猪繁殖与呼吸综合症病毒结构蛋白GP5-M腺病毒载体的构建及其在山羊乳腺中的表达
Construction of PRRS GP5-M Adenovirus Vector and Its Expression in Goat Mammary Gland

猪繁殖与呼吸综合症（PRRS）是严重威胁养猪业的病毒性疾病，疫苗免疫是预防该病的最有效途径，PRRSV糖蛋白GP5和基质蛋白M融合后制成的基因工程疫苗能够有效激发机体产生免疫应答。通过在 GP5和M基因的5′ 端依次加上组氨酸标签序列、信号肽序列，利用Linker(GP)序列将 GP5和M基因连接构建融合表达GP5和M蛋白的重组质粒pMD18T-SignalP-His- GP5-M。以该质粒为模板，构建重组腺病毒载体并包装腺病毒。结果表明，用包装的腺病毒感染乳腺上皮细胞，测得最佳感染复数(MOI)为25，定量PCR和Western blot方法均检测到 GP5-M的表达。大量扩增腺病毒并使用不连续氯化铯浓度梯度离心纯化病毒，使用纯化的病毒灌注山羊乳腺，采用Western blot在收集的乳汁中也检测到GP5-M蛋白的表达。结果证实，利用腺病毒载体作为基因转导的媒介，在山羊乳腺中可以表达GP5-M融合蛋白。
Porcine reproductive and respiratory syndrome (PRRS) is a serious threat to the pig industry and vaccination is the most effective way to prevent it. The vaccine manufactured by fusing PRRSV glycoprotein GP5 and matrix protein M can stimulate body's immune response effectively. Through adding the GP5 tag sequence and signal peptide sequence into the GP5 and M gene sequences 5′-end and using the Linker (GP) sequence to connect GP5 and M gene,the recombinant plasmid of pMD18T-SignalP-His- GP5-M that expresses GP5 and M protein was constructed. Using this plasmid as a template,we constructed and packaged recombinant adenovirus which was then used to infect goat mammary epithelial cells. The best MOI was 25,and the expression of GP5-M were detected by real-time quantitative PCR and Western blot. We amplified adenoviruses and use discrete cesium chloride density gradient centrifugation to purify it,and then used purified adenoviruses to perfuse into goat mammary gland. The expression of GP5-M protein was tested in collecting milk by Western blot. The results confirmed that by using adenovirus as a vehicle for gene transfer,the expression of GP5-M fusion protein can be found in goat mammary gland,which laid the foundation for the research of PRRSV-specific genetic engineering vaccine.