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目的克隆食管癌组织和癌旁组织中HPV11型主要衣壳蛋白L1基因,并比较两个序列间的同源性。方法以食管癌组织和癌旁组织纯化的总DNA为模板,根据HPV11型L1基因的保守区分段设计引物。分两段扩增HPVL1基因,克隆入质粒载体中,pGEM-3Zf(-)以双脱氧法双向测定目的片段的序列,并拼接出该片段的全序列,比较两个序列间的同源性,以及与已知基因序列的差异性。结果从1例食管癌患者食管癌组织和癌旁组织中,分别克隆到1株HPV11型L1的编码序列M1和M2,两序列间的同源性极高,仅在个别位点存在差异。与GenBank中登录的HPV序列NC001525.1(HPV-11)、M14119.1(HPV-11)和AF217526.1(HPV-11)相比较大部分相同。结论成功地构建了HPV11型L1基因上游片段pGEM-3Zf(-)和下游片段pGEM-3Zf(-)的重组克隆,为深入研究HPV的感染与食管癌发病机制的关系奠定了基础。
Objective To clone the HPV11 major capsid protein L1 gene in esophageal cancer tissues and adjacent tissues, and to compare the homology between the two sequences. Methods Based on the purified total DNA of esophageal cancer tissues and paracancerous tissues as templates, the primers were designed according to the conserved region of HPV11 L1 gene. The HPVL1 gene was amplified in two sections and cloned into a plasmid vector. pGEM-3Zf(-) was bidirectionally sequenced to determine the sequence of the target fragment and the entire sequence of the fragment was spliced to compare the homology between the two sequences. And differences from known gene sequences. Results From 1 esophageal cancer patient and esophageal cancer tissue, one coding sequence of HPV11 type L1, M1 and M2, was cloned. The homology between the two sequences was extremely high, and there was only difference in individual sites. They are mostly the same as the registered HPV sequences NC001525.1 (HPV-11), M14119.1 (HPV-11) and AF217526.1 (HPV-11) in GenBank. Conclusion The recombinant clones of the HPV11 L1 gene upstream segment pGEM-3Zf(-) and the downstream segment pGEM-3Zf(-) were successfully constructed, which laid a foundation for further study on the relationship between HPV infection and the pathogenesis of esophageal cancer.