Characteristics Of Transcription And Expression Of Trichinella Spiralis Stage-specific Gene

As a zoonotic parasitic disease, trichinellosis shows a worldwide distribution and has a high prevalence in China, could infect over 150 mammals besides human, not only lead to enormously economic loss of husbandry and meat industry but also severely threat to public health. Two main factors impede its prevention and treatment. First factor from immune rejection of host to bacteria and virus is lower, second factor from parasitic self-protection system in the host can help it escape immune surveillance. More and more studies evidence that the signal transduction between parasite and host is fatefully. Currently, the study on signal transduction between parasite and host will contribute to explore new therapy to cure parasitic disease, has become a focus in the field of parasite. In this study, the transcription and expression of Trichinella spiralis stage-specific genes were detected by RT-PCR and immunofluorescent staining. This will be on ground to research molecular machanism of nurse cell formation.T314 cDNA was reverse-transcribed from total RNA that isolated from Trichinella spiralis-infected muscle tissue. RT-PCR analysis showed that transcription of T314 gene appeared and maintained a considerable high level at the first day when the newborn larvae was born. With the increase of age, the level of T314 mRNA reduced. From 5 to 20d.p.i., the transcription of T314 gene maintained a low level.Rabbit anti-T314 recombination protein antibody from our lab was applied in immunofluorescent staining. Immunofluorescence analysis indicated that the T314 protein was only observed in epicuticular surface. From 7 to 20d.p.i. the protein was secreted and only observed in cytoplasm of nurse cell. It could not be observed in enlarged nuclei and invasion cell. It support a postulation that the T314 protein secreted into nurse cell cytoplasm could play an role in cutting mitochondrial DNA. T314 DNA was amplified and cloned into PCR II vector. The recombinant plasmid was transformed into TOP10F'. The recombinant plasmid was digested by spe I and Eco V. SP6 and T7 RNA pclymerase was used to transcribe in vitro. siRNA was purified by digestion, washing and column. siRNA was introducted into Trichinella spiralis to silence T314 gene by soaking in dsRNA solution. The result showed that T314 gene silencing lead to fall down in total number of Trichinella spiralis and nurse cell vacuolization.T668 cDNA was reverse-transcribed from total RNA. RT-PCR analysis showed that transcription of T668 gene appeared at the first day when the newborn larvae was born. With the increase of age, the level of T668 mRNA reduced. From 11 to 14 d.p.i., there was barely detectable transcription.T668 protein was assaied with rabbit anti-T668 recombination protein antibody by immunofluorescent staining. The result showed that T688 protein was expressed at 1d.p.i.. From 6 to 13d.p.i., T668 protein was secreted into enlarged nuclei. From 14 to 20d.p.i., T668 protein was hardly observed in nurse cell but strong expressed in larvae.