摘要
目的探讨原核表达HPV16L1蛋白的变性、纯化及复性条件,并对复性效果进行评价。方法将重组质粒pET28a-L1转化E.coliBL21(DE3),经放大培养诱导表达后,收集菌体,超声破碎后提取包涵体,并对其进行洗涤、变性,离子交换层析纯化,然后稀释复性。经电镜观察、红细胞凝集试验以及免疫原性分析,对复性效果进行评价。结果L1蛋白在8mol/L尿素中溶解不完全,SDS、硫脲和高pH值可提高其溶解度;纯化后的L1蛋白纯度可达90%;电镜可观察到VLP,并能凝集小鼠红细胞。免疫家兔后,可产生高滴度的抗体。结论复性的HPV16L1蛋白在体外可自我折叠形成具有正确空间构象的VLP,且具有较好的免疫原性,为进一步研制HPV16预防性疫苗及诊断试剂盒奠定了基础。
Objective To investigate the condition for denaturation, purification and renaturation of human papillomavirus type 16 （HPV16）L1 protein and evaluate the renaturation efficacy. Methods Recombinant plasmid pET28a-L1 was transformed to E. coli BL21 （DE3）for scaled-up culture, induction and expression. The harvested bacteria was ultrasonicated, from which inclusion bodies were extracted, washed, de-naturalized and purified by ion exchange chromatography, then re-naturalized by dilution. The renaturation efficacy was evaluated by electron microscopy, red cell agglutination test and analysis on immunogenicity. Results L1 protein was incompletely dissolved in 8 mol / L urea, while SDS, thiourea and high pH value increased the solubility. L1 protein reached a purity of 90% after purification, and showed agglutination reaction with mouse red cells. Virus-like particles （VLPs）were observed by electron microscopy. High antibody titers were induced in the rabbits immunized with L1 protein. Conclusion The renaturalized HPV16 L1 protein formed VLPs with correct spatial conformation by self-folding in vitro and showed good immunogenicity, which laid a foundation of further development of prophylactic HPV16 vaccine and diagnostic kit for HPV16.
出处
《中国生物制品学杂志》
CAS
CSCD
2009年第12期1203-1205,1209,共4页
Chinese Journal of Biologicals
关键词
人乳头瘤病毒16型
L1蛋白
包涵体
变性
纯化
复性
病毒样颗粒
Human papillomavirus type 16
L1 protein
Inclusion body
Denaturation
Purification
Renaturation
Virus-likeparticles
