Study On The Diagnostic Antigens For Evaluating The Therapeutic Efficacy Of Schistososomiasis Japonica

Schistosomiasis is a zoonotic parasitic disease,threatening the health of the nearly700million people around the world. China is the main endemic areas for schistosomiasisjaponica， which has been controlled effectively post50years of comprehensiveprevention and control work. The data of epidemiological survey in2010showed thatthere were still seven provinces,453counties in the transmission of schistosomiasis,there were325,000of schistosomiasis and370,000hectares of land with snails,schistosomiasis is still an important public health issues harm human healthy in China.Endemic areas in China are mainly distributed in lakes and marshes area of Jiangsu,Anhui, Jiangxi, Hubei, Hunan Province, and mountain hills area of Sichuan, YunnanProvince. The water level changes of water network area along the Yangtze river israpid，or the terrain in mountainous areas is complex in these schistosomiasis-endemicareas. The features of schistosomiasis prevailing in these areas are low infection degreeof susceptible population with atypical clinical symptoms; snail distribution is complexand it is difficult to be eliminated; there are a wide variety of schistosome reservoirhosts in nature world. It would be very difficult through the traditional control measuresto achieve complete control of schistosomiasis in these areas. If the prevention andcontrol work is slightly less vigilant, the amount of snails and schistosomiasis would beincreased quickly, the large-scale outbreak of schistosomiasis is likely to stage acomeback.Control of the infectious agents has become the focus of schistosomiasis prevention andcontrol work, and the control strategies of schistosomiasis in China has shifted to theeradication of infectious agent as center of integrated control measures. So, it would bethe key to achieve the target of eliminating infectious agent of schistosomiasis bydeveloping an effective diagnostic method to find the infectious agent ofschistosomiasis， subsequently to treat it effectively, which is also still the focus ofresearching work of preventing schistosomiasis.Examination eggs, hatching miracidia in stool, and intestinal biopsy are the classicmethods of pathogenic diagnosis of schistosomiasis, but these methods are relativelytime-consuming, laborious. The decline of infection degree of schistosomiasis and mucosa thickening of chronic patient’s intestinal wall made the amount of egg enteringintestine reduction, the sensitivity of these methods become lower and lower, which cannot meet the needs of prevention and control work in the field.Schistosome parasitizes in the human blood circulation system,and its proteins areexcreted or secreted into the host blood circulation system during the process ofdevelopment and metabolism, which switches the immune responses of immune systemof host, the specific antibody response against schistosome antigen is one of mainmanifestations of immune responses. The schistosome proteins or antibodies againstschistosome antigen specifically, especially, the antibodies involved in short livedantibody response, presented in the blood circulation system of schistosomiasis, is theobjective indications of existing schistosome infection. Detection of these specificantigens, antibodies or its dynamics would be valuable for diagnosis and evaluating thechemotherapeutic effect of schistosomiasis. The development of sensitive and specificmethods detecting schistosome circulating antigen or antibodies involved in short-livedantibody response to meet the needs of schistosomiasis in routine clinical diagnosis andcontrol work in fields has become the hot topic of current schistosomiasisimmunological diagnosis research.To develop a immunological diagnostic method with the value of diagnostic andtherapeutic evaluation of schistosomiasis by detecting antibodies involved in the ofshort-lived antibody response or circulating antigen, in this study, the large hydrophilicdomain of Schistosoma japonicum23kDa membrane protein(Sj23HD) was expressed inSaccharomyces cerevisiae in exocrine expression, and the purified recombinantSj23HD-HSA fusion protein was prepared. The type of antibody response induced bySj23HD and its value of schistosomiasis diagnosis were investigated. At the same time,the epitopes of high abundance excreted and secretary proteins of Schistosomajaponicum were predicted by bioinformatics method and verified by peptide microarray.An artificial protein composed of multiple epitope peptides was designed and expressed,and a batch of specific monoclonal antibodies against these epitopes were prepared inthis study, which laid a good foundation for the further