乙肝核心抗原病毒样颗粒呈现HPV 16L1抗原表位及特异抗体诱导

发布时间：2018-06-01 08:21

本文选题：人乳头瘤病毒 + 表位　；参考：《中国生物工程杂志》2017年03期

【摘要】：目的:构建呈现HPV 16L1抗原表位的病毒样颗粒(VLPs),为新型HPV疫苗及特异抗体制备提供新的思路。方法:将编码HPV 16L1抗原表位QPLGVGISGHPLLNKLDDTE寡聚核苷酸片段克隆于HBc Ag基因编码第78、79位氨基酸序列之间,重组质粒转化大肠杆菌DH5α。重组蛋白经IPTG诱导后以SDS-PAGE分析表达情况,并以Western blot鉴定重组蛋白中HPV 16L1表位的免疫反应性。菌体超声破碎后经硫酸铵盐析法和蔗糖密度梯度离心进行纯化,并经凝胶层析Sepharose G25脱盐,最后以电子显微镜及高效液相(HPLC)凝胶过滤色谱鉴定VLPs的存在并分析纯度。纯化的病毒样颗粒分别于0周、2周、4周经皮下注射免疫BALB/c小鼠,以Western blot分析血清特异识别L1蛋白的能力。结果:HBc Ag/L1肽嵌合蛋白获得成功表达,能够被商业化L1抗体特异识别,经密度梯度超速离心及HPLC分析显示其与HBc Ag行为一致,电子显微镜观察进一步确定其以HBc Ag病毒样颗粒形式存在。VLPs免疫小鼠获得的抗血清能够特异识别酵母表达的重组L1蛋白。结论:HBc Ag VLPs成功呈现HPV16L1蛋白并有效激发特异抗体应答。
[Abstract]:Aim: to construct the virion like particles of HPV16 L1 epitope and to provide a new idea for the preparation of new HPV vaccine and specific antibodies. Methods: the QPLGVISGHPLLNKLDDTE oligonucleotide fragment encoding HPV16L1 epitope was cloned into the amino acid sequence of HBC Ag gene and transformed into Escherichia coli DH5 伪. The expression of recombinant protein was analyzed by SDS-PAGE after induced by IPTG, and the immunoreactivity of HPV16 L1 epitope was identified by Western blot. The bacteria were purified by ammonium sulfate salting out method and sucrose density gradient centrifugation, and desalted by gel chromatography Sepharose G25. Finally, the existence and purity of VLPs were identified by electron microscope and high performance liquid chromatography (HPLC) gel filtration chromatography. The purified virus-like particles were injected subcutaneously into BALB / c mice at 0 week and 2 weeks and 4 weeks, respectively. The ability of serum specific recognition of L1 protein was analyzed by Western blot. Results the 1: HBc AgP / L1 peptide chimeric protein was successfully expressed and could be specifically recognized by commercial L1 antibody. The results of density gradient ultracentrifugation and HPLC analysis showed that the chimeric protein was consistent with the behavior of HBc Ag. It was further confirmed by electron microscopy that the antiserum obtained by immunizing mice with HBc-Ag virus-like particles could specifically recognize recombinant L1 protein expressed by yeast. Conclusion the HPV16 L1 protein was successfully present in the VLPs of 10% HBc Ag and the specific antibody response was effectively stimulated.
【作者单位】：中国医学科学院/北京协和医学院医学生物学研究所;
【基金】：云南省对外科技合作计划项目(2013IA005) 云南省应用基础研究计划重点项目(2016FA049) 云南省应用基础研究面上项目(2013FZ137、2010ZC232)资助项目
【分类号】：R392

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