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目的探讨共转染与筛选技术获得稳定携带有HPV6全基因细胞的可能性,为HPV6全基因体外培养模型的建立奠定基础。方法质粒p EGFP-1用限制性内切酶切下EGFP段,凝胶电泳,纯化取掉EGFP段的DNA片段,T4 DNA连接酶进行线状DNA的自身环化,转染DH5a细胞,抗生素筛选,获去掉EGFP片段的新质粒p EGFP-△EGFP,多组内切酶酶切鉴定。限制性内切酶切下质粒p SP65-HPV6的HPV6全长基因,纯化后获HPV6全长线性基因。将线性HPV6基因和质粒p EGFP-△EGFP电转法共转染h TERT细胞,抗生素筛选、传代,Southern blot方法检测细胞内HPV6 DNA。结果HPV6基因组DNA转至h TERT细胞,经G418筛选1d,培养皿内可见对G418抗性的阳性细胞克隆出现,其形态与普通h TERT细胞相似。用Southern blot检测细胞内HPV11基因组的存在,传3代后,细胞内依然检测到HPV6 DNA存在。结论 HPV6基因组DNA可通过电转法成功导入h TERT细胞内,通过筛选可获得阳性细胞,HPV6 DNA在细胞内可保持3代以上。
Objective To investigate the possibility of co-transfection and screening to obtain stable genomic cells carrying HPV6, so as to lay a foundation for the establishment of HPV6 whole-gene in vitro culture model. Methods The EGFP fragment of plasmid pEGFP-1 was excised by restriction endonuclease, and the DNA fragment of EGFP fragment was removed by gel electrophoresis. DNA linearized by T4 DNA ligase was self-circularized and transfected into DH5a cells. Antibiotic screening , Get a new plasmid EGFP EGFP fragment removed EGFP, multiple endonuclease digestion. The full-length HPV6 gene of plasmid pSP65-HPV6 was excised by restriction enzyme, and the full-length HPV6 gene was obtained after purification. The hTERT cells were co-transfected by the linear HPV6 gene and plasmid p EGFP- △ EGFP, and the HPV6 DNA was detected by antibiotic selection, passage and Southern blot. Results The HPV6 genomic DNA was transferred to h TERT cells and screened by G418 for 1 day. The positive cells clones showing resistance to G418 were found in the culture dish, and their morphology was similar to that of ordinary h TERT cells. Southern blot was used to detect the presence of intracellular HPV11 genome. After passage 3, HPV6 DNA was still detected in the cells. Conclusion HPV6 genomic DNA can be successfully introduced into h TERT cells by electroporation, and positive cells can be obtained by screening. HPV6 DNA can be maintained in cells for more than 3 generations.