development of efficientdetection method of antibody involved in short-lived antibody response, or circulatingantigen. Part I: Construction of yeast expression vector for expressing the Sj23HD-HSAfusion protein composed of the large hydrophilic domain of Schistosomajaponicum23kDa membrane protein and human serum albumin,preparation and immunologic characterization of Sj23HD-HSA fusionproteinI) Construction of yeast expression vector for expressing the Sj23HD-HSA fusionprotein composed of the large hydrophilic domain of Schistosoma japonicum23kDa membrane protein and human serum albuminThe gene encoding the Sj23HD-HSA fusion protein, composed of Sj23HD gene andgene of the matured peptide of HSA, was prepared by overlapping PCR, and clonedinto the intermediated plasmid pGEMT-MCS directionally to construct the recombinantplasmid Sj23HD-HSA/pGEMT-MCS. The DNA sequencing showed that the fusionprotein gene was inserted into the yeast expression cassette aright, and fused in framewith the signal peptide of HSA. The expression cassette DNA fragment containing theSj23HD-HSA gene was prepared by digesting the recombinant plasmidSj23HD-HSA/pGEMT-MCS DNA with Not1restrictive enzyme, and connected withthe linear yeast expression plasmid pWX530digested by Not1to form a recombinantyeast expression plasmid Sj23HD-HSA/pWX530successfully.II) Expression in Saccharomyces cerevisiae, purification and immunologiccharacterization of Sj23HD-HSA fusion proteinA large number of recombinant plasmid Sj23HD-HSA/pWX530DNA were prepared,and transformed into host cell of Saccharomyces cerevisiae by electric pulse. Thetransformed products were seeded on the Leu deficient SD agar plates to screen theautotrophic transformants containing Sj23HD-HSA/pWX530plasmid. Theseautotrophic transformants were inoculated into YEPD medium, grew in shaker of30℃for one week,and the supernatant of expression products presented a protein moleculeabout73kDa by SDS-PAGE, similar to the prediction value Sj23HD-HSA fusionprotein. The pure Sj23HD-HSA fusion protein was prepared by ion exchangechromatography of SP Sepharose XL sepharose from the supernatant of expressionproducts. This fusion protein could be recognized by sera of schistosome infectedhuman, mouse and rabbit, but can not be recognized by sera of healthy, mouse and rabbit in immunoblotting analysis, which indicated that the recombinant Sj23HD-HSAfusion protein has a natural immunogenicity, and a better specificity for diagnosis.III) The immunodiagnostic value of Sj23HD-HSA fusion proteinThe mice and rabbits were infected with schistosome, and the sera before infection,during infection at different time, and post chemotherapy at different time werecollected. The antibody IgG against recombinant Sj23HD-HSA fusion protein weredetected by ELISA, the antibody level rose continually during the first two weeks postchemotherapy, then decreased gradually. The antibody level of some mice, rabbitsreached the negative threshold in sixteen weeks post chemotherapy， the antibodyagainst SEA decreased slowly post chemotherapy also. No evident decrease of antibodyagainst Sj23HD-HSA fusion protein or SEA was observed in untreated mice, or rabbits.These results indicated that the antibody response induced by Sj23HD is a shorted livedantibody response, detecting this antibody involved the shorted lived antibody responsewith a potential for diagnosis and evaluating the chemotherapy efficacy ofschistosomiasis.The sera antibody was detected by ELISA with recombinant Sj23HD-HSA fusionprotein, the positive rate of132cases of schistosomiasis was eighty nineteen percent,the cross reaction rate of30cases of clonorchiasis was naught percent, the crossreaction rate of10cases of paragonimiasis was fifty percent, and the false positive rateof80cases of healthy was naught percent. Meanwhile, The sera antibody was detectedby ELISA with SEA, the positive rate of antibody of132cases of schistosomiasis wasninety seventeen point seven three percent, the cross reaction rate of30cases ofclonorchiasis was sixteen percent, the cross reaction rate of10cases of paragonimiasiswas ninety percent, and the false positive rate of80cases of healthy was naught percent.Compare to SEA, the recombinant Sj23HD-HSA fusion protein has a better specificityfor schistosomiasis diagnosis, but it is noticed for differential diagnosis of the patientscoming from paragonimiasis endemic areas.Part II Identification of epitopes of high abundance excreted and secretaryproteins of Schistosoma japonicum, design and expression of multipleepitope protein, and monoclonal antibody preparationI) Identification of epitopes of high abundance excreted and secretary protein of Schistosoma japonicumThe epitopes of high abundance excreted and