【作者】 胡元生；

【导师】 沈继龙； 汪学龙；

【作者基本信息】 安徽医科大学 ， 病原生物学， 2005， 硕士

【摘要】 目的:从日本血吸虫（中国大陆株）成虫cDNA中扩增出日本血吸虫信号蛋白14-3-3（Sj14-3-3）和日本血吸虫26kDa谷胱甘肽-S-转移酶（SjGST）编码基因,扩增小鼠白介素-12（mIL-12）编码序列,克隆至真核表达载体pcDNA3.1（+）,并验证其能表达出重组蛋白;利用该重组质粒免疫BALB/c小鼠,观察其对小鼠抗血吸虫感染的保护作用,以及对特异性IgG抗体、脾细胞分泌的细胞因子及CD₄⁺和CD₈⁺比率的影响。 方法:提取日本血吸虫成虫总RNA,纯化出mRNA,逆转录生成cDNA,以其为模板扩增出Sj14-3-3和SjGST编码基因,另从带mIL-12的质粒（pSEC:p30-p40）扩增出mIL-12编码基因,将扩增产物克隆入真核表达载体pcDNA3.1（+）。用RT-PCR、间接免疫荧光和Western blotting等方法验证其在体外（COS-7细胞）表达,制备无内毒素小鼠DNA疫苗,准备免疫小鼠。 BABC/c小鼠（6-8周龄,体重18-22g）随机分成5组,每组5只,雌雄兼用。除对照组外,每组均给予日本血吸虫DNA疫苗pcDNA3.1（+）-Sj14-3-3 20μg/次/鼠,后腿肌肉注射（i.m.）,共免疫2次,更多还原

【Abstract】 Objective: To develop a new molecular candicate vaccine and find adjuvants for immuno-prevention of schistosomiasis.the Sj14-3-3 protein and 26kDa SjGST (Schistosoma japonicum glutathione-S-transferase) coding genes from cDNA of S.japonicum adult worms was amplified and cloned into eukaryotic expression vector(pcDNA3.1 (+)). The mouse IL-12 coding gene from recombinant plasmid pSEC:p30-p40 with mIL-12 was amplified and cloned into eukaryotic expression vector (pcDNA3.1 (+)). The recombinant plasmid (pcDNA3.1(+)-Sjl4-3-3, SjGST and mIL-12) were transfected into cultured COS-7 cells and the successful transfection was confirmed by RT-PCR ,Western blotting and Indirect immunofluorescent assay( IIF) .To estimate the immuno-protection of native antigen DNA vaccine and/or with SjGST and different adjuvants in mice challenged with schistisome cercaria,and to observe the specific IgG induced by Sj14-3-3, and the cytokines(IFN-γ and IL-4) secreted by splenocytes and percentage of CD4+/CD8+ T lyphocytes in vaccinated mice. Methods : Thetotal RNA of S. japonicum was extracted and mRNA was purified to prepare for cDNA by RT-PCR, from which the Sj 14-3-3 and SjGST encoding genes were amplified.The recombinant DNA of pSEC-mIL-12 was extracted from which the mIL-12 encoding gene was amplified.Both Sjl4-3-3and SjGST genes were cloned into eukaryotic expression vector(pcDNA3.1(+)) and expression was identified in vitro and in vivo by RT-PCR ,IIF, and Western blotting . BALB/c mice( 6-8w,weight 18-22g) were randomly divided into 5 groups( 5 mice each). All mice, except the control, were immunized twice with 3-week’s intervals by muscle injection scheduled as follows: 20 |xg of DNA vaccine /dose/mouse;(DpcDNA3.1(+)-Sjl4-3-3 group ; ?pcDNA3.1(+)-Sj 14-3-3+ pcDNA3.1(+)-S/GSTgroup ;(DpcDNA3.1(+)-Sj 14-3-3+ pcDNA3.1(+)-mIL-12group@pcDNA3.1(+)-Sjl4-3-3+CpG group (5) control group. The first group was immunized with pcDNA3.1(+)-Sj 14-3-3 alone .The fifth group injected intramuscularly with the same volume of normal saline as a control.Six weeks after final shot with pcDNA3.1(+)-Sjl4-3-3,the vaccinated and control mice were challenged percutaneously with 40±l cercariae of S.japonicum .The immune protection was tested by adult worm and tissue egg reductions in the portal vein and liver at 6th weeks after challenge.The levels of serai specific IgG, IgGj and IgG2a antibodies against Sj 14-3-3 protein were titered by ELISA and the mouse IFN-y and IL-4 in supernant of cultured splenocytes were quantified using a commercial ELISA kit following the manufacture ’ s instructins .In addition,we analyzed the percentage of CD4+/CDs+ T cell subsets and their ratios in splenocytes by flow cytometry. Results: The size of PCR products were approximately 760bp,670bp and 1700kb respectively. And the insert of pcDNA3.1(+)-Sjl4-3-3, SjGST and mIL-12 digested with BamH I and Xho Iwere the same in length as the PCR products.The recombinant DNA was identified with completed ORF by DNA sequencing and transfected into COS-7 cells and screened by Neo+ antibiotic resistance.Expression was confirmed in vitro and in vivo by RT-PCR,Western blotting, and IIF.The results showed that SjGST and the adjuvants CpG and mIL-12, and as well could significantly enhance the protective immunity of DNA vaccine pcDNA3.1(+)-Sj 14-3-3 for schistosomaiasis in mice,both worms and eggs burdens were markedly reduced .Worm reduction was elevated from 28.7%( Sj 14-3-3 39.2%(Sj 14-3-3+ SjGST),respectively;The egg reduction was raised from 19.9% to 52.6%, 41.2%, and 39.6%,respectively.The levels of serai specific IgG, IgGj and IgG2a antibodies against Sj 14-3-3 protein were dramatically elevated in mice immunized with Sj 14-3-3 + mIL-12, CpG or SjGST .The concentrations of mouse IFN-y and mouse IL-4 in supernatant of cultured splenocytes stimulated by ConA or Sj 14-3-3 were as follows : Sj 14-3-3 + mIL-12 and SJGST+ CpG were significantly higher than Sj 14-3-3 alone .However, The concentrations of mouse IL-4 in supernatant of cultured splenocytes stimulated by Sj 14-3-3 were no significantly change.There is less significantly difference .The percentages of CD4+ T cell subsets in Sjl4-3-3+SjGST,CpG or mIL-12 were not significantly different from Sj 14-3-3 alone except Sj 14-3-3 +mIL-12 group.Its percentages was higher than Sj 14-3-3 alone ;but their percentage of CDg+ T cell subsets was significantly higher than of Sj 14-3-3 alone .Therefore,the ratios of CD4+/ CDs+ T cell subsets in these three groups were less than those of Sj 14-3-3 groups alone.Conclusion: The Sj 14-3-3 , SjGST and mIL-12 were successfully amplified,cloned and expressed.The immuno-protection of DNA vaccine (pcDNA3.1(+)-Sj 14-3-3) with SjGST and different adjuvants (CpG , mIL-12)suggested that SjGST,更多还原