【作者】 陈欲晓；

【导师】 易新元； 曾宪芳；

【作者基本信息】 中南大学 ， 病原生物学， 2003， 博士

【摘要】 本研究着重于对日本血吸虫(Sj)雄虫源性抗原候选疫苗分子的筛选鉴定和保护性免疫的研究，以及细胞因子对核酸疫苗免疫的调节研究二大部分。 为获得Sj雄虫特异性抗原疫苗候选分子，开展了Sj雄虫天然组分抗原的分离鉴定及保护性免疫研究。采用SDS-聚丙稀酰胺凝胶电泳(SDS-PAGE)比较分析Sj雄性、Sj雌性的抗原组分，Western blot检测组分蛋白的抗原性和性别特异性，动物实验评估抗原的免疫保护效果。结果发现Sj雄虫可溶性抗原含一高丰度带，为44.6kDa，Sj雌虫可溶性抗原中未见类似带。通过SDS-PAG胶切法，获高纯度44.6kDa蛋白。Western blot检测44.6kDa蛋白具抗原性和雄虫特异性，并诱导小鼠产生39.31％的减虫率和41.98％的减卵率，与对照组比差异有显著性(p＜0.001)。有关其抗雄虫特异性免疫值得今后研究。 为寻找雄虫抗原表位模拟肽分子，从表位分子水平构建Sj新型疫苗，作者进行了Sj雄虫抗原表位模拟肽的筛选及其保护性免疫研究。用抗Sj雄虫抗原的IgG筛选噬菌体随机肽库，获得多个阳性噬菌体克隆，Dot-ELISA鉴定其具有抗原性。用混和阳性噬菌体克隆免疫小鼠获31.72％的减虫率和51.54％的减卵率，与对照组比差异有显著性(p＜0.001)，表现为抗Sj感染和抗生殖作用。 为发现雄虫特异性的基因及其编码的靶抗原分子，从基因水平揭示V性别特异性差异，采用U雄虫免疫血清筛选对成虫CDNA文库，对部分阳性克隆的插入片段进行 PCR鉴定及测序。结果获得 11个阳性克隆，通过互联网进行核着酸序列同源性分析，发现三个新基因，百 分M、Cps和＃ps（GenBan 注册号分别为AF519808、AF524896和 AF517843入 它们的预测编码蛋白可能为存在于胞浆和胞核内的信息传递分子，三个新基因是否为雄虫性别特异性值得进一步探讨。对SfMA进行了亚克隆及原核表达，并用表达产物免疫小鼠。结果获得SJ－MA原核表达的重组体，并在ECOli中高效表达。SDS－PAGE和Western blot显示融合蛋白为54．skDa具良好的抗原性。融合蛋白与FCA混合免疫小鼠可诱导高滴度的特异性格G抗体，免疫组的减虫率及减卵率分别为34．29％与48．76凡 与对照组比差异有显著性 （／0．001人表明新基因 SfMA表达蛋白能诱导小鼠抗 SJ的保护性免疫，提示其作为U疫苗候选分子的潜在价值。 为研究细胞因子DNA对核酸疫苗免疫的调节，作者分别构建了小鼠 IFN个和小鼠几对真核表达重组质粒，并首次分别联合 SJ组织蛋白酶B DNA疫苗免疫小鼠，通过免疫酶组织化学技术观察两种质粒DNA在小鼠肌肉细胞的表达，通过减虫率减卵率评估免疫保护性作用。结果两种细胞因子质粒和 DNA疫苗在小鼠肌肉细胞分别同时表达，其中 IFN一下白显著增强 DM疫苗的减虫率（27．37％，P＜0，05），而几一4能显著增强＊A疫苗的减虫率不减卵率（43．200和 76．63O，／0，01）。为研究 Th型和 ThZ型应答优势分别对 DNA疫苗免疫的调节奠定基石出。更多还原

【Abstract】 Our study has aimed at the identification of Sj male worm derived new vaccine candidates and the immunoregulation of cytokines on the nucleic acid vaccine.In order to investigate gender-specific antigen and its immunoprotection against Sj, profile of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was studied to compare the protein components among two antigen origins: male adult worm soluble antigen and female adult worm antigen. The antigenicity and gender specificity of the protein fraction were determined by Western blot, and the immunoprotection was assessed by the worm and egg reduction rates. Subsequently SDS-PAGE disclosed a male specific rich band at first time with molecular weight of 44.6kDa that could not be seen in female adult worm soluble antigen. This band was excised from SDS-PAGE and purified followed by mouse immunization. We found that 44.6kDa antigen recognized with immunized sera in Western blot was male worm specific. And the test group vaccinated with 44.6kDa antigenplus FCA revealed significant worm and egg reduction rates as 39.31 % and 41.98 % respectively (p<0. 001) , compared with control groups. Further investigation on its immunoprotection against Sj male worm is of importance.To select epitope mimics of Sj male adult worm soluble antigen and explore the immunoprotection against Sj in mice, phage random peptide library was screened with purified IgG from anti-5/ male worm antigen sera. Positive phage clones collected through three rounds of biopanning were detected by Dot-ELISA. As a result the specific phages binding to IgG were enriched after three rounds of biopanning. Positive clones detected by Dot-ELISA were reacted with the specific IgG. Sera from mice immunized with mixed positive phage clones gave a rise to a specific antibody and the immunoprotection against Sj was induced by immunization of mixed positive phage clones with worm reduction rate of 31.72% and egg reduction rate of 51.54%, indicating the anti-reproduction action to Sj. Therefore the antigenic epitope mimics of Sj male adult worm antigens are obtained by phage peptide-display library and reveal the immunoprotection against Sj, providing new foundation on the molecular basis for further search of the specific .male worm antigen epitope composition and for artificial synthesis of peptidevaccine.In order to obtain male-specific gene and its encoded protein thatcould be a target for anti-male worm development, Sj adult worm cDNA library was immunoscreened with anti-male adult worm antigen sera. Positive clones were identified firstly by PCR and further by sequencing and data analysis through Internet. Consequently eleven positive clones were obtained and finally three novel genes designated as 5/-MA, Cp8 and S/-p8 respectively were testified by GenBank (GenBank accession number are AF519808, AF524896 and AF517843). The deduced proteins of the three novel genes predictably encoded three possibly important nucleic and cytoplasmic signal transduction molecules. Further investigation on whether they are male worm gender-specific genes is of great attraction. Moreover the novel gene Sj-MA cDNA was firstly subcloned into a prokaryotic expression vector to construct recombinant plasmid, which was secondly transformed into E.coli. The recombinant fusion protein was highly expressed as insoluble inclusion bodies in the E.coli and was applied to mice for assessment of protective immunity against Sj. Our study showed that the subcloned novel gene was effectively expressed in the transformed E.coli. A 54.8kDa fusion protein was revealed by SDS-PAGE and its antigenicity was confirmed with the recognition by both anti-male adult worm sera and anti-GST sera in Western blot. Mice vaccinated with the fusion protein revealed significant worm reduction rate ( 34.29% p<0. 001 ) and egg reduction rate ( 48.76% p<0. 001 ), compared with the control groups. Taken together, the novelgene Sj-MA can be expressed in E.coli as a fusion protein that can elicit immunity against Sj, indicatin更多还原