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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); 江苏高校优势学科建设工程项目资助;

Construction and characterization of attenuated recombinant Listeria monocytogenes delivering HPV16 E7

Yanyan Jia1 Yuelan Yin1 Weijun Tan1 Feifei Duan1 Zhiming Pan1 Xiang Chen1 Xin′an Jiao1

(1.Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, Jiangsu, China 225009)

【Abstract】 Listeria monocytogenes (LM) is an excellent tumor vaccine vector. In this study, recombinant LM expressing human papillomavirus type 16 (HPV16) E7 protein, a vaccine candidate was constructed and the biological characteristics were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 hly promoter-signal sequence, and introduced into the LM4 genome. The recombinants, constructed as LM4△hly::E7, were plasmid- and antibiotic-resistant gene-free. LM4△hly::E7 could express and secrete a 66 kDa E7-LLO fusion protein, which had immunological activities. Proliferation of LM4△hly::E7 in RAW264.7 macrophages was observed under confocal laser scanning microscope. Specific anti-E7 antibodies could be detected by ELISA in mice immunized with LM4△hly::E7. The LD50 of LM4△hly::E7 was 3.863 × 109 CFU in C57BL/6 mice with intraperitoneal immunization, which was 104 times higher than that of wild type LM4. No obvious pathological changes were detected in the immunized mice. These results suggest that LM4△hly::E7 expressing E7-LLO fusion protein may be a safe and promising vector-vaccine candidate for cervical cancer, and may also be important biological materials for anti-tumor research.

【Keywords】 attenuated Listeria monocytogenes; HPV16 E7; macrophage cell line; safety;

【DOI】

【Funds】 National Natural Science Foundation of China (Nos. 31472193, 31101841); Priority Academic Program Development of Jiangsu Higher Education Institutions;

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 05, Pages 683-692

May 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and analyses
  • 3 Discussion
  • References