CAJ | 학술논문

目的构建尖锐湿疣患者人乳头瘤病毒6b型晚期基因(HPV6b L1)的双顺反子表达载体,并使其在哺乳动物细胞内表达,以期建立含有HPV6b L1的细胞模型.方法表达质粒pEGFP-HPV6bL1经双酶切纯化后与经过相同双酶切的真核表达质粒pIRES2-EGFP连接,酶切鉴定,挑选阳性克隆进行测序.重组质粒pIRES2-HPV6bL1-EGFP转染进小鼠成纤维细胞(NIH3T3),荧光显微镜下观察EGFP蛋白的表达.RT-PCR检测HPV6b L1 mRNA的生成.结果成功构建含HPV6b L1的重组质粒pIRES2-HPV6bL1-EGFP.重组体成功转染进NIH3T3细胞,并用G418筛选.同时荧光倒置显微镜下可观察到细胞内有绿色荧光蛋白的表达.进一步进行RT-PCR,检测到HPV6b L1 mRNA的生成.结论成功构建携带HPV6b L1的重组体pIRES2-HPV6bL1-EGFP并转染入NIH3T3细胞.经荧光倒置显微镜观察及RT-PCR方法检测证明HPV6b L1在NIH3T3细胞内成功表达.
목적구건첨예습우환자인유두류병독6b형만기기인(HPV6b L1)적쌍순반자표체재체,병사기재포유동물세포내표체,이기건립함유HPV6b L1적세포모형.방법표체질립pEGFP-HPV6bL1경쌍매절순화후여경과상동쌍매절적진핵표체질립pIRES2-EGFP련접,매절감정,도선양성극륭진행측서.중조질립pIRES2-HPV6bL1-EGFP전염진소서성섬유세포(NIH3T3),형광현미경하관찰EGFP단백적표체.RT-PCR검측HPV6b L1 mRNA적생성.결과성공구건함HPV6b L1적중조질립pIRES2-HPV6bL1-EGFP.중조체성공전염진NIH3T3세포,병용G418사선.동시형광도치현미경하가관찰도세포내유록색형광단백적표체.진일보진행RT-PCR,검측도HPV6b L1 mRNA적생성.결론성공구건휴대HPV6b L1적중조체pIRES2-HPV6bL1-EGFP병전염입NIH3T3세포.경형광도치현미경관찰급RT-PCR방법검측증명HPV6b L1재NIH3T3세포내성공표체.
Objective To construct a bicistronic expression vector containing HPV type 6b L1 gene, to express the recombinant vector in mammalian cells, and to establish a cell strain stably expressing HPV6b L1 gene. Methods After endonuclease digestion and purification, the gene fragment of HPV6b L1 was cloned into the eukaryotic expression vector pIRES2-enhanced green fluorescent protein (EGFP). The identification of the recombinant was realized via endonuclease digestion and sequence analysis. Then, the recombinant plasmid pIRES2-HPV6bLl-EGFP was transfected into NIH3T3 (a mouse embryonic fibroblast cell line) cells. Subsequently, the expression of EGFP was observed by fluorescent inverted microscopy, and HPV6b L1 mRNA expression by reverse transcription (RT)-PCR. Results The recombinant plasmid pIRES2-HPV6bLl-EGFP was successfully constructed, transfected into N1H3T3 cells, and selected by G418. The expression of EGFP was seen under an inverted fluorescence microscoy. RT-PCR proved the expression of HPV6b LI mRNA in transfected cells. Conclusions The recombinant plasmid pIRES2-HPV6bLl-EGFP was successfully constructed and transfected into NIH3T3 cells. Inverted fluorescent microscopy and RT-PCR confirmed the successful expression of HPV6b L1 in NIH3T3 cells.