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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 construct a non-replicating recombinant vaccinia virus human vaccine strain expressing human papillomavirus type 16 (HPV16) structural protein. Methods Polymerase chain reaction (PCR) was used to amplify and clone the major structural protein L1 and minor structural protein L2 of HPV16. The L1 and L2 genes were inserted into the vaccinia virus expression vector. After homologous recombination, the recombinant vaccinia virus that co-expressed L1 L2 protein was screened and identified. Results DNA sequence analysis confirmed that the L1 and L2 cloned genes obtained by PCR amplification were correct. Dot blot results showed insertion of L1 and L2 genes in the recombinant virus genome. The recombinant virus did not replicate or replicate in human cells at low level L1 protein with a relative molecular mass of 57000 and L2 protein with a relative molecular mass of 90000 can be stably expressed. Conclusion A non-replicating recombinant vaccinia virus human vaccine strain stably expressing HPV16 L1 L2 protein was obtained.