Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients ...Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.展开更多
To investigate the functions of HPV-16 E6E7 protein in the development of cervical cancer,a recombinant plasmid pEGFP-HPV16E6E7,containing green fluorescent protein and HPV-16 E6E7 proteins was constructed and was tra...To investigate the functions of HPV-16 E6E7 protein in the development of cervical cancer,a recombinant plasmid pEGFP-HPV16E6E7,containing green fluorescent protein and HPV-16 E6E7 proteins was constructed and was transfected into HPV-negative cell line C33A mediated by liposome.The efficiency of tranfection was determined by inverted fluorescent microscope and the expression of E6E7 was detected by Western blot.It showed that transfection of C33A cell and Caski cell by HPV-16 E6E7 gene established a pair of transfected cell types to study the carcinogenesis of E6 and E7 proteins.展开更多
基金Supported by National Natural Science Foundation of China(No.3 970 0 13 1)
文摘Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.
文摘To investigate the functions of HPV-16 E6E7 protein in the development of cervical cancer,a recombinant plasmid pEGFP-HPV16E6E7,containing green fluorescent protein and HPV-16 E6E7 proteins was constructed and was transfected into HPV-negative cell line C33A mediated by liposome.The efficiency of tranfection was determined by inverted fluorescent microscope and the expression of E6E7 was detected by Western blot.It showed that transfection of C33A cell and Caski cell by HPV-16 E6E7 gene established a pair of transfected cell types to study the carcinogenesis of E6 and E7 proteins.
文摘目的利用人乳头瘤病毒(Human papillomavirus,HPV)58突变型E6E7(Mutant type E6E7,mE6E7)融合基因为靶点,巨细胞病毒株(Towne株)细菌人工染色体(SW102 Towne bacterial artificial chromosome,SW102-T-BAC)为载体,构建携带HPV58 mE6E7融合基因的重组病毒,探讨HPV58mE6E7的转化活性。方法PCR扩增带有50 bp Towne开放读码框(open reading frame,ORF)75左右同源臂的GalK、mE6E7及野生型E6E7(Wild type E6E7,wE6E7)融合基因片段,切胶回收纯化;将GalK片段电转到(SW102-T-BAC)感受态细胞,经过同源重组、GalK及氯霉素抗性筛选,获得SW102-T-ORF75-Galk-BAC克隆;然后分别将纯化的mE6E7及wE6E7电转到SW102-T-ORF75-Galk-BAC感受态细胞,经脱GalK及氯霉素抗性筛选,获得SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC克隆;提取所得克隆质粒DNA,转染ARPE-19细胞(以转染SW102-T-BAC的细胞及未转染细胞为对照),逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达状况;观察重组病毒T-ORF75-mE6E7及T-ORF75-wE6E7对转染ARPE-19细胞的影响,通过软琼脂克隆分析mE6E7及wE6E7转化活性。结果获得了SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC的克隆。逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达正确。转染SW102-T-ORF75-wE6E7-BAC的细胞生长失去接触抑制,出现重叠生长现象,其形态由原来梭形变为变圆、肿胀、胞浆颗粒增多;并且在软琼脂中能够形成克隆;而转染SW102-T-ORF75-mE6E7-BAC、SW102-T-BAC及未转染细胞未出现上述细胞的特点。结论获得了T-ORF75-mE6E7及T-ORF75-wE6E7重组病毒,并且重组病毒T-ORF75-mE6E7的转化活性已消除,为HPV58型治疗性疫苗的研究奠定了基础。
