【作者】 马广源；

【导师】 殷国荣； 张建中；

【作者基本信息】 山西医科大学 ， 病原生物学， 2008， 硕士

【摘要】 目的刚地弓形虫是人和动物感染率极高的一种机会致病原虫,其中速殖子阶段是主要的致病阶段。速殖子的大规模全蛋白质组学分析仍然还在进行,但目前尚无弓形虫的完全蛋白质组图。所以本研究应用蛋白质组学技术分析刚地弓形虫可溶性速殖子抗原(soluble tachyzoite antigen,STAg)组分,寻找具有免疫原性的抗原,为弓形虫疫苗有效候选抗原研究提供理论和实验依据。方法常规复苏弓形虫速殖子,腹腔接种BALB/c小鼠,转种3次恢复其活力,收集腹腔冲洗液,纯化,离心,上清液即为STAg,用三氯醋酸丙酮沉淀法制备2-DE样品。STAg样品,通过等电聚焦和SDS-PAGE电泳技术分离获得弓形虫STAg组分全蛋白点,用兔抗弓形虫血清和兔纯血清(阴性对照)做免疫印迹获得免疫原性的蛋白点。切取这些点、进行胶内酶解,ABI4700质谱仪对蛋白点进行鉴定分析。结果2-DE凝胶图显示1227个蛋白斑点,经质谱对其鉴定,得到230个弓形虫蛋白点,Western blot检测显示,16个蛋白点具有免疫原性。这些蛋白点中,主要表面抗原1(SAG1, 4个斑点)曾有报道具有免疫原性,热休克蛋白70(Hsp70)的免疫原性尚存在争议,其余的这些蛋白点:假定的蛋白质二硫键异构酶(putative protein disulfide isomerase),冠蛋白(coronin),热休克蛋白60(Hsp60),丙酮酸激酶(pyruvate kinase)2个点斑点,谷氨酸脱氢酶(glutamate dehydrogenase, putativ),,蛋白激酶C受体1(receptor for activated C kinase 1),苹果酸脱氢酶(malate dehydrogenase),尿苷磷酸化酶(uridine phosphorylase)2个斑点和过氧化氧化还原蛋白(peroxiredoxin)都具有免疫原性均为本研究新发现。结论对所得到的16个阳性蛋白点进行分析,除SAG1(P30)和Hsp70外,其余9种具有免疫原性的抗原均为本研究新发现。这些免疫原性的蛋白点均非表面抗原或排泄-分泌抗原,而是存在于速殖子内。此结果提示,弓形虫速殖子内存在一些尚未证实的免疫原性抗原,可能具有更强的保护性,有深入研究的必要性,可作为弓形虫免疫诊断和疫苗研发新的候选分子。更多还原

【Abstract】 Objective The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. There is presently no complete proteome map of T. gondii, although a number of large-scale whole proteomic analyses of tachyzoites are currently underway. So we have applied proteomic technique to analyze T. gondii soluble tachyzoite antigen (STAg) components, and to search antigen of immunogenicity and provide theoretical and experimental basis for effectively vaccine candidate antigen in T.gondii.Methods Conventional recovery T. gondii tachyzoites, intraperitoneal immunization of BALB/c mice, three species to restore its vitality, peritoneal fluid collection, purification, centrifugation, which supernatant is STAg. Then prepared 2-DE sample using trichloroacetic acid and acetone precipitation method. Samples STAg of preparated was separated by isoelectric focusing electrophoresis and SDS-PAGE electrophoretic technique, and obtained T.gondii whole protein spots, we obtained 21 interesting spots through rabbit- T.gondii serum and pure rabbit serum (negative control) reaction with corresponding antigen by Western blot. Then cut the spots, enzyme, ABI4700 mass spectrometry identification protein points.Results 2-DE gel shows 1227 protein spots, which identified by mass spectrometry, T. gondii proteins have 230 points, showed that 16 protein spots with immunogenicity by Western blot analysis. Among these proteins, a major surface antigen (SAG1, four spots) has been reported with immunogenicity, Heat shock protein 70(Hsp70) has controversy in the immunogenicity, and the rest of these proteins which on newly discovered have all immunogenicity. They are putative protein disulfide isomerase,coronin,Heat shock protein(Hsp60),two spots pyruvate kinase,glutamate dehydrogenase, putativ,receptor for activated C kinase 1,malate dehydrogenase,two spots uridine phosphorylase and peroxiredoxin.Conclusion 16 positive protein points has been analyzed, in addition to SAG1 (P30) and Hsp70, this study of the remaining nine kinds immunogenicity antigen were newly discovered. These protein points of immunogenicity are not surface antigen or excretion - secretory antigen, but they exist tachyzoites within. The results suggest that T.gondii tachyzoites has not been confirmed in a number of the antigen of immunogenicity, may have a stronger protection, and the need for depth study. As T.gondii immunodiagnosis and development of a new vaccine candidate molecule.更多还原