表达人乳头瘤病毒58型L1、L2壳蛋白的非复制型重组痘苗病毒疫苗株的构建

Construction of the Replication-deficient Recombinant Vaccinia Virus Expressing HPV58 L1 and L2 Capsid Proteins

采用PCR定点突变方法 ,对HPV5 81L1基因中痘苗病毒早期基因转录终止信号TTTTTNT结构进行修饰 ,并保留氨基酸不变。选用非复制型重组痘苗病毒为载体 ,将修饰的L1基因 1 5kb和L2基因 1 4kb分别插入痘苗病毒表达载体 pJSD的 7 5k和H6早期启动子之后 ,使之与非复制型重组痘苗病毒在TK区重组。经单斑筛选纯化 ,获得共表达HPV5 8L1、L2晚期蛋白的非复制型重组痘苗病毒疫苗实验株。该病毒在CEF细胞上连续传至第15代 ,经斑点杂交分析 ,重组痘苗病毒基因组中有L1和L2基因插入 ;经Westernblot检测 ,重组病毒能稳定表达HPV5 81L1及L2蛋白。此结果为HPV5

The sequence motifs(TTTTTNT) from the HPV58 L1 gene was removed by amplification method of ovelap extension and maintained the amino acid coding sequence which would otherwise have caused transcription termination when expressed from vaccinia virus early promoter The mutational 1 5kb L1 gene and 1 4kb L2 gene were inserted into vaccinia virus vector pJSD,under the control of vaccinia virus early promoters 7 5K and H6 respectively After CEF cells was transfected with the recombinant plasmid pJSDML1L2LacZ in the presence of infectious NTVTK + vaccinia virus,the recombinant vaccinia virus NTVJmL1L2LacZ was achieved by selection of blue plagues from the white ones DNA hybridization confirmed that the HPV58L1 and L2 genes were integrated into vaccinia virus DNA.Western blot result showed that full-length L1 and L2 proteins were co-expressed in CEF cells infected with the recombinant virus