表达E7蛋白的重组减毒李斯特菌在小鼠宫颈癌模型中的治疗效果  

Therapeutic Effect of Recombinant Attenuated Listeria monocytogenes Expressing E7 Protein in a Mouse(Mus musculus)Model of Cervical Cancer

作　　者：潘晓欣 崔明珠 罗亚如 孙静 程昌勇 王晶 宋厚辉 PAN Xiao-Xin;CUI Ming-Zhu;LUO Ya-Ru;SUN Jing;CHENG Chang-Yong;WANG Jing;SONG Hou-Hui(Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province,Zhejiang Provincial Engineering Laboratory for Animal Health Inspection&Internet Technology,Zhejiang International Science and Technology Cooperation Base for Veterinary Medicine and Health Management,China-Australia Joint Laboratory for Animal Health Big Data Analytics,College of Animal Science and Technology&College of Veterinary Medicine of Zhejiang A&F University,Hangzhou 311300,China)

机构地区：[1]浙江农林大学动物科技学院·动物医学院/浙江省畜禽绿色生态健康养殖应用技术研究重点实验室/动物健康互联网检测技术浙江省工程实验室/动物医学与健康管理浙江省国际科技合作基地/中澳动物健康大数据分析联合实验室,杭州311300

出　　处：《农业生物技术学报》2024年第5期1206-1215,共10页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(LY23C180002);国家自然科学基金(31972648,32172849);浙江省属高校基本科研业务费专项资金(2020KJ004);宁波市公益类科技项目(2022S006)。

摘　　要：单核细胞增多性李斯特菌(Listeria monocytogenes)是一种能在细胞间迁徙繁殖并能促进抗原特异性CD4^(+)和CD8^(+)T细胞增殖产生适应性细胞免疫的优质候选疫苗活载体。本课题组发现新型减毒李斯特菌(attenuated Listeria monocytogenes,LAM)较野生型李斯特菌的溶血能力和增殖能力均显著降低的同时仍可正常分泌李斯特菌溶血素O(Listeriolysin O,LLO)蛋白。为了研究LAM的安全性及其对宫颈癌特异性抗原E7蛋白的递送与免疫效果,本研究在小鼠(Mus musculus)模型上检测了LAM的毒力,并对LAM-E7重组菌株在小鼠宫颈癌免疫治疗和预防中的作用进行了评价。结果显示,LAM对小鼠的毒力是野生型李斯特菌毒力的1/8709,且小鼠脏器中的LAM可在一周内被清除。本研究通过同源重组的方法将E7基因整合到LAM基因组上,PCR鉴定结果显示,LAM-E7菌株构建成功。Western blot结果显示,E7蛋白能够与LLO在重组菌株中融合表达并释放。通过对LAM-E7的体外生长能力和溶血活性分析发现,融合后对LAM的生长能力和溶血能力无显著影响,表明E7蛋白的融合不会影响LAM的安全性。通过腹部皮下注射方式构建小鼠宫颈癌模型发现,LAM-E7组小鼠肿瘤平均体积(161.1202 mm^(3))显著小于LAM组(349.4625 mm^(3))和PBS组(512.4149 mm^(3))(P<0.01),说明接种LAM-E7对肿瘤生长具有显著的抑制作用。经免疫预防试验发现,LAM-E7组小鼠肿瘤平均体积(3.86 mm^(3))显著小于LAM组(172.67 mm^(3))和PBS组(57.38 mm^(3))(P<0.001),表明LAM-E7对宫颈癌有一定的预防效果。本研究证实LAM-E7对小鼠宫颈癌具有良好的治疗和预防效果,为减毒李斯特菌作为疫苗载体用于宫颈癌治疗提供了新的思路,也为今后宫颈癌治疗疫苗的研发提供数据支持。Listeria monocytogenes is a high-quality vaccine candidate live vector that can migrate between cells and stimulate the body to promote the proliferation of antigen-specific CD4^(+) and CD8^(+)T cells to produce adaptive cellular immunity.This research group found that attenuated Listeria monocytogenes(LAM)had significantly lower hemolytic and proliferative ability than wild-type Listeria.At the same time,LAM could normally secrete Listeriolysin O(LLO)protein.In order to study the safety of LAM and its delivery and immune effect on the cervical cancer specific antigen E7 protein,this study tested the virulence of LAM in a mouse(Mus musculus)model,and to evaluate the role of attenuated Listeria monocytogenes-E7(LAM-E7)in the immunotherapy and prevention of cervical cancer in mice.The results showed that the virulence of LAM to mice was 1/8709 of that of wild-type Listeria,and LAM could be eliminated from the organs of mice within a week.In this study,E7 gene was integrated into LAM genome by homologous recombination method.PCR identification results showed that LAM-E7 strain was successfully constructed,and Western blot results showed that E7 protein could be fused with LLO and expressed and released in the recombinant strain.The analysis of the growth ability and hemolytic activity of LAM-E7 in vitro showed that the fusion had no significant effect on the growth ability and hemolytic ability of LAM,indicating that the fusion of E7 protein would not affect the safety of LAM.In the cervical cancer treatment trial,the average tumor volume of the LAM-E7 group(161.1202 mm^(3))was notably smaller than that of the LAM group(349.4625 mm^(3))and the PBS group(512.4149 mm^(3))(P<0.01),indicating a significant inhibitory effected on tumor growth.The immunoprevention test showed that the average tumor volume of the LAM-E7 group(3.86 mm^(3))was significantly smaller than that of the LAM group(172.67 mm^(3))and the PBS group(57.38 mm^(3))(P<0.001),demonstrating the preventive effect of LAM-E7 on cervical cancer.LAM-E7 has demo

关 键 词：减毒李斯特菌 宫颈癌 免疫治疗 载体疫苗 

分 类 号：S857.4[农业科学—临床兽医学]