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Study on the Immuno-Target of Specific Single Chain Antibody Against SIEA26-28 Ku and Its Efficacy as Therapeutic Vaccine Against Schistosomiasis

Author: GaoDongMei
Tutor: WangShiPing
School: Central South University
Course: Pathogen Biology
Keywords: Schistosoma japonicum Affinity chromatography Targetted location Combined immunization Therapeutic vaccine
CLC: R392
Type: PhD thesis
Year: 2010
Downloads: 61
Quote: 2
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Abstract


BackgroundSchistosomiasis japonicum remains to be a serious zoonosis and seriously endanger people’s health and affect social and economic development of epidemic areas. Heretofore, though great achievments have obtained through the work of schistosomiasis prevention in the past 50 years, schistosomiasis japonicum remains a serious public health problem which is prevalent in Hunan, Hubei, Jiangxi, Anhui, Jiangsu 5 provinces around lake and 2 provinces at mountain areas in our country because its popular links and effect factors are complex. In the past several years, though the integrate control strategy that control source of infection primary which we adopted have shown a comparatively effect in the epidemic controlled areas, this strategy dose not relieve from the thought of Communication Ecology Theory. The practice shows that though the integrate control strategy designed by Communication Ecology Theory is effective, the control task is being challenged by the change of natural circumstance and decreased financial aid. The significant role of vaccines have been proved in the prevention and control of many infectious diseases. So it is one of the common goals of the scientists in both China and the world in recent 20-30 years to develop an effective anti-schistosomiasis vaccine in order to make up for short-term effects of conventional chemotherapy to achieve long-term control on schistosomiasis. It is the purpose of the WHO/TDR to coordinate the development of schistosomiasis vaccines.It is well known that mature egg of Schistosoma is the major causative agent to the development of schistosomiasis and is responsible for the transmission and prevalence of this disease. With years of efforts, our laboratory has proved that a strong anti-embryonation and anti-fecundity immunity in host can be induced after being immunized with soluble immature egg antigen (SIEA) of Schistosoma japonicum. Protective humoral immunity induced by SIEA is the main mechanism of the protective efficacy. SIEA immuno-serum can react with the vitellaria and the lining membrane tissue of the gut lumen of female worms as well as with the embryo of immature eggs, which inhibited the embryo development and the fecundity of female worms markedly. We also found in further studies that 26-28 ku components extracted from SIEA were the major antigens inducing the anti-schistosomiasis protective humoral immune response. During the former research, our team constructed SIEA single-chain Fv antibody library by phage display technic and obtained highly specific SIEA26-28 ku antibody. It provides the foundation for the therapeutic vaccine research for schistosomiasis.ObjectiveSIEA26-28 ku-scFv and EGFP-scFv were constructed and highly expressed. The expressed proteins were purified by chromatography, to research its antibody specificity and immuno-target efficacy. Purified SIEA26-28 ku-scFv were also used together with DNA bivalent vaccine and its correspondence protein vaccines which constructed by our team, to observe the protective efficacy and to investigate the immune response mechanism preliminary.Methods1. Highly express and purify SIEA26-28 ku-scFv and EGFP-scFv, and evaluate the antibody’s specificity.Large scale culture E.coli BL21 (DE3) which contained plasmid pET32a/SIEA26-28 ku-scFv or pET32a/EGFP-scFv was induced by IPTG. The target proteins which contained six histidine were purified by 2 steps. The purified protein was identified by SDS-PAGE, Western blot, and HPLC. Affinity constant of scFv was measured by Non-competitive enzyme immunoassay.2. Location of specific SIEA26-28 ku-scFvThe enzyme immunohistochemistry and indirect immuno-fluorescence were used to observe the location of specific antigen of female adult worm and eggs of Schistosoma japonicum by SIEA26-28 ku-scFv.3. Research the immunity efficacy of specific SIEA26-28 ku-scFv as therapeutic antibody, and the efficacy of combined immunization with DNA vaccine and its corresponding protein vaccines which we had constructive before.4. Research the effect of anti-schistosomiasis and immunity mechanism of specific SIEA26-28 ku-scFv as therapeutic antibody combined immuning procines. Results1. Recombinant plasmid pET32a/SIEA26-28 ku-scFv was transfected into E.coli BL21(DE3). The fusion protein was purified by Ni2+ column affinity chromatography and DEAE Anion-exchange chromatography. The purity of scFv was 98.2% by two steps purification. Western blot and ELISA showed that scFv and SIEA had high degree of specificity.2. The specific scFv could combine with immature eggs, mature eggs and reproductive system of female adult worm of S.japonicum. It was mainly located on egg embryo, vitellaria of female adult worm, ovary, eggs in uterus and intima of gut wall near reproductive system and had no cross reactions with normal hepar tissue of human and mice.3. Specific SIEA26-28 ku-scFv as therapeutic antibody’s immunity efficacy to Kunming mice is as follows. The group of scFv immunized Kunming mice with DNA vaccine and protein vaccines had a significant protective immunity. The serum titer was 1:6400, worm reduction rate was 61.4%, liver egg reduction was 51.2%, intestine egg reduction was 56.6%, and uterus egg reduction of per female worm was 54.2%. All value forms of this group were higher than other groups, while diameter and area of hepatic schistosomiasis granuloma were lower than other groups.4. CD4+ T cell counter and CD4+/CD8+ ratio were significantly higher in blood and spleens of procines which were immuned by scFv combined with DNA and protein vaccines. It also increased the expression of IFN-y, but had no obvious effect on IL-4. The group SIEA26-28 ku-scFv had a limited anti-schistosomiasis effect on procines and this effect was not as good as the group combined immunization.Conclusion1. SIEA26-28 ku-scFv engineering strain was successfully constructed. The purity of SIEA26-28 ku-scFv reached 98.2%.2. SIEA and SIEA26-28 ku-scFv have high degree of specificity. SIEA26-28ku-scFv mainly located on egg embryo, vitellaria of female adult worm, ovary, eggs in uterus and intima of gut wall near reproductive system.3. SIEA26-28 ku-scFv combined with DNA vaccine and protein vaccines to immunize Kunming mice and procines had a significant protective immunity. Its anti-schistosomiasis effect might be related to its specificity of immuno-target.4. CD4+T cell counter and CD4+/CD8+ratio were significantly higher in blood and spleens of procines. It could also increase the expression of IFN-y, but had little effect on IL-4. It mainly stimulated Th1 type immunity.5. SIEA26-28 ku-scFv has significant immuno-target effect. The effect of anti-schistosomiasis can be increased by combining immuno-target effect with vaccines. Meanwhile, AHFP drug combined immuning with vaccines has the similar effect.

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