猪精子与慢病毒作用机制的探讨  

Mechanism exploration of interaction of porcine sperm and lentivirus

作　　者：周均云 习欠云[1] 关立增[2] 江青艳[1] 吴珍芳[1] 张永亮[1] 

机构地区：[1]华南农业大学动物科学学院/国家生猪种业工程技术研究中心,广东广州510642 [2]延边大学农学院动物科学学院,吉林延吉133000

出　　处：《中国兽医学报》2014年第4期658-663,共6页Chinese Journal of Veterinary Science

基　　金：转基因生物新品种培育科技重大专项重点资助项目(2014ZX08009-048B);转基因生物新品种培育科技重大专项重大课题资助项目(2014ZX08006-004)

摘　　要：在制备转基因动物的方法中,精子载体法(与质粒结合)因操作简单而备受关注,但整合效率低,遗传稳定性较差,而慢病毒载体具有良好的感染效率和整合效果,慢病毒与精子孵育制备转基因动物是转基因方法的有益尝试。本研究采用这一新方法成功制备出转基因猪。为了探索精子与慢病毒的相互作用机制,首先制备慢病毒,经梯度稀释法测定其滴度为1.2×105 ifu/mL。然后,用DNA酶消化去除慢病毒液中载体质粒,将慢病毒液与精子在17℃下共孵育2h后,再用RNA酶消化慢病毒颗粒,对精子进行RT-PCR、PCR和FISH检测。结果提示,慢病毒颗粒可能进入精子中。该发现为这一转基因新方法的建立提供了理论依据。Sperm mediated transgenic method is highly concerned in transgenic animal preparation, due to its easy operation and low cost. But the method is limited by its unstability and low integra- tion. Lentivrus has high infection rate and integration, and thus is considered to be an efficient transgenic tool. Our group has been succesful in preparing transgenic pigs by using incubation of lentivirus and sperm. To explore the interaction between pig sperm and lentivirus, in the present study,we prepared lenitirus particles by cotransfection of four kinds of plasmid,and the titer was 1.2×105 ifu/mL. After digestion with DNAase to remove plasmid DNA,lentivirus was incubated with pig sperm. DNA and RNA were extracted from the sperm,and RT-PCR and PCR were used to detect target gene in the lentivirus. FISH was also involved to detect antisens strand of lentivir- us in sperm. Results showed lentivirus may possibly enter into the sperm and retro-transcriped to DNA strand. The findings here are helpful to understand interaction mechenism between sperm and lentivirus.

关 键 词：精子 转基因猪 慢病毒 作用机制 

分 类 号：S828.3[农业科学—畜牧学]