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

doi:10.13523/j.cb.20170308

中国生物工程杂志

2017, Vol. 37

Issue (3): 58-64 DOI: 10.13523/j.cb.20170308

技术与方法

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

孙文佳, 姚宇峰, 杨旭, 黄惟巍, 刘存宝, 龙琼, 褚晓杰, 马雁冰

中国医学科学院/北京协和医学院医学生物学研究所昆明 650118

Presentation of HPV 16L1 Peptide-based HBcAg Virus-like Particle and Induction of Specific Antibody

SUN Wen-jia, YAO Yu-feng, YANG Xu, HUANG Wei-wei, LIU Cun-bao, LONG Qiong, CHU Xiao-jie, MA Yan-bing

Institute of Medical Biology, Chinese Academy of Medical Science & Peking Union Medical College, Kunming 650118, China

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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HBcAg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：HBcAg/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HBcAg行为一致，电子显微镜观察进一步确定其以HBcAg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：HBcAg VLPs成功呈现HPV16L1蛋白并有效激发特异抗体应答。

关键词： 表位; 病毒样颗粒; 人乳头瘤病毒; 乙肝核心抗原; 原核表达

Abstract:

Objective:To construct virus-like particles(VLPs) presenting QPLGVGISGHPLLNKLDDTE epitopes of HPV 16 L1, and detect its antigenic specificity. Methods:The reported effective HPV 16L1 epitopes was selected and used to be presented by HBcAg VLPs. The oligonucleotides encoding for the peptide was inserted into plasmid pHBcAg. The recombinant plasmid was transformed into DH5α cells, and the expression of the chimeric proteins was induced with IPTG and identified by SDS-PAGE. The proteins was purified with a procedure consists of ammonium sulfate precipitation and sucrose density gradient centrifugation, and the presence of VLPs was detected with HPLC of size-exclusion chromatography and electron microscopy. Western blot showed the specific bands of the expressed recombinant protein. Mice were immunized with the mixed VLPs, and the serum to identify HPV 16L1 protein was measured by Western blot. Results:The constructed recombinant plasmid was proven to be correct by restriction enzyme digestion and DNA sequencing. The recombinant protein was expressed efficiently, and presented as VLPs. Western blot showed that the antiserum of VLPs immunizied mice could be recogniazed specifically by the recombinant yeast expression of L1 protein. Conclusion:HBcAg VLPs could present HPV 16L1 epitope and the recombinant HBcAg/16L1 VLPs could stimulate special immune response.

Key words: Human papillomavirus(HPV) Antigenic epitope Hepatitis B virus core antigen (HBcAg) Prokaryotic expression Virus like particle(VLP)

收稿日期: 2016-09-22 出版日期: 2017-03-25

ZTFLH:

Q816

基金资助:

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

通讯作者: 马雁冰 E-mail: may@imbcams.com.cn

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引用本文:

孙文佳, 姚宇峰, 杨旭, 黄惟巍, 刘存宝, 龙琼, 褚晓杰, 马雁冰. 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[J]. 中国生物工程杂志, 2017, 37(3): 58-64.

SUN Wen-jia, YAO Yu-feng, YANG Xu, HUANG Wei-wei, LIU Cun-bao, LONG Qiong, CHU Xiao-jie, MA Yan-bing. Presentation of HPV 16L1 Peptide-based HBcAg Virus-like Particle and Induction of Specific Antibody. China Biotechnology, 2017, 37(3): 58-64.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170308 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I3/58

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