截短型人乳头瘤病毒58型L1蛋白在昆虫细胞中的表达

发布时间：2019-04-03 07:43

【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。
[Abstract]:Aim to construct recombinant baculovirus containing truncated human papillomavirus (human papilloma virus,HPV) type 58 major capsid protein L1 gene using insect baculovirus expression system and express it in Sf9 cells. Methods the sequence of HPV 58 L1 gene was obtained from NCBI. The sequence alignment of HPV 58 L1 gene was carried out by Clustal X2 software, and a relatively conservative target gene sequence was determined. HPV58 L1 gene fragment was synthesized and truncated HPV58 L1 gene was obtained by PCR method, and cloned into p Fast Bac-1 vector to construct recombinant donor plasmid p FB-HPV 58 L1 / 1, which was transformed into E. coli DH10Bac, to construct recombinant Bacmid-HPV 58 L 1 / 1, which was transformed into E. coli DH10Bac, to construct recombinant Bacmid-HPV 58 L 1, which was transformed into E. coli DH10Bac, and cloned into p Fast Bac-1 vector. The first generation (P1) recombinant baculovirus BV-HPV 58 L1 / 1 was obtained by transfection of Sf9 cells. The titer of the recombinant baculovirus was determined by rapid titration after amplification. Sf9 cells were infected with recombinant baculovirus, and HPV 58 L1 / 1 protein was expressed. Western blot analysis and transmission electron microscopy were used to observe the expression of the recombinant baculovirus. Results Bacmid-HPV 58 L1 / 1 was correctly constructed by PCR and sequencing, and the titer of P2 recombinant baculovirus was up to 1.0 脳 108 pfu/ml;. The expression product of HPV 58 L1 / 1 in Sf9 insect cells was detected by Western blot, and the specific reaction band with relative molecular weight of 55 000 was found. The self-assembled L1 protein was observed by electron microscope to form virus-like particles (virus-like particles,VLPs). Conclusion HPV 58 L1 / 1 protein was successfully expressed in insect cells, and the self-assembly of VLPs, laid a foundation for the study of preventive HPV vaccine. [WT5 "HZ] conclusion [WT5" BZ] [WT5 "BZ]
【作者单位】：长春生物制品研究所有限责任公司细胞因子室;吉林大学第二医院呼吸与危重症医学科;
【分类号】：R392

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