secretary proteins of Schistosomajaponicum were predicted by bioinformatics, twelve epitopes with high score and lowidentity were selected following the prediction score of bioinformatics prediction andits identity to homologue proteins of human，and the epitopes extended to twenty byamino acid overlapping design. The twenty epitopes were synthesized vertically onprotein chip matrix to form peptide microarray, reacting with sera of schistosomiasis,clonorchiasis and healthy respectively, and detected by immunochemi-luminometricassays. Seven epitopes were selected, which were high response to sera ofschistosomiasis, but negative or lowest response to sera of clonorchiasis or healthy.These epitope peptide are overlapping peptide belonging to Sj Enolase、Sj GAPDH�'�Sj GST28protein respectively, finally, three epitopes were selected. The results ofprotein conformation homologous modeling showed that these three epitopes are on thesurface of spatial structure of the original protein respectively.II) Gene design of multiple epitope protein, vector construction of single epitope,multiple epitope protein and fusion protein preparationA gene encoding a protein composed of three selected epitopes and a epitope derivedfrom Schistosoma japonicum23kDa membrane protein hydrophilic domain (Sj23HD)was designed, and each epitope was linked with a flexible spacer of four glycines toform a artificial Schistosoma japonicum multiple epitope protein （Sj MEP）.The geneDNA fragments encoding the Sj MEP and single epitope were synthesized. Therecombinant expression plasmid Sj MEP/pGEX-5X-1was constructed by inserting theSj MEP DNA fragment into expression plasmid directionally, making the the Sj MEPDNA fragment fused with GST gene of plasmid pGEX-5X-1in frame.The recombinantexpression plasmid was transformed into Ecoli.BL21to screen transformants containingplasmid Sj MEP/pGEX-5X-1on LB plate with ampicillin. The transformants wereinduced with IPTG to express the fusion protein GST-Sj MEP. The soluble GST-Sj MEPfusion protein was purified by affinity chromatography of glutathione sepharose4B.The fusion protein reacted strongly with sera of schistosomiasis in ELISA, indicatingnatural immunogenicity of schistosome.The expression plasmids expressing fusion protein of Trx-Sj Enolase epitope, Trx-Sj GAPDH epitope and Trx-Sj GST28epitope respectively were constructed by insertingsingle epitope gene DNA fragment into the expression plasmid pET32c+directionally,making the single epitope gene fused with the Trx gene of plasmid pET32c+in frame.The recombinant expression plasmids were transformed into Ecoli.BL21, and thepurified single epitope fusion protein was prepared by Ni+ion affinity chromatography.III) Preparation and preliminary identification of monoclonal antibody againstepitopes of high abundance excreted and secretary protein of SchistosomajaponicumThe BALB/c mice were immunized with GST-Sj MEP fusion protein. The titer of seraantibody against GST-Sj MEP was higher than1:200,000post three boostimmunizations. The single spleen cells of immunized mouse were prepared and fusedwith myeloma cells SP2/0to form hybridoma. The hybridoma secreting antibodyagainst GST-Sj MEP was screened by ELISA with recombinant GST-Sj MEP.205wellsof hybridoma were obtained, these hybridoma were subcloned by a Limited dilutionmethod, and screened with single epitope fusion protein of Trx-Sj Enolase epitope,Trx-Sj GAPDH epitope and Trx-Sj GST28epitope further, finally,8clones secretedspecific antibody against Sj Enolase,3clones secreted specific antibody against SjGAPDH,1clone secreted specific antibody against Sj GST28.The value of thesemonoclonal antibodies for schistosomiasis diagnosis by constructing a circular antigensdetection method remains investigation further.ConclusionThe exocrine expression system of Saccharomyces cerevisiae for expressing the23kDamembrane protein hydrophilic domain (Sj23HD) was constructed successfully, and thepure recombinant Sj23HD-HSA fusion protein with natural structure and immunogenicactivity was prepared in this study. The experimental results suggested preliminarilythat the Sj23protein induces a short lived antibody response in mice and rabbit. Itwould be potent for diagnosis and evaluating the chemotherapeutic efficacy ofschistosomiasis by detecting the antibody involved with short lived antibody responseagainst Sj23protein.Three antigen epitopes derived from high abundance excreted andsecretary protein of Schistosoma japonicum have been identified, and a batch ofmonoclonal antibodies against these epitopes were also obtained in this study. This research laid a good foundation for further development of an efficient method forassessing chemotherapeutic efficacy of schistosomiasis.