基于牛XY精子差异表达基因的性控DNA疫苗研究

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 genes will be located in bovine leukocyte metaphase chromosome; the DNA vaccine candidate gene cloning and construction of expression vector and eukaryotic expression will be developed perfectly, at least one DNA vaccine of Y sperm specific candidate gene for sex control in bovine will be harvested.

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。

本项目按照研究计划进行，设计了探针进行原位North Blot验证和XY精子RNA测序分析差异表达基因;二维电泳XY精子差异蛋白; 性控DNA疫苗候选基因真核表达载体构建与真核表达和小鼠免疫试验。当前这些方面的计划研究内容都已完成或基本完成。.对已经筛选得到的牛Y-X精子低丰度差异表达基因设计探针，分别在X、 Y和常规精液，采用上游法获得纯净的精子进行原位North Blot验证； FISH杂交对SRY 基因进行染色体定位。通过高纯度分选XY精子利用RNA-seq获得X,Y精子的转录组，进行差异表达富集分析，共有 539 个差异表达基因注释到 GO 功能类别中，244 个差异表达基因注释到12个KEGG代谢信号通路中。对X\Y精子转录组进行差异表达基因分析，共筛选出27 条差异表达基因,为DNA性控疫苗的研究提供理论依据。.对XY精子蛋白二维电泳分析，得到 57个蛋白点存在差异，其中18个蛋白点在X精子表达上调，且其中8个蛋白点仅在X精子表达，39个蛋白点下调表达，13个蛋白缺失；39个蛋白点在Y精子中表达上调，其中13个蛋白点仅在Y精子上表达，18个蛋白点下调表达，8个蛋白点缺失。经酶切质谱鉴定和生物信息学分析其中的6个差异蛋白点，其中3个蛋白质为：组蛋白H2B、14-3-3α/β蛋白、载脂蛋白A-I，它们与精子生成和代谢有关。 .候选基因SRY重组质粒构建成基因真核表达载体pcDNA3.1/Zeo(+/-)并进行酶切，菌落PCR，测序鉴定后确定真核高效表达的Y精子特异性抗原候选基因的DNA疫苗，重组质粒pcDNA3.1-His-C-SRY肌肉注射小鼠后，产生了抗SRY抗体；免疫雌性实验组后代仔鼠的雌雄比例78：66（1.2：1）发生了向雌性的偏移。动物实验的结果为进一步提高疫苗的性控效果奠定了实验基础。