减毒李斯特菌载体运送HPV16 E7基因重组疫苗的构建及其生物学特性

贾艳艳1 殷月兰1 谈卫军1 段斐斐1 潘志明1 陈祥1 焦新安1

(1.扬州大学江苏省人兽共患病重点实验室江苏省动物重要疫病和人兽共患病防控协同创新中心, 江苏扬州 225009)
【知识点链接】吞噬细胞; 吞噬体; 溶血素; CD4; 穿梭载体

【摘要】单核细胞增生李斯特菌(Listeria monocytogenes,LM)是一种理想的肿瘤疫苗载体,本文旨在构建表达人乳头瘤状病毒(HPV)16型E7蛋白的减毒重组李斯特菌疫苗候选株并分析其生物学特性。利用同源重组技术将HPV16 E7基因定点整合至LMhly基因信号肽序列下游,获得减毒重组李斯特菌LM4△hly::E7,并对该重组菌进行生物学特性研究。实验结果表明,重组菌能够分泌表达具有免疫学活性的E7-LLO融合蛋白,大小约66 k Da;通过激光共聚焦扫描显微镜观察到重组菌能够在RAW264.7细胞胞内增殖。ELISA测定结果显示减毒重组菌能够诱导小鼠产生E7特异性抗体。重组菌在C57BL/6小鼠中LD50为3.863×10~9 CFU,低于亲本株4个数量级,通过组织切片观察到重组菌对小鼠肝脏及脾脏无明显病理变化。以上表明,本研究构建的分泌表达E7-LLO融合蛋白的减毒重组李斯特菌具有较好的安全性,为下一步研究其抗肿瘤作用提供了材料,并为研发宫颈癌的免疫治疗型候选疫苗奠定重要基础。

【关键词】 减毒单核细胞增生李斯特菌; 人乳头瘤状病毒16型E7蛋白; 巨噬细胞系; 安全性;

【DOI】

【基金资助】 国家自然科学基金(Nos.31472193,31101841) National Natural Science Foundation of China(Nos.31472193,31101841) 江苏高校优势学科建设工程项目资助 Priority Academic Program Development of Jiangsu Higher Education Institutions

Download this article

    References

    [1]Bruni L,Diaz M,CastellsaguéX,et al.Cervical human papillomavirus prevalence in 5 continents:meta-analysis of 1 million women with normal cytological findings.J Infect Dis,2010,202(12):1789–1799.

    [2]Ferlay J,Shin HR,Bray F,et al.Estimates of worldwide burden of cancer in 2008:GLOBOCAN2008.Int J Cancer,2010,127(12):2893–2917.

    [3]Zhang HY,Fei MD,Jiang Y,et al.The diversity of human papillomavirus infection among human immunodeficiency virus-infected women in Yunnan,China.Virol J,2014,11(1):202,doi:10.1186/s12985-014-0202-3.

    [4]Honegger A,Schilling D,Bastian S,et al.Dependence of intracellular and exosomal micro RNAs on viral E6/E7 oncogene expression in HPV-positive tumor cells.PLo S Pathog,2015,11(3):e1004712,doi:10.1371/journal.ppat.1004712.

    [5]Zacapala-Gómeza AE,Del Moral-Hernándeza O,Villegas-Sepúlveda N,et al.Changes in global gene expression profiles induced by HPV16 E6oncoprotein variants in cervical carcinoma C33-A cells.Virology,2016,488:187–195.

    [6]Mitchell G,Ge L,Huang QY,et al.Avoidance of autophagy mediated by Plc A or Act A is required for Listeria monocytogenes growth in macrophages.Infect Immun,2015,83(5):2175–2184.

    [7]Pillich H,Chakraborty T,Mraheil MA.Cell-autonomous responses in Listeria monocytogenes infection.Future Microbiol,2015,10(4):583–597.

    [8]Rothman J,Paterson Y.Live-attenuated Listeria-based immunotherapy.Expert Rev Vaccines,2013,12(5):493–504.

    [9]Wood LM,Paterson Y.Attenuated Listeria monocytogenes:a powerful and versatile vector for the future of tumor immunotherapy.Front Cell Infect Microbiol,2014,4:51,doi:10.3389/fcimb.2014.00051.

