title: [Comparison of the rescue efficiency of Sendai virus minigenome mediated by CMV and T7 promoter].
abstract: In this study, we constructed the plasmid of Sendai virus (SeV) BB1 strain minigenome with Gaussia luciferase (Gluc) as reporter and compared the rescue efficiency of SeV minigenome mediated by T7 promoter with that by CMV promoter. Firstly, the sequence was designed and synthesized which contained hammerhead ribozyme, sequence composed of the trailer, untranslated region of L gene, untranslated region of N gene, and the leader from SeV, and mutant hepatitis delta virus ribozyme sequence. Then, the synthesized sequence was inserted into pVAX1 containing CMV and T7 promoters and the general vector for SeV minigenome pVAX-miniSeV was obtained. Furthermore, pVAX-miniSeV-Gluc (+) and pVAX-miniSeV-Gluc(-) were obtained by inserting Gluc gene into pVAX-miniSeV. From the supernatant of BHK-21 cell transfected with pVAX-miniSeV-Gluc(+), high level of Gluc expression was detection indicating the normal transcription function of CMV promoter. pVAX-SeV-miniGluc(-) and plasmids expressing N,P and L protein of SeV were co-transfected into BST T7/5 cell which derived from BHK-21 and expressed T7 RNA polymerase stably. And high expression of Gluc was found, which indicated that SeV minigenome was efficiently rescued. However, we failed to repeat the result on BHK-21 cell, implying that T7 promoter and CMV promoter may have different effects on the rescue efficiency of SeV minigenome. Therefore, we further constructed SeV minigenome vectors pT7-miniSeV-Gluc (-) and pCMV-miniSeV-Gluc(-) with single promoter of T7 or CMV. Then, these vectors were transfected into BSR T7/ 5 cells respectively accompanied with the N, P, and L protein expression vectors. The result demonstrated that high expression of Gluc was found in the group of pT7-miniSeV-Gluc(-), which failed in the group of pCMV-miniSeV-Gluc(-). It indicated that T7 promoter significantly increased the rescue efficiency of SeV minigenome. We successfully constructed a SeV minigenome vector with secreted luciferase gene as report er and proved T7 promoter can enhance the rescue efficiency of SeV minigenome, which provides basis for construction of infectious clone containing SeV full-length genome.
authors: Department of Microbiology, Harbin Medical University, Harbin 150081, China.
authors: Guo-Chao
authors: GC
authors: Wei
authors: Wen-Hong
authors: WH
authors: Tian
authors: Gang
authors: G
authors: Wang
authors: Yun-Fan
authors: YF
authors: Liu
authors: Chi-Jie
authors: CJ
authors: Yu
authors: Xiao-Yan
authors: XY
authors: Dong
authors: Hong
authors: H
authors: Ling
authors: Xiao-Bing
authors: XB
authors: Wu
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D016679:Genome, Viral; D006801:Humans; D011401:Promoter Regions, Genetic; D010253:Respirovirus Infections / Q000821:virology*; D029082:Sendai virus / Q000235:genetics* / Q000502:physiology; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases