RNA Interference Of The Zinc Finger Protein Gene SjZFP1of Schistosoma Japonicum

The life cycle of Schistosoma japonicum (S. japonicum) is very complicated. During its development, the parasite is exposed to different environments and undergoes many morphological and physiological transformations as a result of changes in gene expression. The regulators of gene expression of S. japonicum should be the key regulatory molecules of the parasite’growth and development. Zinc finger proteins are proteins that have zinc finger motifs (ZnF) and widely exist in eukaryotic cells. They can activate/inhibit the expression of target genes through selectively binding to DNA or RNA or the interaction between zinc fingers. In our previous studies, the full-length sequence of S. japonicum zinc finger protein (SjZFPl) was obtained and bioinformatics analysis showed that SjZFP1might be an important gene expression regulator of S. japonicum. In this study, we knocked down the expression of SjZFP1through RNAi silencing and observed the influence on the growth, development and the expression of other genes of S. japonicum worm. Meanwhile, we evaluated their protective efficiency of recombinant protein vaccine and the DNA vaccine prepared with the partial fragment of SjZFPl which contains most antigenic sites.1. The SjZFP1mRNA expression patterns at the stages of S. japonicumFluorescent real-time quantitative PCR was used to analyse the SjZFPl mRNA expression patterns at the stage of egg, cercaria, and worms collected at day7,13,18,23,31and42post infection. SjZFP1was expressed in all studied stages of S. japonicum, with highest level in cercaria. After infection, the SjZFPl expression level graduallly decrease to the lowest at day13post infection, then slightly increase in the18-day worms when the male and female worms are paired, the second-lowest level was found at day23post infection and there was another rise in the subsequent period. In the adult worms, female has slightly higher SjZFP1mRNA expression level than male. The results revealed that the SjZFP1mRNA expression level was closely related to the main physiological activities such as infection of cercaria, early development of shistosomula, pairing, spawning and egg embryo development.2. RNA silencing of SjZFP1gene in vivo and in vitroAccording to the sequence of SjZFP1,3specific siRNAs (AY359, AY546and AY770) and a negative siRNA were designed, synthetised and used to perform RNAi test in vitro with schistosomula transformed from cercariae. Using real-time PCR to detect the SjZFPl mRNA level of worms cultured for72hours in vitro, we screened2pairs of specific siRNAs (AY546and AY770), which respectively had the better interference effect with71.8%and73.7%silence, compared with the negative siRNA group.Then, the specific siRNAs, AY546and AY770, were applied to perform5independent RNAi trails in BALB/c mice. The worms were treated with siRNAs by intravenous administration of mice in the early stages of infection for the trials1-4and in the mid-to late of infection for trial5. The SjZFP1mRNA levels of worms collected at day38-42post infection were detected as above. Compared with NC (negative siRNA control) group, the SjZFP1mRNA level at day-42post infection were significantly increased in the trials1-4in which the specific siRNAs was mainly given in the early stages of infection, while significantly decreased (56%for AY546and38.3%for AY770) in trial5in which the specific siRNAs was given in the the mid-to late of infection. The results showed that the RNA silencing of SjZFPl in mice might be transient. Compared with the blank group,11.9%-39.0%worm reduction rates (overall25.0%, P<0.01) were obtained from AY546group, in the five experiments, while-2.5%-23.2%(overall13.1%, P<0.05) were obtained from AY770group. Compared to blank group, the egg hatching reduction rates in the third to fifth trials were45%,66.7%and30.2%from AY546group, and49.8%,37.3%and38.4%from AY546group, respectively. The liver egg counting results showed that the liver egg numbers of AY546group or AY770group increased in trials1-4(it might be due to the increasement of the expression of SjZFP1in the late infection period), and significantly reduced in trial5.The expressions of other genes in worms of blank and AY546groups from the fifth trail were analysed with gene chip technique. After silencing of SjZFPl gene, some genes participated in transcription and microtubule movement were down-regulated, and some genes related to metabolism, immune response etc. were up-regulated.3. Evaluation of protective efficiency of recombinant SjZFPl-1.The181bp-786bp of SjZFPl (SjZFPl-1) containing most antigen sites, was selected to prepare recombinant protein antigen rSjZFPl-1/His and construct the recombinant eukaryotic expression plasmid pcDNA-SjZFPl-1. Vaccination tests were carryed out in BALB/c mice. In two independent trials, no significant differences were found in the average worms and liver eggs btween the vaccination groups (with rSjZFPl-1/His or with pcDNA-SjZFPl-1and rSjZFPl-1/His) and their corresponding controls. The results suggest that rSjZFPl-1/His can not induce effective anti-infection immunity.Conclusion:Our studies illustrated the expression patterns of SjZFP1at the devlopment stages of S. japonicum, confirmed that the SjZFPl gene played an important role on the schistosomula growth development, spawning and Egg embryo development of S. japonicum through RNAi silencing experiments, and demonstrated that the products of181bp-786bp nucleotide sequence of SjZFPl can not induce effective anti-infection immunity. Those studies have provided a foundation for developing SjZFPl as an efficient candidate vaccine or therapeutic drug target.