【作者】 张蕴斌；

【导师】 张富春；

【作者基本信息】 新疆大学 ， 动物学， 2002， 硕士

【摘要】 草原兔尾鼠是威胁新疆畜牧业发展的重要害鼠，其种群的相对过剩不利于草原生态系统的可持续发展。通过免疫不育方法控制鼠害已成为目前国际上的研究热点。卵透明带3（ZP3）蛋白是哺乳动物生殖过程中介导精卵结合的关键因子，已被用作免疫不育控制鼠害研究的主要靶抗原。本论文研究工作采用RT-PCR与RACE相结合的方法，首次克隆了草原兔尾鼠ZP3 全长cDNA并进行了序列分析，构建了真核表达质粒，为草原兔尾鼠免疫不育的研究奠定了基础。首先根据已发表的五种啮齿目动物ZP3 cDNA序列同源性分析结果设计特异性引物，RT-PCR扩增草原兔尾鼠ZP3（lZP3）cDNA保守序列。将扩增产物连接到pMD18-T载体上进行测序后将其与Gene Bank中多种哺乳动物ZP3 cDNA进行同源性比较，确定已克隆出了lZP3 cDNA保守序列。根据已测序的lZP3保守序列设计特异性引物，使用3’和5’RACE的方法扩增lZP3 cDNA的3’末端和5’末端序列并进行测序，所测序列经与lZP3保守序列相互重叠、核对与拼接，获得了lZP3全长cDNA序列。根据已拼接出的lZP3 全长cDNA序列设计基因特异性引物，利用RT-PCR方法扩增lZP3 全长cDNA。将扩增产物连接到pMD18-T载体上进行测序，序列分析表明已克隆出lZP3 全长cDNA。将其与Gene Bank中多种哺乳动物ZP3 cDNA进行同源性比较后，发现草原兔尾鼠与布氏田鼠ZP3 cDNA编码的蛋白前体的某些位点显著不同于其它哺乳动物，其后续研究可能有助于哺乳动物受精机理的阐明与免疫不育疫苗的设计。获得lZP3全长cDNA后，RT-PCR扩增lZP3 cDNA核心片段，构建pGEX-lZP3cf原核表达质粒；用lZP3全长cDNA构建pCMV-lZP3真核表达质粒。经双酶切鉴定后，证实两种质粒均已构建成功，可用于表达重组蛋白和进行免疫不育实验。本工作首次克隆了草原兔尾鼠卵透明带3全长cDNA，已向GeneBank申请注册为新基因，保护了我国的野生动物基因资源，为哺乳动物生殖机理的研究提供了新的资料。构建了lZP3原核与真核表达质粒，为草原兔尾鼠ZP3免疫不育控制鼠害的研究提供了实践基础。更多还原

【Abstract】 Lagurus lagurus is a serious rodent pest not just because it can destroy local pasture in Xinjiang, but more importantly because its overpopulation changes the overall structure and composition of native grassland and other ecosystems. As one of the most promising population control tools in the world today, immuno- contraception using zona pellucida glycoprotein antigens, specifically ZP3, may be used to control Lagurus population.In this study, by combining RT-PCR and RACE, a full-length cDNA encoding the Lagurus lagurus ZP3 (lZP3) was isolated from the lagurus’s ovary and cloned into a prokaryotic expression vector and a eukaryotic expression vector for further immunocontraceptive investigations.With a pair of degenerate primers designed according to the conserved region among the known cDNA sequences of five rodents, a cDNA fragment was generated by RT-PCR from total RNA isolated from ovaries of Lagurus lagurus. The cDNA fragment was cloned into a cloning vector, pUCm-T. Positive clones were analyzed with restriction enzyme digestions and further identified with sequence analysis. The sequencing results show that the cDNA fragment was homologous identical with the published sequences derived from five rodents.With a set of primers designed according to the sequence of the cloned cDNA fragment, the 3’ and 5’ ends of the cDNAs were obtained by 5’ and 3’ rapid amplification of cDNA ends (RACE), respectively. Combining the sequences of the 3’ ends, 5’ ends and cloned partial cDNA, the full-length sequence of the Lagurus lagurus ZP3 (lZP3) cDNA was assembled. To obtain full-length lZP3 cDNA, a pair of primers was designed according to the assembled lZP3 cDNA sequence. After RT-PCR of total RNA isolated from the ovaries of Lagurus lagurus, the lZP3 cDNA was cloned into pUCm-T and further identified with sequence analysis. The results of sequencing analysis show that the open reading frame (ORF) of the cDNA encodes a protein of 418 amino acids (45 kDa) that is significantly different from other published mammal ZP3 sequences at a <WP=7>few sites. The result may have important implications for the elucidation of mechanisms of fertilization and the development of contraceptive vaccine based on lZP3.For construction of lZP3 prokaryotic expression plasmid, pGEX-lZP3cf, a lZP3 cDNA core fragment (lZP3cf) excluding the regions encoding the leader sequence and the C-terminal transmembrane domain was obtained by RT-PCR and cloned into prokaryotic expression vector, pGEX-4T-1. In addition, for construction of prototype DNA vaccine, pCMV4-lZP3, the cloned lZP3 full-length cDNA was subcloned into a eukaryotic expression vector, pCMV4.These preliminary results could lead to further investigation for the development of immunocontraceptive vaccine based on lZP3 for Lagurus population control.更多还原