The Preliminary Study On The Biological Characteristics Of N-acetyltransferase 13 In Schistosoma Japonicum

N-acetyltransferase 13(NAT13)protein is also called N-alpha-acetyltransferase 50,which is a Nterminal acetyltransferase.It's important for chromosome resolution and segregation in the process of mitosis.In this study,we initially characterized and explored the biological function of Schistosoma japonicum NAT13(SjNAT13).The primers were designed according to the cDNA sequence of Schistosoma japonicum NAT13 gene(SjNAT13,FN317573.1)in GenBank.The SjNAT13 gene was successfully cloned and expressed in Escherichia coli BL21 cells.The length of SjNAT13 gene was 621 bp,and encoded 206 amino acids.The molecular weight of rSjNAT13 was 27 kDa.The transcription of SjNAT13 in S.japonicum at different development stages,and in the male and female worms was detected using Real-time PCR(qRT-PCR).The results revealed that SjNAT13 can be expressed at all tested developmental stages.The transcription level was the highest in 21 d worms and the lowest in 14 d worms.The transcription level was no significant difference in male and female on 21 d,but it was significantly higher in males at 24 d,28d,35 d and 42 d than that in females.The rSjNAT13 and soluble 42 d worm protein(SWAP)were analyzed by Western blotting analysis.The anti-rSjNAT13 mouse serum was used as the primary antibody,and the horseradish peroxidase(HRP)-conjugated goat anti-mouse IgG was used as the secondary antibody.The results revealed that rSjNAT13 and SWAP could be recognized by the anti-rSjNAT13 sera.In two independent immunoprotection trials,the group of BALB/c mice vaccinated with rSjNAT13 produced 12.30 %(P>0.05))and 16.36%(P>0.05)reduction in worm burden compared with PBS control,respectively.rSjNAT13 was also induced 24.23 %(P<0.05)and 24.47%(P<0.05)reduction in hepatic egg of per pair worms compared with the blank control group.The rSjNAT13 could not induce a significant worm reduction effect,however,the reduction in eggs of per pair worms was significant.Indirect enzyme-linked immunosorbent assay(ELISA)was used to detect the changes of specific IgG antibody levels in the three groups before and after immunization.The results showed that the specific IgG antibody level was significantly increased in the rSjNAT13 vaccinated group after the second immunization.The highest level was at the time of after the third immunization.There was no significant change in the 206 adjuvant group and the PBS group before and after infection and during the killing.The 28 d worm was subjected to immunohistochemistry to observe the distribution of the protein.The results showed that it was widely distributed all over the worm.In vitro RNA interference(RNAi)experiments were carried out on 42 d adults by electroporation.After qPCR detection and screening,siRNA-514 produce a good interference effect.Then,siRNA-514 was chosen for further RNAi to 42 d adults in vitro,and egg reduction rate were calculated after RNAi.The results showed that the siRNA-514 group produced 38.91%(P < 0.05)egg reduction rate compared with the blank control group.In vivo RNA interference experiments siRNA-514 were injected into the tail vein of BALB/c mice 18 days after infection.The results revealed that the siRNA-514 group produced 10.91%(P > 0.05)reduction in worm burden and 37.66%(P < 0.05)reduction in hepatic egg of per pair worms compared with the blank control group.In this study,SjNAT13 gene was successfully cloned and expressed,and the effect of rSjNAT13 immunoprotection experiment was evaluated.The gene transcription level was detected at different developmental stages and in different genders of schistosomes.The RNAi of SjNAT13 were operated in vitro and in vivo,and the effect were evaluated.This paper provides a basis for better understanding the characterization of SjNAT13,and exploring the biological functions of SjNAT13,especial in the influence for spawning of schistosomes.