting of MppZL4binding to S.j:The mice, infected with S.j3rd day and10th day, were injected with MppZL41012pfu through tail vein, respectively, and were killed after15min. The phage MppZL4were eluted and counted from the lung and liver organs of the infected mice. At the same time, the control group and blank group were established. The mice, infected with S.j24th day, were injected with MppZL41012pfu through tail vein and killed after15min. The phage MppZL4were eluted and counted from the lung, liver organs and the worms of the infected mice. MppZL4phage lethal effect in vitro:S.j schistosomula were treated with MppZL4in axenic condition and observated every24h for72h through methylene blue stain, while the worms’ mortality was counted. The positive control, negative control and blank control group were established. The mice, infected with S.j24th day, were injected with MppZL41012pfu through tail vein and sacrificed after15min. The S.j cells were manufactured used the worms from the mice in sterile condition and detected MppZL4by immunocytochemistry. The S.j cells were treated by MppZL4for one day whose inhibition rate was studied by MTT kit through IC50. The Rhodamine-labeled peptide RhB-ZL4were synthetic and detected the binding of S.j schistosomula in vitro and adult in vivo and the killing function of S.j schistosomula in vitro by the same ways.Result:The results of elution rate showed, that the abundance of MppZL4in the lung tissue was0.869±0.018and the liver MppZL4abundance was7.211±4.818at the3rdday, whlie the abundance of MppZL4in the lung tissue was0.239±0.065lower than the3rd day and the liver MppZL4abundance was15.833±8.224at the10th day higher than the3rd day, but the abundance of phage eluted lung or liver tissue showed no significant difference among each group while the abundance of MppZL4eluted from worm was17.256±9.588and much higher than M13KE eluted from worm was0.202±0.056at the24th d. The death rate of MppZL4lethal effect in vitro is higher than fortis serum positive control group (P=0.000) and its mortality rate had reached100%after48hr. Immunocytochemical stain displaied that MppZL4phage mainly deposited the cell envelope of big round cells, triquetrous cells and irregular cells of Sj. MTT test illustrateed that MppZL41016pfu possessed significant cytotoxicity (P=0.000), and with the higher concentration of MppZL4, the cytotoxicity enhanced; IC50≈1021. The results showed:RhB-ZL4could specifically binded to the tegument of live S.j schistosomulum in vivo and adult worms in vitro of mouse. The death rate of MppZL4lethal effect in vitro showed no significant difference between the MppZL4positive control group and its mortality rate had reached100%after72h.Conclusion:The specific MppZL4possess targeting and cytotoxicity for S.j. RhB-ZL4could specifically binded to the tegument of live S.j schistosomulum in vivo and adult worms in vitro of mouse, and possessed the cytotoxicity for S.j schistosomulum, too Section Three:Construction and functional identification of ZLW/pEGFP-C2 Objective:To research the efficiency of ZLW/pEGFP-C2plasmid transfection into Schistosoma japonicum (Sj) schistosomula and observe the survival of the worms in vitro.Method:After plasmids of ZLW/pEGFP-C2was constructed and transfected into Sj schistosomula, worms off the end of mechanical, using the immersion method containing0.75%DMSO and the high concentration of plasmid in vitro. The GFP as positive marker, the worm was observed under inverted fluorescence microscope; the presence of the transgenes ZLW and EGFP in Sj were confirmed by RT-PCR and western blot, respectively, with total RNA and proteins from transfect schistosomula and cultured for48hours. After transfect, the worms cultured24,48,72and96hours were respectively stained with methylene blue, observed determining and counted dead or live worm based on color availability. All experiments contained two parallel control groups of pEGFP-C2empty plasmid and TBS.Result:The plasmid of ZLW/pEGFP-C2was constructed successfully. The transfect efficiency showed that the transfect rate was about10%and the fluorescence of EGFP was mainly localized in tegument of the worms especially predominate around the oral sucker and ventral sucker on the worms. The expected size of259bp fragment was successfully amplified by RT-PCR and its result of sequencing was consistent with the ZLW sequence, and the EGFP gene expression in worms was confirmed with western blot analysis. The results of affecting the worms to survive showed that the mortalities between experimental group and control groups showed no significant difference at24and48hours (P24=0.600, P48=0.508), but the mortality rate of experimental group was significantly higher than control groups at72hours (P72=0.000) and was100%after96h.Conclusion:This study proved that ZLW/pEGFP-C2plasmid has been successfully introduced into the juvenile Schistosoma japonicum using the high concentration of plasmid immersion under0.75%DMSO in vitro and the survival of transfect parasites was enable certain effects.