| Trichinellosis remains uncontrolled because of the complicated route of transmission,a wide geographic distribution of Trichinella spp.Strengthened diagnostic capacity and blocking Trichinella transmission through immunization of livestock with an effective vaccine is a practical strategy to prevent human and domestic animals trichinellosis.Cystatins in parasitic nematodes not only have the unique inhibition activity on cysteine proteases but also modulate the host immune response,and have an important role in the immune evasion from host response and the adaptation to parasitism.TsStefin,TsCystatin1 and TsCystatin2,members of cysteine protease inhibitors of Trichinella spiralis,were successfully cloned and expressed in the prokaryotic expression system.The purified recombinant proteins showed protease inhibitory activity under a wide pH and temperature.TsCystatins were expressed at detectable level in all the stages of T.spiralis,including muscle larvae,newborn larvae and adult.In addition,immunohistochemical staining and indirect immunoinfluscent assay showed that TsCystatis mainly localized in the cuticle and stichosome.We investigated the effects of rTsCystatins on the IFN-?-induced activated RAW264.7 in vitro.The results showed that rTsCystatins inhibit IFN-?-induced NO production in a dose-dependent manner.Detection of cytokine levels in cell culture supernatants showed that rTsCystatins inhibited the secretion of pro-inflammatory cytokines(IL-6,IL-12 and TNF-?)in IFN-?-induced RAW264.7,while rTsCystatins promoted the secretion of antiinflammatory cytokines(IL-10)in RAW264.7.These results suggest that TsCystatins are immunomodulatory molecules in the course of Trichinella spiralis infection,and evade the hosts' immune response by inhibiting the inflammatory response of the host macrophages,so as to achieve the goal of long-term parasitization in the host.The results of experimental infection demonstrated that TsCystatins can stimulate the immunized mice to produce high levels of specific antibodies.However,recombinant proteins cannot effectively reduce the amount of larvae in the muscle of immunized mice.In order to ascertain the immunological protection of TsCystatins against T.spiralis infection,TsCystatin2 was successfully engineered into the avirulent and no cyst-forming RH?GRA17 strain and integration of the DNA sequence into the parasite genome do not alter the fitness and immunogenicity of the transgenic parasites.The mice immunized with RH?GRA17::TsCystatin2 produce high levels of toxoplasma specific IgG.Splenocytes stimulated with Toxoplasma antigen lysis produced high levels of IL-12 and IFN-?.Celiac infected with virulent Toxoplasma found that the mice immunized with RH?GRA17::TsCystatin2 can resist Toxoplasma infection.Mice immunized with a single dose of transgenic parasites expressing T.spiralis protein developed a strong anti-Trichinella humoral immune response and Th1-dominant cellular immune responses characterized by increased level of IgG antibody titers(the predominance of IgG2 a production),and higher levels of Th1-type cytokines(IFN-? and IL-12).However,the Th2-type cytokines(IL-4 and IL-10)were not significantly increased in the immunized mice,and this Th1-dominant immune response does not confer immunized mice for a subsequent T.spiralis infection.That is,RH?GRA17::TsCystatin2 cannot effectively reduce the amount of larvae in the muscle of immunized mice.These results suggest that TsCystatins may not be an ideal vaccine candidate.In the present study,the combining recombinase polymerase amplification and a lateral flow strip assay(LF-RPA),targets Trichinella spp.mitochondrial small-subunit ribosomal RNA(rrnS)gene,was developed to detect Trichinella spp.infection.The LF-RPA assay can detect as low as 100 fg DNA of Trichinella strains and be performed within 10-25 min with a wide range of temperatures(25-45?)with the characteristics of simpleness,rapidness,accuracy,specificity and sensibility. |
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