小鼠白细胞介素IL-35基因慢病毒表达载体的构建及鉴定  

Construction and identification of lentivirus vector expressing the mouse IL-35 gene

作　　者：潘秀和 孙俊[1] 刘超波[1] 高巧艳[1] 李燕[1] 李明才[1] 

机构地区：[1]宁波大学医学院免疫学研究室,浙江宁波315211

出　　处：《生物技术》2016年第1期6-11,共6页Biotechnology

摘　　要：[目的]构建小鼠白细胞介素IL-35基因慢病毒表达载体并鉴定。[方法]通过PCR法扩增出小鼠IL-35基因片段,将其与经XhoⅠ和XbaⅠ双酶切的慢病毒载体pLVX-IRES-ZsGreen1连接,构建慢病毒重组质粒pLVX-IL-35-IRES-ZsGreen1。用无内毒素试剂盒提取重组质粒后,将其与病毒包装所需的3个辅助质粒共转染到人肾胚(HEK)293T细胞中包装能表达IL-35的慢病毒颗粒。该病毒培养上清经浓缩后,梯度稀释法检测其滴度。用浓缩后的病毒感染HEK293T细胞,然后通过荧光显微镜观察荧光蛋白的表达并结合RT-PCR法检测IL-35基因在该细胞中的表达。[结果]IL-35慢病毒表达载体构建成功,包装的慢病毒滴度为1×109TU/m L,通过荧光显微镜和RTPCR检测发现IL-35在HEK293T细胞中表达。[结论]成功构建IL-35慢病毒表达载体且IL-35蛋白能在HEK293T细胞中过表达。[ Objective ] To construct and identify lentivirus vector expressing the mouse interleukin IL- 35 gene. [ Methods ] The mouse IL- 35 gene fragment was amplified by polymerase chain reaction （PCR） and connected into the lentivirus vector pLVX - IRES - ZsGreenl linearized By Xho [ and Xba ] to construct recombinant plasmids pLVX - IL - 35 - IRES - Zs- Greenl. The constructed plasmids and other three lentivirus - packing plasmids which were extracted by endotoxin free kit were co- transfected into （human embryonic kidney,HEK） 293T cells to package IL -35 lentivirus. The viral titer was tested by gradient dilution method. After HEK293T cells were infected by lentivirus, IL - 35 fluorescent protein and mRNA expression was detected through fluorescence microscope and RT- PCR,respectively. [ Results] Mouse IL- 35 lentivirus vector was suc- cessfully constructed and titer of lentivirus is 1×10^9 TU/mL. Fluorescence observation and RT - PCR showed that IL - 35 pro- tein could be expressed in HEK293T cells. [ Conclusion] Mouse IL -35 lentivirus vector was successfully constructed and IL - 35 protein could be expressed in HEK293T cells.

关 键 词：IL-35 慢病毒 构建 共转染 鉴定 

分 类 号：R392.1[医药卫生—免疫学]