减毒单增李斯特菌具有成为疫苗活载体的潜力，可同时引起MHCⅠ和MHCⅡ类抗原递呈系统，具有强烈激发CD8+和CD4+细胞免疫的能力。构建毒力基因缺失菌株进而评估其生物活性对于其作为疫苗活载体的开发尤为重要。本文采用同源重组技术构建单缺失菌株Lm-△actA和双缺失菌株Lm-△actA△/inlB ，从生长状态、毒力基因表达和对HepG2细胞侵袭能力方面探讨减毒菌株生物学特性。生长活性测定显示两种减毒菌株和野生型Lm（EGD-e）三者生长状况无差异；实时定量PCR结果显示actA基因缺失后，inlA基因表达水平上升两倍；actA和inlB基因双缺失后，plcB和hly基因的表达水平都有较大幅度的上升；侵袭HepG2细胞结果显示actA基因缺失后侵袭能力增强、actA和inlB基因共同缺失后侵袭能力减弱。因此，减毒菌株的构建及其生物特性研究不仅对食源性致病菌Lm致病机理具有重要意义，也为构建预防人类和动物疫病的疫苗载体奠定了基础。

Attenuated Listeria monocytogenes (Lm) has the potential to become a live vaccine vector. It can also produce major histocompatibility complex (MHC)Ⅰ and MHC Ⅱ antigen presentation systems, and significantly stimulate immunity in cluster of differentiation (CD)8+ and CD4+ cells. The construction of avirulent gene deletion strains and the subsequent evaluation of their biological activity are crucial for the development of live vaccine vectors. Homologous recombination technology was used to construct Lm-?actA and Lm-?actA/?inlB strains, and the biological activities of attenuated strains were explored in terms of growth, virulence gene expression, and the capacity to invade HepG2 cells. The results showed that no difference in growth was found among the attenuated strains Lm-?actA, Lm-?actA/?inlB, and wild type Lm (EGD-e). Real time quantitative polymerase chain reaction (PCR) results showed that inlA expression was enhanced two-fold after the actA gene was deleted. The expression of plcB and hly genes was significantly elevated when actA and inlB genes were simultaneously deleted. The HepG2 cell invasion assay showed that the invasive capacity was enhanced after the actA gene was deleted and was weakened after deleting actA and inlB genes. Therefore, the construction of attenuated strains and study of their biological properties is not only important for understanding the pathogenesis of food-borne Lm, but also lays a foundation for the construction of vaccine vectors for the prevention of human and animal diseases.

国家自然科学基金项目(31371776);上海市研究生教育创新计划