| pIRES2-HPV18E2-EGFP质粒重组及其在HeLa细胞中的表达 |
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| 引用本文： | 汪云,李力,李元朝,吕凤林.pIRES2-HPV18E2-EGFP质粒重组及其在HeLa细胞中的表达[J].第三军医大学学报,2006,28(12):1280-1282. |
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| 作者姓名： | 汪云 李力 李元朝 吕凤林 |
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| 作者单位： | 1. 第三军医大学大坪医院野战外科研究所妇产科,重庆,400042
2. 第三军医大学大坪医院野战外科研究所第一研究室,创伤、烧伤与复合伤研究室,重庆,400042 |
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| 摘 要： | 目的构建人乳头瘤病毒18型E2基因片段(HPV18E2)重组表达质粒并予以表达,为进一步研制HPV18相关疾病提供材料.方法以重组质粒(pBR322-HPV18)为模板,PCR方法扩增HPV18E2DNA片段,将HPV18E2DNA与加强绿色荧光质粒(pIRES2-EGFP)重组构建重组质粒(pIRES2-HPV18E2- EGFP).用酶切电泳及测序检查质粒重组后序列正确性.重组质粒转染Hela细胞.RT-PCR鉴定转染细胞中E2基因.结果 PCR扩增DNA片段约为1 kb,与预期结果相同.克隆重组质粒pIRES2-HPV18E2- EGFP酶切后显示的酶切图谱与预期相同,而且测序验证插入片段全序列无改变.转染并用G418筛选后,在荧光显微镜下可见绿色荧光细胞的表达.转染细胞RT-PCR出现特异性条带.结论成功构建表达HPV18 E2蛋白的靶细胞模型.
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| 关 键 词： | 人乳头瘤病毒18型 E2基因 基因重组 转染 |
| 文章编号： | 1000-5404（2006）12-1280-03 |
| 收稿时间： | 2005-12-12 |
| 修稿时间： | 2005-12-122006-04-12 |
Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells |
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| WANG Yun,LI Li,LI Yuan-chao,LU Feng-lin.Construction of recombinant plasmid PIRES2-HPV18-EGFP and its expression in HeLa cells[J].Acta Academiae Medicinae Militaris Tertiae,2006,28(12):1280-1282. |
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| Authors: | WANG Yun LI Li LI Yuan-chao LU Feng-lin |
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| Institution: | 1 Department of Gynaecology and Obstetrics; 2 State Key Laboratory of Trauma, Burns and Combined Injury, Department 1, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China |
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| Abstract: | Objective To construct a recombinant plasmid for expressing the HPV18 E2 gene and develop some materials for HPV18 related disease. Methods The E2 gene of HPV18 was amplified by PCR from PBR322-HPV18 and cloned into PIRES2-EGFP. The sequence of cloned HPV18E2 was confirmed by restriction analysis and DNA sequencing, then transfected into HeLa cells. E2 gene was detected by RT-PCR. Results HPV18 E2 gene fragment was amplified by PCR and ligated to PIRES2-EGFP, then transfeeted into HeLa cells successfully. The expression of green fluorescence cells was observed by fluorescence microscopy. Specific band could be seen in transfeeted cells by RT-PCR. Conclusion Recombinant plasmid PIRES2-EGFP is effectively expressed after being transfected into HeLa cells in vitro. |
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| Keywords: | human papillomavirus type 18 E2 gene plasmid construction gene transfection |
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