基于牛XY精子差异表达基因的性控DNA疫苗研究

项目名称： 基于牛XY精子差异表达基因的性控DNA疫苗研究

项目编号： No.31260551

项目类型： 地区科学基金项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 高庆华

作者单位： 塔里木大学

项目金额： 54万元

中文摘要： 基于自然基金项目"牛XY精子差异表达基因筛选"（30760170）获得的4个Y精子特异表达基因片段(SRY和3个未知基因)，作为性控DNA疫苗的候选基因开展研究。采用RACE法分析未知基因全长，进行X、Y和常规精液的原位Northern Blot验证和牛白细胞中期分裂相FISH染色体定位，高纯度X、Y精子蛋白二维电泳和质谱分析，结合Genebank比对、确定Y精子特异性抗原DNA疫苗的候选基因；连接pcDNA载体、脂质体法转染真核细胞，经SDS-PAGE和Western Blot及RNA印迹检测，确定其高效表达；构建DNA疫苗并免疫小鼠，抗血清体外特异性抑制Y精子实验和后代性别比例测定。获得3个Y-X差异表达基因片段全长序列，4个基因的Y精子定位和3个未知基因的牛染色体定位；完善性控DNA疫苗候选基因克隆与表达载体构建及真核表达，筛选获得至少1个牛性控DNA疫苗的Y精子特异性候选基因。

中文关键词： XY精子；差异表达基因；牛；性别控制；DNA疫苗

英文摘要： The last Natural Science Fund Project"Research on screening of differentially expressed genes in bovine XY sperm" (30760170) have been completed. The four Y-sperm specific expressed gene fragments including Sry gene and three unknown genes which can be researched as the DNA vaccine candidate genes for sex control, have been obtained. The full-length of the unknown genes are analyzed using RACE method, in situ Northern Blot are done in X, Y and conventional sperm and FISH in bovine leukocyte of metaphase chromosome location. The proteins from the flow cytometric-sorted X or Y sperm are analyzed using two-dimensional electrophoresis and mass spectrometry analysis, combined with Genebank comparison, the Y sperm-specific antigen DNA vaccine candidate genes are determined. With pcDNA vector, liposome mediated transfection of eukaryotic cells, SDS-PAGE and Western by Blot and RNA blotting detection, the effective expression for the four genes are researched and optimized; and then the DNA vaccines are constructed, immunization of mice, antiserum specific inhibition of Y sperm and offspring sex ratio determination experiment in vitro are carried out. Finally, the full-length sequences of three Y-X differentially expressed gene fragments will be obtained,the four genes will be oriented in Y sperm and the three unknown g

英文关键词： XY sperm；differentially expressed genes；bovine；sex control；DNA vaccine