【作者】 郝知友；

【导师】 黄复深；

【作者基本信息】 湖南农业大学 ， 基础兽医学， 2007， 硕士

【摘要】 疫苗是防治传染病的有效手段，作为一种相对便宜且长效的防治措施，研制出血吸虫病的疫苗一直是科学家们追求的目标。血吸虫病疫苗的研究经历了从死疫苗、致弱活疫苗、亚单位疫苗到基因工程疫苗的探索过程。核酸疫苗是血吸虫疫苗研究的重要内容。现阶段研制的血吸虫核酸疫苗候选分子所诱导的保护力比较弱，保护不完全，因此有必要提高疫苗分子的免疫诱导力和寻找新的疫苗候选分子。1．本研究是通过由日本血吸虫cDNA文库筛选出的新基因（SjIR3）与真核表达载体pCDNA3.0构建出重组质粒SjIR3／pC，并以生理盐水（100μl）、空白质粒pCDNA3.0（50μg）为对照免疫KM小鼠（每组18只），免疫方案是0-2-4-6，每次免疫前及攻击感染前于尾静脉采血进行ELISA实验检测抗体滴度。末次免疫后2w，各组剖杀任意6只小鼠取脾细胞，分别用ConA或rSjIR3重组蛋白诱导，MTT法检测淋巴细胞增殖。各组余下小鼠经腹部感染日本血吸虫尾蚴40±1条，45d后宰杀，观察实验组小鼠的减虫率和减卵率。其结果显示，SjIR3／pC可诱导小鼠产生较高抗体水平（0.78±0.05），ConA或rSjIR3重组蛋白诱导淋巴细胞增殖结果分别为0.57±0.02、0.68±0.01，减虫率和减卵率分别是29.4％、36.6％。2．首次构建了鼠粒细胞-巨噬细胞集落刺激因子（mGM-CSF）与血吸虫基因的共表达载体（SjIR3-GMCSF／pC），并设计了生理盐水（100μl）、空白质粒pCDNA3.0、SjIR3／pC、mGM—CSF／pC和SjIR3-GMCSF／pC（50μg）五组小鼠实验，其免疫方案、攻击感染、检测方法同前。结果显示，SjIR3／pC组与SjIR3-GMCSF／pC诱导小鼠产生的抗体水平分别为0.78±0.04、0.91±0.03；ConA或rSjIR3重组蛋白诱导淋巴细胞的转化率明显增加，其A₅₇₀分别为0.56±0.07和0.68±0.05、0.56±0.07和0.77±0.04；减虫率和减卵率分别是39.81％、34.75％和33.96％、28.43％。结论：SjIR3 DNA疫苗能够诱导受攻击感染的小鼠产生体液免疫和细胞免疫而具有部分抗攻击感染的免疫保护作用。mGM-CSF可增强SjIR3DNA疫苗诱导小鼠产生的体液免疫和细胞免疫应答和抗攻击感染的免疫保护作用。SjIR3 DNA疫苗免疫诱导家畜等产生的免疫应答和抗攻击感染的保护效果值得进一步研究，SjIR3 DNA疫苗可能成为血吸虫“鸡尾酒疫苗”的组成成分。更多还原

【Abstract】 Vaccine is an efficient measure to prevent and control infectious disease. Usingschistosomasis japonicum vaccine for prophylaxis as a relatively cheap and long-effectivemeasure, is a goal for people to pursue. The study of schistosomasis japonicum vaccineundergoes three stages: inactivated vaccines, attenuated live vaccines, subunit vaccines andgenetic engineering vaccines. DNA vaccine is an important field of schistosomasis japonicumvaccine research. Because of low immunoprotection of studied candidate-molecule vaccines,it is necessary to study how to improve their immune efficacy and find new candidatemolecule vaccines with high immune efficacy against challenge infection.1. To detect the protection in mice induced by DNA vaccine encoding SjIR3 against thechallenge of Sj in this research, SjIR3 was amplified by PCR from adult worm cDNA libraryand the recombinant plasmid SjIR3/pC was constructed by routine methods. Fifty-four femalemice were randomly divided into 3 groups (each 18 immunized with normal saline （100μl）,pcDNA3.0 and SjIR3/pC （50μg） respectively. The all groups immunized three times withinterval of two weeks. The mice were infected by （40±1） Sj cercariae per mouse 2w after finalimmunization. Forty-five days later, mice were sacrificed and perfused, the adult worms andeggs were counted. Sera were collected before the every immunization and challengeinfection.The serum 1gG were detected by ELISA, and the spleenocytes of mice werecollected before challenge infection. The proliferation activity of spleen T lymphocyteinduced by ConA or rSjIR3 was detected by MTT assay. The results of ELISA showed nosignificant change of sera 1gG level group A and B, but considerable increase in experimentalgroup immunized by SjIR3/pC （0.78±0.05）. And the proliferation activity of spleen Tlymphoeytes detected by MTT assay increased with induction of ConA or recombinantproteins rSjIR3 after final immunization. The A₅₇₀ is 0.57±0.02, and 0.684±0.01, respectively,with significant difference compared with two control groups （P＜0.01）. The worm reductionrate and the egg reduction rate in experimental group were 29.42％and 36.56％respectively.2.mGM-CSF was amplified by PCR with specific primers. The recombinant plasmidSjIR3-GMCSF/pC and mGM-CSF/pC was constructed by routine methods. Ninety femalemice were randomly divided into 5 groups immunized with normal saline （100μl）,pcDNA3.0（50μg）, SjIR3/pC（50μg）, mGM-CSF/pC（50μg） and SjIR3-GMCSF/pC （50μg）respectively. Protocal of immunization and animal challenge infection experiment was same as the above. The results of ELISA showed the level of 1gG in sera of two control groups hadno changes, and the level of sera 1gG in groups immunized by SjIR3/pC or SjIR3-GMCSF/pCincreased （0.78±0.04、0.91±0.03） and had significant difference compared with that in controlgroups （P＜0.01）. And the proliferation activity of spleen T lymphocytes detected by MTTassay increased after induction of ConA or recombinant proteins rSjIR3. A₅₇₀ is 0.56±0.07 and0.68±0.05, 0.56±0.07 and 0.77±0.04, respectively, with significant difference compared withthree control groups （P＜0.01）. The worm reduction rate and the egg reduction rate in groupsimmunized by SjIR3/pC or SjIR3-GMCSF/pC were 39.81％and 34.75％, 33.96％and 28.43％,respectively. In conclusion, DNA vaccine encoding SjIR3 induced humoral and cell-mediatedimmune response and partial immune protection against the challenge infection in mice.mGM-CSF improved the humoral and cell-mediated immune response induced by DNAvaccine encoding SjIR3 and immunoprection against challenge infection in mice. It was worthfurther studying induction of immune response and immunoprotection against challengeinfection in livestock. This data also showed SjIR3 DNA vaccine become possible asingredient of Schistosomasis "cocktail vaccine".更多还原