目的:制备人乳头状瘤病毒(human papilloma virus,HPV)18型阳性的肿瘤疫苗,并观察其体外活性。方法:利用昆虫杆状病毒(简称Bac to Bac)表达系统,将HPV18L1基因重组入穿梭质粒pFastBac-Htb,构建HPV18L1-Htb,通过转座反应,将目的基因片段重组入杆状病毒基因组,分离重组的Bacmid DNA,并转染Sf-9昆虫细胞进行表达;透射电镜观察病毒样颗粒(virus-like particle,VLP)的形成;用Ni-NTA系统纯化表达蛋白;以小鼠红细胞凝集试验鉴定蛋白生物活性。结果:收集被转染的Sf-9细胞,提取细胞蛋白,SDS-PAGE检测在相对分子质量大约63000处可出现一新生蛋白条带,Western blotting证实为HPV18L1蛋白;透射电镜观察证实L1蛋白可自我组装成VLP,且主要定位于细胞核;小鼠红细胞凝集试验证实纯化的蛋白在0.5～4ng/μl范围可介导小鼠红细胞凝集。结论:Bac to Bac表达系统可高效地制备HPVVLP,并具有体外生物学活性;Ni-NTA系统能高效简便地纯化带有6×His短肽的HPV18L1蛋白。
Telomerase activity was examined in invasive cervical carcinoma to assess whether it is activated during cervical malignant transformation and to look for its possible association with human papillomavirus （HPV） infection. Histologically confirmed invasive cervical carcinomas and benign cervices were assayed for telomerase activity by using a modified telomere repeat amplification protocol （TRAP）. The same cases were subjected to polymerase chain reaction （PCR） detection of HPV by using consensus primers and type-specific （HPV types 16 and 18） primers. Telomerase activity was detected in 40 of 45 （88.9%） invasive cervical carcinomas and 2 （all chronic cervicitis） of 50 （4%） benign cervical lesions. HPV was detected in 36 （24 HPV-16 and 4 HPV-18 cases） of 45 （80%） invasive cervical carcinomas and 20 （11 HPV-16 and 1 HPV-18 cases） of 50 （40%） benign cervical changes. There was a significant correlation between the expression of telomerase with histological grade （φ=0.44, P〈0.005）, but no correlation was found between telomerase expression and HPV-18 （P〉0.05）. Although larger sample studies are needed, there seems to be a clear association between telomerase upregulation and HPV status, mainly HPV-16 infection.
By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...
