摘要
目的利用昆虫细胞杆状病毒表达系统构建含截短型人乳头瘤病毒(human papilloma virus,HPV)58型主要衣壳蛋白L1基因的重组杆状病毒,并在Sf9细胞中表达。方法从NCBI中获得HPV 58 L1基因序列,应用Clustal X2软件进行序列比对后,确定相对保守的目的基因序列;人工合成HPV 58 L1基因片段,PCR法获得截短型HPV58 L1-1基因,克隆至p Fast Bac-1载体中,构建重组供体质粒p FB-HPV 58 L1-1,转化大肠埃希菌DH10Bac,构建重组Bacmid-HPV 58 L1-1,转染Sf9细胞,获得第1代(P1)重组杆状病毒BV-HPV 58 L1-1,扩增后采用快速滴定法测定病毒滴度;将重组杆状病毒感染Sf9细胞,表达HPV 58 L1-1蛋白,并进行Western blot分析和透射电镜观察。结果Bacmid-HPV 58 L1-1经PCR及测序鉴定构建正确;P2重组杆状病毒的滴度可达1.0×108 pfu/ml;HPV 58 L1-1在Sf9昆虫细胞中的表达产物经Western blot检测,可见相对分子质量约55 000的特异性反应条带,电镜观察可见L1蛋白自组装形成病毒样颗粒(virus-like particles,VLPs)。结论成功在昆虫细胞中表达了HPV 58 L1-1蛋白,并自组装为VLPs,为预防型HPV疫苗的研究奠定了基础。
Objective To construct a recombinant baculovirus with truncated human papillomavirus 58 L1 gene by using Bac-to-Bac expression system and express in Sf9 cells. Methods HPV 58 L1 gene was collected from NCBI and aligned by Clustal X2 software to obtain the relatively conserverd target gene sequences. The gene was synthesized, based on which truncated HPV58 L1-1 gene was amplified by PCR and cloned into vector p Fast Bac-1. The constructed recombinant plasmid p FB-HPV 58 L1-1 was transformed to E. coli DH10 Bac. The obtained recombinant strain Bacmid-HPV 58 L1-1was transfected to Sf9 cells to obtain the passage 1（P1） of recombinant baculovirus BV-HPV 58 L1-1 which was determined for titer by rapid titration after propogation. The recombinant baculovirus was infected to Sf9 cells, and the ex-pressed HPV58 L1-1 protein was identified by Western blot and transmission electron microscopy. Results PCR and sequencing proved that Bacmid-HPV58L1-1 was constructed correctly. The recombinant baculovirus of P2 reached a titer of1. 0 × 108 pfu / ml. The expressed HPV 58 L1-1 showed a specific band with relative molecular mass of about 55 000 on Western blot profile, and assembled spontaneously into virus-like particles（VLPs）under electron microscope. Conclusion HPV 58 L1-1 was successfully expressed in insect cells and assembled spontaneously into VLPs, which laid a foundation of study on prophylactic HPV vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第3期233-238,244,共7页
Chinese Journal of Biologicals
