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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 (LM) is an ideal tumor vaccine vector, and the aim of this paper is to construct an attenuated recombinant Listeria vaccine candidate expressing human papillomavirus (HPV) type 16 E7 protein Analysis of its biological characteristics. The homologous recombination technique was used to integrate the HPV16 E7 gene downstream of the LMhly gene signal peptide sequence to obtain an attenuated recombinant Listeria monocytogenes LM4Δhly :: E7. The biological characteristics of the recombinant strain were studied. The results showed that the recombinant strain secreted E7-LLO fusion protein with an immunological activity of about 66 kDa. The recombinant bacteria could proliferate in RAW264.7 cells by laser confocal scanning microscopy. The results of ELISA showed that attenuated recombinant bacteria could induce mice to produce E7 specific antibodies. The LD50 of recombinant bacteria was 3.863 × 10 ~ 9 CFU in C57BL / 6 mice, which was 4 orders of magnitude lower than that of the parent strain. The histological sections of the recombinant bacteria showed no significant pathological changes in the liver and spleen of mice. The above shows that the constructed Listeria monocytogenes secreting E7-LLO fusion protein has good safety and provides a material for further study on its anti-tumor effect and is an immunotherapy candidate for developing cervical cancer Vaccines lay an important foundation.