CAJ | 학술논문

目的体外快速繁殖人巨细胞病毒（ HCMV）。方法用试剂盒抽提、纯化重组HCMV细菌人工染色体（BAC），以重组HCMV细菌人工染色体（BAC-HCMV）为模板扩增UL82基因，酶切后连接至pcDNA载体，构建pcDNA-pp71重组质粒，转化感受态宿主菌，抽提及鉴定重组质粒。通过电穿孔技术将BAC-HCMV与鉴定成功的pcDNA-pp71重组质粒共转染人包皮成纤维细胞，通过多重PCR法及观察细胞病变效应、绿色荧光蛋白等方法鉴定培养后获得的HCMV。结果（1）获得pcDNA-pp71重组质粒，经PCR及双酶切后，片段大小与预期相符，进一步测序鉴定成功。（2） BAC-HCMV与pcDNA-pp71共转染人包皮成纤维细胞，出现细胞病变效应，经荧光显微镜观察可见绿色荧光。（3）以培养出的病毒DNA为模板进行多重PCR扩增，获得两条与预期大小相符的条带，鉴定HCMV体外繁殖成功。结论应用BAC技术成功在体外快速繁殖HC-MV，为HCMV研究提供实验材料，也为进一步研究HCMV分子机制奠定基础。
목적체외쾌속번식인거세포병독（ HCMV）。방법용시제합추제、순화중조HCMV세균인공염색체（BAC），이중조HCMV세균인공염색체（BAC-HCMV）위모판확증UL82기인，매절후련접지pcDNA재체，구건pcDNA-pp71중조질립，전화감수태숙주균，추제급감정중조질립。통과전천공기술장BAC-HCMV여감정성공적pcDNA-pp71중조질립공전염인포피성섬유세포，통과다중PCR법급관찰세포병변효응、록색형광단백등방법감정배양후획득적HCMV。결과（1）획득pcDNA-pp71중조질립，경PCR급쌍매절후，편단대소여예기상부，진일보측서감정성공。（2） BAC-HCMV여pcDNA-pp71공전염인포피성섬유세포，출현세포병변효응，경형광현미경관찰가견록색형광。（3）이배양출적병독DNA위모판진행다중PCR확증，획득량조여예기대소상부적조대，감정HCMV체외번식성공。결론응용BAC기술성공재체외쾌속번식HC-MV，위HCMV연구제공실험재료，야위진일보연구HCMV분자궤제전정기출。
Objective To rapidly propagate human cytomegalovirus in vitro.Methods BAC-HCMV were extracted and purified by using a plasmid DNA purification kit .The HCMV UL82 gene fragments were amplified from the template BAC-HCMV by polymerase chain reaction ( PCR) and digested by BamHⅠand XhoⅠwhich were also used on vector pcDNA3.1(+).The digested UL82 gene fragments and pcDNA3.1 were connected by T4 ligase, forming the combinant plasmid pcDNA-pp71 which was transformed into competent E .coli DH5α.After amplifying in Amp + LB liquid medium , combinant plasmid pcDNA-pp71 was extracted , purified and confirmed by DNA sequencing .Human foreskin fibroblast ( HFF ) cells were grown in Dulbecco′s modified Eagle′s medium ( DMEM ) supplemented with 10%fetal calf serum and cotransfected with bacterial artificial chromosome-human cytomeglovirus ( BAC-HCMV) and pcDNA-pp71 by electroporation .By multiplex PCR method and observation of cytopathic effect (CPE),green fluorescent protein and other methods were used to identify human cytomegalovirus .Results The combinant plasmid pcDNA-pp71 was successfully constructed , confirmed by PCR , the digestion of enzymes and sequencing .CPE and green fluorescence were observed under microscope after the HFF cells were cotransfected with BAC-HCMV and pcDNA-pp71 .The virus produced by the transfected HFF cells was confirmed to be Towne strain by multiple PCR.Conclusions Human cytomegalovirus was rapidly propagated in vitro as a result of electroporation , providing essential material and foundation for further research on molecular mechanism of HCMV .