Analysis And Identification Of Gene Functions Of Schistosoma Japonicum Adenylate Kinase 1

Objective:Schistosomiasis is a serious and widespread zoonosis.It is reported that host factors could influence the growth and development of schistosome.The gene function of Schistosoma japonicum adenylate kinase 1(SjAK1)gene,which is differentially expressed under the influence of host factors,was investigated in this study.Methods:We cloned and expressed SjAK1,and tested the immune protective effects and mechanism.Real time quantitative PCR and Western blot were used to detect the expression in different developmental stages.Immunohistochemistry was performed to test the distribution of SjAK1 in different tissue of schistosome.RNA interference experiments were used to testify the gene function and mechanism of SjAK1 in growth and development of schistosomula and egg formation of schistosome.Results:1.The recombinant plasmid pET28a(+)-SjAK1 was successfully constructed.Solube recombinant SjAK1 protein(rSjAK1)was obtained from prokaryotic expression system.The molecular weight of rSjAKl was about 26 kDa which was consistent with the predicted value.rSjAK1 was purified by nickel column affinity chromatography and the enzyme activity was tested to be 135.2 U/mg.Anti-rSjAK1 polyclonal antiserum was prepared by recombinant protein immunization in New Zealand male rabbits and the titer of the antiserum reached 1:25600 by ELISA.2.The immunohistochemistry result showed that SjAKl was mainly distributed in the tegument and partial parenchyma of schistosomula,in the vitellarium and tegument of the female worms and in partial parenchyma of male worms.The results of real time quantitative PCR showed the expression level of SjAK1 was highest in the egg and 21 days old liver-stage schistosomula,and was lowest in the 3 days old lung-stage schistosomula.3.The results of immune protection research showed that immunization with recombinant Sj AKl reduced worm burden by 50%,decreased the density of eggs deposited in the host liver by 40%,and reduced granulomas in the host liver by 56%.ELISA results showed that recombinant SjAK1 might elicit immune protection effect by inducing the expression of Thl cytokines such as IL-2 and IFN-γ.4.SjAK1 dsRNA was added into the culture medium of schistosomula to perform the interfere experiment.The optimal RNAi concentration of SjAK1 dsRNA was 2×10-5 mg/ml,and the optimal RNAi duration was 6 days.There was no significant difference in size between the schistosomula of SjAK1-knockdown group and the control group.Scanning electron microscope of schistosomula showed that the body surfaces of schistosomula from the SjAK1-knockdown group were with few tegumental spines.Transmission electron microscope of schistosomula indicated that the degeneration of sensory papilia and the mean tegument thickness was 1.25±0.25μm which decreased significantly compared to those in the control group.TUNEL results showed that the rate of apoptosis in SjAK1-knockdown schistosomula was significantly higher than that in the control group.5.SjAK1 dsRNA was added into the culture medium of paring adult worms.The optimal RNAi concentration of SjAK1 dsRNA was 4×10-5 mg/ml,and the optimal RNAi duration was 5 days.The egg number of females spawning of SjAK1-knockdown schistosoma was less than that of the control group,the reduction rate was 35%.Immature egg percentage accounted for 46%which was significantly higher than the control group.Scanning electron microscope of female worms showed that there was no significant difference on the body surfaces of schistosomula between the SjAK1-knockdown group and the control group.Transmission electron microscope of female worms indicated that the number of vitelline globules in the droplets was significantly less than that of control group and partial vitelline cells had expansion.There were more immature oocytes in the ovaries of SjAK1-knockdown worms.Partial oocytes showed swelling or degradation and there were less cortical granules around the oocytes.TUNEL results showed that the rate of apoptosis in the ovary,vitellarium,testis et al.tissue of SjAK1-knockdown schistosoma were significantly higher than that in the control group.Further real time qPCR was performed to test the Sj AK1 expression in vitellarium before and after oviposition.The result showed that SjAK1 expression in vitellarium reached a peak on 24 oviposition day,then reduced drastically and recovered on 28 day.The expression levels of SjAK1 in 35 d immature eggs and 42 d mature eggs were comparable,with no significant difference.Conslusion:Through the quantitative and localization,detection of immune protection effect and the RNAi experiments during the growth and development of schistosomula and the egg formation,SjAK1 was found that could elicit partial protective immunity against S.japonicum infection through the induction of Thl cytokines.The reduction rates of worm and egg in liver were 50%and 40%,respectively.SjAK1-knockdown influenced the tegument development of schistosomula and the development of yolk cells and occytes.The study reveals the important function of SjAK1 in the growth and development of S.japonicum and egg formation and provides scientific data reference for the growth and development mechanism of S.japonicum.