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目的:扩增出单纯疱疹病毒I型(HSV-I)Stocker株胸苷激酶(tk)基因,并将其克隆到真核表达质粒中,构建一个含有tk基因片段的高效真核表达载体.方法:根据已发表的HSV-I CL101株tk基因的核苷酸序列,设计并合成了一对引物,以HSV-I Stocker株核酸为模板,进行PCR扩增,并将扩增产物连接到pUCll9中,进行序列分析,将此基因进一步克隆到含巨细胞病毒极早期启动子的真核表达质粒pCR3-Uni中,并对重组子进行酶切鉴定.结果:PGR扩增出1.427Kb大小的片段,通过酶切和序列分析证明含完整的tk基因序列,此序列与CL101株tk基因的同源性为98.5%.酶切证实了构建的真核表达载体株的同源性很高,重组子pCR3-tk含tk基因并获得了正向重组子.结论:克隆的HSV-I Stocker株tk基因与CL101株的同源性很高,重组于pCR3-tk是一种高效真核表达载体,这为今后应用非病毒载体特别是脂质体进行基因转导来研究HSV-tk/CCV系统在肿瘤基因治疗中的应用打下了基础.
Objective: To amplify thymidine kinase (tk) gene of herpes simplex virus type I (HSV-I) Stocker strain and clone it into eukaryotic expression plasmid to construct a highly efficient eukaryotic expression vector containing tk gene fragment. : According to the published nucleotide sequence of the tk gene of HSV-I CL101 strain, a pair of primers was designed and synthesized. The HSV-I Stocker strain nucleic acid was used as a template for PCR amplification, and the amplification product was ligated into pUCll9. The sequence analysis was performed and the gene was further cloned into the eukaryotic expression plasmid pCR3-Uni containing the very early promoter of cytomegalovirus, and the recombinant was subjected to enzyme digestion. Results: The PGR amplified a 1.427 Kb fragment. The complete tk gene sequence was proved by restriction enzyme digestion and sequence analysis. The homology of this sequence to the tk gene of CL101 strain was 98.5%. The homology of the constructed eukaryotic expression vector strain was high and the recombinant pCR3 was confirmed by enzyme digestion. -tk contains the tk gene and obtains the forward recombination. Conclusion: The cloned HSV-I Stocker strain tk gene is highly homologous to the CL101 strain. Recombination with pCR3-tk is an efficient eukaryotic expression vector. In the future, non-viral vectors, particularly liposomes, will be used for gene transduction to study HSV-tk/CCV systems. Application of gene therapy laid the foundation.