Gene Cloning, Expression And Its Immunobiological Characteristics Analysis Of Saposin-like Protein Of Schistosoma Japonicum

Schistosomiasis is a great serious public health problem. About 200 million people are infected,779 million people are at risk of infection. Every year,about 250 000 die of schistosomiasis worldwide. China is the most serious endemic area of schistosomiasis japonica. For more than 60 years of control effects, the great achievements have made, but there are still about 413 000 patients located in five Lake provinces (Jiangsu, Anhui, Jiangxi, Hubei and Hunan) and two mountain provinces (Sichuan, Yunnan). Praziquantel,the only effective drug for Schistosoma japonicum, can not control the rapid re-infection, Using praziquantel chemotherapy for long-term, large-scale and repetitious in the epidemic areas could cause the potential resistance to praziquantel. In recent years, a number of water conservancy projects, climate change, as well as natural disasters caused by environmental degradation, could bring more difficulties in control of schistosomiasis. Development of schistosome vaccines as a supplementary measure for control of schistosomiasis has become a consensus for researchers. In recent years, a large number of vaccines candidates of schistomiasis has been developed, such as 23 kDa membrane protein(Sm,Sj), triose- phosphate isomerase(TPI)(Sm,Sj), glutathine-s-transferase(GST) (Sm,Sj), IrV-5(Sm), fatty acid binding protein (Sm,Sj), 14-3-3 signal transduction protein (Sj),etc. could induce partial protective efficacy against schistosome challege, but their protective effects are not satisfactory, almost all below 40%. Seeking for the other potential candidates should be one of the important research approaches.Saposin-like protein is a large protein family, Their existence is conserved in phylogenetically most distant organisms from primitive protozoa to mammals and play the different biological roles. Such as saposin-like protein(SLP) of Fasciola hepatica, the mice were immunized with the recommbinant Fasciola SLP protein (rFhSAP-2), 81.2% of worm reduction rate and 73.0% and 83.8% egg reduction rate in the liver and fecal respectively were induced. It indicated that rFhSAP-2 could be as a vaccine candidate for Fasciola hepatica infection. Saposin-like protein of Clonorchis sinensis (clonorin) used for immunodiagnosis,34% of sensitivity and 99% of specificity could be obtained. Saposin-like protein of Schistosoma mansoni could be recognized not only by infected mice sera, also by infected human sera. However, it has not reported on saposin-like protein of Schistosoma japonicum. In this study,the Saposin-like protein (Sj-SLP) of Schistosoma japonicum was screened from schistosome GenBank by using bioinformatics methods, then cloned and expressed.The immunogenicity and antigenicity of the expressed product(rSj-SLP) were analyzed. And also the immunoprotective efficacy of the product was evaluated.Part 1 Gene cloning and expression of saposin-like protein of Schistosoma japonicumAccording to the sequence of saposin-like protein(SLP) of Clonorchis sinensis, the homologous EST fragment of Schistosoma japonicum (S.j) was found from the schistosome Genbank using the tblastn technique, and then the gene sequences of Sj-SLP were obtained with DNAStar and SignalP 3.0. analysis. The gene of Sj-SLP was amplified with PCR from cDNA of S.japonicum. The gene was cloned into plasmid pET15b and expressed in E.coli BL21. The expressed product was purified,and the purified rSj-SLP was obtained.Part 2 Analysis of immunogenicity and antigenicity for recombinant Sj-SLPTwo rabbits were immunized with the purified rSj-SLP, and after 3 times immunization, the anti-rSj-SLP antibody in immunized rabbits'sera was tested with ELISA assay. The results showed that the antibody titer to rSj-SLP was high, at least 1:12800. The rSj-SLP could be recognized by infected rabbit sera, but not by normal rabbit sera with western blot analysis. The hemolytic activity of rSj-SLP was tested with healthy people red blood cells. The hemolysis was occurred(5%) when rSj-SLP(500μg/ml) was added into the red blood cells suspension after 6h and increased graduately with time continued, at 62h after adding rSj-SLP the hemolysis rate was up to 50%.Part 3 Evaluation of the immunoprotective efficacy of rSj-SLP of Schistosoma japonicum against cercariae challenge in miceSixty BALB / c female mice were randomly divided into 5 groups, group A (natural infection), group B (Freund's adjuvant control group), group C (ISA206 control group), group D (Freund's adjuvant + Sj-SLP protein), group E (ISA206 + Sj-SLP protein). Each mice were immunized by subcutaneous injection with multipoint on back of mouse with the corresponding purified reagent respectively, for three times at week 0, 2 and 4 with the same dosage and the same method. Two weeks after the 3 rd immunization, all mice were challlenged with (40±1) cercariae of a Schistosoma japonicum by abdominal skin penetraton. Forty-two days post-challenge, the mice were sacrificed and perfused, and the numbers of recovered worm and hepatic eggs were counted. The blood was collected from the tail veins of all mice 2 days before immunization and challenge, respectively. Serum was prepared for detection of IgG, IgG1 and IgG2a. Two days before challenge, the spleen cell of 3 mice from each group were cultured and stimulated with Con A and rSj-SLP, the supernatant was collected after 72h culture and used for detection of IL-4 and IFN-γwith ELISA assay. The results show that the worm reduction rate in group D and E were 29.6% and 25.9%, and the egg reduction rate were 27.8% and 28.5% compared with control group. The special antibodies, IgG, IgG1 and IgG2a to rSj-SLP in experiment group (group D and E) were increased. But the cytokine ( IL-4 and IFN-γ)did not show any increase. The results indicated that rSj-SLP might be a potential vaccine candidate of Schistosoma japonum, it could induce high humoral immunity response.