【作者】 宋震宇；

【导师】 刘金明； 岳城；

【作者基本信息】 新疆农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 血吸虫病是世界性分布的、危害最为严重的人畜共患寄生虫病之一。东方田鼠对血吸虫具有天然抗性。探索东方田鼠抗日本血吸虫机理,筛选东方田鼠抗日本血吸虫抗性相关靶基因,对研制抗日本血吸虫疫苗和治疗药物具有重要借鉴意义。刘金明组课题组在前期研究中利用东方田鼠血清筛选日本血吸虫成虫噬菌体展示cDNA文库,获得10个阳性克隆,为寻找其中具有开发成疫苗潜力的抗原编码基因,为研制疫苗创造条件。本文对其中的8个噬菌体克隆的免疫预防效果进行观察,并对具有较高保护效果的锌指蛋白编码基因进行克隆、表达和重组蛋白免疫预防效果的研究。以8个噬菌体克隆单独或联合免疫BALB/c小鼠,和空白对照组相比,展示日本血吸虫锌指蛋白的1号噬菌体克隆免疫组在两次实验中分别获得了32.10%和31.25%的减虫率、61.14%和47.31%的肝脏减卵率,虫体合抱率分别由92.59%、57.39%下降到69.09%、41.03%;其它噬菌体克隆单独免疫组或联合免疫组在第一次实验中获得了8.02%-32.72%的减虫率和40.19%-69.53%的减卵率,但在第二次实验中未能得到验证。以RT-PCR技术从血吸虫成虫mRNA中成功克隆日本血吸虫锌指蛋白编码基因(我们将其命名为Sjznf1)的全长ORF序列,其ORF全长1014bp,编码337个氨基酸,与GenBank登陆号为AY222909和EZ000159的日本血吸虫锌指蛋白基因序列比对,核苷酸同源性分别为99.7%和99.4%;氨基酸序列同源性分别为99.4%和98.5%。其中含有一个锌指蛋白结构域。将该全长序列连接到pGEX-4T-2表达载体上,构建重组表达质粒并用大肠杆菌BL21表达,经IPTG诱导,获得表达产物为63 KDa的融合蛋白。应用重组蛋白免疫BALB/c小鼠,获得了54.62%的减虫率和75.39%的肝脏减卵率。本研究首次通过实验验证日本血吸虫环型锌指蛋白编码基因的表达产物具有良好的免疫预防效果。研究结果提示日本血吸虫环型锌指蛋白编码基因在疫苗方面具有重要研究价值。更多还原

【Abstract】 Schistosomasis is one of the word-wide spread parasitic zoonosis that causes serious healthy problem to both human and animals. It was reported that there are 40 species of mamals in the endemic area could be naturally infected with Schistosoma japonicum (S. japonicum),and Microtus fortis (M. fortis) is the only mamalian animals with native resistance to S. japonicum. Exploring the anti-schistosoma mechanism of M.fortis, screening the genes of M. fortis or target genes of S.japonicum that related to the resistance could provide some useful information for the development of anti-schistosomula vaccines and drugs.Ten positive clones were obtained in precious work of the liujinming`s group, by screening a adult worm phage display cDNA library from worms of S. japonicum with fresh sera of Microtus fortis. In order to find the candidate vaccine antigen encoding genes, we used 8 of positive clones alone or as cocktail vaccines, to vaccinate BALB/c mice and observe the preventive effects to schistosomiasis japanica. Compared with the blank control group, vaccination with clone 1 which displayed the ring zinc finger protein of S. japonicum induced reductions of 32.10% and 31.25% for mean worm burdens, 61.14% and 47.31% for liver egg burdens, and decreases of Paired worm ratios from 92.59% and 57.39% to 69.09% and 41.03%, respectively, over two independent trials. 8.02%-32.72% worm reductions and 40.19%-69.53% liver egg reductions were obtained from groups vaccinated with other clones in trial 1,but these could not be confirmed in trial 2.The full-length ORF sequence of the gene encoding a zinc finger protein (Here we named it Sjznf1) was obtained by RT-PCR from the mRNA of S.japonicum S.japonicum adult worm. The ORF is composed of 1014 nucleotides and encodes 195 amino-acid resides. Compared with the squences of zinc finger protein encoding gene of S. japonicum reported in GENEBANK with accession numbers AY222909 and EZ000159 , there were 99.7% and 99.4% identities in nucleotide sequence, and 99.4% and 98.5% identities in deduced amino acid sequence. There was a zinc finger protein domain (Ring-type or PHD-type) in the deduced amino acid sequence. After subcloning the cDNA of Sjznf1 into PGEX-4T-2 expression vector, the gene was expressed in E. coli BL21 when induced with IPTG. The expression product was a 63KDa fusion protein. The recombinant protein was used to vaccinate BALB/c mice, and 56.62% worm reduction and 75.39% liver egg reduction were obtained from the vaccinated group.It was the first reported that the phaged-displayed antigen and recombinant protein antigen of a ring zinc finger protein encoding gene from S. japonicum could induce high protective level in mice. The results revealed that this S. japonicum was worth further investigation on the development as a vaccicne.更多还原