title: [Construction and identification of mouse BTLA lentiviral expression vector].
abstract: 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 pMD18-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 (TCID50;) 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 sequencing 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 1.3×10(8); pfu/mL. RT-PCR and Western blotting demonstrated that the Lenti-mBTLA vector could effectively express mBTLA mRNA and protein.
  
  
  CONCLUSIONS
  The lentiviral vector which can effectively express mBTLA mRNA and protein has been successfully constructed.
authors: Institute of Laboratory Medicine, Guangdong Medical College, Dongguan, China. zengjincheng83@126.com
authors: Jincheng
authors: J
authors: Zeng
authors: Jingjing
authors: J
authors: Bao
authors: Hongmei
authors: H
authors: Wang
authors: Hui
authors: H
authors: Zhang
authors: Junai
authors: J
authors: Zhang
authors: Wandang
authors: W
authors: Wang
authors: Xiangqian
authors: X
authors: Guan
authors: Junfa
authors: J
authors: Xu
mesh_terms: D000818:Animals; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011971:Receptors, Immunologic / Q000235:genetics*; D019562:Viral Load
chemical_list: C488868:BTLA protein, mouse; D011971:Receptors, Immunologic; D004262:DNA Restriction Enzymes