摘要
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。
Objective： To prepare human papilloma virus type 18 （HPV 18）-positive tumor vaccine and to examine its in vitro activity. Methods： A Bac to Bac expression system was employed in the present study. HPV18L1 gene was inserted into shuttle plasmid pFastBac-Hth to construct HPV18 L1-Htb; the latter was used to transform DH10Bac cells to construct a recombinant baculovirus to infect Sf-9 insect cells. The protein of interest was identified by observing self-assemble into VLPs under transmission electron microscope and was purified by Ni-NTA purification system. The bioactivity of the purified protein was analyzed by mouse erythrocyte hemagglutination assay. Results： The infected Sf-9 cells were collected and total cellular proteins were extracted. SDS-PAGE assay revealed a roughly 63 000 D protein, which was confirmed to be HPV18 L1 by Western blotting. Transmission electron microscope showed that the proteins formed into VLP by self-assemble in vitro and located mainly in the nuclei. It was found that the protein induced mouse erythrocyte hemagglutination. Conclusion： Bac to Bac expression system can effectively prepare HPV VLP with in vitro bioactivity. The Ni-NTA purification system can purify the protein with 6 × His tag efficiently.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2007年第3期220-224,共5页
Chinese Journal of Cancer Biotherapy
基金
国家重点基础研究发展(973)计划资助项目(No2002CB513100)
国家自然科学基金资助项目(No30672227)~~
