论文部分内容阅读
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。
Objective To construct and express the recombinant plasmid of human papillomavirus type 18 E2 gene (HPV18E2) and provide the material for further development of HPV18 related diseases. Methods The recombinant plasmid (pBR322-HPV18) was used as a template to amplify the HPV18E2 DNA fragment by PCR. Recombinant plasmid (pIRES2-HPV18E2-EGFP) was constructed by recombining HPV18E2 DNA and enhanced green fluorescent plasmid (pIRES2-EGFP). The correctness of the sequence after plasmid recombination was checked by restriction enzyme digestion and sequencing. Recombinant plasmids were transfected into Hela cells. E2 gene was identified by RT-PCR in transfected cells. Results The DNA fragment amplified by PCR was about 1 kb, which was the same as expected. The digestion map of the cloned recombinant plasmid pIRES2-HPV18E2-EGFP showed the same expectation as expected, and the sequencing confirmed that there was no change in the entire sequence of the inserted fragment. After transfection and screening with G418, the expression of green fluorescent cells was observed under a fluorescence microscope. RT-PCR transfected cells showed specific bands. Conclusion The target cell model expressing HPV18 E2 protein was constructed successfully.