摘要
利用PCR技术克隆截短型HPV5 8L1基因并重组入杆状病毒表达系统穿梭质粒pFastBac 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BacmidDNA ,并转染Sf 9昆虫细胞 ,收集被转染的Sf 9细胞 ,提取细胞蛋白 ,SDS PAGE检测可见在大约 5 8Kda处出现一新生蛋白条带 ,Westernblot证实为HPV5 8L1蛋白。用ProBondTM 纯化系统纯化所表达的蛋白。小鼠红细胞凝集试验证实纯化的蛋白可介导小鼠红细胞凝集 ,透射电镜观察证实纯化蛋白可自组装成VLP。结果表明昆虫杆状病毒表达系统可高效表达截短型HPV5 8L1蛋白 ,纯化后的截短型HPV5 8L1蛋白在体外可自组装VLP 。
To prepare carboxyl terminus truncated human papillomavirus type 58 L1 protein,and study on its in vitro bioactivity. PCR was used to amplify carboxyl terminus truncated HPV 58L1 gene, the product was inserted into the PUCMT cloning vector, preparing recombinant PFastBacHTb containing carboxyl terminus truncated HPV 58L1 gene. Further more,the recombinant plasmid PfastbacHTb was used to transform DH10Bac cells,constructing recombinant Baculovirus,then the recombinant virus was successfully used to infect Sf 9 insect cells. After incubating at 27℃ for 72 hours, the infected cells were collected and total cellular proteins were extracted. The target protein with MW 58KD was revealed by SDS PAGE and confirmed by Western blot.The interested protein was purified by ProBond TM purification system. The purified interested protein was identified to self assemble into VLPs by Transmission electron microscope, and induce murine erythrocyte hemagglutination,indicating that the given proteins had the conformation of VLPs,collecting,HPV58 L1 proteins with carboxyl terminus truncation could be efficiently expressed in baculovirus Sf 9 cells expression system, it has identical in vitro bioactivity to the wild type HPV58 L1,The present study is fundmental for preparing HPV58 L1 prophylactic vaccine.
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第4期536-539,共4页
Chinese Journal of Biotechnology
基金
国家自然科学基金资助项目 (No .3 0 2 71184)~~
关键词
截短型HPV58
L1蛋白
昆虫-杆状病毒表达系统
蛋白纯化
病毒样颗粒
Hpv58 L1protein of carboxyl terminus truncation, baculovirus expression system, protein purification, virus-like particles
