摘要
目的研究麻疹病毒(measles virus,MV)沪191株载体中,外源基因插入位置对基因表达及病毒复制的影响。方法将新型冠状病毒S1蛋白表达序列克隆至MV反基因组不同基因间隔区(N基因前、P和M基因之间、H和L基因之间),与分别表达T7聚合酶及N、P、L蛋白的辅助质粒共转染293T细胞,转染后细胞裂解上清感染Vero细胞,以获得重组病毒。通过RT-PCR、免疫荧光法、Western blot和ELISA检测S1蛋白,并对重组病毒的增殖特性进行检测。结果S1克隆至MV N基因前转染未得到重组病毒,克隆至P和M基因之间、H和L基因之间成功获得了重组病毒MV-M-S1、MV-L-S1,并可检测到S1蛋白的表达。MV-M-S1的S1表达量高于MV-L-S1,但病毒滴度低于MV-L-S1。结论将外源基因插入MV基因组中不同位置,对基因表达及病毒复制将产生不同影响,为后续MV载体的改造提供了经验和参考。
Objective To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus(MV)vector on gene expression and virus replication.Methods The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome(the upstream of the N gene,between P and M genes,between H and L genes),and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N,P,and L proteins,respectively.The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses.S1 proteins expressed by the recombinant viruses were identified by RT-PCR,indirect immunofluorescence assay,Western blot and ELISA.Growth kinetics of the recombinant viruses were analyzed.Results Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene.Two recombinant viruses,MV-M-S1 and MV-L-S1,were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes,or H and L genes,and could express S1 protein.MV-M-S1 expressed more S1 protein than MV-L-S1,but the titer of MV-M-S1 was lower.Conclusions Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication.This study provided reference for the subsequent construction of MV vector.
作者
苏文浩
任秀秀
赵婷婷
王轶男
李实实
黄秋芳
王晓杰
张晓焕
卫江波
Su Wenhao;Ren Xiuxiu;Zhao Tingting;Wang Yinan;Li Shishi;Huang Qiufang;Wang Xiaojie;Zhang Xiaohuan;Wei Jiangbo(Laboratory of Wei Jiangbo,National Vaccine&Serum Institute,Beijing 101111,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2022年第12期924-930,共7页
Chinese Journal of Microbiology and Immunology