    [10]Lin QQ,Zhou MY,Xu ZK,et al.Construction of two Listeria ivanovii attenuated strains expressing Mycobacterium tuberculosis antigens for TB vaccine purposes.J Biotechnol,2015,196–197:20–26.

    [11]Abdallah DS,Bitar PA,Oliveira F,et al.A Listeria monocytogenes-based vaccine that secretes sand fly salivary protein LJM11 confers long-term protection against vector-transmitted Leishmania major.Infect Immun,2014,82(7):2736–2745.

    [12]Kang MQ,Cai XX,Tan WJ,et al.Construction and characterization of Listeria monocytogenesΔhfq mutant strain.Acta Microbiol Sin,2015,55(4):433–439(in Chinese).

    [13]Yin YL,Tian DB,Jia YY,et al.Attenuated Listeria monocytogenes,a Mycobacterium tuberculosis ESAT-6 antigen expression and delivery vector for inducing an immune response.Res Microbiol,2012,163(8):540–549.

    [14]Zamoiskii EA.Evaluation of Reed-Muench method in determination of activity of biological preparations.Zh Mikrobiol Epidemiol Immunobiol,1956,27(1):77–83.

    [15]Kennedy GT,Judy BF,Bhojnagarwala P,et al.Surgical cytoreduction restores the antitumor efficacy of a Listeria monocytogenes vaccine in malignant pleural mesothelioma.Immunol Lett,2015,166(1):28–35.

    [16]Jia YY,Yin YL,Duan FF,et al.Prophylactic and therapeutic efficacy of attenuated Listeria monocytogenes-based vector vaccine delivering HPV16 E7 antigen in a murine model.Int J of Mol Med,2012,30(6):1335–1342.

    [17]van Pijkeren JP,Morrissey D,Monk IR,et al.A novel Listeria monocytogenes-based DNA delivery system for cancer gene therapy.Hum Gene Ther,2010,21(4):405–416.

    [18]Keenan BP,Saenger Y,Kafrouni MI,et al.A Listeria vaccine and depletion of T-regulatory cells activate immunity against early stage pancreatic intraepithelial neoplasms and prolong survival of mice.Gastroenterology,2014,146(7):1784–1794.e6

    [19]Hannan R,Zhang HG,Wallecha A,et al.Combined immunotherapy with Listeria monocytogenes-based PSA vaccine and radiation therapy leads to a therapeutic response in a murine model of prostate cancer.Cancer Immunol Immunother,2012,61(12):2227–2238.

    [20]Le DT,Brockstedt DG,Nir-Paz R,et al.A live-attenuated Listeria vaccine(ANZ-100)and a live-attenuated Listeria vaccine expressing mesothelin(CRS-207)for advanced cancers:phase1 studies of safety and immune induction.Clin Cancer Res,2012,18(3):858–868.

    [21]Walls ZF,Goodell S,Andrews CD,et al.Mutants of listeriolysin O for enhanced liposomal delivery of macromolecules.J Biotechnol,2013,164(4):500–502.

    [22]Hernández-Flores KG,Vivanco-Cid H.Biological effects of listeriolysin O:implications for vaccination.Biomed Res Int,2015,2015:360741–360749.

    [23]Seveau S.Multifaceted activity of listeriolysin O,the cholesterol-dependent cytolysin of Listeria monocytogenes.Subcell Biochem,2014,80:161–195.

    [24]Neeson P,Pan ZK,Paterson Y.Listeriolysin O is an improved protein carrier for lymphoma immunoglobulin idiotype and provides systemic protection against 38C13 lymphoma.Cancer Immunol Immunother,2008,57(4):493–505.

    [25]Hamon MA,Ribet D,Stavru F,et al.Listeriolysin O:the Swiss army knife of Listeria.Trends Microbiol,2012,20(8):360–368.

This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 683-692

May 2016

Downloads:1

Share
Article Outline

知识点

摘要

  • 1 材料与方法
  • 2 结果与分析
  • 3 讨论
  • 参考文献