Study On The Immune Protection,Growth And Development Of TAP Gene Of The Schistosoma Japonicum

Objective:Schistosoma japonicum tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein(SjTAP)is known to be a potential immune protective gene with anti schistosomiasis infection;At the same time,SjTAP is the up-regulated gene in mother sporocyst of S.japonicum which were co-cultured with protein extracts of Oncomelania hupensis head-foot tissue(PHF),it may also be the key gene that influences the growth and development of mother sporocyst.Accordingly,adult and mother sporocyst of S.japonicum was targeted to research the expression and the histological distribution of TAP protein,and the function of TAP protein,inculdingimmune protective effect of TAP protein against S.japonicum infection,and the effect and mechanism of TAP protein on growth and development of mother sporocyst of S.japonicum in co-culture condition,was further studied.Methods:The recombinant PET28a(+)-SjTAP plasmid was constructed by molecular biology method,and the recombinant TAP protein was expressed and purified to immunize mice to obtain TAP immune serum..Immunofluorescence histochemical staining was used to detect the distribution and localization of TAP in adult and mother sporocyst of S.japonicum.The mice,regularly immunized for 6 weeks with recombinant TAP protein,was attacked with schistosoma cercariae.42 days later,the number of worms in each mouse and the egg load per gram of liver were counted to calculate the worm reduction rate and egg reduction rate which was used to evaluated the immune protective effect of SjTAP protein.The expression level of TAP mRNA in mother sporocyst that were co-cultured with 1%PHF for 1,3,4 and 8 days were detected with real-time PCR.;SjTAP specific siRNA was designed to feed mother sporocyst to silent TAP gene expression,then the expression level of TAP mRNA,the growth and development indexes such as the body length,body width,body volume and survival rates of mother sporocyst were observed and measured.After that,mother sporocysts co-cultured with PHF were treated with TAP siRNA again,TAP gene and protein expression level,the growth and development indexes and survival rate of each group of mother sporocyst were observed and measured at the same time.Results:PET28a(+)-SjTAP recombinant plasmid containing 774 bp SjTAP was successfully constructed,and 35 kDa TAP recombinant expression protein was obtained.Immunofluorescence staining showed that the TAP protein specific red fluorescent particles were widely distributed in the cortex,subcutaneous layer,muscular layer and parenchyma of male and female adults In most tissue,TAP fluorescent granules were diffuse distribution with partly fluorescent enhancement,however,in cortex and around ovary of the female,in cortex and subcutaneous layer of the male,TAP were clustered and linearly distributed.The immunoprotective results of the recombinant TAP protein showed that the rate of worm reduction in the immune group was 39.9%,the egg reduction rate was 71.8%,the rate of worm reduction in the adjuvant control group was 10.8%,the rate of egg reduction was 59.7%,and the difference between the two groups was significant(p<0.05),indicating that the recombinant protein of TAP had some protective immunity against infection of S.japonicum.The results of real-time PCR showed that compared with the control group,the expression level of the TAP mRNA of mother sporocyst co-cultured with PHF increased significantly 6.64,2.64 and 6.27 times(p<0.01),respectively on the 3rd,4th,and 8th days.Immunofluorescence histochemical staining showed that TAP protein was mainly expressed in the body wall and cytoplasm of mother sporocyst.The results of RNAi interference test showed that 30nM SjTAP siRNA could significantly reduce 93.45%TAP mRNA expression level in mother sporocyst.On the 6th day after RNAi interference,the body length,body width,volume and survival rate of the mother sporocyst in SjTAP siRNA group(siTAP)were 87.23±6.29μm,47.52±3.15μm,128276.36±26920.07μm3 and 89.2%respectively,compared with the mother sporocyst in siRNA control group(sictrl)which body length was112.45±11.6pm,body width was 46.74±3.13μm,volume was 16900.73±40326.01μm3 and survival rate was 98%,in group siTAP,the size of the mother sporocyst was obviously shortened,the volume and the survival rate were decreased(p<0.05).After adding PHF,mother sporocysts were divided into four test groups:sictrl,siRNA control group+1%PHF group(sictrl+1%PHF),siTAP group and SjTAP siRNA+1%PHF group(siTAP+1%PHF);on the 6th day after siRNA interference,expression level of TAP mRNA of mother sporocyst in sictrl+l%PHF group was significantly higher than that in sictrl group(p<0.05),and the TAP mRNA expression level of mother sporocyst in group siTAP+1%PHF was significantly lower than that in group sictrl+1%PHF(p<0.05)after TAP siRNA was silent on TAP gene.The immunofluorescent histochemical staining showed that the fluorescent expression intensity of the TAP in mother sporocyst of four groups were in accordance with the TAP mRNA expression level in four groups.Under the light microscope,on the 6th day after RNAi interference,the body length,width,volume and survival rate of mother sporocyst in siTAP group compared with those of sictrl group decreased significantly(P<0.05).sictrl+1%PHF group compared with sictrl group,sictrl+l%PHF group compared with siTAP+1%PHF group,siTAP+1%PHF group compared with siTAP group,the body length,width,volume and survival rate of mother sporocyt in the former groups were all significantly higher than those in the latter groups(P<0.05).Conclusion:PET28a(+)-SjTAP recombinant plasmid containing 774 bp SjTAP was successfully constructed,and 35 kDa TAP recombinant protein was obtained.TAP protein is mainly expressed in the cortex,subcutaneous layer,myometrium and parenchyma of male and female adults;in most tissues,TAP was diffuse distribution,but in the cortex,it was clustered and linearly distributed.The recombinant TAP protein has certain immune protective effect on against schistosoma infection.In mother sporocyst,TAP protein is mainly expressed in body wall and cytoplasm,and the expression of TAP in body wall increases with the prolongation of the culture time.The TAP gene promotes the growth and survival of mother sporocyst.In the co-culture system,through up-regulated TAP gene expression level,PHF could further promote the growth and viability of mother sporocyst.