【作者】 李志伟；

【导师】 王志钢； 扈廷茂；

【作者基本信息】 内蒙古大学 ， 微生物学， 2007， 硕士

【摘要】 细粒棘球蚴病(CE)又称囊性包虫病是由细粒棘球绦虫的续绦期幼虫寄生于人和动物体内引起的一类重要的、具有地方流行性的人畜共患寄生虫病。CE呈全球性分布，畜牧业发达的地方往往是此病的流行区。我国CE主要分布于西部和北部的内蒙古、新疆、甘肃、宁夏、青海、四川和西藏等地。疫苗是防治包虫病的一种重要途径，该研究领域涉及天然蛋白疫苗、重组蛋白疫苗、合成肽疫苗、核酸疫苗和重组伤寒沙门氏杆菌疫苗等。Eg95抗原基因在细粒棘球绦虫六钩蚴期表达，具有株系间的高度保守性，是一种可以诱导机体免疫保护的有效的疫苗候选分子。利用PCR技术从细粒棘球蚴中扩增出Eg95基因DNA，构建了pMD19-T／Eg95 DNA重组克隆载体，转化大肠杆菌DH5α感受态细胞，经抗生素筛选获得重组子，进行DNA测序，利用GenBank／BLAST功能对Eg95-NM DNA序列与GenBank中已有的Eg95基因家族的成员进行序列比较分析，结果表明编码区完全一致。利用RT-PCR技术从细粒棘球蚴中扩增了Eg95基因cDNA，构建了重组克隆载体pMD19-T／Eg95 cDNA，转化E.coli DH5α感受态细胞，经抗生素筛选获得重组子并测序，结果发现Eg95-NM基因cDNA序列与基因DNA序列编码区出现5个碱基差异，编码的氨基酸有4个发生改变。因此，以DNA序列编码区推导的理论氨基酸为基础，对Eg95蛋白的相关特性及抗原表位加以分析，结果表明变异的58和122位氨基酸处于抗原表位形成区，所以选定编码这两个氨基酸的变异碱基进行PCR定点突变，以确保Eg95蛋白的正确表达。PCR定点突变后测序结果表明，预期位点碱基已被修正。得到的Eg95-NM基因cDNA序列长度为471bp，表达16.9 Kda的Eg95抗原。将定点突变后修正后的Eg95基因cDNA序列克隆至原核表达载体pET44a(+)上，构建了重组原核表达载体pET44a(+)／Eg95。载体pET44a(+)上大小约66.4KDa的标签NusA蛋白与Eg95-NM融合，预期表达83.3 KDa的融合蛋白。经测序鉴定后转化E.coli BL21(DE3)菌株在IPTG诱导下，重组菌株表达了融合蛋白质。经SDS-PAGE检测结果显示，重组质粒表达了约83.3 KDa的目的条带。Western-blotting试验证明，该融合蛋白表达产物可与囊性包虫病人血清特异结合，说明该融合蛋白得到了正确的表达。更多还原

【Abstract】 Cystic Echinococcus(CE) is a serious zoonosis caused by infection with the metacestode stage of the typeworm Echinococcus granulosus in humans and many livestock such as sheep, goat, cattle and horse. CE is a global verminosis and is particularly epidemic in the developed region of animal husbandry. In China, CE mainly occurs in the western and northern areas such as Inner Mongolia, Xinjiang, Gansu, Ningxia, Qinghai, Sichuan and Xizang. Vaccinotherapy using CE vaccines, such as natural protein vaccine, recombinant protein vaccine, synthetic peptide vaccine, DNA vaccine and recombinant Salmonella typhimurium vaccine, is important way to control this disease. Eg95 genes are highly conservative among all strains of Echinococcus granulosus, and are expressed at the oncosphere stage. Thus Eg95 is an effective antigen candidate which can trigger a high immunoreaction to preventpeople from CE. The Eg95 genomic DNA was amplified from the hydatid cyst By PCR and ligated with pMD19-T plasmid to construct pMD19-T/Eg95 DNAvector for sequencing. The coding regions of Eg95-NM sequence was found to be identical to those of Eg95 genes reported in GenBank.The Eg95 cDNA was also amplified by RT-PCR from the hydatid cyst and sequenced. Sequence analysis showed that 5 bp of cDNA sequence were different from the coding regions of Eg95-NM genomic DNA and henceled to 4 amino acid variation. Antigen epitope analysis of the amino acid sequence deduced from the coding regions of genomic DNA sequence demonstrated that the varied amino acid 58 and 122 located in the epitopes. So the varied base pairs which coding the amino acid 58 and 122 were rectified by PCR based site-directed mutagenesis to assure the correct structure of Eg95 protein.Finally a corrected 493bp cDNA fragment carrying an open reading frame(ORF) of 471bp, which encodes a protein of about 16.9KDa, was cloned and inserted into the prokaryotic expression vector pET44a(+). A putative NusA fusion protein of about 83.3KDa, in which NusA tag is 66.4KDa and Eg95 is about 16.9KDa, was induced to be expressed by IPTG in E.coli BL21(DE3). SDS-PAGE and western blotting analysis with the positive sera of echinococcosis patients as the primary antibody confirmed that the target band of about 83.3KDa was NusA-Eg95 fusion protein. Our work may provide an alternative way to generate recombinant Eg95 antigen for inoculation against CE.更多还原