【摘要】 根据GenBank中SjFer序列设计1对特异引物,以日本血吸虫(Schistosoma japonicum)cDNA文库为模板扩增SjFer基因,常规方法构建质粒pcDNA3.0/SjFer、pcDNA3.0/GM-CSF及pcDNA3.0/SjFer-GM-CSF。85只昆明小鼠分A、B、C、D和E组,分别于0、2和4周通过左后肢股四头肌注射生理盐水50μL/只,质粒50μg/只。感染前和每次免疫前采血收集血清,ELISA检测特异性IgG水平。末次免疫后2周每组随机宰杀小鼠5只无菌取脾,MTT法检测淋巴细胞增殖情况,取免疫部位的肌肉组织通过免疫组化检查重组质粒在小鼠肌细胞内表达情况。末次免疫后2周每只小鼠贴壁感染40±1条尾蚴,45d后宰杀观察减虫率和减卵率。结果表明,重组质粒能在小鼠肌细胞内表达;A、B和C组IgG水平变化不明显,D组和E组可诱导小鼠IgG水平升高,且E组高于D组。淋巴细胞转化试验结果A、B和C组OD570变化不明显,彼此无显著差异,D组和E组脾淋巴细胞转化率明显增加,与对照组差异显著,且E组OD570明显高于D组。D组和E组可诱导小鼠产生36.27%和39.22%的减虫率、36.10%和49.04%的减卵率。GM-CSF可增强体液和细胞免疫应答并显著增强pcDNA3.0/SjFer的免疫保护力。更多还原

【Abstract】 To test immunoprotection elicited by eucaryotic expression vector of co-encoding SjFerritin gene(SjFer)and granulocyte macrophage colony-stimulating factor(GM-CSF)against challenge infection by Schistosoma japonicum in mice,the special primers were designed according to the sequence of SjFer in GenBank,and SjFer was amplified from S.japonicum adult worm cDNA library.Eighty-five female mice were randomly divided into 5 groups,immunized with 50 μL normal salt,50 μg pcDNA3.0,50 μg pcDNA3.0/GM-CSF,50 μg pcDNA3.0/SjFer and 50 μg pcDNA3.0/SjFer-GM-CSF per mouse in 0,2nd and 4th week,respectively.Sera were collected before immunization and challenge infection,respectively.Levels of specific antibody were detected by ELISA.Lymph-spleencytes of mice were collected two weeks after final immunization.Activity of Lymph-spleencyte proliferation was detected by MTT assay.Expression of recombinant plasmids in murine muscular tissue was detected with immunohistochemistry.Mice were challenged with 40±1 S.japonicum cercaria per mouse 2 weeks after the final vaccination.Forty-five days later,mice were killed and perfused,and the adult worms and eggs were counted.Results showed that pcDNA3.0/SjFer,pcDNA3.0/GM-CSF and pcDNA3.0/SjFer-GM-CSF expressed in murine muscular tissue.The serium IgG level did not change obviously in control groups(P >0.05),but increased significantly in experimemtal groups(P <0.01).Furthermore,the serium IgG level in group immunizated with pcDNA3.0/SjFer-GM-CSF was higher than that with pcDNA3.0/SjFer(P <0.01).The proliferation activity of spleen T lymphocytes detected by MTT assay did not change obviously in control groups(P >0.05)and increased significantly in experimental groups after inducation of ConA or rSjFer(P <0.01).The proliferation activity of spleen T lymphocytes in the group immunizated with pcDNA3.0/SjFer-GM-CSF were higher than that with pcDNA3.0/SjFer(P <0.01).The worm reduction rate and egg reduction rate was 36.27 %,39.22 % and 36.10 %,49.04 % in groups immunization with pcDNA3.0/SjFer and pcDNA3.0/SjFer-GM-CSF,respectively.In conclusion,GM-CSF can improve humoral,cellular immunity and immunoprotection elicited by eucaryotic expression vector encoding SjFer in mice.更多还原