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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 mouse BTLA lentivirus expression vector and identify its expression product. Methods The plasmid pET-28a-mBTLA was used as a template to construct pMD18-T-mBTLA plasmid by PCR. The full-length coding sequence of mouse BTLA gene was cloned into lentiviral vector and transfected into 293T cells by lipofectamine BTLA Lentiviral Particles. Recombinant plasmids were identified by PCR and gene sequencing. The morphological changes of 293T cells infected by recombinant lentivirus were observed by fluorescence microscopy. The expression of BTLA in 293T cells infected with recombinant lentivirus was identified by RT-PCR and Western blot. 50% tissue culture infection dose method (TCID50 method) detection of recombinant lentivirus titer. Results The constructed pMD18-T-mBTLA plasmid and pSL6-mBTLA plasmid showed a size of about 1 kb after double enzyme digestion electrophoresis, which was consistent with the size of mB-TLA coding sequence. Gene sequencing and alignment analysis further confirmed that the mBTLA coding sequence was successfully integrated into the plasmid vector. PCR amplification of virus supernatants and morphological observation of 293T cells confirmed that Lenti-mBTLA lentivirus packaging was successful. The titer of Lenti-mBTLA lentivirus was 1.3 × 108pfu / mL by TCID50 method. The Lenti-mBTLA lentivirus could effectively express mBTLA mRNA and protein by RT-PCR and Western blot. Conclusion The lentivirus expressing mouse BTLA was successfully constructed.