乳腺炎是由病原体等多种因素引起的复杂炎症，给奶牛业造成了巨大损失。课题组前期通过乳腺组织转录组测序发现CD46和CD14基因存在差异表达及异常剪切模式，后续实验验证CD46和CD14基因分别存在异常剪切生成的转录本CD46-AS和CD14-AS，及可能参与剪切的单核苷酸多态（SNP）。CD46是细胞膜上的一种受体，可结合入侵的链球菌，通过细胞内吞作用将其吞噬；而CD14可作为革兰氏阴性菌LPS受体参与大肠杆菌免疫反应。本项目拟在前期研究基础上，利用外显子捕获载体pSPL3验证候选SNPs是否参与CD46-AS和CD14-AS的生成,利用RT-PCR、RT-qPCR、Western Blot、原核表达、免疫荧光、真核表达和攻毒实验等技术，研究异常剪切体CD46-AS在乳腺不同生理状态下的差异表达、细胞定位及抗链球菌的作用；解析CD46和CD14基因表达调控的分子机制及其在乳腺炎抗性中的作用。

Bovine mastitis is the most complex disease mostly caused by pathogens and it causes great losses in dairy farming. It is an inflammation of dairy mammary glands mainly in response to pathogens such as Streptococcus, Escherichia coli and Staphylococcus aureus. Recently, using genetic selection of related resistance genes to improve dairy cow mastitis resistance is considered to be the best long-term strategy to solve the problem..Previously, we found that the differential expression and aberrant splice variants of CD46 and CD14 genes in the mammary gland tissues between healthy and mastitis-infected cows using transcriptome sequencing.It is reported that CD46, a ubiquitous cell surface pathogen receptor, can bind Streptococcus to trigger cell autophagy, which is a critical step to control infection; while CD14 as the receptor of Gram-negative bacterium LPS is involved in the Escherichia coli immunity. Abundant expression of splice variant CD46-AS and CD14-AS in mammary tissues has been confirmed using the RT-PCR, qRT-PCR and clone sequencing methods. Therefore, they maybe play roles in the mammary gland infection of pathogens. As a prominent mechanism for gene posttranscriptional regulation, alternative splicing is of significance in cell development, differentiation, physiologic function and pathogenesis and has been reported as many critical roles in human diseases. Interestingly, we also found a single nucleotide polymorphism (SNP) in CD46 gene and CD14 genes,respectively, which is separately located in the exon splicing enhancer (ESE) and branch site motif and maybe cause the production of splice variant CD46-AS and CD14-AS..Based on the previous experiment findings and other reports regarding the CD46 and CD14 genes, we hypothesize that CD46-AS splice variant acts as important role in the resistance of dairy cow mastitis caused by Streptococcus, and candidate functional SNPs may be the reason producing the CD46-AS and CD14-AS splice variants. So as to testify the hypothesis, we construct CD46-AS prokaryotic and eukaryotic expression systems to analyze its expression, tissue position and antibacterial activity in dairy cow mammary gland tissues; we also introduced pSPL3 plasmid, the exon capturing vector, to verify the relationship between the SNPs and splicing mechanism..These experiments will make sure the role of CD46-AS in the mastitis resistance, and certify the SNP function in splicing mechanism. The study will provide evidence that the alternative splicing of CD46 and CD14 has important roles in the infection process of mammary tissues,and new ideas for molecular breeding in dairy farming.