表达HPV16型结构蛋白的非复制型重组痘苗病毒的构建与鉴定  

Construction and identification of the replication\|deficient recombinant vaccinia virus co\|expressing HPV type 16 L1 and L2 proteins

作　　者：韩立群[1] 任皎[1] 梁雨[1] 田厚文[1] 职慧军[1] 骆卫锋[1] 陆振华 魏兰兰[1] 阮力[1] 

机构地区：[1]中国预防医学科学院病毒学研究所遗传免疫研究室,北京100052

出　　处：《中华实验和临床病毒学杂志》2002年第3期256-260,共5页Chinese Journal of Experimental and Clinical Virology

基　　金：国家"九五"科技攻关重点项目基金资助 (96 90 6 0 1 11)

摘　　要：目的　构建表达人乳头瘤病毒 16型 (HPV16 )结构蛋白的非复制型重组痘苗病毒人用疫苗株。方法　采用聚合酶链反应技术 (PCR)扩增并克隆HPV16主要结构蛋白L1和次要结构蛋白L2基因 ;将经过结构修饰的L1和L2基因插入痘苗病毒表达载体 ,通过与痘苗病毒在宿主细胞中同源重组后 ,筛选共表达L1 L2蛋白的重组痘苗病毒并对其进行鉴定。结果　DNA序列分析证实PCR扩增所获L1和L2克隆基因是正确的 ;斑点杂交结果表明重组病毒基因组中有L1和L2基因插入 ;该重组病毒在人源细胞中不复制或低水平复制 ,但可稳定表达相对分子质量为 5 70 0 0的L1蛋白和相对分子质量为 90 0 0 0的L2蛋白。结论　获得 1株稳定表达HPV16L1 L2蛋白的非复制型重组痘苗病毒人用疫苗株。Objective To generate an HPV16 prophylactic vaccine candidate for cervical cancer. Methods HPV16 major capsid protein L1 gene and minor capsid protein L2 gene were amplified using PCR.These genes were mutated by PCR site\|directed mutagenesis for removal of sequence motifs(TTTTTNT)which would cause transcription termination when expressed from a vaccina virus early promoter,then inserted into a vaccina virus expression vector.A strain replication\|deficient recombinant vaccinia virus containing the mutant sequences was obtained through a homologous recombination and identified. Results The nucleotide sequence remained the correct amino acid sequence of the L1 and L2 proteins after mutated.Full\|length L1 and L2 proteins were generated in cells infected with the recombinant virus.The virus strain propagated at very low titer or could not reproduce in some kinds of cell derived from different human tissues.Conclusion The authors have generated a strain replication\|deficient recombinant vaccinia virus expressing HPV16 L1 plus L2 proteins as an HPV16 prophylactic vaccine candidate for cervical cancer.

关 键 词：宫颈肿瘤 乳头状瘤病毒 子宫颈癌 非复制型重组痘苗病毒 HPV16型结构蛋白 

分 类 号：R392[医药卫生—免疫学]