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  

Construction and characterization of an attenuated recombinant Listeria monocytogenes vector vaccine delivering HPV16 E7

作　　者：贾艳艳 殷月兰 谈卫军 段斐斐 潘志明 陈祥 焦新安 

机构地区：[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

出　　处：《生物工程学报》2016年第5期683-692,共10页Chinese Journal of Biotechnology

基　　金：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~~

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。Listeria monocytogenes（L. monocytogenes, LM） is an excellent tumor vaccine vector. In this study, recombinant LM vaccine candidate expressing human papillomavirus type 16（HPV16） E7 protein was constructed and its charactericts were determined. Through homologous recombination, E7 gene was cloned in frame with the LM4 Phly promoter-signal sequence, and introduced into the chromosome of LM4. The recombinant strain named LM4△hly：：E7 with the plasmid-free and antibiotic-resistant gene-free was constructed. LM4△hly：：E7 could express and secrete E7-LLO fusion protein; its size is 66 k Da and has immunological activity. Furthermore, LM4△hly：：E7 could multiply in RAW264.7 macrophages by confocal laser scanning microscope. Additionally, LM4△hly：：E7 could induce specific antibodies against E7 in immunized mice in ELISA. Also, the 50% lethal dose（LD50） of LM4△hly：：E7 strain was 3.863×10^9 CFU（Colony-Forming Units） in C57BL/6 mice with intraperitoneal immunization, which was more attenuated than wild type LM4. Mice immunized with LM4△hly：：E7 did not show obvious pathological change. These data show that LM4△hly：：E7 expressing E7-LLO fusion protein has good safety, which may provide the materials for research of antitumor effect and would be a promising vaccine candidate for cervical cancer.

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 

分 类 号：R730.51[医药卫生—肿瘤]