CAJ | 학술논문

目的研究HPV2变异株上E2蛋白不同功能区域的点突变对其转录作用的影响.方法根据HPV2突变株E2上的突变点设计引物,采用延伸法构建不同的E2突变体,并插入到真核表达质粒pcDNA3.1中.表达不同突变体E2的pcDNA3.1-E2与带有HPV原毒株LCR的CAT报道基因载体进行共转染Hela细胞.通过检测CAT的表达量对不同E2突变体的转录抑制作用进行评估.结果与全长的原毒株E2相比较,去除N端及C端功能区的E2蛋白均降低了E2蛋白对启动子活性的抑制作用.位于E2蛋白转录激活区(nt 3037),铰链区(nt 3387)及DNA结合区(nt 3697)的点突变明显影响了其转录抑制功能.结论 HPV2 E2蛋白的转录调节功能与其转录激活区以及DNA结合区密切相关.HPV2突变株上的不同的E2的单个突变点能够明显降低E2蛋白对启动子活性的抑制作用.
목적 연구HPV2변이주상E2단백불동공능구역적점돌변대기전록작용적영향.방법 근거HPV2돌변주E2상적돌변점설계인물,채용연신법구건불동적E2돌변체,병삽입도진핵표체질립pcDNA3.1중.표체불동돌변체E2적pcDNA3.1-E2여대유HPV원독주LCR적CAT보도기인재체진행공전염Hela세포.통과검측CAT적표체량대불동E2돌변체적전록억제작용진행평고.결과 여전장적원독주E2상비교,거제N단급C단공능구적E2단백균강저료E2단백대계동자활성적억제작용.위우E2단백전록격활구(nt 3037),교련구(nt 3387)급DNA결합구(nt 3697)적점돌변명현영향료기전록억제공능.결론 HPV2 E2단백적전록조절공능여기전록격활구이급DNA결합구밀절상관.HPV2돌변주상적불동적E2적단개돌변점능구명현강저E2단백대계동자활성적억제작용.
Objective To study the potential transcriptionai depression activities of HPV2 E2 proteins with mutations in different functional domains. Methods The primers for constructing various E2 mutants were synthesized based on a HPV2 isolate containing several point mutations within E2 open reading frame. Different E2 mutations were generated by the method of extending PCR and inserted into plasmid pcDNA3. 1. Various recombinant mammalian expression plasmids pcDNA3. 1-E2 were co-transfected into HeLa cells together with a CAT-reporter plasmid pBLCAT-LCR containing HPV-2 prototype LCR, respectively. The transcriptionai repression activities of the E2 mutants were evaluated by detection of CAT expression values. Results Compared with the full-length prototype E2, removals of both N- and C-terminal domains abolished E2 transcriptionai repressive activities. The point mutations in the transactivation domain ( nt 3037 ) , the internal hinge region ( nt 3387 ) and DNA binding domain ( nt 3697 ) showed remarkable inhibition on its transcriptionai depression function. Conclusion The transcriptionai regulation activity of HPV2 E2 is related with its DNA binding and transactivation domains. The exchanges of the single amino acid within E2, derived from a HPV2 isolate, abolish significantly the repressive effect on viral promoter in the context of full-length E2.