小鼠BTLA慢病毒表达载体的构建及鉴定

Construction and identification of mouse BTLA lentiviral expression vector

目的构建小鼠BTLA慢病毒表达载体,并对表达产物进行鉴定。方法以pET-28a-mBTLA质粒为模板,通过PCR技术构建pMD18-T-mBTLA质粒,将小鼠BTLA基因全长编码序列克隆到慢病毒转移载体,通过脂质体转染293T细胞包装成小鼠BTLA慢病毒颗粒。PCR技术和基因测序对重组质粒进行鉴定。荧光显微镜观察重组慢病毒感染293T细胞形态学变化。RT-PCR和Western blot法鉴定BTLA在重组慢病毒感染293T细胞中的表达。50%组织培养感染剂量法(TCID50法)检测重组慢病毒滴度。结果成功构建的pMD18-T-mBTLA质粒和pSL6-mBTLA质粒,经双酶切电泳后均出现大小约为1 kb的条带与mB-TLA编码序列的大小相符。基因测序并比对分析进一步证实mBTLA编码序列成功整合到质粒载体。病毒上清PCR扩增和293T细胞形态学观察证实Lenti-mBTLA慢病毒包装成功。TCID50法测定Lenti-mBTLA慢病毒滴度为1.3×108pfu/mL。RT-PCR和Western blot法证实Lenti-mBTLA慢病毒能有效表达mBTLA mRNA和蛋白质。结论成功构建了表达小鼠BTLA的慢病毒。

Objective To construct and identify a lentiviral vector for mBTLA （ mouse B and T lymphocyte attenuator） expression. Methods The entire coding sequence of mBTLA gene was amplified from pET-28a-mBTLA plasmid, and then mBTLA gene was transferred into pMDlS-T plasmid before cloning into a lentiviral transfer vector. Liposome was used to package lentiviral particles in 293T cells. After lentiviral particles packaging, morphological changes of 293T cells were observed by fluorescence microscope. The recombinant plasmid was identified using RT-PCR and sequencing. Lentiviral titer was detected by 50% tissue culture infectious dose （ TCIDso ） assay. RT-PCR and Western blotting were used to detect mBTLA mRNA and protein expression. Results pMD18-T-mBTLA and pSL6-mBTLA plasmids were successfully constructed and digested for electrophoresis appearing a near 1 kb strip which matched the size of mBTLA coding sequence. Gene se- quencing and alignment analysis further confirmed mBTLA coding sequence to be successfully integrated into the plasmid vector. Morphological observation and supernatant PCR amplification of 293T cells confirmed that Lenti-mBTLA lentiviral packaging was successful, and the Lenti-mBTLA lentiviral titer was 1.3 x 108. pfu/mL. RT-PCR and Western blotting demon- strated that the Lenti-mBTLA vector could effectively express mBTLA mRNA and protein. Conclusion The lentiviral vector which can effectively express mBTLA mRNA and protein has been successfully constructed.